Abstracts (complete list) - Wissenschaft Online

Abstracts (complete list) 

Luciana Berod, Oliver Frey, Johannes Norgauer, Thomas Kamradt 

SHIP1 phosphatase controls different aspects of dendritic cell biology 

Natalia Zietara, Marcin Lyszkiewicz, Stefan Lienenklaus, Siegfried Weiss 

"Lack of IFN-β impairs antigen presentation capacity of splenic dendritic cells" 

Hendrik Poeck, Cornelius Maihoefer, Robert Besch, David Anz, Shizuo Akira, Simon Rothenfusser, Veit Hornung, 

Stefan Endres, Thomas Tueting, Gunther Hartmann 

5´triphosphate siRNA as a novel therapeutic tool against tumors 

Hans KUENG, Victoria LEB, Daniela HAIDERER, Graca RAPOSO, Clotilde THERY, Klaus SCHMETTERER, Alina 

NEUNKIRCHNER, Christian SILLABER, Brian SEED, Winfried PICKL 

A General Strategy for Decoration of Enveloped Viruses with Functionally Active Lipid-Modified 

Cytokines 

Claudia Brandt, Margitta Worm, Ria Baumgrass 

Drug induced modulation of T cell activation and differentiation in atopic dermatitis patients 

Frank Autschbach, Felix Lasitschka, Thomas Giese, Nikolaus Gassler, Benjamin Funke, Jutta Schroeder-Braunstein, Ulf 

Brunnemer, Stefan C. Meuer, Bernd Sido 

Expression of the cystine transporter subunit xCT is a critical determinant of the immune 

response in human inflammatory bowel disease 

Katharina Becher 

In vivo persistence of Anaplasma phagocytophilum after depletion of CD4 + T cells, but not in the 

absence of Th1-type cytokines and effector mechanisms 

Manuela Brenk, Marina Scheler, Helene Wilms, Thomas Bieber, Dagmar von Bubnoff 

Induction of human CD4 + CD25 + Foxp3 + T regulatory cells by tryptophan-deprived tolerogenic 

dendritic cells 

Kristina Röschmann, Artur J. Ulmer, Torsten Goldmann, Arnd Petersen 

Interaction of Timothy grass pollen major allergen Phl p 1 with human respiratory epithelial 

cells 

Andreas Brandl, Juergen Wittmann, Michael Boesl, Hans-Martin Jaeck 

MicroRNA-Mediated Control of B Cell Activation 

Savita Nair, Ulf Dittmer 

Role of CD4 + T cells in acute Friend Retroviral infections 

Alexia Nass, Hans-Willi Mittruecker, Alf Hamann, Jochen Huehn 

Transfer of in vivo operating inflammation-specific Tregs does not interfere with the host´s 

response against bacterial infection 

Björn Stork, Ingo Goldbeck, Konstantin Neumann, Thilo Kähne, Michael Naumann, Michael Engelke, Jürgen Wienands 

‘Membrane zip codes’ for Grb2 determine differential Ca2+ signaling in B lymphocytes 

Stefanie Kunz, Karin Oberle, Anna Sander, Christian Bogdan, Ulrike Schleicher 

Bartonella-Induced Cat Scratch Disease-like Lymphadenopathy in Mice Involves Cell 

Proliferation and Altered Lymphocyte Recruitment 

Georg Peschel, Nuria Rodriguez, Christine Cirl, Nina Wantia, Tanja Erl, Susi Dürr, Hermann Wagner, Thomas Miethke 

Chlamydophila pneumoniae follows different strategies to downregulate surface MHC II 

expression depending on the cell type 

Annabelle Schnaith, Sonja A. Leggio, Martin Krönke, Oleg Krut

Staphylococcus aureus Subverts Autophagy for Induction of Caspase-independent Host Cell 

Death 

Stella Eugenie Autenrieth, Dirk Middendorf, Ingo Birger Autenrieth 

Yersinia enterocolitica induces cell death in splenic dendritic cells: evidence for YopP dependent 

and independent mechanisms 

Heribert Appelhans, Michael Walther, Andrea Friße, Michael A. Harvey, Stephen A. Judice, Brett A. Stillman, Hans 

Peter Seelig 

A CE-Certified Autoimmune Disease Diagnostic Protein Microarray 

Martina Papadopoulos, Frank Momburg 

A complex sequence motif of tapasin stabilizes the TAP2 protein 

Andreas Oliver Weinzierl, Dominik Maurer, Florian Altenberend, Nicole Schneiderhan-Marra, Karin Klingel, Oliver 

Schoor, Thomas Joos, Hans-Georg Rammensee, Stefan Stevanovic 

A cryptic VEGF T-cell epitope: Identification and characterization by mass spectrometry and Tcell 

assays 

Susanne Tartz, Holger Ruessmann, Jana Kamanova, Peter Sebo, Angelika Sturm, Volker Heussler, Bernhard Fleischer, 

Thomas Jacobs 

A heterologous prime/boost immunization using recombinant Salmonella and Bordetella 

adenylate cyclase induces complete protection against P. berghei malaria 

Nousheen Zaidi, Timo Herrmann, Daniel Baechle, Sabine Schleicher, Jeannette Gogel, Christoph Driessen, Wolfgang 

Voelter, Hubert Kalbacher 

A new approach for distinguishing cathepsin E and D activity in antigen processing organelles 

Wiebke Hartmann, Markus M. Simon, Bernhard Fleischer, Simone Korten 

A new role for granzymes: Suppression of defence against helminths 

Elisa Kieback, Jehad Charo, Daniel Sommermeyer, Thomas Blankenstein, Wolfgang Uckert 

A new safeguard eliminates TCR gene-modified autoimmune-reactive T cells after adoptive 

therapy 

Matthias Bros, Frank Jährling, Andrea Renzing, Nadine Wiechmann, Ngoc-Anh Dang, Arne Sutter, Ralf Ross, Jürgen 

Knop, Stephan Sudowe, Angelika B. Reske-Kunz 

A newly established murine immature dendritic cell line exerts tolerogenic function at its 

immature state and upon alternative activation in the presence of glucocorticoid 

Iris Watermann, Jeannette Gerspach, Matthias Lehne, Klaus Pfizenmaier, Harald Wajant 

A novel CD95L-Prodrug Fusion Protein with tumorselective antitumoral activity 

Ines Gütgemann, Jasmin Teresa Ney, Qi Zhou, Christian Kurts, Hubert Schorle, Andreas Limmer, Martina Berg, 

Reinhard Büttner 

A novel model of antigen recognition in a hepatic tumor microenvironment: c-myc/OVA x 

LAPtTA transgenic mice 

Peggy Riese, Thomas Ebensen, Claudia Link, Kai Schulze, Michael Morr, Carlos Guzmán 

A pegylated derivative of alpha galactosylceramide exhibits improved immune stimulatory 

activities 

Eva Schlosser, Carolina Otero, Benedikt Kessler, Mariola Edelmann, Rene Brunisholz, Daniel Legler, Marcus Groettrup 

A prostate carcinoma antigen reveals a new cytosolic class I processing pathway for 

endoplasmic reticulum targeted proteins 

Mathias Konstandin, Guido Wabnitz, Hülya Aksoy, Martin Klemke, Thomas Dengler, Yvonne Samstag 

A sensitive assay for the quantification of integrin-mediated adhesiveness of human stem cells 

and leukocyte subpopulations in whole blood 

Heiko Bruns, Frank Stegelmann, Steffen Stenger 

Abl-tyrosine kinase modulates immune responses to Mycobacterium tuberculosis

Angelika Stöcklinger 

Ablation of Epidermal Langerhans Cells has no Impact on Gene-Gun mediated Immune 

Responses 

Brigitte Santner-Nanan, Nabila Seddiki, Cindy Zhu, Verena Quent, Anthony Kelleher, Barbara Fazekas de St. Groth, 

Ralph Nanan 

Accelerated age-dependent transition of human regulatory T cells to effector memory phenotype 

Jasmin Herz, Julian Pardo, Hamid Kashkar, Erik Bos, Reinhard Wallich, Peter J. Peters, Elmon Schmelzer, Martin 

Krönke, Markus M. Simon, Olaf Utermöhlen 

Acid sphingomyelinase is a critical regulator of cytotoxic granule secretion by primary T 

lymphocytes 

Manuela Rossol, Undine Meusch, Holm Häntzschel, Sunna Hauschildt, Ulf Wagner 

Activated CD4 T cells induce TNF production in human monocytes via reverse signalling of 

membrane TNF 

Milena Josefina Tosiek, Marcus Gereke, Jan Buer, Dunja Bruder 

Activation and regulation of autoreactive CD8 T cells in the lung 

Xuefang Ren, Marion Schneider, Ying Wang, Lin Xu, Zhenggang Jiang, Fei Ye 

Activation induced cell death of CD4+CD25+Foxp3+ T cells by TCR re-stimulation involves a 

CD95/CD95L dependent mechanism 

Alexei Gratchev, Julia Kzhyshkowska, Sheila Kannookadan, Miriam Ochsenreiter, Anna Popova, Xiaolei Yu, Isabelle 

Muller-Molinet, LiMing Gooi, Sergij Goerdt 

Activation of a TGF-β-specific multistep gene expression program in mature M2 macrophages 

requires glucocorticoid-mediated surface expression of TGF-β receptor II 

Umme Amara, Miriam Kalbitz, Andreas Klos, Markus Huber-Lang 

Activation of Complement by the Coagulation System 

Luisa Cervantes-Barragan, Ulrich Kalinke, Roland Züst, Constantino Lopez-Macias, Volker Thiel, Burkhard Ludewig 

Activation of myeloid cells through plasmacytoid dendritic cell-derived type I interferon secures 

control of murine coronavirus infection 

Alexander Donald McLellan, Patrizia Stoitzner, Jacquie Harper, Sarah Saunderson, Ralph Jack, Anthea Bouwer 

Activation of natural killer cells by dendritic cells stimulated with gram positive bacteria 

Dimitra Kotsougiani, Birgit Prior, Gertrud Maria Hänsch, Christof Wagner 

Activation of T lymphocytes in bacterial infection: production of interferon 4gamma, expression 

of the chemokine receptor CXCR6, and up-regulation of Toll-like receptors (TLR) in patients with 

implant-associated posttraumatic osteomyelitis 

Eva Distler, Catherine Wölfel, Sylvia Köhler, Marion Nonn, Elke Schnürer, Ralf G. Meyer, Christoph Huber, Thomas 

Wölfel, Udo F. Hartwig, Wolfgang Herr 

Acute myeloid leukemia (AML)-reactive cytotoxic T lymphocyte clones rapidly expanded from 

CD8+ CD62L(high)+ T cells of healthy donors prevent AML engraftment in NOD/SCID IL2Rγnull 

mice 

Sarvari Velaga, Stephan Halle, Sabrina Dähne, Oliver Pabst 

Adaptation of Solitary Intestinal Lymphoid Tissue in response to microbial stimulation 

Nancy Brewig, Adrien Kissenpfennig, Bernard Malissen, Alexandra Veit, Bernhard Fleischer, Uwe Ritter 

Adaptive immune response to Leishmania parasites is induced in the absence of epidermal 

Langerhans cells 

Gaubert Sophie, Zimmermann Andra, Arbach Olga, Rossa Simone, Thiel Andreas, Voigt Sebastian, Ebell Wolfram 

Adenovirus-specific T cell therapy in paediatric stem cell transplants: Isolation and expansion of 

donor-derived T lymphocytes

Iris Bellinghausen, Barbara Häringer, Beatrice Lafargue, Tamara Hilmenyuk, Heinz Decker, Joachim Saloga 

Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 

3. 

Ingrid Schuster, Elfriede Eppinger, Christoph Salat, Bernhard Frankenberger, Joachim Ellwart, Elfriede Nößner, Dirk 

Busch, Angela Krackhardt 

Allorestricted T cells with high specificity for the FMNL1-derived peptide PP2 show tumor 

reactivity but also limited alloreactivity against other MHC alleles – implications for adoptive T 

cell therapy using allorestricted T cell receptors 

Andrey Bogdanov, Tatyana Rybakova, Nikolai Belyaev 

Alpha-fetoprotein impair recovery of cell-mediated immunity normal function after tumor 

ablation 

Connie Schulze, Petra Heyder, Sandra Franz, Kerstin Sarter, Luis Munoz, Stefan Pöhlmann, Hanns-Martin Lorenz, 

Martin Herrmann, Martin Schiller 

Alteration of glycocalyx and exposition of mannose residues during apoptosis are essential for 

the clearance of apoptotic marterial 

Sabine Höpner, Katharina Dickhaut, Jamina Eckhard, Shashank Gupta, Kirsten Falk, Olaf Rötzschke 

Amplification of CD4 T cell responses by catalysing antigen-loading through MHC-loading 

Enhancer (MLE) 

Stefanie Scheu, Philipp Dresing, Richard M. Locksley 

An IFNβ Reporter Mouse Model for the Visualization of the Initiation of the Type I Interferon 

Response in vivo 

Frank Schmitz, Antje Heit, Tobias Haas, Hermann Wagner 

An mTOR dependent transport mechanism of cytosolic receptors licenses TLR-independent 

recognition of nucleic acids 

Linda Sender, Zoe Waibler, Camilla Merten, Roland Hartig, Matthias Gunzer, Peter Reichardt, Ulrich Kalinke, Burkhart 

Schraven 

An unusual signalling signature suggests a molecular basis for the adverse side effects of antihuman 

CD28 superagonistic antibodies 

Lorena Martinez Gamboa, Henrik Mei, Karin Reiter, Kristin Kemnitz, Arne Hansen, Florian Emmerich, Abdulgabar 

Salama, Thomas Dörner 

Analysis of B cell subpopulations in patients with autoimmune thrombocytopenia: effects of 

splenectomy and implications for therapy 

Timo Lischke, Andreas Hutloff, Richard A. Kroczek 

ANALYSIS OF CD4+ T CELL IMMUNITY IN VIVO 

Katja Maurer, Ellen Harrer, Andreas Goldwich, Kathrin Eismann, Silke Bergmann, Birgit Schätz, Sandra Müller, Thomas 

Harrer 

Analysis of CTL mediated immune selection in a dominant HLA B8-restricted CTL Epitope in Nef 

Simone Wüst, Denise Tischner, Anna Kleimann, Ralf Gold, Jan P. Tuckermann, Holger M. Reichardt, Fred Lühder 

Analysis of Glucocorticoid action in Experimental Autoimmune Encephalomyelitis (EAE) 

Anett Schulz, Manuela Rossol, Matthias Pierer, Sybille Arnold, Holm Häntzschel, Christoph Baerwald, Ulf Wagner 

Analysis of polymorphisms in the TNFR2 gene in rheumatoid arthritis and possible functional 

relevance 

Sonja Kothlow, Benjamin Schusser, Nicola Penski, Georg Kochs, Peter Staeheli, Bernd Kaspers 

Analysis of potential antiviral Mx activity in the chicken 

Andreas Jeron, Susanne Pfoertner, Tanja Toepfer, Jan Buer, Robert Geffers, Andres J Schrader 

Analysis of regulatory T cells in renal cell carcinoma 

Nicole Warnecke, Burkhart Schraven, Luca Simeoni

Analysis of TCR-mediated MAPK activation in primary T cells. 

Eva Billerbeck, Hubert E Blum, Robert Thimme 

Analysis of virus specific FoxP3+ regulatory CD8+ T cells 

Julia Hoffmann, Ralf-Holger Voss, Ruth Frommolt, Matthias Theobald, Udo Hartwig 

Analyzing GVL-immune responses of p53-specific CD8+ cytotoxic T cells to leukemia cell lines 

and acute myeloid leukemia in vitro and in vivo in humanized NOD/SCID/IL-2Receptor gammachain 

null mice 

Heiko Weyd, Lucie Doerner, Andrea Mahr, Nadine Eberhardt, Dagmar Riess, Bjoern Linke, Christine S. Falk, Peter H. 

Krammer 

Annexin I – an anti-inflammatory signal on the surface of apoptotic cells 

Nicole Gerlach, Cassandra James, Ulf Dittmer 

Anti-retroviral effects of type I interferon subtypes in vivo 

Christina Stöckle, Timo Burster, Thomas Rückrich, Alexander Beck, Christoph Driessen, Arthur Melms, Eva Tolosa 

Antigen Processing in the Human Thymus 

Patrick C. Rämer, Susanne Haemmerling, Mathias H. Konstandin, Thomas Giese, Thomas J. Dengler, Sivanandam 

Vijayshankar 

Antigen-presenting capacity and T cell costimulation of endothelial progenitor cells is 

comparable to monocytes 

Marcela Fajardo-Moser, Christine Hambrecht, Heidrun Moll 

Antigen-pulsed dendritic cell-derived exosomes as cell-free vaccines against infection 

Christian Schütz, Andreas Mackensen, Jonathan P. Schneck, Jürgen Schölmerich, Mathias Oelke, Martin Fleck 

Antigen-specific CD8+ T cell depletion mediated by apoptosis-inducing HLA-A2-Ig based 

artificial APCs 

Heike Koehler, Andreas Hombach, Hinrich Abken 

Antigen-specific T cell activation is repressed by TGF-b which can be overcome by CD28 

costimulation 

Nonsikelelo Mpofu, Konstantinos Iordanidis, Matthias Hardtke-Wolenski, Micheal. P Manns, Elmar Jaeckel 

Antigen-specific, Foxp3 transduced T cells for therapy of type 1 diabetes 

Anjana Singh, Miri Blank, Yehuda Shoenfeld, Harald Illges 

Antiphospholipid syndrome patients display reduced titers of soluble CD21 in their sera 

irrespective of circulating anti-beta-2-glycoprotein-I autoantibodies. 

Stefan Lienenklaus, Marcin Lyszkiewicz, Jadwiga Jablonska, Siegfried Weiss 

ANTIVIRAL AND ILLUMINATED A NEW MOUSE LINE TO MONITOR β-INTERFERON INDUCTION 

Carola Pongratz, Benjamin Yazdanapanah, Hamid Kashkar, Martin Kroenke 

Antiviral implications of a convertible HIV-1 directed siRNA library 

Cosima Kretz, Bartlomiej Berger, Lucie Dörner, Heiko Weyd, Ingo H. Tarner, Ulf Müller-Ladner, Hanns-Martin Lorenz, 

Peter H. Krammer, Annegret Kuhn 

Apoptosis in Systemic Lupus Erythematosus: Influence on Immune Response and Peripheral 

Tolerance 

Katharina Randers, Telja Pursche, Doreen Finke, Kirsten Jacobsen, Robert Hoerster, Christian Brockmann, Holger 

Hennig, Tony Marion, Sigfried Goerg 

Apoptotic DNA is able to induce in vivo IFN α production within the splenic marginal zone via a 

TLR dependent pathway. 

Katharina Weibhauser, Bernd Kaspers, Sonja Kothlow

APPLICATION OF THE RCAS RETROVIRAL VECTOR SYSTEM FOR FUNCTIONAL IN VIVO STUDIES 

OF THE CYTOKINE BAFF IN THE CHICKEN 

Johanna Oberlies, Carsten Watzl, Thomas Giese, Claudia Luckner, Stefan Meuer, Markus Munder 

Arginine depletion by human granulocyte arginase impairs NK cell function 

Katrin Drögemüller, Martina Deckert, Ulrike Hellmuth, Monika Sakowicz-Burkiewicz, Dirk Reinhold, David Gutmann, 

Werner Müller, Dirk Schlüter 

Astrocyte gp130-expression is critical for astrocyte survival, downregulation of intracerebral 

immune responses and survival of experimental autoimmune encephalomyelitis and Toxoplasma 

encephalitis 

Mihály Józsi, Stefanie Strobel, Hans-Martin Dahse, Wei-shih Liu, Peter F. Hoyer, Martin Oppermann, Christine Skerka, 

Peter F. Zipfel 

Autoantibodies block C-terminus of factor H in atypical hemolytic uremic syndrome 

Martin Schiller, Isabelle Bekeredjian-Ding, Petra Heyder, Norbert Blank, Klaus Heeg, Hanns-Martin Lorenz 

Autoantigens are translocated into apoptotic bodies during apoptosis 

Karin Loser, Sandra Balkow, Kerstin Klimmek, Claus Kerkhoff, Wolfgang Nacken, Thomas A. Luger, Stefan Beissert 

Autoreactive CD8+ T cell development in CD40L-mediated autoimmunity is controlled by S100A8 

and A9 proteins 

Bernd Lepenies, Klaus Pfeffer, Michelle Hurchla, Theresa Murphy, Kenneth Murphy, Juliane Oetzel, Bernhard Fleischer, 

Thomas Jacobs 

B and T lymphocyte attenuator (BTLA) ligation prevents cerebral malaria during P. berghei 

ANKA infection 

Janine Suffner, Kristin Hochweller, Natalio Garbi, Günter Hämmerling 

BAC-transgenic mice for depletion of Foxp3+ regulatory T cells 

Matthias Peiser, Juliana Koeck, Burghardt Wittig, Reinhard Wanner 

Bacterial lipopeptides activate human Langerhans cells via Toll-like receptor 2 

Konrad Bode, Klaus Heeg, Alexander Dalpke 

Bacterial origin histone deacetylase inhibitor butyric acid inhibits dendritic cells 

Marta Rizzi, Ulrich Salzer, Klaus Warnatz, Stefanie Hamm, Sigune Goldacker, Beate Fischer, Hans Hartmut Peter, 

Hermann Eibel 

BAFF Receptor expression in CVID patients 

Jenny Dieckmann, Susanne Rehfeld, Bernd Kaspers, Sonja Kothlow 

BAFF regulates the expression of bcl-2 family members in chicken B cells 

Astrid Karbach, Evelyn Rossmann, Veronique Kitiratschky, Heidelore Hofmann, Markus M. Simon, Peter Kraiczy, 

Reinhard Wallich 

BbCRASP-1 of the Lyme disease spirochetes induces antibodies to nondenatured structural 

determinants in humans 

Evelyn Rossmann, Peter Kraiczy, Pia Herzberger, Christine Skerka, Michael Kirschfink, Markus M. Simon, Peter F. 

Zipfel, Reinhard Wallich 

BhCRASP-1 of the relapsing fever spirochete Borrelia hermsii is a factor H and plasminogen 

binding protein 

Xin Ding, Niklas Beyersdorf, Gregor Blank, Fred Lühder, Kevin Dennehy, Ralf Gold, Thomas Kerkau, Thomas Hünig 

Blockade of CD28-B7 interactions by anti-CD28 antibodies protects from immunopathology in 

vivo 

Tea Gogishvili, Beate Geyer, Susanne Grunewald, Thomas Hünig 

Blockade of CD28-mediated co-stimulation ameliorates allergic airway inflammation in mice 

Mostafa Jarahian, Carsten Watzl, Yasmin Issa, Peter Altevogt, Frank Momburg

Blockade of natural killer cell-mediated lysis of NCAM140 expressed on tumor cells 

Nicole Bethke, Matthias Böthe, Siegfried Kohler, Matthias Niedrig, Andreas Thiel 

Bystander activation of recall antigen-specific CD4+ T-cells during primary immunization with 

live attenuated yellow fever virus 

Olaf Gross, Andreas Gewies, Katrin Finger, Martin Schäfer, Tim Sparwasser, Christian Peschel, Irmgard Förster, Jürgen 

Ruland 

Card9 controls a non-TLR signaling pathway for innate anti-fungal immunity 

Christina Stöckle, Vinod Sommandas, Hubert Kalbacher, Athur Melms, Eleni Adamopoulou, Ekkehard Weber, Christoph 

Driessen, Bernhard Boehm, Eva Tolosa, Timo Burster 

Cathepsin distribution in primary human antigen presenting cells 

Marion Leick, Tanja Hartmann, Susann Ewers, Andrea Diefenbacher, Robert Nibbs, Meike Burger 

CCL5 upregulates surface expression of the orphan atypical chemokine receptor CRAM-A/B in a 

heparin sulphate dependent manner in pre-B acute lymphoblastoid leukemia cells 

Uta Elisabeth Höpken, Susann Winter, Kerstin Krüger, Armin Rehm, Martin Lipp 

CCR7 REGULATES LYMPHOCYTE EGRESS AND RECIRCULATION THROUGH BODY CAVITIES 

Tim Worbs, TR Mempel, J Bölter, UH von Andrian, R Förster 

CCR7-ligands stimulate the intranodal motility of T lymphocytes in vivo 

Matthias Krusch, Tina Baessler, Katrin Miriam Baltz, Helmut Rainer Salih 

CD137 ligand expression on acute myeloid leukemia cells modulates immune surveillance of 

human NK cells identified to express CD137 upon activation 

Holger Hoff, Zulema Cabail, Karin Knieke, Marion Rudolph, Heike Hirseland, Barbara Bröker, Monika Brunner-Weinzierl 

CD152 (CTLA-4) controls CD28-independently cell cycle progression and resistance against 

apoptosis of human T lymphocytes 

J. Kolja Hegel, Pushpa Pandiyan, Paula Kolar, Karin Knieke, Steven L. Reiner, Monika Brunner-Weinzierl 

CD152 (CTLA-4) utilizes Eomesodermin, but not T-bet, for regulating effector function of 

individual CD8 T lymphocytes: Implication for tumor therapy 

Karin Knieke, Holger Hoff, Frank Maszyna, Paula Kolar, J. Kolja Hegel, Alf Hamann, Gudrun F. Debes, Monika Brunner- 

Weinzierl 

CD152 (CTLA-4)- signalling promotes homing to secondary lymphoid organs 

Baerbel Keller, Mirzokhid Rakhmanov, Sylvia Gutenberger, Sigune Goldacker, Dirk Holzinger, Elisabeth Nikolopoulus, 

Paul Fisch, Hermann Eibel, Hans-Hartmut Peter, Klaus Warnatz 

CD21low B Cells Represent a Polyclonally Activated B Cell Population Exposed to Type I 

Interferons 

Isis Ludwig-Portugall, Emma E. Hamilton-Williams, Christian Kurts 

CD25+ regulatory T cells induce peripheral B cell tolerance against non-lymphoid tissue 

autoantigens 

Christian Becker, Tobias Bopp, Jan Kubach, Franz-Josef Schneider, Edgar Schmitt, Helmut Jonuleit 

CD4-mediated, TCR-independent functional activation of human CD4+CD25+ regulatory T cells 

Alexander Donald McLellan, Sarah Charlotte Saunderson, Petra Schuberth, Lilija Miller, Amy Charlotte Dunn, Philippa 

Anne MacKay, Ralph Wilson Jack 

CD40/IL-4-stimulated B cells release exosomes enriched in components of the B cell receptor 

complex. 

Rachid Marhaba, Margot Zöller 

CD44 blocking induces apoptosis by uncoupling Ezrin from the Ras pathway 

Annalena Bollinger, Zane Orinska, Silvia Bulfone-Paus

CD8+CD38+ T cells: An newly defined IL-15 dependent regulatory T cell population with effects 

in allergic asthma 

Katja Lüthje, Svenja Ehrlich, Bernhard Fleischer, Minka Breloer 

CD83 expression level affects spleenic B cell maturation 

Wiebke Hansen, Simone Reinwald, Astrid M. Westendorf, Carsten Wiethe, Jan Buer 

CD83 is involved in the immunosuppressive function of regulatory T cells 

Birte Kretschmer, Katja Lüthje, Andreas H. Guse, Svenja Ehrlich, Friedrich Koch-Nolte, Friedrich Haag, Bernhard 

Fleischer, Minka Breloer 

CD83 modulates B cell function in vivo and in vitro 

Thomas Bickert, Andrea Horst, Christoph Wagener, Bernhard Fleischer, Uwe Ritter 

CEACAM1 is crucial for lymphvasculogenesis during inflammatory immune responses 

Ulf Alexander Wenzel, Sonja Möller, Werner Solbach, Tamas Laskay, Ger van Zandbergen 

Cell death regulation controls Leishmania disease development 

Norbert Koch, Alexander McLellan, Jürgen Neumann 

Chaperone Controlled Assembly and Matched Isotype Pairing of Major histocompatibility 

complex Class II Subunits 

Johann Poetzl, Catherine Botteron, Daniela Maennel, Anja Lechner 

Characterisation of long-lived CCR6 expressing TH cells in the immune response 

Bernhard Fleischer, Juliane Ladhoff, Elke Effenberger, Cornelia Doebis, Hans-Dieter Volk, Martina Seifert 

Characterisation of NK cell attacks towards MHC class I deficient rat aortic endothelial cells in 

vitro 

Mirjam Peter, Klaus Heeg, Alexander Dalpke 

Characterization of a guanosine-rich suppressive oligodesoxynucleotide which inhibits Toll-like 

receptor-9 signaling 

Andrea Kiessling, Dagmar Riemann, Susanne Fuessel, Esther Kamphausen, Barbara Seliger 

Characterization of expression pattern and regulation of ISGylation system in tumors 

Christoph Treese, Franziska Lange, Nadja Hilger, Anja Mittag, Attila Tarnok, Andreas Lösche, Frank Emmerich, Ulrich 

Sack 

Characterization of fibroblasts eroding cartilage in arthritis 

Jan Leipe, Alla Skapenko, Hendrik Schulze-Koops 

Characterization of human Interleukin 17 (IL-17)-producing T helper cells (Th17 cells) in 

healthy individuals and in patients with rheumatoid arthritis 

Michael Mihlan, Mario Hebecker, Markus Huber-Lang, Peter F. Zipfel, Mihály Józsi 

Characterization of ligand binding of the human Factor H-related protein 4 (FHR-4) provides 

insight into function 

Sandra Klein, Cosima Kretz, Nina Oberle, Martin Hartmann, Alexander Enk, Peter H. Krammer, Elisabeth Suri-Payer, 

Annegret Kuhn 

Characterization of regulatory T cells in human autoimmune diseases with skin manifestations 

Björn Kolbe, Ulrike Kolrep, Roland Wagner, Jürgen Schmitz, Ian C.D. Johnston 

Characterization of the natural ligand of the plasmacytoid dendritic cell-specific marker CD303 

Stefanie Gross, Uwe Trefzer, Wolfram Sterry, Peter Walden 

Characterization of tumor-specific versus virus-specific tumor-infiltrating and peripheral blood T 

cells from melanoma patients 

Marco Frentsch, Regina Starke, Sarah Meier, Gebhardt Friedemann, Dirk Busch, Chiara Romagnani, Andreas Thiel

Characterization of versatile CD8 memory T-cells with potent APC helper function 

Shipra Gupta, Sebastian Rieder, Sylvia Escher, Aleksandra Heitland, Wolf-Georg Forssmann, Jörn Elsner, Ulf 

Forssmann 

Chemokine receptor-mediated intravascular inactivation of leukocytes by a nonglycosaminoglycan(GAG)-binding 

variant of NNY-CCL14. 

Silvia Capellino, Peter Angele, Werner Falk, Maurizio Cutolo, Rainer H. Straub 

Chromaffin-like cells and catecholamine production: role on the inflammatory response in 

rheumatoid arthritis (RA) patients 

Thomas Bollinger, Monika Bajtus, Annalena Bollinger, Stojan Dimitrov, Werner Solbach 

Circadian Rhythm of Regulatory T Cell distribution and function 

Nicholas Schwab, Christoph Leder, Chi Wang Ip, Antje Kroner, Klaus-Armin Nave, Klaus Dornmair, Rudolf Martini, 

Heinz Wiendl 

Clonal expansions of pathogenic CD8+ effector T cells in the CNS of myelin mutant mice 

Andreas Grahnert, Steffi Richter, Sunna Hauschildt 

Cloning and characterization of an enzymatically active ADP-ribosyltransferase 4 (ART4) from 

chicken 

Jens Wuerfel, Alina Smorodchenko, Elena Pohl, Johannes Vogt, Eva Tysiak, Robert Glumm, Sven Hendrix, Robert 

Nitsch, Frauke Zipp, Carmen Infante-Duarte 

CNS-irrelevant T cells enter the brain, cause blood-brain barrier disruption but no cellular 

neuropathology 

Maria Lawrenz, Alexander Visekruna, Thorsten Joeris, Nicole Schmidt, Anjo Kroesen, Stefan H.E. Kaufmann, Ulrich 

Steinhoff 

Coherence or Coincidence ? - ERK activation and Immunoproteasomes in Crohn Disease 

Marcus Gereke, Jan Buer, Dunja Bruder 

Collaboration of the innate and adaptive immune system leads to immunological tolerance in the 

lung 

Jan Hendrik Niess, Frank Leithauser, Guido Adler, Jörg Reimann 

Commensal-driven local TH17 responses trigger inflammatory bowel disease 

Maria Diedrichs-Möhring, Georges M.G.M. Verjans, Seerp G. Baarsma, Gerhild Wildner 

Comparison of antigen-specific cytokine secretion and proliferation of T lymphocytes in vitro 

Vera Jakobi, Swen Wagner, Michael Loos, Franz Petry 

Complement activation of Cryptosporidium parvum (Apicomplexa, Protozoa) via the classical 

and lectin pathways 

Feng Guo, Debra Weih, Elke Meier, Falk Weih 

Constitutive alternative NF-?B signaling promotes marginal zone B cell development but disrupts 

the marginal sinus and induces HEV-like structures in the spleen 

Christian Koble, Jens Derbinski, Bruno Kyewski 

CONSTITUTIVE CROSS-PRESENTATION OF ENDOGENOUS SELF- ANTIGENS BY THYMIC 

DENDRITIC CELLS 

Katja Sabel, Oleg Krut, Martin Krönke, Alexander Klimka 

Construction of a recombinant intrabody library to select for inhibitors of intracellular pathogens 

Vladimir Kocoski, Norbert Tautz, Eberhard Burkhardt 

CONSTRUCTION OF A STABLE TRANSFECTED, PERMANENTLY SECRETING BHK Tet-On CELL LINE 

CARRYING THE SINGLE-CHAIN CANINE IL-12 FOR APPLICATION IN THE TUMOR 

IMMUNOTHERAPY IN DOG

Alexey Popov, Julia Driesen, Zeinab Abdullah, Claudia Wickenhauser, Tomo Saric, Svenja Debey-Pascher, Trinad 

Chakraborty, Martin Krönke, Olaf Utermöhlen, Joachim L. Schultze 

Containment of pathogens and induction of the local immune privilege by IDO + denritic cells in 

granulomatous infections and its implication for human disease 

Kathrin Westphal, Sara Leschner, Holger Loessner, Siegfried Weiss 

Containment of tumor colonizing bacteria by host neutrophils 

Martin Schlee, Michael Bscheider, Veit Hornung, Andrea Ablasser, Stefan Endres, Gunther Hartmann 

Contrasting roles of p38 in TLR signaling 

Bishnudeo Roy, Oliver Pabst, Swati Shukla, Sandra Düber, Siegfried Weiss 

Contribution of B-1 cells to gut associated humoral immunity 

Susanne Kirschnek, Robert Paul, Bianca Obermaier, Georg Häcker, Uwe Koedel 

Contribution of cell death in phagocytes and resident cells to the outcome of pneumococcal 

meningitis in mice 

Gabriele Weintz, Michael Hammer, Ilona Moßbrugger, Leticia Quintanilla-Martinez, Christian Stemberger, Dirk H. 

Busch, Roland Lang 

Control of inflammation and host resistance during Listeria infection by the MAPK-Phosphatase 

DUSP1 

Caspar Ohnmacht, Nico van Rooijen, David Voehringer 

Cooperation between innate and adaptive immunity during type 2 immune responses in vivo. 

Kathrin Schönberg, Gesine Kögler, Johannes Fischer, Markus Uhrberg 

Correlation of KIR expression and presence of HLA-C ligands in adult but not neonatal NK cells: 

transition from a naïve to an adult NK cell repertoire 

Zoe Waibler, Martina Anzaghe, Abdo Konur, Shizuo Akira, Werner Müller, Ulrich Kalinke 

CpG 1668 treatment stimulates an anti-inflammatory environment that abrogates CpG 2216 

induced type I IFN responses by pDC 

Viktor Kölzer, David Anz, Michaela Golic, Cornelia Wurzenberger, Stefan Endres, Carole Bourquin 

CpG Oligonucleotide Treatment Alters the Morphological Distribution and Phenotype of 

Regulatory T Cells 

Wolfgang Kastenmüller, Georg Gasteiger, Ingo Drexler 

Cross-competition of CD8+ T cells shapes the immunodominance hierarchy during recall 

vaccination 

Nanette von Oppen, Linda Diehl, Rene Tolba, Percy Knolle 

CROSS-PRESENTING LIVER SINUSOIDAL ENDOTHELIAL CELLS ESTABLISH ANTIGEN-SPECIFIC 

ADHESION OF NAÏVE CD8 T CELLS LEADING TO T CELL TOLERANCE IN VIVO 

Heinke Conrad, Kerstin Gebhard, Julia Neudorfer, Christian Peschel, Helga Bernhard 

Cross-reactivity of HER2p369-377 reactive CTL clones against other HER family members 

Mandy Pierau, Engelmann Swen, Thomas Drewes, Thabo Lapp, Dirk Reinhold, Burkhart Schraven, Ursula Bommhardt 

Cross-talk between PKB/Akt and TGFβ signalling in T cell activation 

Sven Burgdorf, Christian Kurts 

Current models and mechanisms of antigen crosspresentation 

Jörg Rossbacher, Frank Wilde, Gerd Müller, Martin Lipp 

CXCR5 as a therapeutic target in Non Hodgkin lymphomas and autoimmune disease 

Sven Hartmann, Antje M. Wengner, Uta E. Hoepken, Peter K. Petrow, Uta Schurigt, Rolf Braeuer, Martin Lipp 

CXCR5- and CCR7-dependent lymphoid neo-genesis in a chronic model of antigen-induced 

arthritis (AIA)

Tanja Nicole Hartmann, Bretton Summers, Valentin Grabovsky, Eilon Woolf, Ziv Shulman, Eike Buss, Tom Schall, 

Marcus Thelen, Ronen Alon 

CXCR7 blockage inhibits in human hematopoietic progenitor cells and T cells a CXCR4 subset 

specialized in integrin activation by CXCL12 under shear stress conditions 

Tobias Bopp, Christian Becker, Matthias Klein, Stefan Klein-Heßling, Alois Palmetshofer, Edgar Serfling, Marc Becker, 

Jan Kubach, Schmitt Steffen, Sabine Stoll, Hansjoerg Schild, Martin Staege, Michael Stassen, Helmut Jonuleit, Edgar 

Schmitt 

Cyclic AMP: The decicive component of naturally occuring regulatory T cell-mediated 

suppression 

Bianca Paul, Linda Diehl, Alexander Knorre, Percy Knolle, Marc Beyer, Waldemar Kolanus 

Cytohesin-3 Links B7H1 mediated Shut-down of the PI3 Kinase Pathway to the Repression of IL- 

2 Synthesis in Anergic T cells 

Sabrina Hoffmann, Michael Winkler, Marcus Gutscher, Helmut Fickenscher, Carsten Watzl 

Cytomegalovirus infected fibroblasts downregulate ligands for the Natural Cytotoxicity receptors 

NKp30 and NKp44 

Doris Urlaub, Sven Mesecke, Hauke Busch, Roland Eils, Carsten Watzl 

Decision making in NK cells 

Jochen Maul, Susanne Pförtner, Robert Geffers, Kerstin Kapp, Jan Buer, Martin Zeitz, Rainer Duchmann 

Decreased expression of CCR4 on CD4+CD25high regulatory T cells as a possible mechanism for 

impaired migration to inflamed mucosa in Crohn´s disease 

Mahmoud Sadeghi, Gerhard Opelz, Volker Daniel, Cord Naujokat, Rainer Zimmermann, Angela Huth-Kühne, Caner 

Süsal 

Decreasing Soluble CD30 and Increasing IFN-γ Plasma Levels are Indicators of Effective Highly 

Active Antiretroviral Therapy. 

Nadine Voelxen, Sylvia Gutenberger, Hans-Hartmut Peter, Hermann Eibel, Klaus Warnatz 

Defective activation of B cells in persistent polyclonal B cell lymphocytosis (PPBL)? 

Christine Skerka, Nadine Lauer, Claudia N Keilhauer, Lars Fritsche, Bernhard H.F. Weber, Peter F. Zipfel 

Defective Binding of Factor H (Y402H) and FHL-1 to CRP and Collagen in Age Related Macular 

Degeneration 

Florian Börncke, Beatrix Pollok-Kopp, Mladen V. Tzvetkov, Martin Oppermann 

Defective Binding to C-Reactive Protein and Impaired Cofactor Activity of the Allotypic Y402H 

Variant of Complement Factor H 

Frank Guenther, Gertud Maria Hänsch, Christof Wagner 

DEFENCE AGAINST BACTERIAL BIOFILMS: ROLE OF POLYMORPHONUCLEAR NEUTROPHILS 

(PMN) 

Tobias Schulze, Katrin Räbel, Sven Golfier, Martin Lipp 

Deficiency in Sphingosine-1-phosphate receptor 4 (S1P4) results in deviated humoral immune 

responses 

Anja Erika Hauser, Tobias Junt, Thorsten R. Mempel, Michael W. Sneddon, Steven H. Kleinstein, Sarah E. Henrickson, 

Ulrich H. von Andrian, Mark J. Shlomchik, Ann M. Haberman 

Definition of Germinal Center B Cell Migration In Vivo Reveals Predominant Intra-zonal 

Circulation Patterns 

Christine Skerka, Mihály Józsi, Stefanie Strobel, Stefan Heinen, Matthew Edey, Svante L. H. Zipfel, Judith A Goodship, 

Timothy H.J. Goodship, Christoph Licht, Peter F. Zipfel 

Deletion of CFHR1 and CFHR3 correlates with presence of Factor H autoantibodies in hemolytic 

uremic syndrome 

Thorsten Feyerabend, Annette Tietz, Herve Luche, Freddy Radtke, Hans Joerg Fehling, Hans Reimer Rodewald

DELETION OF NOTCH-1 IN KIT+ PRO-T CELLS BLOCKS T CELL DEVELOPMENT, BUT DOES NOT 

CONVERT T CELL PROGENITORS INTO THYMIC B CELLS 

Carsten Wiethe, Alexander Steinkasserer, Manfred Lutz, Andre Gessner 

Dendritic cell differentiation state and their interaction with NKT cells determines Th1/Th2 

differentiation in the murine model of Leishmania major infection 

Stefan A. Kaden, Juergen Schmitz, Gregor Winkels 

Dendritic Cell immuno-activating receptor 1 – Characterization of a novel member of the C-type 

Lectin family 

Marcin •yszkiewicz, Natalia Zi•tara, Manfred Rohde, Kurt Dittmar, Jadwiga Jab•o•ska, Siegfried Weiss 

Dendritic cell like function of ER-TR9 + marginal zone macrophages 

Kristin Hochweller, Jörg Striegler, Günter J Hämmerling, Natalio Garbi 

Dendritic cells control awareness of T lymphocytes for antigen 

Mathias Lucas, William Schachterle, Karin Oberle, Peter Aichele, Andreas Diefenbach 

Dendritic Cells Prime Natural Killer Cells by trans-Presenting Interleukin 15 

Anja Saalbach, Claudia Klein, Ulf Anderegg, Jan C. Simon 

Dermal fibroblasts induce maturation of dendritic cells 

Elke Pogge von Strandmmann, Boris Böll, Daniel Re, Andreas Engert, Venkateswara Simhadri 

Detection of HLA-B associated transcript 3 (BAT3) in sera from Hodgkin Lymphoma patients and 

its release from Hodgkin lymphoma cells 

Leander Grode, Hans-Heinrich Henneick v. Zepelin, Albrecht Laeufer, Bernd Eisele 

Developing a TB vaccine for human use 

Stefan Porubsky, Anneliese O. Speak, Bruno Luckow, Vincenzo Cerundolo, Frances M. Platt, Hermann-Josef Gröne 

Development and function of invariant natural killer T cells in mice with 

isoglobotrihexosylceramide (iGb3) deficiency 

Nina Wantia, Tanja Ertl, Christine Cirl, Nuria Rodriguez, Hermann Wagner, Thomas Miethke 

Development of a protein and CpG-based vaccination against Chlamydophila pneumoniae 

Augustin J Kerkdijk, Gerhard Held, Antje Mueller, Wolfgang L Gross, Michael Pfreundschuh, Jan Voswinkel 

Development of a System to Test for Specificity of B-cell Receptors found in Granulomatous 

Lesions of Wegeners Granulomatosis patients 

Nadja Hilger, Rico Hiemann, Jörg Michel, Ursula Anderer, Martin Weigert, Ulrich Sack 

Development of an completely automatized system for image aquisition and detection of HEp-2 

immunofluorescence patterns 

Anne Brüstle, Sylvia Heink, Magdalena Huber, Christine Rosenplänter, Christine Stadelmann, Philipp Yu, Enrico Arpaia, 

Tak W. Mak, Thomas Kamradt, Michael Lohoff 

Development of inflammatory Th17 cells requires interferon regulatory factor 4 

Christian Menge, Evelyn A. Nystrom 

Dexamethasone depletes γδT cells and alters the activation state and responsiveness of bovine 

peripheral blood lymphocyte subpopulations 

Christian Menge, William C. Stoffregen, Joachim F.L. Pohlenz, Evelyn A. Nystrom 

Dexamethasone differentially down-regulates L-Selectin (CD62L) expression by bovine 

lymphocyte subsets in vivo and depletes the intestinal mucosa of intraepithelial γδT cells 

Eva Rieser, Monika Braun, Barbara Simm, Barbara Mosetter, Christine S. Falk 

Differences between cytotoxicity and cytokine expression are due to different phosphorylation 

patterns

Marcel Andre Krüger, Kathrin Kopplin, Nadine Unterwalder, Christian Meisel, Hans-Dieter Volk, Gerald Grütz 

Differences in Lipopolysaccharide and Lipid A desensitisation 

Jan Diekmann, Olaf Beck, Georg Rauser, Hansjörg Schild, Hermann Einsele, Max S. Topp 

Different mechanisms contribute to the immune evasion of Epstein-Barr virus latent membrane 

protein 1 

Diana Dudziak, Alice O'Kamphorst, Gordon F. Heidkamp, Veit R. Buchholz, Christine Trumpfheller, Chae Gyu Park, 

Ralph M. Steinman, Michel C. Nussenzweig 

Differential Antigen Processing and Presentation by Dendritic Cell Subsets in vivo 

Seray Cetin, Niels Kruse, Andrew Chan, Ralf Gold, Fred Lühder 

Differential expression of BDNF mRNA splice variants in mouse brain and immune cells 

Ildiko Boross, Christine Lux, Hans-Anton Lehr 

Differential expression of IL-17F and IL-17A in the lung of patients affected with 

bronchioalveolar cell carcinoma 

Markus Kleinewietfeld, Giovanna Borsellino, Adamo Diamantini, Alexander Sternjak, Luca Battistini, Olaf Rötzschke, 

Kirsten Falk 

Differential expression of VLA-4 by functional Treg and effector CD4+ T cells 

Florian Reißfelder, Jutta Schröder-Braunstein, Thomas Giese, Carolin Reiser, Stefan C. Meuer, Bernd Sido 

Differential inhibition of human intestinal lamina propria T-lymphocyte activation versus 

peripheral blood T cells by the gold-compound auranofin 

Svetlana Karakhanova, Karsten Mahnke, Alexander Enk 

Differential modulation of B7-H1 expression in pDCs and mDCs upon maturation of dendritic 

cells (DCs). 

Daniel Engel, Ulrich Dobrindt, Juliane Maurer, Frank Tacke, Christian Kurts 

Differential role of CCR2 on Gr1HI and Gr1LO monocyte migration in response to bacterial 

infection 

Maik Moermann, Mareike Thederan, Christof Wagner, Inaam Nakchbandi, Gertrud Maria Hänsch 

Differentiation of the promonocytic cell-line U 937 to osteoclasts by bacterial 

lipopolysaccharides: a link between infection and pathological bone resorption. 

Kristine Kohl, Sylvia Schnautz, Elisabeth Klein, Thomas Bieber, Susanne Koch 

DIFFERENTIATION SIGNALS FOR HUMAN LANGERHANS CELL PRECURSORS IDENTIFIED BY 

SEQUENTIAL MIGRATION OF MONOCYTES 

Tim Meyer, Susann Beetz, Daniela Wesch, Ina Martens, Dieter Kabelitz 

Direct Costimulatory Effect of TLR3 Ligand Poly(I:C) on T cells 

Chiara Massa, Christiane Kellert, Esther Kamphausen, Barbara Seliger 

Disparate modulation of antigen processing components during maturation of the different 

human DC subsets. 

Andreas Hombach, Markus Chmielewski, Tobias Riet, Caroline Kopecky, Patrick Schmidt, Nadin Fein, Claudia Ederer, 

Anja Hombach, Heike Koehler, Hinrich Abken 

Dissecting and modulating a redirected anti-tumor T-cell response for adoptive immunotherapy: 

second generation of recombinant immunoreceptors. 

Marc A. Blank, Olaf Utermohlen, Holger M. Reichardt, Marco J. Herold 

Dissecting the apoptotic pathways induced by Glucocorticoids in T-cells 

Annelies Verbrugge, Adelheid Cerwenka 

Dissecting the molecular mechanisms involved in the synergy of TREM-1 with TLRs

Sven Burgdorf, Andreas Kautz, Volker Böhnert, Percy Knolle, Christian Kurts 

Distinct antigen uptake and intracellular routing mechanisms for activation of CD4+ and CD8+ T 

cells 

Astrid Menning, Uta Hoepken, Kerstin Siegmund, Martin Lipp, Alf Hamann, Jochen Huehn 

Distinctive role of CCR7 in migration and functional activity of naïve- and effector/memory-like 

Treg subsets 

Julia Polansky, Jennifer Freyer, Stefan Floess, Karsten Kretschmer, Harald von Boehmer, Alf Hamann, Jochen Huehn 

DNA methylation controls foxp3 gene expression 

Manije Sabet, Maja Frankuski, Anja Reutzel-Selke, Andreas Pascher, Peter Neuhaus, Johann Pratschke, Katja Kotsch 

Donor pretreatment with Simvastatin reduces graft immunogenicity following prolonged cold 

ischemia in an experimental model of kidney transplantation 

Xiaoqian Wang, Luca Simeoni, Jonathan A. Lindquist, Julio Saez-Rodriguez, Ernst D. Gilles, Stefanie Kliche, Burkhart 

Schraven 

Dynamics of proximal signaling events after TCR/CD8-mediated induction of proliferation or 

apoptosis in mature CD8+ T-cells 

Börge Arndt, Burkhart Schraven, Luca Simeoni 

Dynamics of TCR signaling events leading to human T-cell proliferation 

Stephan Meinke, Philipp Eissmann, Carsten Watzl 

Early events in NTB-A signaling 

Claudia N. Detje, Hauke Schmidt, Thomas Meyer, Marco Prinz, Ulrich Kalinke 

Early type I interferon responses exclude nerotropic viruses from central nervous system 

Andrey Bogdanov, Tatyana Rybakova, Anna Nizkorodova, Nikolay Belyaev 

Effect of alpha-fetoprotein-activated hematopoietic stem cells on monocytes functional activity 

Pablo Ariel Casalis, Martin Griebenow, Maria Laura Zenclussen, Ana Claudia Zenclussen, Hans-Dieter Volk, Christian 

Woiciechowsky, Ulrich-Wilhelm Thomale 

Effect of injury severity on the local and systemic cytokine expression in a rat model of 

traumatic brain injury 

Michael Schramm, Ulrike Karow, Albert Haas, Martin Krönke, Olaf Utermöhlen 

Effects of Acid SphingoMyelinase on listeriocidal activiy of macrophages 

Markus Janke, Jens Poth, Thomas Giese, Gunther Hartmann 

Effects of immunostimulatory RNA on human granulocyte populations 

Özen Sercan, Günter J. Hämmerling, Bernd Arnold, Thomas Schüler 

EFFECTS OF INTERFERONγ ON CD8 + T CELL HOMEOSTASIS 

Besir Okur, Rainer Glauben, Arvind Batra, Thorsten Stroh, Inka Fedke, Jeannette Pietsch, Martin Zeitz, Britta Siegmund 

Effects of Leptin on T helper cell polarisation 

Stephan Paxian, Markus P. Kummer, Lars Tatenhorst, Klaus Pfeffer, Frank Kirchhoff, Roland Schmid, Michael T. Heneka 

Effects of neuronal and microglial disrupted RelA(p65) in the CNS during neuroinflammatory 

disorders 

Fanny Edele, Cindy Reinhold, Stefan F. Martin 

Efficiency of T cell defence against melanoma depends on the DC immunization route 

Prajeeth Chittappen Kandiyil, Thomas Ebensen, Carlos Guzmàn, Reinhold Schmidt, Georg Behrens 

Efficient cross-priming induced by the toll-like receptor 2/6 agonist MALP-2 

Julius Hafalla, Ana Rodriguez, Fidel Zavala

Efficient development of Plasmodium liver stage-specific memory CD8+ T cells during the course 

of blood stage malaria infection 

Stefanie Hoyer, Katrin Birkholz, Verena Wellner, Ina Müller, Erwin Schultz, Gerold Schuler, Niels Schaft, Jan Dörrie 

Electroporation of TCR-encoding RNA into CD4+ T cells in order to provide T-cell help 

Stefanie Helm, Patrick Pankert, Stefanie Eikelmeier, Edward Shang, Hans Ulrich Weltzien, Martina Schnoelzer, 

Hermann-Josef Thierse 

Elements of the innate immune barrier: Proteomic identification of allergen-protein interactions 

in the human epidermis 

Benjamin Frey, Luis E. Munoz, Friederike Pausch, Ernst Pöschl, Klaus von der Mark, Martin Herrmann, Udo S. Gaipl 

Endogenous AnnexinA5 is Involved in the Immune Reaction Against Allogeneic Cells 

Christina Hartwig, Miriam Mazzega, Thomas Tschernig, Detlef Neumann 

Endogenous IL-18 in experimentally induced asthma affects cytokine serum levels but is 

irrelevant for clinical symptoms 

Niklas Engels, Jürgen Wienands 

Enhanced signaling of the IgG-BCR is accomplished by tyrosine-phosphorylation of the 

cytoplasmic mIgG tail 

Fanny Edele, Rosalie Molenaar, Cindy Reinhold, Dominique Gütle, Jan C. Dudda, Reina Mebius, Mathias Hornef, Stefan 

F. Martin 

Environmental instruction of dendritic cells for T cell homing receptor imprinting 

Jana Zeitvogel, Thomas Werfel, Miriam Wittmann 

Epidermal stem cells differ in their response to IFN&gamma from other proliferative 

keratinocytes 

Ellen Andresen, Joern Bullwinkel, Christoph Lange, Holger Heine 

Epigenetic regulation of defensin gene expression in lung epithelial cells and COPD 

Anke Schütz, Hongqi Lue, Jürgen Bernhagen 

ERK1/2-MAPK signaling induced by macrophage migration inhibitory factor (MIF) is influenced 

by its CXXC motif 

Winfried Barchet, Vera Wimmenauer, Leonid Gitlin, Susan Gilfillan, Marina Cella, Marco Colonna, Gunther Hartmann 

Essential Role of MDA-5 in Type I IFN Responses to Poly (I:C) and Encephalomyocarditis 

Picornavirus 

Roman Karwot, Joachim Maxeiner, Steffen Schmitt, Petra Scholtes, Michael Hausding, Ildiko Boross, Hans Lehr, 

Susetta Finotto 

Essential role of NFATc2 in CD8+ cells in a murine model of allergic sensitization 

Andre Tittel, Daniel Engel, Ulrich Dobrindt, Christian Kurts 

Establishing a murine model system to investigate the adaptive immune response against 

urinary tract infection 

Marion Nonn, Shamsul A. Khan, Eva Distler, Ralf G. Meyer, Leonard D. Shultz, Rupert Handgretinger, Christoph Huber, 

Wolfgang Herr, Udo F. Hartwig 

Establishment of a NOD/SCID/IL2Rγcnull hematopoietic stem cell transplantation model to study 

graft-vs-host and graft-vs-leukemia immune responses of ex vivo modified human T lymphocyte 

grafts. 

Rainer Wurth, Angelika Bold, Thomas Keller, Ulrike Trahorsch, Peter Voigt, Stefan Schubert, Ulrich Sack 

Evaluation and validation of a manual low-cost assay for the monitoring of CD4 counts in HIVinfected 

individuals in non-OECD countries 

Mathias Fousse, Robert Dinser, Urban Sester, Katinka Albrecht, Mahavir Singh, Hans Köhler, Ulf Müller-Ladner, 

Martina Sester

Evaluation of latent tuberculosis infection in patients with inflammatory arthropathies before 

treatment with tumour necrosis factor-α blocking drugs using a novel flow-cytometric interferonγ 

release assay 

Katherina Sewald, Maja Henjakovic, Simone Switalla, Norbert Krug, Armin Braun 

Ex vivo Immunomodulatory Testing using Precision Cut Lung Slices: Focus on Dendritic Cells 

Ingo Irmler, Mieczyslaw Gajda, Rolf Bräuer 

Exacerbation of Antigen induced Arthritis in IFN-γ-deficient Mice as a Result of Unrestricted IL- 

17 Response 

Gasteiger Georg, Kastenmuller Wolfgang, Sutter Gerd, Drexler Ingo 

Exclusive cross-priming of cytotoxic T-cells dictates antigen requisites for MVA vector vaccines 

daomin gong 

Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory 

function but does not abrogate the suppressive effect of CD4+CD25+ regulatory T cells 

Katjana Klages, Anja Stirnweiss, Jörg Reimann, Hansjörg Hauser, Andrea Kröger 

EXPRESSION OF INTERFEON REGULATORY FACTOR-1 IN CT26 COLON CARCINOMA CELLS 

INDUCES ANTI-TUMOR ACTIVITY 

Thorsten Stroh, Arvind Batra, Rainer Glauben, Inka Fedke, Stephen Girardin, Martin Zeitz, Britta Siegmund 

Expression of NOD2 but not NOD1 is modulated by pro-inflammatory cytokines in murine 

preadipocytes 

Cemil Korcan Ayata, Cinthia Farina, Markus Krumbholz, Florian Weisel, Thomas Winkler, Andreas Rosenwald, Reinhard 

Hohlfeld, Edgar Meinl 

Expression of p75 neurotrophin receptor (p75NTR) and brain derived neurotrophic factor 

(BDNF) in germinal centers 

Matthias M Gaida, Frank Guenther, Martin Loos, Christof Wagner, Gertrud Maria Hänsch, Helmut Friess, Nathalia 

Giese, Wente Moritz 

Expression of the chemokine receptor CXCR6 on polymorphonuclear neutrophils (PMN) in 

pancreatic tumour specimen and in acute, localised bacterial infections 

Eric Keil, Nana Ueffing, Linda Clayton, Ellis Reinherz, Klaus Schulze-Osthoff, Ingo Schmitz 

Expression profiling identifies Gadd45β as a novel mediator of negative selection 

Silke Meister, Kirsten Neubert, Kai Herrmann, Renate Burger, Martin Gramatzki, Sabine Hahn, Sandra Schreiber, 

Ulrich Schubert, Hans-Martin Jäck, Reinhard Voll 

Extensive immunoglobulin production sensitizes myeloma cells for proteasome inhibition 

Carina Klein, Anja Grahnert, Sunna Hauschildt 

Extracellular NAD+ triggers transient [Ca2+]i changes in LPS-activated human monocytes via 

P2Y receptors 

Anja Grahnert, Erik Schilling, Carina Klein, Sunna Hauschildt 

Extracellular NAD + triggers transient [Ca2+ ] i changes in human monocytes via P2X-receptors 


Feed-back control of dendritic cell homeostasis 

Karsten Kretschmer, Alexander Marson, Garrett M. Frampton, Julia Polansky, Richard A. Young, Harald von Boehmer 

Foxp3-dependent gene regulation requires T cell activation 

Thomas Quast, Barbara Tappertzhofen, Cora Schild, Waldemar Kolanus 

Function of CD81 in dendritic cell migration 

Sebastian Dütting, Wolfgang Schuh, Kai Hermann, Christiane Lang, Hans-Martin Jäck, Dirk Mielenz 

Function of Swiprosin-2/EFhd1 during B cell development

Cornelia Rosner, Lutz Walter 

Functional analysis of MHC class I genes in the rhesus monkey (Macaca mulatta) 

Simone Abel, Jan Buer, Wiebke Hansen 

Functional analysis of Neuropilin1 in regulatory T cells 

Gamze Kabalak, Torsten Matthias, Reinhold E. Schmidt, Torsten Witte 

FUNCTIONAL CHARACTERISATION OF ILT6 AS GENETIC RISK FACTOR FOR MULTIPLE 

SCLEROSOS AND SJÖGREN’S SYNDROME 

Kathrin Gube, Inga Gebuhr, Katrin Vogt, Erik Kwidzinski, Christine Brandt, Hans-Dieter Volk, Birgit Sawitzki 

FUNCTIONAL CHARACTERISATION OF THE TOLERANCE ASSOCIATED GENE (TOAG-1) 

Susanne Stutte, Sabine Brauer, Irmgard Förster 

FUNCTIONAL ROLE OF CCL17 IN ALLERGIC IMMUNE REACTIONS OF THE SKIN 

Daniel Reim, Kay Westenfelder, Simone Kaiser-Moore, Sylvia Schlautkötter, Bernhard Holzmann, Heike Weighardt 

Functional role of T cells during mixed bacterial peritonitis 

Jan Kubach, Petra Lutter, Tobias Bopp, Sabine Stoll, Christian Becker, Jürgen Knop, Stefan Müllner, John Wijdenes, 

Edgar Schmitt, Helmut Jonuleit 

Galectin-10, a previously unnoted protein essential for the functional activity of human CD4 

+CD25+ regulatory T cells 

Kerstin Sarter, Connie Schulze, Sandra Franz, Benjamin Frey, Luis Munoz, Udo Gaipl, Martin Herrmann 

Galectins contribute to the recognition and clearance of apoptotic cells 

Nadja Hilger, Frank Emmrich, Ulrich Sack 

Gene expression in microdissected invasive fibroblast isolated from arthritic joints from patients 

with RA 

Alexander Gerbaulet, Julia Scholten, Thomas Krieg, Karin Hartmann, Axel Roers 

Generation of a Mouse Model for Mastocytosis 

Dafne Müller, Bettina Meißburger, Katharina Frey, Anette Karle, Ines Höfig, Roland Stork, Roland E. Kontermann 

Generation of an improved recombinant bispecific antibody molecule and B7 fusion proteins for 

targeted cancer immunotherapy 

Kathrin Hofer, Holger Kroenig, Heinke Conrad, Barbara Kast, Christian Peschel, Helga Bernhard 

Generation of Th1 lymphocyte clones against an immunodominant epitope of NY-ESO-1 

Johannes Stephani, Ronald Naumann, Hermann Wagner, Tim Sparwasser 

Generation of TLR- „humanized“ Mice with Bacterial Artificial Chromosome-Technology 

Sandra Ehser, Jing-Jing Chuang, Lucian Jiga, Christian Kleist, Flavius Sandra-Petrescu, Gerhard Opelz, Peter Terness 

Generation of tolerogenic dendritic cells by treatment with Mitomycin C 

Ann-Kristin Mueller, Martina Deckert, Kirsten Heiss, Kristin Goetz, Kai Matuschewski, Dirk Schlüter 

Genetically Attenuated Plasmodium berghei Liver Stages Persist and Elicit Sterile Protection 

Primarily via CD8 T Cells 

Marc Beyer, Sabine Classen, Daniela Eggle, Alexey Popov, Svenja Debey-Pascher, Elmar Endl, Percy A. Knolle, Jim 

Riley, Joachim L. Schultze 

Genomic screening reveals new proteins specifically expressed by human regulatory CD4+ 

CD25high FOXP3+ CD127low T cells 

Charles Andrew Stewart, Thierry Walzer, Scott Hamilton Robbins, Bernard Malissen, Eric Vivier, Immo Prinz 

Germline and rearranged Tcrd transcription distinguish bona fide NK cells and NK-like γδ T cells

Benjamin Wilde, Xin Cai, Sebastian Dolff, Andreas Kribben, Jan Dürig, Christof Specker, Thomas Philipp, Oliver Witzke 

GITR and CD134 expression on T-lymphoctyes is associated with disease activity in Wegener's 

Granulomatosis 

Adjobimey Tomabu, Arndts Kathrin, Satoguina Judith, Hörauf Achim 

GITR-GITRL interactions regulate the IgG induction by regulatory T cells 

Matthias Krusch, Katrin Miriam Baltz, Tina Baessler, Helmut Rainer Salih 

Glucocorticoid-Induced TNF Related Protein (GITR) ligand is spontaneously released by tumor 

cells and diminishes anti-tumor reactivity of NK cells 

Denise Tischner, Nora Müler, Jens van den Brandt, Andreas Weishaupt, Holger Reichardt 

Glucocorticoids exert distinct effects on Experimental Autoimmune Encephalomyelitis 

Sabine Stegmaier, Christof Wagner, Gertrud Maria Hänsch 

Granzyme B expression in mature polymorphonuclear neutrophils (PMN) and in their precursor 

cells 

Praxedis Martin, Julian Pardo, Reinhard Wallich, Klaus Ebnet, Sandra Iden, Aynur Ekiciler, Arno Muellbacher, Michael 

Huber, Markus M. Simon 

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death 

independent of perforin 

Gerhard Wingender, Jonathan Braun, James Borneman, Mitchell Kronenberg 

Gut derived antigens trigger the final steps of Vα14 iNKT cell differentiation 

Dagmar Quandt, Hubert Ludwiczak, Barbara Seliger 

Heterogeneous B7-H molecule expression and regulation in RCC and melanoma 

Christian Stemberger, Katharina Huster, Martina Koffler, Florian Anderl, Matthias Schiemann, Hermann Wagner, Dirk 

Busch 

Heterogeneous subset generation from a single naïve CD8+ T cell upon in vivo priming 

Wibke Bayer, Simone Schimmer, Dennis Hoffmann, Ulf Dittmer, Oliver Wildner 

Heterologous Prime-Boost Vaccination with Ad5 and Fiber Chimeric Adenoviral Vectors Enhances 

Immune Protection against Friend Virus 

Maren Mönkemeyer, Hans Heiken, Rachel Thomas, Reinhold E. Schmidt, Torsten Witte 

Higher risk of CMV reactivation in HIV-1 infected patients homozygous for MICA5.1 

Konrad Alexander Bode, Klaus Heeg, Alexander H. Dalpke 

Histone deacetylase inhibitor butyric acid of bacterial origin as mediator of tolerance in the 

intestinal mucosa 

Mareike Schmudde, André Braun, Ulrike Klier, Daniela Pende, Jürgen Sonnemann, Lorenzo Moretta, James F. Beck, 

Barbara M. Bröker 

Histone deacetylase inhibitors sensitize tumour cells for cytotoxic effects of natural killer cells 

Vanessa Witte, Andreas Baur 

HIV-1 Nef enhances viral gene expression by linking transcriptional derepression and activation 

events 

Karsten Gülow, Marcin Kaminski, Peter H. Krammer 

HIV-Tat induced generation of Reactive Oxygen Species sensitizes T cells towards Activation- 

Induced Cell Death 

Claudia Sievers, Kasia Nasilowska, Kerstin Wolk, Robert Sabat, Hans-Dieter Volk, Christian Meisel 

HO-1 inhibits constitutive and IFNg-induced HLA-DR expression on myeloid antigen-presenting 

cells via inhibition of IFNg receptor signalling and down-regulation of CIITA expression 

Nadja Brachwitz, Maria Laura Zenclussen, Andre Sollwedel, Ritschel Stefanie, Hans-Dieter Volk, Ana Claudia 

Zenclussen

HO-1 up-regulation increases the number of uNK at the fetal-maternal interface 

Jan C. Dudda, Nikole Perdue, Mary Beauchamp, Daniel J. Campbell 

Homing receptors CD62L and CD103 mark different subsets of development and function of 

Regulatory T cells for suppression of autoimmunity 

Otilia Postea, Christian Weber, Andreas Ludwig 

Homocysteine-induced adhesive and scavenger activity of endothelial cells involves 

upregulation of the transmembrane chemokine CXCL16 by a PPAR-gamma dependent 

mechanism 

Matthias von Herrath, Christophe Filippi 

How viral infections prevent type 1 diabetes by augmenting Treg function 

Stefan Welte, Kathrin Pietschmann, Lothar Marischen, Susann Beetz, Ina Martens, Daniela Wesch, Dieter Kabelitz 

Human γδ T lymphocytes express pattern recognition receptors 

Susann Beetz, Tim Meyer, Ina Martens, Thomas Stempfl, Daniela Wesch, Dieter Kabelitz 

Human γδ T lymphocytes can initiate an anti-viral immune response to double-stranded RNA 

Thi Thu Hoai Nguyen, Silva Holtfreter, Thi Thu Hong Le, Harald Kusch, Michael Hecker, Susanne Engelmann, Alex van 

Belkum, Uwe Völker, Heiman Wertheim, Barbara M. Bröker 

Human antibody response to experimental colonization with Staphylococcus aureus 

Anja Mayer, Holger Bartz, Fabian Fey, Alexander Dalpke 

Human bronchial epithelial cells modify function and phenotype of dendritic cells in 

inflammatory settings 

Caroline Maas, Shenchu Jin, Oliver Germandi, Gerd Otto, Peter Galle, Dennis Strand, Susanne Strand 

Human Chorionic Gonadotropin protects against T cell-mediated liver injury in mice by 

downregulating Bim and Puma 

Clarissa Mindnich, Sonja Bonness, Kristine Kohl, Sylvia Schnautz, Dagmar von Bubnoff, Dagmar Wilsmann-Theis, 

Susanne Koch, Thomas Bieber 

HUMAN IN VITRO GENERATED DENDRITIC CELLS EXPRESS THE INDUCIBLE NITRIC OXIDE 

SYNTHASE (iNOS) 

Annette Paschen, Mostafa Jarahian, Antje Sucker, Sandra Striegel, Iris Moll, Dirk Schadendorf, Frank Momburg 

Human Natural Killer (NK) Cells Effectively Kill Autologous Melanoma Cells In Vitro but Limited 

NK Cell Infiltration into Tumor Metastasis might Interfere with an Effective Anti-Tumor 

Immunity In Vivo 

Anja A. Kuehl, Jürgen Westermann, Nina N. Pawlowski, Katja Grollich, Martin Zeitz, Jörg C. Hoffmann 

Human peripheral γδ T Cells posses regulatory Potential 

Anja Saalbach, Jacqueline Lessig, Jan C Simon, Jürgen Arnhold, Ulf Anderegg 

Human Thy-1 induces secretion of matrix metalloproteinase-9 and CXCL8 from neutrophils 

Sabrina Laing, Mareike Pilz, Michel Seman, Friedrich Koch-Nolte, Friedrich Haag 

Human TNF&alpha is a substrate for modification by ADP-ribosyltransferase-1 (ART1) 

Petra Richl, Martin Albers, Henner Morbach, Stephanie Brändlein, H. Peter Vollmers, Hermann Girschick 

Humoral immunity against malignant gastric carcinoma cells: molecular characterization and 

age-related frequency of SC-1 antibody positive B cells 

Gordon Grochowy, Michelle Hermiston, Arthur Weiss, Michael Huber 

Hyperactivation of mast cells from CD45 E613R („wedge“) mice 

Ursula Ellinghaus, Rudolf Rupec, Oliver Pabst, Ralf Ignatius, Reinhold Förster, Bernd Dörken, Franziska Jundt 

IκBα is crucial for marginal zone B cell development

R. Riedl, J. Sommer, K. Prinz, A. Egyed, C. Schellack, A. von Gabain, E. Nagy, K. Lingnau 

IC31TM: a novel adjuvant that potently activates Type I immune responses 

Alexandra Doerr, Carsten Watzl, Michael Kirschfink 

iC3b binding to Raji cells modulates Rituximab- induced antibody-dependent cellular cytotoxicity 

(ADCC) 

Yvonne Burmeister, Timo Lischke, Anja C. Dahler, Hans-Werner Mages, Kong-Peng Lam, Anthony J. Coyle, Richard A. 

Kroczek, Andreas Hutloff 

ICOS controls the pool size of effector-memory and regulatory T cells 

Eva Nina Huter, Sabine Stoll, Julia Horn, Juergen Knop, Bodo Grimbacher, Helmut Jonuleit 

ICOS plays an essential role in the development of anergic and suppressive CD4+ T cells 

Johann Röhrl, Thomas Hehlgans 

Identification and Biological Characterization of Mouse Beta Defensin 14 – an Ortholog of 

Human Beta Defensin 3 

Laura Rivino, Federica Sallusto, Antonio Lanzavecchia, Jens Geginat 

Identification and characterization of human "context-dependent Tr1/memory" cells. 

Andrea Baetz, Christoph Koelsche, Alexander Dalpke 

Identification of a nuclear localization signal (NLS) in SOCS1 

Theresa Tretter, Ram Kumar Venigalla, Volker Eckstein, Hanns Martin Lorenz 

Identification of human B cells with immunoregulatory properties 

Tereza Havlova, Anja Tessarz, Vaclav Horejsi, Adelheid Cerwenka 

Identification of Key-Players in TREM1/DAP12 Signaling Pathway 

Katja Kotsch, Vera Merck, Kristina Kunert, Anja Reutzel-Selke, Andreas Pascher, Hans-Dieter Volk, Peter Neuhaus, 

Johann Pratschke 

Identification of molecular candidate marker in zero kidney biopsies are indicative for graft 

quality 

Katja Kotsch, Vera Merck, Kristina Kunert, Anja Reutzel-Selke, Andreas Pascher, Hans-Dieter Volk, Peter Neuhaus, 

Johann Pratschke 

Identification of molecular candidate marker in zero kidney biopsies are indicative for graft 

quality 

Wolfram Osen, Mingxia Song, Sabine Soltek, Barbara Leuchs, Julia Steitz, Xuan Duc Ngyuen, Dirk Schadendorf, 

Annette Paschen 

Identification of novel CD4+ T cell epitopes from human tyrosinase related protein 2 (TRP-2) by 

a combinatorial approach based on the immunisation of HLA-transgenic mice with recombinant 

Adenovirus and antigen peptide library screening 

Josip Zovko, Marco Herold, Christa Kraus, Andrea Peters, Ingolf Berberich 

Identification of proteins that influence stability and functionality of the anti-apoptotic Bcl-2 

family member A1/Bfl-1 

Susanne Berchtold, Edda Fahl, Mathias Hornef, Julia Geisel, Julia-Stefanie Frick, Erwin Bohn 

IFIT-2 – a putative novel negative regulator of proinflammatory responses 

Uwe Müller, Werner Stenzel, Gabriele Köhler, Gesine Hansen, Nicole Schütze, Reinhard Straubinger, Manfred Blessing, 

Andrew McKenzie, Frank Brombacher, Gottfried Alber 

IL 13 induces disease promoting type 2 cytokines, alternatively activated macrophages and 

allergic inflammation during pulmonary infection of mice with Cryptococcus neoformans 

Annette Erhardt, Markus Biburger, Gisa Tiegs 

IL-10 AND REGULATORY T CELLS – THE MAIN MEDIATORS OF IMMUNOLOGICAL TOLERANCE 

AGAINST CONCANAVALIN A

Anne Schumacher, Paul Ojiambo Wafula, Ana Teles, Hideo Yagita, Hans-Dieter Volk, Ana Zenclussen 

IL-10 but not TGF-ß is essential for the suppressor function of Treg cells in murine pregnancy 

Hyun-Dong Chang, Jun Dong, Andreas Thiel Thiel, Andreas Radbruch 

IL-10 Expression in Th lymphocytes is conditional 

Manuel N. D. M. Guerreiro, Anne Marie Asemissen, Gianna Schulz, Il-Kang Na, Jochen Hühn, Sandra Bauer, Eckhard 

Thiel, Ulrich Keilholz, Carmen Scheibenbogen 

IL-2 induces IL-10 producing regulatory CD3+ T cells in vitro and in vivo 

Kerstin Wolk, Ellen Witte, Ute Hoffmann, Wolf-Dietrich Döcke, Stefanie Endesfelder, Khusru Asadullah, Wolfram 

Sterry, Hans-Dieter Volk, Bianca Maria Wittig, Robert Sabat 

IL-22 Induces Lipopolysaccharide-Binding Protein in Hepatocytes: A Potential Systemic Role of 

IL-22 in Crohn's Disease 

Daniel Hebenstreit, Elisabeth Maier, Jutta Horejs-Hoeck, Min Li-Weber, Albert Duschl 

IL-4 suppresses the Gene Expression of TCF-1 in T cells in a STAT6 dependent way 

Manuel Otte 

IL-4R – signaling through an alternative signal transduction pathway Manuel Otte, Mario Zaiss, 

Susanne Bürgis, Anja Thiel, Georg Schett and André Gessner Institute of Medical Microbiology, 

Immunology and Hygiene; University of Erlangen-Nuremberg 

Julia-Stefanie Frick, Julia Geisel, Frauke Kahl, Hermann Wagner, Carsten Kirschning, Ingo Autenrieth 

IL-6 and maturation govern TLR2 and TLR4 induced TLR agonist tolerance and cross-tolerance in 


Marsilius Mues, Marco Mank, Oliver Griesbeck, Hartmut Wekerle, Florian Kurschus 

Imaging Activation: FRET-based Calcium Biosensors in T-Lymphocytes 

Thomas G. Berger, Hendrik Schulze-Koops, Michaela Schäfer, Ester Müller, Manfred B. Lutz 

Immature and maturation-resistant human dendritic cells generated from bone marrow under 

GMP conditions induce allogeneic T cell anergy in vitro 

Daniel Nickel, Sven Poppert, Tatjana Zelenski, Nicole Kästner, Heiko Bruns, Axel Schubert, Axel Spahr, Steffen Stenger 

Immune modulation mediated by Aggregatibacter actinomycetemcomitans as a possible 

mechanism for the development of periodontitis 

Jessica Butz, Cordula Fuchs, Barbara Kessler, Heiner Voigt, Daniel Wienhold, Mathias Buettner 

Immune reaction of swine after repeated intra-muscular (i. m.) immunization with avian 

influenza virus H5 

Marcin Wlodarski, Zachary Nearman, Alan Lichtin, Hans-Dieter Volk, Jaroslaw Maciejewski 

IMMUNODOMINANT CYTOTOXIC T LYMPHOCYTE EXPANSIONS IN PATIENTS WITH 

UNEXPLAINED NEUTROPENIA. 

Doreen Haase, Anne Marie Asemissen, Carmen Scheibenbogen 

Immunogenic epitopes of the PAX2 transcription factor recognized by colon carcinoma patients 

Lukas Frenzel, Zeinab Abdullah, Anja Kriegeskorte, Rebecca Borsutzky, Manoj K. Gupta, Olaf Utermöhlen, Dirk H. 

Busch, Martin Krönke, Jürgen Hescheler, Tomo Saric 

Immunological properties of murine embryonic stem cell-derived cardiomyocytes 

Felix Heymann, Emma E. Hamilton-Williams, Isis Ludwig-Portugall, Susan Quaggin, Jürgen Floege, Hermann-Josef 

Gröne, Christian Kurts 

Immunopathology of T cell-mediated glomerulonephritis 

Nadine Nippe, Katja Gutsche, Mechthild Jung, Gerald Grütz 

Immunoregulation of IL-10 induced Autotaxin 

Wolfgang G Bessler, Karola Puce, Carsten Kirschning, Maria Huber

Immunostimulating effects of the bacterial extract OM-89 

Kerstin Annika Sauer, Joachim Heinrich Maxeiner, Petra Scholtes, Roman Karwot, Hans-Anton Lehr, Mark Birkenbach, 

Richard Steven Blumberg, Susetta Finotto 

Immunosurveillance of lung melanoma metastasis in EBI-3 (-/-) mice by NK-DCs- induced CD8+ 

T cells 

Henoch Hong, Nupur Bhatnagar, Maren Mönkemeyer, Hans Heiken, Reinhold E. Schmidt, Dirk Meyer-Olson 

Impact of HIV-1 Vpr on type I and type II interferon secretion by plasmacytoid dendritic cells 

and natural killer cells 

Nadine Kämper, Claudia Wegscheid, Jörg Keßler, Norbert Koch 

Impact of HLA encoded BAT3 splice variants on MHC class I and class II expression 

Diana Fleissner, Jan Buer, Astrid Westendorf 

Impact of intestinal dendritic cells for the induction of tolerance or pathology 

Johannes Lutz, Werner Müller, Chander Raman, Hans-Martin Jäck 

Impaired B Cell Development in the Presence of a Non-Coding IgM mRNA 

Verena Moos, Kristina Allers, Thomas Schneider 

Impaired innate functions of monocytes and macrophages in Whipple`s disease 

Kittan Nicolai A., Bergua Antonio, Haupt Sabrina, Donhauser Norbert, Schuster Philipp, Korn Klaus, Harrer Thomas, 

Schmidt Barbara 

Impaired plasmacytoid dendritic cell (PDC) innate immune responses in patients with 

herpesvirus-associated severe acute retinal necrosis (ARN) 

Daniela Wesch, Philine Wrobel, Hamed Shojaei, Hans-Heinrich Oberg, Monika Kunz, Dieter Kabelitz 

Implications for the design of γδ T cell-based cancer immunotherapy 

Inga Gebuhr, Kathrin Gube, Katrin Vogt, Christian Meisel, Sandra Naundorf, Hans-Dieter Volk, Birgit Sawitzki 

Importance of cell surface N-glycosylation for activation of T cell subpopulations 

Lydia-Mareen Köper, Andrea Schulz, Hans-Jürgen Ahr, Hans-Werner Vohr 

In vitro Differentiation of Skin Sensitizers by Cell Signaling Pathways 

Sabine Ring, Karsten Mahnke, Alexander Enk 

In vivo activation of injected Tregs precedes the suppression of the elicitation phase of Contact 

hypersensitivity reactions independent from spleen and lymph nodes 

Sonja Schallenberg, Sabine Ring, Tanja Bedke, Sabrina Schmitt, Kurt Schönfeld, Karsten Mahnke, Elisabeth Suri- 

Payer, Alexander H. Enk 

In vivo depletion of CD4+CD25+Foxp3+ regulatory T cells does not affect the growth of 

established B16 tumors 

Alla Skapenko, Joachim R. Kalden, Peter E. Lipsky, Hendrik Schulze-Koops 

In vivo function of IL-4-induced Tregs 

Marcus Gereke, Karsten Mahnke, Elmar Jäckel, Jan Buer, Dunja Bruder 

In vivo induction of tolerance via DEC-205 mediated antigen delivery – therapeutic safety in the 

context of infection 

Andreas Wieland, Markus Denzel, Jörg Reimann, Reinhold Schirmbeck 

In vivo produced complexes of antigen with stress proteins are potent immunogens 

Undine Meusch, Manuela Rossol, Holm Häntzschel, Christoph Baerwald, Sunna Hauschildt, Ulf Wagner 

Increased Infliximab-induced monocyte apoptosis via reverse signalling of membrane TNF in 

patients with rheumatoid arthritis

Joachim Heinrich Maxeiner, Kerstin Annika Sauer, Roman Karwot, Petra Scholtes, Rainer Wiewrodt, Hans-Anton Lehr, 

Susetta Finotto 

Increased lung tumor in NFATc2 (-/-) mice mediated by defective CD8+ T cells and increased 

CD4+CD25+ lung T cells 

Matthias Kresse, Ingo Uthe, Heike Weighardt, Irmgard Förster 

Inducible ablation of CCL17 positive DC in vivo 

Katharina A. Remer, Calin Apetrei, Tobias Schwarz, Heidrun Moll 

Induction of a local Th1 response after plasmacytoid dendritic cell-based vaccination protects 

mice against infection with Leishmania major 

Christine Warmbold, Arthur Ulmer, Thomas Roeder 

Induction of inflammatory signal transduction pathways in a Drosophila-derived cell line 

Julia Strebovsky, Claus Kaiser, Alexander Dalpke, Klaus Heeg, Holger Bartz 

Induction of regulatory T-cells by Toll-like receptor-ligand derived deviant dendritic cells 

Ria Baumgrass, Vladimir Pavlovic, Britta Lamottke, Maria Lexberg, Joachim Grün, Uwe Niesner, Andreas Radbruch 

Induction of Treg cells by manipulation of TCR signalling 

Stephan Fricke, Nadja Hilger, Manuela Ackermann, Peter Ruschpler, Lutz Uharek, Guido Hildebrandt, Jan Matthias 

Braun, Frank Emmrich 

Induction of xenogenic acute graft versus host disease (aGvHD) in mice 

Uta Bussmeyer, Arup Sarkar, Kirsten Broszat, Ger van Zandbergen, Christian Bogdan, Werner Solbach, Friederike von 

Loewenich, Tamás Laskay 

Infection with Anaplasma phagocytophilum inhibits IFN-&gamma signaling in human 

neutrophils 

Vilma Urbonaviciute, Barbara G. Fürnrohr, Silke Meister, Petra Heyder, Martin Herrmann, Joachim R. Kalden, Reinhard 

E. Voll 

Inflammation and immune activation by HMGB1-nucleosome complexes – implications for the 

etiopathogenesis of systemic lupus erythematosus 

Simone Vallbracht, Birthe Jessen, Sonja Mrusek, Anselm Enders, Peter L. Collins, Christine D. Krempl, Stephan Ehl 

Influence of a single viral epitope on T cell response and disease after infection of mice with 

respiratory syncytial virus 

Torsten Lowin, Rainer H. Straub, Olga Wiesner, Ulf Müller-Ladner, Jörg Schedel 

Influence of cortisol on the expression of integrins and intracellular signaling molecules in 

synovial fibroblasts in rheumatoid arthritis 

Veronika Lukacs-Kornek, Verena Semmling, Christian Kurts 

Influence of Cross-Presentation via CCR7 

Birgit Weinberger, Ilka Weiskirchner, Beatrix Grubeck-Loebenstein 

Influence of latent infection with Cytomegalovirus on Epstein-Barr viral parameters in healthy 

elderly individuals: possible interaction of different herpesviruses in immunosenescence 

Sandra Martina Dittrich, Elfriede Noessner, Hans Demmelmair, Gernot Desoye, Dolores Schendel, Berthold Koletzko, 

Susanne Krauss-Etschmann 

Influence of n3/n6 polyunsaturated fatty acids on placental immune responses 

Stephan Sudowe, Karina Gisch, Nadine Gehrke, Matthias Bros, Christina Priesmeyer, Angelika B. Reske-Kunz 

Inhibition of allergic sensitization and suppression of Th2-mediated airway inflammation by 

application of formalin-fixed Staphylococcus aureus-particles 

Tobias Frankenberg, Susanne Kirschnek, Hans Häcker, Uwe Koedel, Georg Häcker 

Inhibition of apoptosis conserves neutrophil effector function and can contribute to 

inflammation in vivo

Martin Raftery, Günther Schönrich 

Inhibition of CD1 Antigen Presentation by Human Cytomegalovirus 

Dorit Fabricius, Sue O’Dorisio, Sue Blackwell, Bernd Jahrsdörfer, Klaus-Michael Debatin 

Inhibition of IFN-α Secretion and Modulation of Immunophenotype and Stimulatory Capacity of 

Human Plasmacytoid Dendritic Cell by Vasoactive Intestinal Peptide 

Rainer Glauben, Arvind Batra, Thorsten Stroh, Elena Sonnenberg, Inka Fedke, Hans Anton Lehr, Paolo Mascagni, 

Martin Zeitz, Britta Siegmund 

Inhibition of NF-κB by histone deacetylase inhibitors in models of inflammation-related 

tumorigenesis 

Dirk Reinhold, Alexander Goihl, Bianca Guth, Uwe Lendeckel, Ute Bank, Michael Täger, Siegfried Ansorge, Jürgen 

Faust, Klaus Neubert, Stefan Brocke 

Inhibitors of dipeptidyl peptidase IV (DP IV, CD26)-like and of aminopeptidase N (APN, CD13) 

enzymatic activity suppress human T cell activation and IL-17 production in vitro and in vivo 

Simone Klöter, Max v. Holleben, Bernhard Reis, Klaus Pfeffer, Sandra Beer 

Interaction analysis of the adaptor protein SLy2 

Pia Herzberger, Corinna Siegel, Christine Skerka, Volker Fingerle, Ulrike Schulte-Spechtel, Bettina Wilske, Volker 

Brade, Reinhard Wallich, Peter F. Zipfel, Peter Kraiczy 

Interaction of immune regulators factor H and FHL-1 with the Lyme disease spirochete Borrelia 

spielmanii sp. nov. is mediated by a plasmid-encoded complement regulator-acquiring surface 

protein-1 

Nikola Baschuk, Olaf Utermöhlen, Roland Gugel, Gabriele Warnecke, Ulrike Karow, Daniela Paulsen, Frank 

Brombacher, Martin Krönke, Wolfgang Deppert 

Interleukin-4 impairs granzyme-mediated cytotoxicity of Simian virus 40 large tumor antigenspecific 

CTL in Balb/c mice 

Michael Meyer-Hermann, Marc Thilo Figge 

Interpreting two-photon lymphocyte motility data of the germinal centre: Predictions and 

analysis by mathematical models 

Astrid M. Westendorf, Wiebke Hansen, Jan Buer 

Intestinal antigen display promotes the peripheral induction of antigen specific CD8 + Foxp3 + T 

cells 

Ilka Knippertz, Andrea Hesse, Eckhart Kaempgen, Gerold Schuler, Alexander Steinkasserer, Dirk. M. Nettelbeck 

Intracellular expression of CD40L after adenoviral transduction in combination with IFN-gamma 

treatment generates human dendritic cells that both secrete IL-12 and have migratory functions 

Silke Overbeck, Peter Uciechowski, M. Leigh Ackland, Dianne Ford, Lothar Rink 

Intracellular Zinc Homeostasis in Leukocyte Subsets is Regulated by Different Expression of Zinc 

Exporters ZnT-1 to ZnT-9 

Benedikt Fritzsching, Jürgen Haas, Fatima König, Eva Pauly, Johannes Pöschl, Peter Krammer, Wolfgang Brück, 

Elisabeth Suri-Payer, Brigitte Wildemann 

Intracerebral Human Regulatory T Cells: Analysis of CD4+CD25+FOXP3+ T Cells in Brain Lesions 

and Cerebrospinal Fluid of Multiple Sclerosis Patients 

Sebastian Kreiter, Mustafa Diken, Abderraouf Selmi, Abdo Konur, Michael Koslowski, Christoph Huber, Özlem Türeci, 

Ugur Sahin 

Intranodal RNA immunisation – a potent method for the induction of immunity by selective 

transfection of DC 

Stephanie Konrad, Linda Engling, Reinhold E. Schmidt, J. Engelbert Gessner 

INVERSE REGULATION OF THE MURINE FcγRIIB AND FcγRIII GENE EXPRESSION BY C5a IS 

MEDIATED THROUGH DISTINCT DNA-RESPONSE ELEMENTS 

Luise Weigand, Xiaoling Liang, Florian Anderl, Judith van der Griendt, Ingrid Schuster, Andreas Moosmann, Bernhard 

Helga, Elfriede Nößner, Dirk Busch, Angela Krackhardt

Investigation of allorestricted peptide-specific T cell responses against Her2/neu – implications 

for adoptive T cell therapy in solid cancer 

Stefanie Margraf, Carsten Watzl 

Investigation of membrane microdomains surrounding Natural Killer cell receptors 

Vivienne Engelschalt, J. Engelbert Gessner, Richard A. Kroczek 

Involvement of complement and Fc-receptors in the depletion of T cells in vivo 

Alexander Fassold, Werner Falk, Rainer H. Straub 

Is neuropilin-2 a possible target for the treatment of arthritis? 

Selina Christen, Edith Hintermann, Monika Bayer, Urs Christen 

JAM-C and its influence on the pathogenesis of type 1 diabetes 

Nils Schoof, Frederike von Bonin, Lorenz Trümper, Dieter Kube 

Janus kinases are targets of tyrphostin AG17 and HSP90-inhibitor 17-AAG in classical Hodgkin 

Lymphoma 

Eva Schlecker, Isabel Hartmann, Michael Ackmann, Elisabeth Weiss 

KIR2DS2 and interaction with DAP12 

Gleb Turchinovich, Jan Kranich, Sonja Schmid, Jürgen Bachl, Jörg Kirberg 

Kruppel-like factor 3 (KLF3) affects marginal zone B cell differentiation 

Dietmar Zehn, Michael J. Bevan 

Lack of peripheral control of low avidity self reactive T cells 

Gordon Wilke, Gretel Wittenburg, Claudia Berek 

Laser Capture Microdissection and Mircroarray: a Characterisation of Follicular Dendritic Cells 

Katja Farhat, Sabine Riekenberg, Jennifer Debarry, Roland Lang, Jörg Mages, Günther Jung, Karl-Heinz Wiesmüller, 

Artur J. Ulmer 

LIGAND BINDING AND SIGNAL TRANSDUCTION INDUCED BY LIPOPEPTIDES 

Christian Draing, Christoph Rockel, Susanne Deininger, Stefanie Sigel, Oliver Dehus, Tamara Rupp, Artur Ulmer, 

Thomas Hartung, Corinna Hermann, Sonja von Aulock 

Lipoteichoic acid from a lipoprotein diacylglycerol transferase deletion mutant is a potent 

immunobiologically active compound 

Anna Schurich, Silke Hegenbarth, Jan Böttcher, Sven Burgdorf, Andreas Dolf, Elmar Endl, Christian Kurts, Percy A. 

Knolle 

Liver sinusoidal endothelial cells are more efficient in cross-presentation than CD8alpha splenic 

dendritic cells expressing endocytic receptors devoted to cross-presentation 

Matthias Hardtke-Wolenski, Nadja Saal, Konstantinos Iordanidis, Mark S Anderson, Michael P Manns, Elmar Jäckel 

Liver specific immune responses related to AIRE mutations 

Stephan Borte, Uwe Gerd Liebert, Michael Borte, Ulrich Sack 

Long-term cell-mediated immunity following vaccination with live, attenuated measles-mumpsrubella-vaccine 

in children with juvenile idiopathic arthritis under treatment with low-dose 

Methotrexate and/or tumour necrosis factor α receptor antagonist 

Christian Pötschke, Mandy Busse, Annegret Dummer, Tobias Traeger, Wolfram Keßler, Erika Friebe, Marlene Mikulcak, 

Claus-Dieter Heidecke, Stefan Maier, Barbara Bröker 

Long-term effects of polymicrobial sepsis on the adaptive immune system 

Zulema Cabail, Holger Hoff, Heike Hirseland, Steven Nadler, Gerd R. Burmester, Monika C. Brunner-Weinzierl 

Longevity of CD28null T Lymphocytes is abrogated by CTLA-4Ig treatment

Anja Siepert, Birgit Sawitzki, H.M. Reichardt, Jochen van den Brandt, Markus Tiedge, Manfred Lehmann, Hans-Dieter 

Volk, Petra Reinke 

Low dose CNI treatment can control effector function of depletion resistant allo-specific memory 

T cells (financial support by Else-Kröner-Fresenius-Stiftung P14/06//A01/06) 

Markus Kleinewietfeld, Mireille Starke, Thomas Blankenstein, Kirsten Falk, Olaf Rötzschke 

Low dose cyclophosphamide tumor rejection is independent of CD4+ CD25+ regulatory T cells 

(Treg) 

Juliane Ladhoff, Michael Bader, Sabine Brösel, Elke Effenberger, Isabela Schmitt-Knosalla, Hans-Dieter Volk, Martina 

Seifert 

Low immunogenicity of rat embryonic stem cell derivatives 

Anette Brass, Shiyuan Hong, Nicole Schwarz, George Dubyak, Michel Seman, Friedrich Koch-Nolte, Friedrich Haag 

LPS and interferons induce surface expression and activity of ADP-ribosyltransferase ART2.1 on 

murine bone marrow-derived macrophages 

Christian Schiller, John-Christian Eilert, Maximilian Nitschké, Alexander Seidl, Michael Schleicher, Dolores J. Schendel, 

Elisabeth H. Weiss 

LST1: a potential transmembrane adaptor protein that modulates cell morphology 

Niklas Engels, Gökhan Yigit, Christoph Emmerich, Dirk Czesnik, Detlev Schild, Jürgen Wienands 

Lytic replication of Epstein-Barr virus can be induced by LMP2A 

Heiko Johnen, Tamara Kuffner, Andrew Cook, Emma Braine, Ben Wu, Roland Stocker, Samuel Norbert Breit 

Macrophage Inhibitory Cytokine 1(MIC-1) reduces disease severity in mouse models of arthritis 

and atherosclerosis 

Katharina Kronenberg, Beate G. Exner, Christine Sattler, James A. Hutchinson, Gudrun E. Koehl, Stefan Farkas, Hans 

J. Schlitt, Fred Fändrich, Edward K. Geissler 

Macrophages Driven to a Novel State of Activation have Anti-Inflammatory Properties in Mice 

Günes Esendagli, Kirsten Bruderek, Torsten Goldmann, Andreas Busche, Detlev Branscheid, Ekkehard Vollmer, Sven 

Brandau 

Malignant and non-malignant lung tissue areas are differentially populated by natural killer cells 

and regulatory T cells in non-small cell lung cancer 

Christina Janko, Udo S. Gaipl, Sandra Franz, Nina Ebel, Eberhard Schlücker, Roland Meyer-Pittroff, Martin Herrmann, 

Benjamin Frey 

Mammalian cells under pressure – Cell death pathways and immunogenicity of dying cells 

Christoph Lauer, Michael Basler, Susan D. Demo, Marcus Groettrup 

Manipulation of MHC class I antigen presentation by a LMP7-specific inhibitor 

Lisa Bruns, Oliver Frey, Christiane Landgraf, Rudolf Volkmer, Thomas Kamradt 

Mapping of T cell epitopes in Glucose-6-Phosphate-Isomerase induced arthritis 

Peter Kramer, Frank Siebenhaar, Marcus Maurer, Sven Hendrix 

Mast cell deficient mice display increased brain inflammation, neurodegeneration and 

astrogliosis 

Yves Montier, Axel Lorentz, Sigrid Krämer, Stephan C Bischoff 

Mast cell mediators stimulate fibroblasts to produce IL-6 that vice versa supports mast cell 

survival 

Julia Scholten, Alexander Gerbaulet, Giuseppe Testa, Thomas Krieg, Karin Hartmann, Axel Roers 

Mast cell-specific Cre/loxP-mediated mutagenesis in vivo 

ERIETTA STELEKATI, ZANE ORINSKA, ANNALENA BOLLINGER, SILVIA BULFONE-PAUS 

Mast cells modulate CD8+ T cell responses.

Milan Popovic, Ana Teles, Catharina Thuere, Anne Schumacher, Paul Ojiambo Wafula, Hans-Dieter Volk, Ana Claudia 

Zenclussen 

Mast-cell-associated genes Thp1, Mcpt1 and Mcpt5 are up-regulated after Treg-induced 

tolerance at the fetal-maternal interface: new role for mast cells in pregnancy-induced 

tolerance? 

Kristina Wiege, Syed Raza Ali, Stephanie Konrad, Roland Piekorz, Bernd Nürnberg, Reinhold E Schmidt, J Engelbert 

Gessner 

MECHANISM OF CELL AND ISOTYPE SPECIFIC Gαi DEPENDENT SIGNALING IN IMMUNE 

EFFECTOR CELLS 

Marco Wendel, Elisabeth Suri-Payer, Adelheid Cerwenka 

Mechanisms of NK cell migration in response to tumors 

Barbara C. Rütgen, Wilhelm Gerner, Armin Saalmüller, Sabine E. Hammer 

MHC typing in swine: The SLA-haplotype repertoire of Austrian Large White, Landrace, and 

Pietrain breeding stocks 

J. Albrecht, T. J. Boeld, K. Doser, R. Eder, J. Stahl, R. Andreesen, J. Ermann, M. Edinger, P. Hoffmann 

MHC-compatibility between conventional and regulatory T cells is required for suppression of 

allospecific T cell responses 

Romney Haylett, Lothar Rink 

MHC-II Signaling Directs the Activation of NFAT but not NF-κB in B Cells 

Karina Stein, Jennifer Debarry, Anna Hanuszkiewicz, Otto Holst, Jörg Mages, Roland Lang, Holger Heine 

Microarray analysis of human dendritic cells stimulated with four different bacterial strains with 

focus on allergy-protecting mechanisms 

Kristina Allers, Désirée Kunkel, Verena Moos, Martin Eisenblätter, Christiane Stahl-Hennig, Annette Schrod, Ralf 

Ignatius, Franz-Josef Kaup, Thomas Schneider 

Migration patterns of activated versus non-activated non-human primate T lymphocytes: 

preferential homing of activated autologous CD8+ T cells in the rectal mucosa 

Stefan Wiehr, Thomas Herrmann 

Milk-derived immune cells may serve as Trojan Horses in Toxoplasma gondii transmission 

Marcin Kaminski, Peter H. Krammer, Karsten Gülow 

Mitochondria function as oxidative signalling organelles in activation-induced apoptosis of T 

cells. 

Martina Anzaghe, Zoe Waibler, Holger Ludwig, Shizuo Akira, Siegfried Weiss, Gerd Sutter, Ulrich Kalinke 

Modified vaccinia virus Ankara induces Toll-like receptor independent type I interferon 

responses 

Christian Jacobi, Jürgen Roemisch, Stefan Meuer, Thomas Giese 

Modulation of gene expression by IVIG in healthy donors and multiple sclerosis patients 

Anastasia Schneider, Tatiana Binder, Rodica Bernatowicz, Anke Zobywalski, Christine Falk, Anton Hartmann, Dolores 

Schendel, Susanne Krauss-Etschmann 

MODULATION OF HUMAN DENDRITIC CELLS BY SIX DIFFERENT PROBIOTIC BACTERIAL STRAINS 

Irene Wittmman, Diana Aichele, Gerhard Groer, André Gessner, Markus Schnare 

Modulation of innate immune responses by recombinant murine bactericidal permeability / 

increasing protein through neutralizaton of LPS and Gram-negative bacteria 

Stephan Schierer, Andrea Hesse, Ina Mueller, Eckhart Kaempgen, David T. Curiel, Gerold Schuler, Alexander 

Steinkasserer, Dirk M. Nettelbeck 

Modulation of viability and maturation of human monocyte-derived dentritic cells by oncolytic 

adenoviruses 

Philip Kruse, Cornelia Rosner, Lutz Walter

Molecular characterisation of the killer cell immunoglobulin-like receptors (KIR) of the rhesus 

macaque (Macaca mulatta) 

Cary Mac Millan, Alexander Hann, Peter Bannas, Wolfgang Koestner, Friedrich Buck, Friedrich Haag, Friedrich Nolte 

Molecular characterization of ADP-ribosylated T cell membrane proteins 

Viet Bui, Andreas Diefenbach 

Molecular cloning and functional characterization of a novel stimulatory immunoreceptor 

expressed by myeloid cells 

Susan M Schlenner, Lars A Schneider, Thorsten B Feyerabend, Markus Wunderlin, Hans-Reimer Rodewald 

Molecular mechanism of mast cell-mediated innate defense against endothelin and snake venom 

sarafotoxin 

Jennifer Debarry, Anna Hanuszkiewicz, Otto Holst, Holger Heine 

Molecular mechanisms of human dendritic cell stimulation by the Allergy-protective Lactococcus 

lactis strain G121 

Malte Bachmann, Jens Paulukat, Josef Pfeilschifter, Heiko Mühl 

MOLECULAR MECHANISMS OF IFNγ-INDUCED IL-18 BINDING PROTEIN PROMOTER ACTIVATION 

AS DETECTED IN HUMAN DLD-1 COLON CARCINOMA CELLS 

Maria Papatriantafyllou, Thilo Oelert, Günter Hämmerling, Bernd Arnold 

MOLECULAR MECHANISMS OF PERIPHERAL CD8 T CELL TOLERANCE 

Tobias Schwerd, Johannes C. Hellmuth, Andreas Schmidt, Hendrik Poeck, Michael Wenzel, Stefan Endres, Simon 

Rothenfusser 

Molecular mechanisms of virus recognition by Rig-I-like helicases 

Petra Riedl, Kurt Reifenberg, Joerg Reimann, Reinhold Schirmbeck 

Mono-specific, hepatic CD8 T cell responses primed by cationic peptide/oligonucleotide 

complexes suppress viral replication in HBV transgenic mice 

Sonja Schmucker, Mario Assenmacher, Anne Richter 

Monocytes and myeloid dendritic cells are both able to induce primary activation of naïve MART- 

1-specific CD8 + T cells in vitro 

Beatrice Jahn-Schmid, Gottfried Fischer, Gabriele Gadermaier, Matthias Egger, Fatima Ferreira, Christof Ebner, 

Barbara Bohle 

Mugwort pollen allergy as a unique model for the investigation of allergen-specific CD4+T cells 

using HLA classII/peptide tetramers 

Andreas Junker, Jana Ivanidze, Joachim Malotka, Ingrid Eiglmeier, Hans Lassmann, Hartmut Wekerle, Edgar Meinl, 

Reinhard Hohlfeld, Klaus Dornmair 

Multiple Sclerosis: T cell receptor transcriptome in distinct brain regions 

Felix C. Popp, Elke Eggenhofer, Prezemyslaw Slowik, Philipp Renner, Katharina Kronenberg, Hans J. Schlitt, Pompiliu 

Piso, Marc H. Dahlke 

MULTIPOTENT MESENCHYMAL STROMAL CELLS INDUCE LONG-TERM ALLOGRAFT ACCEPTANCE 

MEDIATED THROUGH IDO 

Verena Besche, Christina Glowacki, Nadine Wiechmann, Andrea Renzing, Ngoc-Anh Dang, Stephan Sudowe, Jürgen 

Knop, Angelika B. Reske-Kunz, Matthias Bros 

Murine dendritic cells exert tolerogenic function at their immature state and upon differentiation 

in the presence of dexamethasone 

Ulrich Salzer, Chiara Bacchelli, Stephanie Jennings, Allesandro Plebani, Helen Chapel, Hans D Ochs, Simon Urschel, 

Bernd H Belohradsky, H Bobby Gaspar, Bodo Grimbacher 

Mutations in TACI/TNFRSF13b in patients with CVID – a genetic, immunological and clinical 

study in a large patient cohort 

Max Bastian, Tobias Braun, Heiko Bruns, Martin Röllinghoff, Steffen Stenger

Mycobacterial Lipopeptides Elicit CD4+ Cytolytic T Lymphocytes in Mycobacterium tuberculosis 

Infected Humans 

Peter Reichardt, Bastian Dornbach, Song Rong, Stefan Beissert, Faikah Güler, Karin Loser, Matthias Gunzer 

Naive B cells generate regulatory T cells in the presence of a mature immunological synapse 

Nils Kruse, Arnhild Schrage, Katrin Neumann, Katharina Eulenburg, Friderike Blumenthal-Barby, Simon Fillatreau, 

Percy A. Knolle, Martin Zeitz, Alf Hamann, Katja Klugewitz 

Naive CD4+ T cells primed by Liver Sinusoidal Endothelial Cells (LSEC) do not differentiate into 

cytokine producing effector cells and fail to induce a delayed type hypersensitivity reaction 

(DTH) 

Benedikt Fritzsching, Eva Pauly, Nina Oberle, Navina Kuss, Katrin Hartmann, Peter Ruf, Johannes Pöschl, Peter 

Krammer, Elisabeth Suri-Payer 

Naive regulatory T cells: activation and apoptosis sensitization of this novel Treg subpopulation 

during the human neonatal period 

Manoj Kumar, Norman Putzki, Hans Christoph Diener, Hans Grosse-Wilde, Ernst Kreuzfelder 

Natalizumab seems to effect mainly B lymphocytes in patients with Multiple Sclerosis 

Stephanie Joachim, Martin Wax, Daniela Kraft, Norbert Pfeiffer, Franz Grus 

Neurodegeneration of the optic nerve and changes in antibody profiles after immunization with 

heat shock protein 27 

Barbara Daller, Michaela Jungbeck, Klaus Pfeffer, Daniela N. Männel, Thomas Hehlgans 

Neutralization of LIGHT ameliorates DSS-induced intestinal inflammation 

Katrin Birkholz, Niels Schaft, Jan Dörrie, Michael Schwenkert, Christian Kellner, Georg Fey, Gerold Schuler 

New strategies for antigen loading of human monocyte-derived DC 

Timo Herrmann, Nousheen Zaidi, Hubert Kalbacher 

New strategies for the analysis of processed antigens in dendritic cells 

Hamid Kashkar, Anke Deggerich, Jens-Michael Seeger, Benjamin Yazdanpanah, Katja Wiegmann, Dirk Haubert, Carola 

Pongratz, Martin Krönke 

NF-kappaB independent down-regulation of XIAP by bortezomib sensitizes Hodgkin Lymphoma 

B-cells against cytotoxic drugs 

Michael Kiessling, Peter H. Krammer, Karsten Gülow 

NF-kB suppression sensitizes cells towards ROS-induced apoptosis 

Maren Claus, Sabine Wingert, Carsten Watzl 

NK cell activity is regulated by CD48 - 2B4 (CD244) interaction in cis and in trans 

Matthias Krusch, Sorin Armeanu, Ulrich Martin Lauer, Alexander Steinle, Michael Bitzer, Helmut Rainer Salih 

NK cell lysis of hepatoma cells following specific induction of NKG2D ligands by the proteasome 

inhibitor Bortezomib 

Marion Schneider, Xuefang Ren, Ying Wang 

NK cell targeting of phagocytic dendritic cells 

Cédric VONARBOURG, Andreas DIEFENBACH 

NK CELLS EXPRESSING CLASS II MHC MOLECULES: SEPARATE LINEAGE OR JUST NK CELLS ? 

Petra Prinz, Ainhoa-Marie Figel, Judith Hosse, Peter J Nelson, Julia S Schleypen, Nicole Baur, Dolores J Schendel, 

Christine S Falk, Elfriede Noessner 

NK cells with distinct phenotypic and functional profile in the context of myeloid infiltration and 

renal cell carcinoma milieu 

Rachel Thomas, Maren Mönkemeyer, Gamze Kabalak, Reinhold E. Schmidt, Torsten Witte 

NKG2D polymorphism associated with reactivation of CMV in HIV-1 infected patients

Benjamin Stoelcker, Kirsten Krätzel, Günther Eissner, Michael Pfeifer, Christian Schulz 

NKG2D-triggered effector function of bronchial epithelial cell activated alloreactive CD8+ T cells 

Hansjörg Thude, Kathrin Rebstock, Bärbel Lutz, Jörg Blume, Dagmar Barz 

No association of the CD45 C77G gene polymorphism with inflammatory bowel disease in 

German patients 

Sascha Rutz, Marko Janke, Manuela Krueger, Alexander Scheffold 

Notch induces interleukin-10 production by T helper 1 cells 

Anne Endmann, Dirk Bumann, Susanne zur Lage, Marcin Lyszkiewicz, Siegfried Weiss, Holger Lößner 

Novel Salmonella vaccine strains with in vivo amplifiable antigen expression plasmids 

Britta Schneider, Jeannette Gerspach, Sabine Münkel, Harald Wajant, Peter Scheurich, Klaus Pfizenmaier 

Novel TRAIL fusion proteins as promising candidates for cancer therapy 

Verena Susanne Meyer, Dagmar Sigurdardottir, Wolfgang Kastenmüller, Georg Gasteiger, Ingo Drexler, Stefan 

Stevanovic 

Novel vaccinia virus T-cell epitopes identified by mass spectrometry of HLA ligands 

Kurt Schönfeld, Sabine Ring, Tanja Bedke, Sonja Schallenberg, Karsten Mahnke, Alexander H. Enk 

Nucleofection is an effective method to transduce regulatory T cells with different homing 

receptors. 

Sabrina Schmitt, Karsten Mahnke, Kurt Schönfeld, Svetlana Karakhanova, Alexander H. Enk 

Number and function of regulatory T cells in GvHD increase by Extracorporeal Photopheresis 

Martin Kolev, Marieta Ruseva, Steffen Thiel, Frederik Hansen, Jens Jensenius 

On the role of MBL in a murine model of oral infection with Salmonella typhimurium 

Monika Braun, Christine Sers, Ruprecht Kuner, Barbara Simm, Barbara Mosetter, Dolores Schendel, Christine Falk 

Oncogenic KRAS-signalling and DNMT activity are involved in immune escape of tumor cells 

Varsha Kumar, Jens Stein 

Optical ProjectionTomography as a tool for studying the three-dimensional structure of 

Seconday Lymphoid Organs* 

Mathias Fousse, Ulrich Mack, Tobias Hodapp, Urban Sester, Gerhard W. Sybrecht, Hans Köhler, Martina Sester 

OPTIMIZED DIAGNOSIS OF LATENT TUBERCULOSIS INFECTION BY ANALYSIS OF ESAT-6 AND 

CFP-10 SPECIFIC T-CELL REACTIVITY USING A RAPID FLOW-CYTOMETRIC ASSAY 

Gudrun Szalay, Martina Sauter, Carmen Ruoff, Reinhard Kandolf, Karin Klingel 

Osteopontin is not involved in antiviral immunity but in cardiac fibrosis during chronic 

enterovirus myocarditis 

Stefan Ehlers, Sahar Aly, Klaus Wagner, Sven Malm, Tamas Laskay, Jörg Mages, Roland Lang, Ian Orme, Franz Bange 

Oxygen Status of Lung Granulomas in M. tuberculosis-Infected Mice and Guinea Pigs and Causal 

Involvement of CXCR3-targeted Chemokines in Granuloma Necrosis 

Michal Smida, Anita Posevitz-Fejfar, Burkhart Schraven, Jonathan A. Lindquist 

PAG downregulation results in increased proximal signaling but reduced functional outcomes in 

primary T cells 

Gurumoorthy Krishnamoorthy, Florian C. Kurschus, Klaus Dornmair, Reinhard Mentele, Hans Lassmann, Hartmut 

Wekerle 

Paradoxical autoimmunity: Molecular self mimicry initiates spontaneous autoimmunity in myelinspecific 

T cell receptor transgenic mice deficient of cognate self antigen 

Hanna Erdmann, Paul Crocker, Bernhard Fleischer, Thomas Jacobs

Pathogenic Trypanosoma cruzi interacts with the immune modulatory molecule mouse Siglec-E 

(sialic acid-binding Ig-like lectin-E) 

Jan Dirks, Urban Sester, Daniela Presser, Heinrike Wilkens, Hans Köhler, Martina Sester 

PD-1 expression on CMV specific CD4 T cells is associated with viremia and reversible functional 

anergy in patients after organ transplantation 

Hiroaki Azukizawa, Nobuo Kanazawa, Manfred B. Lutz 

Peripheral de novo induction of CD4+ CD25+ Foxp3+ regulatory T cells by steady state 

migratory dendritic cells 

Fabien Agenes, Jean-Pierre Dangy, Jörg Kirberg 

Peripheral inter-clonal competitiveness requires T cell receptor contact to restricting MHC 

molecules 

Kristina Streyl, Mikhail Nosov, Vijay Kumar Ulaganathan, Hartmut Wekerle, Alexander Flügel 

Peripheral milieus license autoaggressive effector T cells to invade their target organ 

Sebastian Bunk, Iuliana Suznea, Jan Rupp, Matthias Maass, Michael Przybylski, Albrecht Wendel, Corinna Hermann 

Persisting Chlamydia pneumoniae infections are associated with differential immune responses 

Nirmal Robinson, Tom Li Stephen, Sonja Meemboor, Wiltrud M. Kalka-Moll, Georg Plum 

Phagosomal and endosomal trafficking studies by adenoviral Rab-GFP fusion proteins in primary 

cells 

Thomas Giese, Claudia Sommerer, Martin Zeier, Stefan Meuer 

Pharmacodynamic controlled tapering of Cyclosporine A – a new step towards individualized 

immunosuppressive therapy in transplanted patients 

Carmen Scheibenbogen, Anne Letsch, Antonia Busse, Sandra Bauer, Alexander Schmittel, Wolf-Karsten Hofmann, Lutz 

Uharek, Eckhard Thiel, Ulrich Keilholz 

Phase II trial of vaccination with WT1 peptide, GM-CSF, and KLH in patients with acute myeloid 

leukemia: immunological, molecular, and clinical results 

Kai Schledzewski, Klein Diana, Martin Falkowski, Gerhard Moldenhauer, Jo van Ginderachter, Julia Kzhyshkowska, Lou 

de Leij, Gernot Geginat, Bernd Arnold, Sergij Goerdt 

Phenotype analysis, cytokine induction and gene expression profiling identify LYVE-1+ tumour 

associated macrophages to be a specialized subset of M2 macrophages 

Tobias Käser, Wilhelm Gerner, Sabine Hammer, Martina Patzl, Armin Saalmüller 

Phenotypic and functional characterisation of porcine CD4 + CD25high regulatory T cells 

Stefan Maßen, Dirk Jäger, Inka Seil, Barbara Mosetter, Christine Falk 

Phenotypic and functional characterization of KIR-expressing cytotoxic T cells 

Esther Wilk, Tibor Horvath, Katy Kalippke, Nadine Wilke, Reinhold E. Schmidt, Roland Jacobs 

Phenotypical and functional characteristics of CD20 + T cells 

Bernhard Reis, Roland Piekorz, Bernd Nürnberg, Klaus Pfeffer, Sandra Beer 

PI3Kγ and δ play combined roles in B cell development 

Kerstin Schilling, Bin Hu, Karine Missy, Klaus-Dieter Fischer 

Pix proteins in B cell Signalling 

Katrin Moser, Oliver Winter, Nicole Haupt, Martin Szyska, Bimba F. Hoyer, Andreas Radbruch, Falk Hiepe, Rudolf A. 

Manz 

Plasma cell longevity in NZB/W mice 

Oliver Winter, Katrin Moser, Nicole Haupt, Martin Szyska, Andreas Radbruch, Rudolf A. Manz 

PLASMA CELL SURVIVAL AND DEVELOPMENT IN THE BONE MARROW

Julian Pardo, Christin Urban, Arno Müllbacher, Reinhard Wallich, Christoph Borner, Markus M Simon 

Pleiotropism of granzyme B-induced cell death; A means of the host to counter pathogens 

evasion strategies of CTL-mediated recovery 

Mario Fabri, Alessandra Zingarelli, Eva Flenner, Martina Bessler, Helena Hafke, Wiltrud Maria Kalka-Moll 

Polysaccharide modulates CD8+ T cell responses by enhancing TCR cross-linking 

Hans-Willi Mittrücker, Steinhoff Ulrich, Stefan H. E. Kaufmann 

Poor correlation between BCG vaccination-induced T cell responses and protection against 

tuberculosis 

Michael Probst-Kepper, Andrea Kroeger, Robert Geffers, Christian Erck, Miguel Godinho, Vitor Martins dos Santos, 

Hansjörg Hauser, Jan Buer, Rudi Balling, Siegfried Weiss 

POSITIVE FEEDBACK-CONTROL BETWEEN GARP AND FOXP3 IN REGULATORY T CELLS 

Nasr Hemdan, Frank Emmrich, Joerg Lehmann, Ulrich Sack 

Possible induction or exacerbation of autoimmune diseases by heavy metals through changing 

cytokine profiles 

Anja Dahten, Dennis Ernst, Dana Hoser, Margitta Worm 

PPARγ-ligation improves development of allergen-induced skin inflammation in a murine model 

of dermatitis 

Luisa Klotz, Indra Dani, Linda Diehl, Ari Waisman, Thomas Klockgether, Percy Knolle 

PPARgamma ablation in CD4+ T cells augments T cell responses resulting in enhanced T cell 

infiltration of the CNS and increased disease severity during experimental autoimmune 

encephalomyelitis 

Jürgen Haas, Benedikt Fritzsching, Petra Truebswetter, Peter Krammer, Elisabeth Suri-Payer, Brigitte Wildemann 

Prevalence of Newly Generated Naïve Regulatory T-Cells is Critical for Treg Suppressive 

Function and Determines Treg Dysfunction in Multiple Sclerosis 

Reinhard Maier, Rita De Giuli, Veronika Nindl, Simone Miller, Volker Thiel, Roland Züst, Ari Waisman, Birgit 

Ledermann, Burkhard Ludewig 

Preventing autoimmune myocarditis through cardiac myosin-specific tolerance 

Patrick Vollmar, Stefan Nessler, Bianca Wolff, Sudhakar Reddy Kalluri, Hans-Peter Hartung, Bernhard Hemmer 

Preventive and therapeutic effects of the antidepressant venlafaxine on murine experimental 

autoimmune encephalomyelitis 

Ivan Bogeski, Valentin Mirceski, Markus Hoth 

Probing the redox activity of T-lymphocytes deposited at electrode surfaces with voltammetric 

methods 

Lars-Oliver Tykocinski, Anna Sinemus, Esmail Rezavandy, Bruno Kyewski 

Promiscuous gene expression in the thymus - is epigenetic regulation the key? 

Thorsten Joeris, Petra Krienke, Ulrike Kuckelkorn, S.H.E. Kaufmann, Ulrich Steinhoff 

Proteasome assembly: Competitive integration of constitutive and IFNγ inducible catalytic 

subunits 

Kirsten Neubert, Silke Meister, Damian Maseda, Kerstin Amann, Reinhard Voll 

Proteasome inhibition ameliorates lupus symptomes in NZB/W mice 

K. Doser, J. Albrecht, T. J. Boeld, R. Eder, J. Stahl, R. Andreesen, P. Hoffmann, M. Edinger 

Protection from graft-versus-host disease by donor CD4+CD25+ regulatory T cells improves B 

lymphocyte reconstitution after allogeneic bone marrow transplantation 

Aleksandar Backovic, Nikolaus Wick, Georg Wick 

PROTEIN SIGNATURES ON SILICONE SURFACE AS BIOMARKERS FOR IMMUNOLOGIC-FIBROTIC 

SIDE EFFECTS TO IMPLANTS

Bettina Tosetti, Eva Glowalla, Martin Krönke, Oleg Krut 

Proteomics Based Identification of Cell Wall-associated Proteins as Protective Vaccine 

Candidates against Staphylococcus aureus 

Dirk Haubert, Nina Gharib, Francisco Rivero, Katja Wiegmann, Marianna Hösel, Martin Krönke, Hamid Kashkar 

PtdIns(4,5)P-restricted plasma membrane localization of FAN is involved in TNF-induced actin 

reorganization 

Carina Conrads, Ramona Siemer, Mario Assenmacher, Claudia Niemand 

Quality assessment of enriched CD4+CD25+ regulatory T cells 

Tatiana Binder, Rodica Bernatowicz, Michael Rothballer, Michael Schmid, Susanne Krauss-Etschmann, Dolores J. 

Schendel, Anton Hartmann 

QUANTITATION OF LIVE VERSUS DEAD PROBIOTIC BACTERIA 

Guido Wabnitz, Urban Sester, Henning Kirchgessner, Yvonne Samstag 

Ras/PI3Kinase/cofilin independent activation of human CD45RA+ and CD45RO+ T-cells by 

superagonistic CD28 stimulation 

Rebekka Wehner, Bärbel Löbel, Martin Bornhäuser, Ernst Peter Rieber, Marc Schmitz 

Reciprocal activating interaction between native human blood dendritic cells and NK cells in 

antitumor immunity 

Andra Schromm, Jörg Howe, Artur Ulmer, Karl-Heinz Wiesmüller, Tobias Seyberth, Günther Jung, Manfred Rössle, 

Michel H.J. Koch, Klaus Brandenburg 

Recognition of bacterial lipopeptides by human macrophages is critically determined by physicochemical 

parameters 

Ram Kumar Chowdary Venigalla, Theresa Tretter, Stefan Krienke, Regina Max, Norbert Blank, Volker Eckstein, 

Anthony D Ho, Hannes Martin Lorenz 

Reduced CD4+CD25- T Cell sensitivity to the suppressive function of CD4+CD25highCD127-/low 

Regulatory T Cells in active SLE Patients 

Annegret Plege, Katja Borns, Reinhard Schwinzer 

Reduction of human anti-pig T cell responses by transgenic expression of human PD-L1 on pig 

cells 

Niko Föger, Andrew Chan 

Redundant versus unique roles of the actin regulatory proteins coronin-1 and coronin-2 in the 

immune system 

Manuela Ahrendt, Reinhard Pabst, Ulrike Bode 

Regeneration of transplanted lymph node fragments in the mesentery to study effects of the gut 

lymph on lymph node cytokines and chemokines 

Michael Andrzejewski, Nicole Schwarz, Christian Weber, Andreas Ludwig 

Regulated shedding of transmembrane chemokines by the metalloproteinase ADAM10 facilitates 

detachment of adherent leukocytes 

Dirk Brenner, Alexander Golks, Mareike Becker, Christian R. Frey, Rostislav Novak, Friedemann Kiefer, Peter H. 

Krammer, Rüdiger Arnold 

Regulation of Activation-induced Cell Death by T Cell Receptor-Proximal Signalling 

Christina Hartwig, Miriam Mazzega, Hanne Constabel, Georg Behrens, J. Engelbert Gessner, Thomas Tschernig 

Regulation of allergic inflammation in a murine asthma model: Antigen uptake via Fcγ-receptors 

by DC-subsets, impact on tolerance and immunity 

Daniela Sánchez, Sigrid Krämer, Yves Montier, Herbert Schmidt, Stephan C Bischoff 

Regulation of human intestinal fibroblasts by Endotoxins and Shiga toxins 

Norbert Hüser, Annette Fasan, Monika Semmrich, Bernhard Holzmann, Melanie Laschinger

Regulation of LFA-1 activity: Importance for leukocyte recruitment and alloantigenic T cell 

activation in cardiac allograft rejection 

Jens Derbinski, Sheena Pinto, Stefanie Rösch, Klaus Hexel, Bruno Kyewski 

Regulation of promiscuous gene expression within defined chromosomal regions 

Katja Thümmler, Andreas Ramming, Alla Skapenko, Hendrik Schulze-Koops 

Regulation of specific immunity by homotypic T cell/T cell interaction 

Carl Friedrich Classen 

Regulation of the granzyme B inhibitor proteinase inhibitor 9 (PI-9) in monocytes: 

Immunomodulating effects of cotrimoxazole 

Jennifer Freyer, Stefan Floess, Alf Hamann, Jochen Huehn 

REGULATION OF THE MURINE TRANSCRIPTION FACTOR FOXP3 

Janine Wehrhahn, Robert Kraft, Sunna Hauschildt 

Regulation of TRPM2 channels in human monocytes and macrophages 

Christian Erbel, Roland Klingenberg, Sultan Celik, Benjamin Funke, Hardy Schumacher, Nadine Wambsganss, Thomas 

Dengler 

Regulatorische T Zellen und Signalkaskaden über IL-17 und IL-10 als atheroprotektive 

Komponenten in atherosklerotischen Läsionen 

Sabine Riekenberg, Katja Farhat, Jennifer Debarry, Holger Heine, Karl-Heinz Wiesmueller, Roland Lang, Artur J. Ulmer 

Regulators of G-protein signaling are modulated by bacterial lipopeptides and 

lipopolysaccharide 

Arthur Liesz, Elisabeth Suri-Payer, Claudia Veltkamp, Henrike Dörr, Clemens Sommer, Thomas Giese, Roland Veltkamp 

Regulatory T Cells (Treg) are Important Cerebroprotective Immunomodulators in Acute 

Experimental Stroke by an Interleukin-10 Dependent Pathway. 

Paula Kolar, Holger Hoff, Karin Knieke, Kolja Hegel, Dagmar Quandt, Monika Brunner-Weinzierl 

Regulatory T cells require costimulation by CTLA-4 (CD152) 

Ioanna Galani, Marco Wendel, Carola Schellack, Elisabeth Suri-Payer, Adelheid Cerwenka 

REGULATORY T CELLS SUPPRESS IFN-γ DEPENDENT LEUKOCYTE ACCUMULATION AND 

MACROPHAGE ACTIVATION IN LYMPHOMA 

Mario Zaiss, Jochen Zwerina, Karin Polzer, Eva Gückel, Alla Skapenko, Hendrick Schulze-Koops, Nikki Horwood, 

Andrew Cope, Georg Schett 

Regulatory T cells suppress osteoclast formation- a new link between the immune system and 

bone 

Florian Eberle, Mehtap Sirin, Klaus Heeg, Alexander Dalpke 

Relevance of RNA-mediated immunostimulation for RNA-Interference (RNAi) 

Christian Hofmann, Thomas Harrer, Kathrin Eismann, Silke Bergmann, Matthias Schmitt-Haendle, Gerold Schuler, Jan 

Dörrie, Niels Schaft 

REPROGRAMMING T CELLS WITH A HIV-1-SPECIFICTY BY ELECTROPORATION OF TCR- 

ENCODING RNA 

Esther Wilk, Katy Kalippke, Sabine Buyny, Reinhold E Schmidt, Roland Jacobs 

Response of CD56dim and CD56bright NK cells to Interleukin 21 

Elke Scandella, Evelyn Lattmann, Sanjiv Luther, Simone Miller, Tobias Junt, Burkhard Ludewig 

Role for lymphoid tissue inducer-like cells in the restoration of immunopathological destruction 

of the lymphoid stroma cell network 

Adan Chari Jirmo, Claus-Henning Nagel, Beate Sodeik, Reinhold E Schmidt, Georg Behrens

Role of direct versus cross-presentation after acquisition of HSV-1 by murine DC for generating 

antiviral immunity 

Katharina König, Linda Diehl, Carsten Golletz, Thomas Quast, Waldemar Kolanus, Percy Knolle, Reinhard Büttner, 

Lukas C Heukamp 

ROLE OF FHL2 IN THE REGULATION OF DENDRITIC CELL MIGRATION 

Kerstin Juelke, Andreas Thiel, Chiara Romagnani 

Role of inflammatory cytokines in modulating NK cell competence and maturation 

Romy Laugks, Patricia Schmidbauer, Bernhard Holzmann, Melanie Laschinger 

ROLE OF LFA-1 ACTIVITY REGULATION IN CELL CYCLE CONTROL OF PROLIFERATING T 

LYMPHOCYTES 

Chiara Cordiglieri, Werner Dammermann, Bo Zhang, Barry Potter, Andreas Guse, Alexander Flügel 

Role of NAADP-mediated Ca 2+ signalling in encephalitogenic CD4 + T cells 

Annette I. Garbe, Taras Kreslavsky, Harald von Boehmer 

Role of Notch in survival, proliferation and differentiation of alpha beta T cell precursors 

Varsha Pattu, Bin Qu, Eva.C Schwarz, Tanja Mayer, Carolin Bick, Reiko Trautmann, Markus Hoth, Jens Rettig 

Role of SNARE proteins in vesicle fusion at the Immunological Synapse of Cytotoxic T 

lymphocytes. 

Stefano Majocchi, Natalio Garbi, Günter J Hämmerling 

Role of Tapasin-ERp57 heterodimers in the generation of MHC class I/peptide complexes 

Carolin Konermann, Daniel Degrandi, Cornelia Beuter-Gunia, Sandra Beer, Klaus Pfeffer 

Role of the IFN-γ inducible 65kDa GTPases in pathogen defense 

Christiane Habich, Volker Burkart 

Role of Toll-like receptor 4 (TLR4) in heat shock protein 60 (hsp60)-mediated beta cell-directed 

immune reactivity in non-obese diabetic (NOD) mice 

Bernadette Pöllinger, Gurumoorthy Krishnamoorthy, Michael Bösl, Hans Lassmann, Andeas Holz, Hartmut Wekerle, 

Florian C. Kurschus* 

RR Mouse: Spontaneous relapsing remitting EAE in SJL/J mice expressing a MOG responsive T 

cell receptor 

Elisa Monzón-Casanova, Christian Söllner, Nico Westphal, Ingolf Berberich, Tohru Miyoshi-Akiyama, Takehiko 

Uchiyama, Ingrid Müller, Lutz* Walter, Thomas* Herrmann 

RT1Db2: Identification and first functional analysis of a new non-polymorphic MHC class II 

molecule 

Julia Kzhyshkowska, Alexei Gratchev, Liis Krusell, Srinivas Mamidi, Gail Workman, E. Helene Sage, Sergij Goerdt 

Scavenger receptor stabilin-1 links endocytosis and intracellular sorting in alternatively 

activated macrophages 

Tim Sparwasser, Andrea Hartl, Katharina Lahl, Heinz Fehrenbach, Holger Garn, Harald Renz, Hermann Wagner 

Selective depletion of Foxp3+ cells in DEREG mice allows functional analysis of regulatory T cells 

during experimental allergic airway inflammation 

Ulrike Schleicher, Jan Liese, Claudia Kurzmann, Christian Bogdan 

Selective requirement of Toll-like receptor 9 for the activation of natural killer cells in cutaneous 

and visceral leishmaniasis 

Ulrich Salzer, Jennifer Birmelin, Chiara Bacchelli, Torsten Witte, Ulrike Buchegger-Podbielski, Rita Rzepka, H Bobby 

Gaspar, Reinhold E Schmidt, Inga Melchers§, Bodo Grimbacher§ 

Sequence analysis of TNFRSF13b/TACI in patients with Systemic Lupus Erythematosus 

Zeinab Abdullah, Tomo Saric, Hamid Kashkar, Nikola Baschuk, Benjamin Yazdanpanah, Bernd K. Fleischmann, Jürgen 

Hescheler, Martin Krönke, Olaf Utermöhlen

Serpin-6 expression protects embryonic stem cells from lysis by antigen-specific CTL 

Michael Conzelmann, Michael Rieger, Michael Hess, Ulrich Strohhaecker, Axel Benner, Ute Hegenbart, Anthony D. Ho, 

Peter Dreger, Thomas Luft 

Serum cytokeratin-18 fragments as sensitive marker of epithelial apoptosis in intestinal and 

hepatic graft-versus-host disease 

Pia Herzberger, Corinna Siegel, Christine Skerka, Volker Fingerle, Ulrike Schulte-Spechtel, Bettina Wilske, Volker 

Brade, Reinhard Wallich, Peter F. Zipfel, Peter Kraiczy 

Serum resistance of human pathogenic Borrelia spielmanii sp. nov. correlates with binding of 

complement regulators factor H and FHL-1 

Anette J. Bauer, Katharina M. Huster, Verena Labi, Roland M. Schmid, Dirk H. Busch, Andreas Villunger, Georg Häcker 

Several members of the mitochondrial apoptosis pathway control activated T cell death to 

terminate the T cell immune response 

Jessica Prüßmeyer, Rory Koenen, Line Fraemohs, Christian Weber, Andreas Ludwig 

SHEDDING OF THE JUNCTIONAL ADHESION MOLECULE JAM-A BY THE DISINTEGRIN AND 

METALLOPROTEINASE ADAM17 REGULATES NEUTROPHIL TRANSMIGRATION 

Oliver Frey, Lisa Bruns, Andreas Reichel, Lars Morawietz, Thomas Kamradt 

Short-term depletion of regulatory T cells converts self-limiting arthritis into a chronic disease 

Wenhan Li, Stefan Schultz, Nicolas Geis, Michael Kirschfink 

shRNA-mediated knockdown of CD59 sensitises tumour cells to complement attack and inhibits 

proliferation 

David Frommhold, Andreas Ludwig, M. Gabi Bixel, Alexander Zarbock, Annette G. Beck-Sickinger, Alma Zernecke, 

Christian Weber, Dietmar Vestweber, Klaus Ley, Markus Sperandio 

Sialyltransferase ST3Gal-IV interferes with chemokine-dependent leukocyte adhesion during 

inflammation 

Andrea Kießling, Juliane Ladhoff, Mir Farzin Mashreghi, Sabine Brösel, Elke Effenberger, Hans-Dieter Volk, Martina 

Seifert 

Silencing of IFNγ-receptor by siRNA inhibits MHC class II upregulation on endothelial cells 

Verena Boschert, Anja Krippner-Heidenreich, Ingo Grunwald, Marie Kühnle, Klaus Pfizenmaier, Peter Scheurich 

Single chain TNF, a covalently stabilized TNF derivate 

Friedrich Koch-Nolte, Jan Reyelt, Janusz Wesolowski, Kristina Burkert, Nicole Schwarz, Felix Scheuplein, Friedrich 

Haag, Vanina Alzogaray, Fernando Goldbaum 

Single domain antibodies from llama effectively and specifically block an immunoregulatory T 

cell surface enzyme in vivo 

Andrej Mantei, Sascha Rutz, Ioanna Andreou, Martin Weber, Alexander Scheffold 

siRNA-mediated gene knock-down in primary mouse T cells 

Uwe Koelsch, Burkhart Schraven, Luca Simeoni 

SIT and TRIM determine T-cell fate in the thymus 

Anja Hänsel, Michael Meurer, Ernst Peter Rieber, Knut Schäkel 

Slan-dendritic cells (6-Sulfo LacNAc-expressing dendritic cells) in contrast to CD1c+ dendritic 

cells express high levels of functional TLR7- and TLR8- receptors 

Abudula Abulizi, Annika Grabbe, Markus Brechmann, Christian Polaschegg, Nadine Herrmann, Ingo Goldbeck, Kai 

Dittmann, Jürgen Wienands 

SLP-65 signal transduction requires SH2-mediated membrane anchoring and a kinaseindependent 

adaptor function of Syk 

Bernhard Reis, Tanja Scheikl, Norbert Huser, Bernhard Holzmann, Klaus Pfeffer, Sandra Beer 

SLy-an Orphan Adaptor Protein Displaying a Nonredundant Role in Lymphocyte Development 

and Activation

Max von Holleben, Simone Klöter, Bernhard Reis, Klaus Pfeffer, Sandra Beer 

SLy2 represents a new member of a recently identified family of adapter proteins 

Bin Qu, Varsha Pattu, Eva C. Schwarz, Tanja Mayer, Jens Rettig, Markus Hoth 

SNARE proteins are involed in cytotoxic granule secretion at the immunological synapse. 

Simon Frank, Leif Sander, Stephan Bischoff, Axel Lorentz 

SNARE proteins in mature primary human mast cells 

Manfred Hönig, Ansgar Schulz, Catharina Schütz, Paul Fisch, Tatjana Kersten, Ulrich Pannicke, Markus Rojewski, 

Wilhelm Friedrich, Klaus Schwarz 

Somatic reversion of lymphocyte subpopulations after HSCT in a patient with JAK3 deficiency - 

Evidence for independent αβ- and γδ- T-lineage stem cells 

Kevin Thurley, Dorothea Busse, Thomas Höfer 

Spatiotemporal Cytokine Dynamics in T-Cell Regulation - A Mathematical Model of IL-2• 

•Diffusion and Regulatory T-Cells• (•Treg•) 

Ariel H. Achtman, Sven Golfier, Martin Lipp 

SPHINGOSINE-1-PHOSPHATE RECEPTOR-4 IS MAINLY EXPRESSED BY LYMPHOID CELLS 

Shin-Young Na, Heike Eujen, Catherine Toben, Yi Cao, Anneliese Schimpl, Ralf Gold, Thomas Hünig 

Spontaneous development of CD8-mediated EAE in a new transgeneic mouse model 

Melinda Czéh, Gerald Willimsky, Thomas Kammertöns, Jehad Charo, Thomas Blankenstein 

Sporadic immunogenic cancer induces cytotoxic T lymphocyte unresponsiveness against MHCmismatched 

targets 

Cornelia Beuter-Gunia, Daniel Degrandi, Sandra Beer, Klaus Pfeffer 

SSPII, a new IFN gamma upregulated secretory protein 

Maria Lexberg, Hyun-Dong Chang, Andreas Radbruch 

Stability and plasticity of IL-17 producing T cells 

Anita Correll, Andrea Tuettenberg, Christian Becker, Jürgen Knop, Helmut Jonuleit 

Stage-dependent quantification of regulatory T cells and antigen-specific T cell responses in 

melanoma patients 

Christiane Siewert, Sascha Cording, Markus M. Heimesaat, Oliver Liesenfeld, Stefan Bereswill, Christoph 

Loddenkemper, Gunnar Loh, Michael Blaut, Alf Hamann, Jochen Huehn 

Stay with friends: commensal microflora drives expansion of Foxp3+ Tregs in gut-associated 

lymphoid tissue 

Svetlana Karakhanova, Markus Schneider, Johannes Schenkel, Markus Munder 

Steroids modulate the T effector cell / TREG / dendritic cell balance during treatment of Graftversus-Host 

Disease. 

Marcin Wlodarski, Lukasz Gondek, Zachary Nearman, Sandra Zwinger, Hans-Dieter Volk, Jaroslaw Maciejewski 

Strategies for quantitation and in-vivo tracking of clonal cytotoxic T-cell responses. 

Volker Teichgräber*, Surojit Sarkar*, Vandana Kalia, David Masopust, Laurie Harrington, Antonio Polley, Rafi Ahmed, 

E. John Wherry 

Strength of stimulus and clonal competition impact the rate of memory CD8 T cell differentiation 

Sonja Meemboor, Eva Flenner, Alessandra Zingarelli, Martina Bessler, Wiltrud Kalka-Moll 

Streptococcus pneumoniae capsular polysaccharide-mediated CD4+ T cell-dependent immune 

response requires IL-6 

Daniel Schaefer, Anja Janysek, Jürgen Neumann, Norbert Koch 

Strong impact of invariant chain on surface expression of HLA-DQ

Nana Ueffing, Eric Keil, Christian Freund, Ronald Kühne, Klaus Schulze-Osthoff, Ingo Schmitz 

Structural and mutational analyses of c-FLIP R reveal requirements for DISC-recruitment 

Björn Linke, Heiko Weyd, Lucie Dörner, Andrea Mahr, Peter H. Krammer 

Structural Requirements for Annexin 1 Mediated Suppression of Dendritic Cells 

Nina Oberle, Nadine Eberhardt, Christine S Falk, Peter H Krammer, Elisabeth Suri-Payer 

Suppression Mechanisms of cytokine transcription in human CD4+CD25- T cells upon interaction 

with CD4+Foxp3+ regulatory T cells 

Beatrix Schumak, Gerhard Wingender, Schwandt Timo, Frank Juengerkes, Thomas Tueting, Percy Knolle, Bernhard 

Holzmann, Andreas Limmer 

SUPPRESSION OF ADAPTIVE IMMUNE RESPONSES BY TOLL-LIKE RECEPTOR LIGANDS 

Christiane A. Opitz, Tobias Lanz, Christian Lutz, Wolfgang Wick, Michael Platten 

Suppression of antigen-specific T cell immunity by mesenchymal stem cells: implications for 

stem cell therapy of autoimmune diseases. 

Andrea Mahr 

Suppression of Dendritic Cell Activation by Annexin 1 on the Surface of Apoptotic Cells 

Jens Haenig, Manfred B. Lutz 

Suppression of mature dendritic cell function by regulatory T cells in vivo is abrogated by CD40 

licensing 

Yu-Hwa Huang, Eva Tolosa, Heinz Wiendl 

Suppressive action of HLA-G-expressing T cells, a novel population of natural regulatory cells, is 

independent of antigen-presenting cell, facilitated by TCR-engagement, involves HLA-G-ILT-2 

ligation and IL-10 

Kathrin Held, Elke Dauber, Michael Loos, Franz Petry 

Susceptibility of complement deficient mouse strains to systemic infection with Candida albicans 

Liangping Li, Gerald Willimsky, Susanne Seitz, Yaxin Xu, Yongping Li, Lope Estevez Schwarz, Peter Schlag, Thomas 

Blankenstein 

SV40 LT-immortalized human primary normal and cancerous mammary epithelial cells are 

phenotypically similar but can be distinguished in selection medium and 3D culture 

Carmen Kroczek, Athanasia Avramidou, Stephan Feller, Christiane Lang, Hans-Martin Jäck, Dirk Mielenz 

Swiprosin-1 - molecular switch in B cell receptor signaling? 

Anke Osterloh, Ulrich Kalinke, Siegfried Weiß, Bernhard Fleischer, Minka Breloer 

SYNERGISTIC AND DIFFERENTIAL MODULATION OF IMMUNE RESPONSES BY HSP60 AND LPS 

Nousheen Zaidi, Timo Burster, Vinod Sommandas, Timo Herrmann, Bernhard O. Boehm, Wolfgang Voelter, Hubert 

Kalbacher 

Synthesis of novel cell penetrating aspartic proteases inhibitors as the targeted inhibitors of 

antigen processing 

Edith Jasny, Martin Eisenblaetter, Tamara Visekruna, Klara Tenner-Racz, Christiane Stahl-Hennig, Andres Salazar, 

Ralph M. Steinman, Klaus Ueberla, Mariagrazia Uguccioni, Ralf Ignatius 

Synthetic double-stranded RNA, Poly ICLC, enhances the induction of cellular immune responses 

against protein antigens in rhesus macaques 

Stefanie Frey, Christine D. Krempl, Stephan Ehl 

T cell dependent and T cell independent disease after infection with pneumonia virus of mice 

Annette I. Garbe, Jerome P. Jayasekera, Michael C. Carroll, Harald von Boehmer 

T cell vaccination with DEC-205-influenza hemagglutinin fusion antibodies

Christof Iking-Konert, Tim Vogl, Matthias Schneider, Konrad Andrassy, Gertrud Maria Hansch 

T-lymphocytes in patients with primary vasculitides: Expression of CD11b identifies activated T 

cells with the propensity to stimulate polymorphonuclear neutrophils 

Sven Meuth, Stefan Bittner, Ole Simon, Heinz Wiendl 

Tandem pore domain potassium channels TWIK-related acid-sensitive K+ channel 1 (TASK1) 

and TASK3 modulate effector functions of T lymphocytes: a novel therapeutic target for T-cell 

mediated autoimmune diseases 

Kai Dittmann, Anja Uhmann, Ralf Dressel, Heidi Hahn, Jürgen Wienands 

Targeted inactivation of the Hh receptor Ptch abrogates lymphocyte development in mice 

Andreas Goldwich, Sabine Hahn, Sandra Schreiber, Ralf Wagner, Manfred Lutz, Eckhardt Kämpgen, Ulrich Schubert 

Targeting HIV-1 Gag into the DRiP-Pathway results in enhanced CD8 T cell activation 

Stephan Schlickeiser, Katharina Tschimmel, Andreas Meisel, Christian Meisel, Inga Gebuhr, Uwe Pleyer, Hans-Dieter 

Volk, Birgit Sawitzki 

TARGETING N-GLYCOSYLATION AFFECTS APC FUNCTION IN VITRO AND IN VIVO 

Sebastian Temme, Anna Maria Eis-Hübinger, Peter Kuckenberg 

Targeting of the major histocompatibility complex class II pathway by herpes simplex virus 

type-1 glycoprotein B 

Theron Johnson, Karsten Mahnke, Dirk Nettelbeck, Volker Storn, Alexander H. Enk 

Targeting of Tumor antigens to dendritic cells (DCs) using a novel single chain fragment variable 

Tobias Seibel, Florian Schuetz, Christoph Domschke, Mariana Bucur, Christoph Sohn, Philipp Beckhove 

TGFβ and IFN-α regulate Type 1 T-Cell Responses in Primary Breast Carcinomas 

Guido Wabnitz, Philipp Lohneis, Yvonne Samstag 

The actin bundling protein L-plastin is important for LFA-1 accumulation in the pSMAC of the 

immunological synapse 

Anne Sappok, Anna S. Wenning, Melodie-Jo Wolfs, Tanja Mayer, Bettina Strauß, Markus Hoth, Eva C. Schwarz 

The activation and proliferation of primary human CD4+ T-cells following focal stimulation 

Stefanie Kliche, Gael Menasche, Dennis Breitling, Mauro Togni, Xiaoqian Wang, Rico Pusch, Ana Kasirer-Friede, 

Theresia E. B. Stradal, Gary A. Koretzky, Burkhart Schraven 

THE ADAP/SKAP-55 SIGNALING MODULE REGULATES TCR-MEDIATED INTEGRIN ACTIVATION 

THROUGH PLASMA MEMBRANE TARGETING OF RIAM/RAP1 

Stephanie Joachim, Joachim Storf, Norbert Pfeiffer, Franz Grus 

The Analysis of Antibody Patterns in Glaucoma Patients 

Bettina Jux, Markus Frericks, Charlotte Esser 

The arylhydrocarbon receptor is a critical regulator of Langerhans cell maturation 

Sonja Ortler, Christoph Leder, Michel Mittelbronn, Alla Zozulya, Lieping Chen, Antje Kroner, Heinz Wiendl 

The B7-homologue 1 (B7-H1/PD-L1) contributes to confinement of immunopathological CNS 

damage: implications for Multiple sclerosis 

Thomas Ebensen, Kai Schulze, Peggy Riese, Michael Morr, Carlos A. Guzmán 

The bacterial second messenger cdiGMP exhibits promising activity as mucosal adjuvant 

Martin Klemke, Elisabeth Kramer, Guido Wabnitz, Mathias Konstandin, Yvonne Samstag 

The chemokine SDF-1α induces dephosphorylation of the actin remodelling protein cofilin via a 

Gi/Ras/MEK pathway in primary human T cells 

Birgit C Viertlboeck, Sonja Schweinsberg, Matthias A Hanczaruk, Ramona Schmitt, Louis du Pasquier, Friedrich W 

Herberg, Thomas W Göbel

The chicken leukocyte receptor complex encodes a primordial, activating, high affinity IgY Fc 

receptor. 

Matthias A. Hanczaruk, Thomas W. Göbel, Birgit C. Viertlboeck 

THE CHICKEN TRIGGERING RECEPTOR EXPRESSED ON MYELOID CELLS (TREM) 

Barbara Simm, Matthias Witt, Monika Braun, Barbara Mosetter, Rudolf Gruber, Dolores Schendel, Christine Falk 

The correlation of CD6 expression with infiltration of NK cells into synovial fluid of Arthritis 

patients. 

Edgar Serfling, Rene Rost, Wen Chen, Sergei Chuvpilo, Eisaku Kondo 

The Crosstalk between NF-kB and NFATc1/aA, the Inducible Short Isoform of NFATc1, Protects 

B-Lymphocytes against Apoptosis 

Martin Holdener, Edith Hintermann, Eric F. Johnson, Michael P. Manns, Matthias G. von Herrath, Urs Christen 

The CYP450 mouse model for autoimmune hepatitis: Breaking of self-tolerance in transgenic 

CYP2D6 and wildtype FVB-mice by viral infection 

Tarvo Rajasalu, Wolfram Karges, Andreas Spyrantis, Andreas Wieland, Bernhard Böhm, Reinhold Schirmbeck 

The diabetogenic, insulin-specific CD8 T cell response primed in the experimental autoimmune 

diabetes model in RIP-B7.1 mice 

Stephan Thurau, Maria Diedrichs-Möhring, Christiane Hoffmann, Gerhild Wildner 

The distribution of uveitogenic GFP+ T cells in a rat model of uveitis 

Tobias Haas, Jochen Metzger, Frank Schmitz, Antje Heit, Eicke Latz, Hermann Wagner 

The DNA sugar backbone 2-deoxyribose determines Toll-like receptor 9 activation 

Dieter Kube, Diana Pinkert, Nils Schoof, Frederike von Bonin, Jennifer Theiss, Fred Schaper, Johannes Bode, Martina 

Vockerodt 

The EBV oncoprotein Latent Membrane Protein 1 affects the expression of suppressors of 

cytokine signaling (SOCS) in transformed B lymphocytes 

Dong Wang, Melanie Drenker, Thomas Werfel, Miriam Wittmann 

The epidermal inflammatory response in lupus erythematosus shows an autocrine amplification 

loop. Major role of TNF&alpha and IL-18. 

Andreas Vilcinskas, Boran Altincicek 

The greater wax moth Galleria mellonella as a mini-host model for human pathogens and as a 

reservoir for novel peptide antibiotics 

Leslie Elsner, Vijayakumar Muppala, Mathias Gehrmann, Jingky Lozano, Heike Bickeböller, Thomas Herrmann, Lutz 

Walter, Frauke Alves, Gabriele Multhoff, Ralf Dressel 

The heat shock protein HSP70 promotes NK cell activity against tumors which express inducible 

NKG2D ligands 

Aysefa Doganci, Petra Scholtes, Joachim Maxeiner, Roman Karwot, Edgar Schmitt, Hans A. Lehr, I.Cheng Ho, Susetta 

Finotto 

The IL-2R alpha and IL-2R beta chains differentially regulated allergic airway inflammation in an 

experimental in vivo model 

Christine S. Falk, Dominik ter Meer, Iris Bigalke, Alexander Buchner, Barbara Mosetter, Barbara Simm, Monika Braun, 

Dusan Prevalsek, Dolores Schendel, Hans-Jochem Kolb 

The Impact of HLA-C and KIR Genetics on Survival of AML versus ALL Patients following 

Haploidentical Bone Marrow / Stem Cell Transplantation. 

Kristina Kunert, Constanze Schönemann, Hans-Dieter Volk, Ruth Neuhaus, Andreas Pascher, Claudia Wenzel, Peter 

Neuhaus, Johann Pratschke, Katja Kotsch 

The impact of KIR/HLA ligand incompatibility on acute rejection episodes after orthotopic liver 

transplantation 

Mauro Togni, Dirk Reinhold, Burkhart Schraven, Aneegret Reinhold

The importance of adhesion and migration of dendritic cells during the induction of immune 

reaction: A role for the adaptor protein SKAP-HOM 

Rachid Marhaba, Mario Vitacolonna, Dagmar Hildebrand, Michal Baniyash, Pia Freyschmidt-Paul, Margot Zöller 

The importance of myeloid suppressor cells in the regulation of autoimmune effector cells by a 

chronic allergic eczema 

Michal Smida, Sabine Lindquist, Anita Posevitz-Fejfar, Diana Karitkina, Ramnik Xavier, Brian Seed, Burkhart Schraven, 

Jonathan A. Lindquist 

The importance of PAG in regulating proximal signaling 

Lilli Podola, Hans Jürgen Ahr, Hans-Werner Vohr 

THE INDUCTION PHASE OF ALLERGIC ASTHMA INVESTIGATED BY A SHORT-TERM MODEL IN 

BROWN NORWAY RATS 

Lucie Dörner, Heiko Weyd, Andrea Mahr, Björn Linke, Dagmar Riess, Christine S. Falk, Peter H. Krammer 

The influence of apoptotic cells and annexin 1 on peripheral tolerance 

Shafaqat Ali, Michael Huber, Christian Kollewe, Werner Falk, Michael U. Martin 

The Interleukin-1 Receptor Accessory Protein is essential for Interleukin-33 induced activation 

of T cells and mast cells 

Manuela Hessmann, Dominik Rueckerl, Toshiyuki Takai, Stefan Ehlers, Christoph Hoelscher 

The ITAM-bearing adapter protein DAP12 modulates inflammatory immune responses after 

Mycobacterium tuberculosis infection 

Veronika Lukacs-Kornek, Sven Burgdorf, Sabine Specht, Miroslaw Kornek, Christian Kurts 

The kidney – renal LN system contributes to cross-tolerance against innocuous soluble antigen 

Gennadiy Zelinskyy, Sandra Balkow, Simone Schimmer, Tanja Werner, Markus M. Simon, Ulf Dittmer 

The level of Friend retrovirus replication determines the cytolytic pathway of CD8+ T cellmediated 

pathogen control 

Iryna Prots, Alla Skapenko, Jörg Wendler, Stefan Mattyasovszky, Clarisse Yone´, Bernd Spriewald, Harald Burkhard, 

Joachim R Kalden, Peter E Lipsky, Hendrik Schulze-Koops 

The loss-of-function IL4R single nucleotide polymorphism I50V is associated with early erosive 

rheumatoid arthritis 

Petra Hoffmann, Tina J. Boeld, Ruediger Eder, Jochen Huehn, Stefan Floess, Udo Baron, Sven Olek, Reinhard 

Andreesen, Matthias Edinger 

The Naive CD45RA+, But Not The CD127- Subpopulation of Human CD4+CD25high T Cells 

Homogeneously Maintains Regulatory T Cell Characteristics Upon In Vitro Expansion 

Sandra Düber, Martin Hafner, Elias Hobeika, Michael Reth, Ari Waisman, Karsten Kretschmer, Siegfried Weiss 

The origin of B-1a cells: Analysis in mice with inducible B cell development 

Ulrich Steinhoff, Alexander Visekruna, Anjo Kroesen 

The proteasome controls NF-κB-mediated inflammation in IBD patients 

Sandra Kraemer, Regina Krohn, Hongqi Lue, Manfred Dewor, Christian Weber, Jürgen Bernhagen 

The pseudo-ELR motif of MIF is required for its functions as CXC arrest chemokine 

Nicole Dietrich, Nelson Gekara, Siegfried Weiss 

The regulatory mechanisms inviolved in iNOS, TNF-&alpha, and IL-6 induction during host 

defence 

Uwe Kolsch, Christina Weber, Caroline Ritter, Anna Viehbahn, Thomas Brune 

THE REPRODUCTIVITY OF MICE IS INDEPENDENT OF A MHC-I HAPLOTYPE MISMATCH BETWEEN 

THE MOTHER AND THE FETUS 

Andrey Bogdanov, Tatyana Rybakova, Nikolay Belyaev

The role of alpha-fetoprotein in induction of immunological tolerance to tumor growth: direct 

and indirect breaking of tumor immunosurveillance 

Filiz Demircik, Ari Waisman 

The role of B cells and other antigen presenting cells in infections 

Sonja Reißig, Ari Waisman 

The Role of CYLD in T cell Development and Regulation 

Fabia T.L. Rocha, Rüdiger Arnold, Renate Rutz, Michael Kirschfink 

The role of FLIPLong in C5a-modulated spontaneous apoptosis of neutrophils 

Denise Bogdanski, Thomas M. Frangen, Christian Schinkel, Gert Muhr, Manfred Köller 

The role of IL-17 in the post traumatic phase of polytrauma patients. 

Inna Lavrik, Alexander Golks, Dirk Brenner, Julia Hoffmann, Carina Pforr, Peter Krammer 

The role of new c-FLIP proteins in life and death of the T cell. 

Eva Gueckel, Silke Meister, Mario Zaiss, Sankar Ghosh, Reinhard E. Voll 

The role of NF-κB for the development and function of naturally occurring regulatory T cells 

Zoran V. Popovic, Roger Sandhoff, Tjeerd P. Sijmonsma, Eva Kiss, Sylvia Kaden, Edgar Tone, Hermann-Josef Gröne 

The role of sulfatide SM4s in apoptotic cell uptake and macrophage phenotype alteration 

Marko Janke, Michael Peine, Thordis Hohnstein, Lars Morawietz, Alexander Scheffold 

THE ROLE OF TH1 AND TH17 T CELLS IN INFLAMMATORY REACTIONS IN VIVO 

Jan Liese, Ulrike Schleicher, Christian Bogdan 

The role of TLR9 signalling in murine cutaneous leishmaniasis 

Paul Gutwein, Anja Schramme, Liliana Schäfer, Hermann-Josef Gröne, Mohamed Sadek, Ingeborg Hauser, Josef 

Pfeilschifter 

The role of transmembrane and released CXCL16 during inflammatory processes in the kidney 

Kai Schulze, Thomas Ebensen, Karina Watzke, Michael Morr, Carlos Guzman 

The second messenger 5′GMP is a promising adjuvant for vaccine development 

Matthias Eberl, David Vermijlen, Francesco Dieli, Nadia Caccamo, Serena Meraviglia, Giuseppe Cicero, Andrew 

Roberts, Bernhard Moser, Hassan Jomaa, Adrian Hayday 

The unexpected functional plasticity of human γδ T cells: implications for immunotherapy and 

beyond 

Rebekka Geiger, Antonio Lanzavecchia, Federica Sallusto 

THE “T CELL AMPLIFICATION METHOD” AS A NEW TOOL TO ANALYZE THE REPERTOIRE OF 

HUMAN T CELLS 

Stefanie Thiele, Andreas Pahl 

Therapy of chronic airway diseases with nucleic acids 

Arvind Batra, Markus M. Heimesaat, Stefan Bereswill, Jeannette Pietsch, Thorsten Stroh, Rainer Glauben, Inka Fedke, 

Martin Zeitz, Britta Siegmund 

TLR – expression in cells from adipose tissue – a contribution to chronic inflammation? 

Linda Diehl, Anna Schurich, Regina Grochtmann, Silke Hegenbarth, Lieping Chen, Percy Knolle 

Tolerogenic maturation of LSEC after cognate T cell interaction promotes B7-H1-dependent CD8 

+ T cell tolerance 

Elke Gülden, Seiji Imamura, Masaru Ihira, Christiane Habich, Hubert Kolb, Volker Burkart 

Toll-like receptor 4: A key regulator of beta-cell directed autoimmunity in a mouse model of type 

1 diabetes.

Marina Scheler, Manuela Brenk, Helene Wilms, Thomas Bieber, Dagmar von Bubnoff 

Toll-like receptor ligands, and not T cell signals, are strong inducers for Indoleamine-2,3dioxygenase 

(IDO),and lead to T cell suppression 

Hans-Heinrich Oberg, Jan Lenke, Susann Beetz, Norbert Reiling, Dieter Kabelitz, Daniela Wesch 

Toll-like receptor-2 ligands act on Treg to abolish suppression of CD4 + CD25- responder T cell 

proliferation and IL-2 production 

Elena Babich, Ata-Ur Rasheed, Hans-Peter Rahn, Martin Lipp, Gerd Müller 

Tracking follicular B helper T cell differentiation and function by gene expression analysis 

Ana Teles, Catharina Thuere, Milan Popovic, Anne Schumacher, Paul Wafula, Hans-Dieter Volk, Ana Zenclussen 

Trafficking of regulatory T cells during pregnancy: Expression profile of Treg-related chemokine 

and chemokine receptor at the maternal-fetal interface. 

Katharina Gropp, Michael Reuter, Gerhard D. Wieland, Christine Skerka, Peter F. Zipfel 

Transcription factors EGR-2 and NFkappaB induce chemokine synthesis in macrophages 

challenged with the human pathogenic yeast Candida albicans 

Yuriy Shebzukhov, Sergei Grivennikov, Andrey Kruglov, Chang-Yi Cui, Ina Wagner, Anna Litvin, Hans-Joachim 

Mollenkopf, David Schlessinger, Dmitry Kuprash, Sergei Nedospasov 

Transcriptional analysis of lymphoid organs from mice with conditional inactivation of TNF and 

LT 

Monika Lindemann, Alexandra Schumann, Melanie Fiedler, Camino Valentin-Gamazo, Dietmar Knop, Christoph E. 

Broelsch, Michael Roggendorf, Hans Grosse-Wilde 

Transfer of adoptive immunity to hepatitis B virus by liver transplantation* 

Annette Busch, Thomas Quast, Waldemar Kolanus, Percy Knolle, Andreas Limmer 

Transfer of T cell surface molecules to Dendritic cells upon T cell priming involving two distinct 

mechanisms differing in quality and time 

Jacqueline Moebius, Michael Basler, Marcus Groettrup 

TRANSGENIC MICE REVEAL A NOVEL ROLE FOR LMP7 IN TCR TRIGGERED EXPANSION OF T 

CELLS 

Volker Storn, Karsten Mahnke, Sonja Schallenberg, Theron S. Johnson, Tanja Bedke, Natalio Garbi, Bernd Arnold, 

Günter J. Hämmerling, Alexander H. Enk 

Treatment of established RMA-Tag tumors via DC directed targeting of tumor antigens is further 

improved by depletion of regulatory T cells. 

German Salamov, Rupert Holms, Wolfgang G. Bessler, Ravshan Ataullakhanov 

Treatment of Hepatitis C Virus Infection with Human Ezrin Peptide One (HEP1) in HIV Infected 

Patients 

Claudia Stühler, Sarah Lurati, Manik Chatterjee, Ralf C. Bargou, Hermann Einsele, Max S. Topp 

Treatment of T lymphocytes with Hsp90 inhibitors selectively kills activated cells while 

preserving viability and functionality of resting cells 

Kai Zanzinger, Carola Schellack, Gernot Geginat, Adelheid Cerwenka 

TREM-1 in infection and cancer 

Anna-Maria Herr, Olivia Sövegjarto, Lutz Walter 

TRIM proteins: Novel candidates for HIV resistance? 

Christiane Desel, Stefan H.E. Kaufmann 

Tuberculosis: Efficacy of DNA vaccines encoding dormancy-associated antigens 

Jenny Pahne, Subramanya Hegde, Nadine Schröer, Sigrun Smola-Hess

Tumor cell-derived interleukin-6 skews myeloid dendritic cells from an immunostimulatory to a 

pro-tumorigenic phenotype 

Gerald Willimsky, Melinda Czéh, Christoph Loddenkemper, Johanna Gellermann, Harald Stein, Peter Wust, Thomas 

Blankenstein 

Tumor immunogenicity, not tumor burden is the primary cause of general cytotoxic T 

lymphocyte unresponsiveness 

Christoph D. Brenner, Susan King, Imke Wolters, Georg Bornkamm, Martin Röcken, Ralph Mocikat 

Tumour control by natural killer cells in a spontaneous mouse lymphoma model 

Nicolas Sabarth, Louise Chamberlain, John Tite, Sara Brett, Craigen Jenny 

Two-sided effect of T cell help in tolerance breaking and immunodominance 

Christian Wahl, Petra Bochtler, Reinhold Schirmbeck, Jörg Reimann 

Type I IFN-producing CD4 Valpha14i NKT cells facilitate priming of IL-10-producing CD8 T cells 

by hepatocytes 

Markus Krumbholz, Hans Faber, Florian Steinmeyer, Lisa-Ann Hoffmann, Hannah Pellkofer, Tania Kümpfel, Tobias 

Derfuß, Camelia Ionescu, Michaela Starck, Thomas Giese, Gunther Hartmann, Christian Hafner, Reinhard Hohlfeld, 

Edgar Meinl 

Type I interferon therapy increases systemic BAFF expression 

Susanne Rauchmann, Karin Knieke, Marion Rudolph, Holger Hoff, Monika Brunner-Weinzierl 

Ubiquitin-ligases in co-stimulation of T-lymphocytes 

Christine Wolff, Peter Härle, Johannes Wildmann, Hugo O. Besedovsky, Rainer H. Straub, Adriana del Rey 

Uncoupling of the HPA axis and the sympathetic nervous system in rheumatoid arthritis is linked 

to marked changes of norepinephrine levels in the hypothalamus 

Sonja Textor, Rosita Accardi, Matthias Dürst, Massimo Tommasino, Lutz Gissmann, Adelheid Cerwenka 

Up-regulated expression of activating NK cell receptor ligands in human cervical cancer 

Felix Lasitschka, Jutta Schröder-Braunstein, Thomas Giese, Carolin Reiser, Bernd Sido, Stefan C. Meuer 

Uptake of Cystine in T-Lymphocytes following CD2 and CD3 stimulation 

Meike Winter, Roel Schins, Irmgard Förster 

Uptake of nanoparticles in the food by gut-associated dendritic cells 

Sascha Barabas, Tanja Bauer, Regina Gary, Petra Lindner, Juha Lindner, Wolfgang Jilg, Hans Wolf, Ludwig Deml 

Urea-mediated cross-presentation of soluble Epstein-Barr Virus BZLF1 

Emmanuel Prodhomme, Claude Muller 

Vaccination against Tobacco Specific N-nitrosamines: Synthesis of a new NNK hapten for the 

induction of specific antibodies. 

Carsten Alt, Travis Harrison, Akihiro Matsukawa, Charles Litterst, Jon Mirsalis, Annalisa D'Andrea 

Vaginal cytokine production as a biomarker for the safety evaluation of microbicide-induced 

vaginal irritation 

Beatrice Bolinger, Philippe Krebs, Engeler Daniel, Ying Hua Tian, Elke Scandella, Simone Miller, Nicola P. Restifo, 

Pierre-Alain Clavien, Burkhard Ludewig 

Vascular endothelial cells presenting minor histocompatibility antigen do neither activate nor 

tolerize CD8+ T cells 

Michaela Kern, Kai Scholz, Ulrich Kalinke, Joachim Schultze, Ulrich Koszinowski, Gunther Hartmann, Percy A. Knolle 

Viral infection but not pattern recognition induces functional maturation of tolerogenic liver 

endothelial cells leading to induction of effector CD8 T cells 

Dennis Lindau, Dagmar Sigurdardottir, Evelyna Derhovanessian, Thomas Leyhe, Graham Pawelec, Stefan Stevanovic, 

Lars Stoltze 

Visualizing Abeta-specific cytotoxic T cells with HLA tetramers

Patricia Bach, Elisabeth Kamphuis, Bernhard Odermatt, Gerd Sutter, Christian J. Buchholz, Ulrich Kalinke 

VSV-G Displaying Retrovirus-Like Particles Induce a Type I IFN Receptor Dependent Switch to 

Neutralizing IgG Antibodies 

Jessica Nickel, Birgit Löer, Reinhard Bauer, Roland Bornheim, Elisabeth Kremmer, Michael Hoch, Waldemar Kolanus 

Wech, a novel intracellular regulator of integrin-dependent cell adhesion 

Hamid Kashkar, Jens Michael Seeger, Andreas Hombach, Anke Deggerich, Benjamin Yazdanpanah, Olaf Utermöhlen, 

Gerd Heimlich, Hinrich Abken, Martin Krönke 

XIAP targeting sensitizes Hodgkin Lymphoma cells for cytolytic T cell attack 

Isabel Koch, Reinhard Hoffmann 

Yersinia virulence protein YopP inhibits NK cell cytokine production by blocking host cell IL-12 

signaling. 

Hajo Haase, Lothar Rink 

Zinc ions act as a signal in leukocyte activation 

Laura Kahmann, Sabine Warmuth, Birgit Plümäkers, Axel M. Gressner, Marco Malavolta, Eugenio Mocchegiani, Lothar 

Rink, Peter Uciechowski 

Zinc supplementation in the elderly reduces spontaneous inflammatory cytokine release and 

restores T cell functions

Luciana Berod, Oliver Frey, Johannes Norgauer, Thomas Kamradt 

SHIP1 phosphatase controls different aspects of dendritic cell 

biology 

Introduction: The signal transduction events underlying Dendritic cell (DC) maturation 

involve several molecules working in an orchestrated manner. Between them, the 

hematopoietic-restricted protein Src homology 2–containing inositol-5-phosphatase 

(SHIP1) may play a significant role by dephosphorylating phosphatidylinositol-3,4,5trisphosphate, 

the major PI3K substrate. Here, we show that SHIP1 is involved in DC 

differentiation and maturation. 

Results: Despite a higher proliferation rate and a faster differentiation, normal 

percentages of CD11c BMDC SHIP1 -/- are generated in response to GM-CSF + IL-4 

(88.5 vs 85.8 %) compared with WT cells. Moreover, upon LPS or TNF-α-induced 

maturation MHC class II and CD86 up regulation is impaired in SHIP1-/- BMDC. 

Cytokine production in SHIP1-/- BMDC showed a bias towards lower IL-12 and higher IL- 

10 production in response to LPS, as determined by ELISA. Whereas other functional 

changes, like antigen uptake downregulation upon LPS treatment were not significantly 

affected. 

Conclusion: These results indicated that in DC, SHIP1 might act not only as a negative 

regulator of PIP3-activated signalling pathways, but also as an activator of certain 

events during DC life.

Natalia Zietara, Marcin Lyszkiewicz, Stefan Lienenklaus, Siegfried Weiss 

"Lack of IFN-β impairs antigen presentation capacity of 

splenic dendritic cells" 

Type I interferons (I IFNs) constitute a highly pleiotropic, large cytokine family 

composed of a single IFN-β and multiple IFN-α. Thus far, type I IFNs are best known for 

their role in protection against viral infections. However in recent years these cytokines 

have also been shown to have multiple immunomodulatory functions and therefore play 

important role in other diseases including bacterial infections as well as under 

autoimmune conditions. 

The present study was carried out with the aim to investigate the role of type I IFNs in 

antigen presentation. Bone marrow (BM) derived and splenic antigen presenting cells 

(APCs) from WT, IFN-β -/- and IFNAR -/- mice were fed with either ovalbumin (OVA) 

peptide or whole protein and their capacity to present antigen was tested in proliferation 

assay using T cells from OT I or OT II transgenic mice. APCs derived from WT and IFNβ 

-/- BM exhibited a comparable capacity for the antigen presentation to CD8 + T cells. In 

contrast splenic APCs from IFN-β -/- as well as from IFNAR -/- were found to be highly 

impaired in their ability to activate CD4 + T cells and CD8 + T cells. Thus, our results 

provide evidence that type I IFNs play a crucial role in antigen presentation via MHC 

class I and II molecules

Hendrik Poeck, Cornelius Maihoefer, Robert Besch, David Anz, Shizuo Akira, Simon 

Rothenfusser, Veit Hornung, Stefan Endres, Thomas Tueting, Gunther Hartmann 

5´triphosphate siRNA as a novel therapeutic tool against 

tumors 

The cytosolic RNA helicase retinoic acid inducible gene-I (RIG-I) is ubiquitously 

expressed suggesting a broad spectrum of target cells and a high therapeutic efficacy in 

the immunotherapy of tumors and infections. Latest studies further demonstrate that 

uncapped 5'-triphosphate RNA, which can be generated by in–vitro transcription, serves 

as a molecular signature for the detection of viral infection by RIG-I. Thus, “infection” of 

tumor cells with in–vitro transcribed RNA may represent an important tool to elicit direct 

anti-tumor activity. Here we show that the transfection of a 5´-triphosphate siRNA 

molecule not only resulted in direct immune activation, apoptosis and relevant RNAimediated 

gene-silencing in a murine melanoma cell line, but also elicited strong 

immune responses in vitro and in vivo. More importantly, only 5´-triphosphate siRNA, 

but not synthetic siRNA, significantly reduced the number of lung metastases in a 

murine melanoma model. Interestingly, tumor regression by 5´-triphosphate siRNA was 

strongly diminished in NK cell–depleted and IFNAR–deficient mice, but not in TLR7– 

deficient mice. Additionally, the administration of 5´-triphosphate siRNA yielded high 

numbers of apoptotic tumor cells in vivo and resulted in a down-regulation of murine 

Bcl-2 in metastatic lungs of IFNAR-deficient mice indicating that 5´-triphosphate siRNA 

displays direct anti-tumor activity in vivo. In conclusion, our results identify 5´triphosphate 

siRNA as a novel therapeutic approach combining RNAi and direct antitumor 

effects with immunostimulation in one molecule providing the basis for the 

development of new therapeutic reagents in the immunotherapy of tumors.

Hans KUENG, Victoria LEB, Daniela HAIDERER, Graca RAPOSO, Clotilde THERY, Klaus 

SCHMETTERER, Alina NEUNKIRCHNER, Christian SILLABER, Brian SEED, Winfried PICKL 

A General Strategy for Decoration of Enveloped Viruses with 

Functionally Active Lipid-Modified Cytokines 

Viral particles preferentially incorporate extra- and intracellular constituents of host cell 

lipid-rafts, a phenomenon central to pseudotyping. Based on this mechanism we have 

developed a system for the predictable decoration of enveloped viruses with functionally 

active cytokines that circumvents the need to modify viral proteins themselves. Human 

interleukin(hIL)-2, hIL-4, hGM-CSF and murine (m)IL-2 were used as model cytokines 

and fused at their C-terminus to the glycosyl phosphatidyl inositol (GPI) acceptor 

sequence of the human Fc gamma receptor III (CD16b). We here show that genetically 

modified cytokines are all well expressed on 293 producer cells. However, only 

molecules equipped with GPI-anchors but not those linked to transmembrane/ 

intracellular regions of type I membrane proteins are efficiently targeted to lipid-rafts 

and consequently to virus-like particles (VLP) induced by MoMLV Gag-Pol. Human IL-4:: 

GPI and hGM-CSF::GPI co-expressed on VLP were found to differentiate monocytes 

towards dendritic cells. Apart from myeloid committed cell types, VLP-bound cytokines 

also act efficiently on lymphocytes. Human IL-2::GPI strongly co-stimulated TCR/CD3 

dependent T cell activation in vitro and mIL-2::GPI co-activated antigen-specific T cells 

in vivo. On a molar basis, the functional activity of VLP bound hIL-2::GPI was found to 

be comparable to soluble hIL-2. VLP decorated with hIL-2::GPI and co-expressing a 

TCR/CD3 ligand have an IL-2 specific activity of 5 x 10 4 units/mg protein. Virus particles 

decorated with lipid-modified cytokines might help to improve viral strains for 

vaccination purposes, propagation of factor-dependent cell types, as well as gene 

transfer by viral systems in the future.

Claudia Brandt, Margitta Worm, Ria Baumgrass 

Drug induced modulation of T cell activation and 

differentiation 

in atopic dermatitis patients 

Atopic dermatitis (AD) is a T cell dependent chronic relapsing inflammatory skin 

disorder. Cyclosporine A (CsA) has been shown to be an effective treatment for severe 

AD. We studied the effect of low dose CsA therapy on T cell activity and T cell 

differentiation in AD patients. Using a whole blood assay we demonstrated that TCR 

signalling in peripheral blood T cells of CsA treated AD patients is reduced to 42 % ± 18 

but not totally blocked. Such partial inhibition of TCR signalling allowed regulatory T cell 

induction under in vitro conditions. Therefore we asked is there an in vivo regulatory T 

cell induction, too. Indeed AD patients under low dose CsA therapy have higher 

numbers and frequencies of regulatory T cells compared to untreated AD patients. To 

evaluate the causal connection between low dose CsA treatment and higher regulatory 

T cell numbers we studied individual patients before and during CsA therapy. The data 

clearly indicate increased numbers and frequencies of regulatory T cells after onset of 

CsA therapy which remained stable during the therapy. The therapeutic effect of low 

dose CsA therapy in AD patients could be due to both, inhibition of T cell hyperactivity 

and induction of suppressor T cells.

Frank Autschbach, Felix Lasitschka, Thomas Giese, Nikolaus Gassler, Benjamin Funke, 

Jutta Schroeder-Braunstein, Ulf Brunnemer, Stefan C. Meuer, Bernd Sido 

Expression of the cystine transporter subunit xCT is a critical 

determinant of the immune response in human inflammatory 

bowel disease 

Aims: T-cell receptor reactivity of intestinal lamina propria T cells (LP-T) is critically 

dependent on the capacity of local accessory cells, especially mucosal macrophages (LP- 

M), to secrete cysteine. For T cells, cysteine is a limiting precursor for glutathione 

synthesis, a prerequisite for antigen-dependent proliferation. Production of cysteine by 

LP-M requires uptake of the derivative cystine via a membrane transporter. We now 

investigated the expression and functional relevance of the cystine transporter system 

in tissues and isolated immune cells from normal gut and from patients with 

inflammatory bowel disease (IBD). 

Methods: Expression of the cystine transporter subunit xCT was analyzed by 

quantitative RT-PCR, western-blot (WB) and immunohistology (IH). Glutathione (GSH) 

levels were detected by flow cytometry and IH. Cysteine secretion and uptake of [ 35 S] 

cystine were measured by in vitro assays. 

Results: As compared with peripheral blood monocytes, resident LP-M from normal gut 

revealed a defective uptake of cystine which was associated with an absent expression 

of the transporter molecule xCT, both in vitro and in situ. In contrast to normal gut 

tissues, an enhanced expression of xCT was apparent in IBD samples from patients with 

Crohn´s disease and ulcerative colitis (RT-PCR, WB, IH). CD68+ and/or CD14+ 

mononuclear phagocytes were found to coexpress xCT in inflamed tissues. In vitro, such 

macrophages secreted substantial amounts of cysteine, which was associated with an 

enhanced GSH content of LP-T in IBD versus normal gut. 

Conclusions: Enhanced expression of the cystine transporter subunit xCT by mucosal 

macrophages alters the redoxactive microenvironment of LP-T and is a critical 

determinant of the immune response in human IBD.

Katharina Becher 

In vivo persistence of Anaplasma phagocytophilum after 

depletion of CD4 + T cells, but not in the absence of Th1-type 

cytokines and effector mechanisms 

In vivo persistence of Anaplasma phagocytophilum after depletion of CD4 + T 

cells, but not in the absence of Th1-type cytokines and effector mechanisms 

Katharina Becher, Yvonne Kern, Birte Steiner, Christian Bogdan and Friederike von 

Loewenich 

Department of Medical Microbiology and Hygiene, University Clinic of Freiburg, Hermann- 

Herder-Straße 11, D-79104 Freiburg, Germany 

Anaplasma phagocytophilum is a Gram-negative, obligate intracellular bacterium that is 

transmitted by ticks and replicates within neutrophils. In order to further define the 

mechanisms of its control in the mammalian host, we studied the course of infection in 

transgenic mouse strains with various defects of the immune system. The mice were 

systemically infected via i.p. injections of infected SCID mouse blood. At certain time 

points of infection, the bacterial load was determined in the blood, spleen and lung by a 

quantitative PCR technique. Mice deficient for cytokines and effector molecules involved 

in the control of other intracellular pathogens (IL-12p35 -/- , IL-12p35 -/- p40 -/- , IL-4 -/- , 

IFN-gamma -/- , IFN-alphaR -/- , TNF -/- , iNOS -/- , gp91 phox-/- , neutrophil elastase -/- , 

cathepsin G -/- , MCP-1 -/- , perforin -/- , Fas lpr , FasL gld ) were still able to eliminate A. 

phagocytophilum. SCID and Rag1 -/- mice developed strikingly increased bacterial loads 

in the blood compared to wild-type. SCID mice died after about 21 to 30 days of 

infection, but were rescued by the transfer of T cells. Likewise, T cell-deficient nude 

mice were not able to control the pathogen, whereas Igα -/- mice (lacking mature Bcells) 

cleared the infection. Wild-type mice as well as JHT-mice (no B-cell-receptor) 

were resistant against a secondary infection. MHC II -/- mice, CD4-depleted mice or 

Tcrb -/- mice showed a low level persistence of Anaplasma. These findings suggest that a 

CD4 + T cell-dependent Th1 response plays a role in controlling A. phagocytophilum in 

vivo.

Manuela Brenk, Marina Scheler, Helene Wilms, Thomas Bieber, Dagmar von Bubnoff 

Induction of human CD4 + CD25 + Foxp3 + T regulatory cells by 

tryptophan-deprived tolerogenic dendritic cells 

Tryptophan catabolism is a tolerogenic effector system to induce anergic T cells and 

regulatory T cells (Tregs). Given the crucial importance of DCs in the initiation of a T cell 

response, surprisingly little is known about the impact of a high indoleamine 2,3dioxygenase 

(IDO)-activity, i.e., a tryptophan-deprived milieu, on DCs themselves. We 

show that human monocyte-derived dendritic cells (DCs), when differentiated under 

tryptophan-deprived conditions in vitro, acquire potent tolerogenic properties. This 

effect is associated with a decreased surface expression of the costimulatory molecules 

CD40, CD80, and MHC class II-molecules. In contrast, the inhibitory receptors 

Immunoglobulin-like transcript 2 (ILT2), and Immunoglobulin-like transcript 3 (ILT3) 

are increased on these DCs. Functionally, tryptophan-deprived DCs show a reduced 

capacity to uptake antigens and to stimulate T cells in vitro which can be partly restored 

by the addition of an anti-ILT3 Ab. Moreover, we provide evidence that tryptophandeprived 

DCs are able to induce CD4 + CD25 + Foxp3 + T regulatory cells with suppressive 

functions in an ILT3-dependent fashion. The induction of DCs with tolerogenic properties 

by tryptophan deprivation is associated with the induction of an active stress response 

pathway mediated by the GCN2 kinase during differentiation of both, immature and 

mature DCs. Altogether, these results demonstrate that tryptophan starvation 

establishes a regulatory environment for DCs, which, by means of their tolerogenic 

properties, favour the enhancement of regulatory T cells.

Kristina Röschmann, Artur J. Ulmer, Torsten Goldmann, Arnd Petersen 

Interaction of Timothy grass pollen major allergen Phl p 1 

with human respiratory epithelial cells 

The bronchial epithelium as a first line of contact of the immune system with the 

environment including airborne grass pollen functions as physical barrier and serves as 

important immunological regulation system, also involved in allergic reactions like 

asthma. Upon activation epithelial cells release inflammatory mediators and cause the 

attraction of immuno-competent cells. About 20% of the population of industrialized 

countries are suffering from type-I-allergies. Group-I-allergens (e.g. Phl p 1 from 

Phleum pratense) cause IgE-reactivity in about 95% of the allergic subjects and exist in 

all grass species. We are investigating the interaction of Phl p 1 with human respiratory 

epithelium in detail: uptake of the allergen, activation of the cells, route of the allergen 

through the epithelial barrier and the potential role of respiratory epithelium as 

unprofessional APC. We determined that Phl p 1 activates epithelial cells measured by 

release of IL-8 and IL-6. The activity of the allergen cannot be inhibited after preincubation 

with mAb blocking the TLR2- and TLR4-signalling pathways, indicating that 

contaminating bacterial components are not responsible for the activation of the cells. 

However, a complete deletion of the biological activity of Phl p 1 after enzymatic 

digestion indicates that the allergen itself interacts with the epithelium. After preincubation 

with different inhibitors we were able to show the involvement of p38, ERK 

and NFκB in the activation of epithelial cells by the allergen. We could also show that Phl 

p 1 is taken up by human respiratory epithelium and that these cells can express HLA- 

DR. A co-localization of Phl p 1 and HLA-DR and the effective processing of the allergen 

by epithelial cells remains to be determined. (Supported by DFG SFB TR22)

Andreas Brandl, Juergen Wittmann, Michael Boesl, Hans-Martin Jaeck 

MicroRNA-Mediated Control of B Cell Activation 

MicroRNAs (miRNAs) constitute a growing class of small noncoding RNAs that regulate 

gene expression mainly at the posttranscriptional level. The regulatory function of 

miRNAs has recently been shown to play critical roles in the control of hematopoiesis. 

The goal of this project is to determine whether miRNAs function also during the 

antigen-induced activation of B lymphocytes. To determine the miRNA profiles in naive 

B cells before and after polyclonal activation, we stimulated B cells isolated via magnetic 

CD43 beads from spleen with either LPS (simulates T cell-independent activation) or 

with a combination of anti-IgM, anti-CD40 and IL4 (simulates T cell-dependent 

activation) for various times. The analysis of isolated RNA using miRNA microarray- and 

qRT-PCR assays identified about 104 miRNAs in unstimulated B cells. 35 of these were 

mostly downregulated after stimulation. In silico analyses with various miRNA target 

prediction programs revealed a very interesting and promising set of potential 

transcripts whose translation/stability could be controlled by miRNAs during the antigeninduced 

activation phase of mature B cells. Among these targets are BCR signalling 

molecules and transcription factors that control proliferation, IgH class switch as well as 

differentiation in antibody-secreting plasma cells. We will now verify biochemically 

potential targets for each the differentially regulated miRNAs and determine the effect 

of retrovirally transduced miRNAs on B cell development.

Savita Nair, Ulf Dittmer 

Role of CD4 + T cells in acute Friend Retroviral infections 

Retroviruses are extremely pathogenic microbes and have accounted for significant 

mortality and morbidity worldwide. Cytotoxic T lymphocytes (CTLs) are efficient in 

keeping the viral replication under control during acute phase of infections but lose their 

protective function during later stages under the suppressive effect of Regulatory T 

cells. As a result of this, the virus manages to escape immune surveillance to cause 

persistent infections for life. In life-threatening infections such as Human 

Immunodeficiency virus (HIV) or Human T-cell leukemia virus (HTLV-1) immune escape 

becomes critical. Role of CTLs is well defined in various retroviral infection models but 

role of CD4 + T helper cells in retroviral immunity is poorly understood. Hence, we aimed 

at characterising the function of CD4 + T cells during the initial phase of infections using 

the Friend retrovirus (FV) murine infection model. FV infection causes lethal leukemia in 

most strains of mice and the mouse model helps in studying the basic mechanisms of 

immunological control and escape in both acute and persistent retroviral infections. In 

vivo depletion of CD4 + T cells in acutely infected FV resistant mice strain demonstrated 

that CD4 + T cells are important in controlling viral replication in acute FV infections and 

onset of erythroleukemia. An ex vivo MHC-II tetramer kinetic analysis of FV-specific CD4 

+ T cell responses during acute FV infection showed that maximal activation of CD4 + T 

cells are achieved at 10 days post infection correlating with a decrease in viral loads. 

Most significantly, FV-specific CD4 + T cells adoptively transferred into mice acutely 

infected with FV showed that the transferred cells specifically recognised the FV antigen 

in vivo, got activated to express an effector phenotype and produced the anti-viral 

cytokine IFN-γ resulting in decreased viral loads. These results indicate that CD4 + T cell 

response is vital for the control of virus replication during acute FV infection and for the 

resistance of certain mouse strains to virus–induced disease.

Alexia Nass, Hans-Willi Mittruecker, Alf Hamann, Jochen Huehn 

Transfer of in vivo operating inflammation-specific Tregs does 

not interfere with the host´s response against bacterial 

infection 

Regulatory T cells (Tregs) play an essential role in regulating inflammatory immune 

responses in vivo. In several models it has been shown that Tregs can suppress both 

the initiation of immune responses as well as ongoing inflammatory reactions. Little 

knowledge exists about the therapeutic use of antigen-specific Tregs to suppress 

unwanted immune reactions, while allowing an effective immune response against 

invading pathogens at the same time. Here we used a Th1-mediated delayed-type 

hypersensitivity (DTH) model for an inflammatory response and analyzed whether a 

defined subpopulation of antigen-specific ? + 

E Tregs would be able to suppress the DTH 

response in the context of a contemporaneous systemic infection with Listeria 

monocytogenes. Furthermore, we determined if the presence of high numbers of 

inflammation-specific Tregs would influence the immune response against L. 

monocytogenes, which mainly takes place within the spleen and liver. ? + 

E Tregs turned 

out to be potent suppressors of the local inflammation irrespective of the presence or 

absence of a systemic bacterial infection. In addition mice adoptively transferred with 

antigen-specific Tregs still established an effective immune response against L. 

monocytogenes compared to animals not receiving Tregs. Together, our findings 

provide evidence that adoptive transfer of antigen-specific Tregs effectively suppresses 

inflammatory reactions even in the context of a systemic L. monocytogenes infection 

without impairing the host´s pathogen-specific immune response.

Björn Stork, Ingo Goldbeck, Konstantin Neumann, Thilo Kähne, Michael Naumann, 

Michael Engelke, Jürgen Wienands 

‘Membrane zip codes’ for Grb2 determine differential Ca 2+ 

signaling in B lymphocytes 

The intracellular second messenger Ca 2+ is a key determinant of B cell development, 

activation, induction of anergy, or death by apoptosis. The inducible Ca 2+ initiation 

complex downstream of the B cell antigen receptor (BCR) is composed of the adaptor 

protein SLP-65, Bruton's tyrosine kinase (Btk) and phospholipase C-γ2 (PLC-γ2). 

Complex formation allows PLC-γ2 to produce inositol-tris-phosphate (IP3), which 

releases Ca 2+ from ER stores into the cytosol followed by entry of extracellular Ca 2+ 

across the plasma membrane. The relative contribution of intra- and extracellular Ca 2+ 

fluxes to the total Ca 2+ mobilization profile varies in a developmental stage-specific 

manner. This results in differential Ca 2+ signals to ultimately control distinct nuclear 

responses and hence cell fate. Our group has shown that fine tuning of the Ca 2+ 

response involves the intracellular SH2/SH3 domain adaptor protein Grb2, which we 

identified as a negative regulator of BCR-induced Ca 2+ signaling. Using a combination of 

biochemical, gene targeting and imaging techniques we have now elucidated the mode 

of Grb2 action in B cells. We found that following BCR activation, the SH2 domain of 

Grb2 binds the tyrosine-phosphorylated adaptor protein Dok-3 (downstream of kinase- 

3), which permanently localizes at the inner leaflet of the plasma membrane by virtue 

of its PH domain. When recruited to phospho-Dok-3, Grb2 attenuates Btk-dependent 

PLC-γ2 activation and IP3 production by interfering with proper formation of the Ca 2+ 

initiation complex and/or inhibition of Btk activity. However, Grb2 is expressed 

throughout the B cell lineage. Maximal Ca 2+ mobilization in mature B cells can be 

accomplished by inactivation of Grb2 through translocation of Grb2 to an alternative 

transmembrane protein, namely NTAL. Hence, stimulation-dependent relocalization of 

Grb2 to distinct submembranous destinations, i.e NTAL or Dok-3, is a critical parameter 

to orchestrate the interaction efficiency of Ca 2+ -mobilizing enzymes and to balance BCRinduced 

Ca 2+ mobilization.

Stefanie Kunz, Karin Oberle, Anna Sander, Christian Bogdan, Ulrike Schleicher 

Bartonella-Induced Cat Scratch Disease-like 

Lymphadenopathy in Mice Involves Cell Proliferation and 

Altered Lymphocyte Recruitment 

In immunocompetent humans the Gram-negative bacterium Bartonella (B.) henselae, 

which has a feline reservoir host and is known as facultative intracellular pathogen, 

causes cat scratch disease (CSD). The clinical manifestations of CSD are characterized 

by a long-lasting, but self-healing enlargement of the draining lymph node which rarely 

contains cultivable Bartonella. So far, no data about the immunopathogenic mechanism 

leading to this lymphadenopathy exist. 

This prompted us to establish a mouse model of CSD. After subcutaneous infection with 

a high dose of B. henselae the bacteria were rapidly eliminated, but similar to human 

CSD, a striking and long-lasting swelling of the draining lymph node developed. It was 

more severe compared to that induced by other Bartonella species. In vivo-proliferation 

assays using 5-Bromo-2`-deoxyuridine (BrdU) and the transfer of 

carboxyfluoresceinsuccinimidylester (CFSE)-labeled splenocytes revealed that cell 

proliferation and a preferential influx of B cells contributed to the observed lymph node 

enlargement after Bartonella infection. With respect to possible immunostimulatory 

functions of Bartonella, we found that plasmacytoid dendritic cells exposed to Bartonella 

in vitro secreted high amounts of interferon (IFN)-α/β. Compared to B. henselae, the 

rodent pathogen B. grahamii elicited only a mild lymph node swelling in wildtype mice, 

but a severe lymphadenopathy in IFN-α/β receptor-deficient mice. From these data, we 

conclude that lymphoproliferation as well as altered immune cell recruitment are 

involved in the pathogenesis of B. henselae-induced lymphadenopathy in mice. 

Furthermore our data suggest an inhibitory effect of IFN-α/β on the described 

lymphadenopathy in mice.

Georg Peschel, Nuria Rodriguez, Christine Cirl, Nina Wantia, Tanja Erl, Susi Dürr, 

Hermann Wagner, Thomas Miethke 

Chlamydophila pneumoniae follows different strategies to 

downregulate surface MHC II expression depending on the 

cell type 

Chlamydophila pneumoniae (CP), an obligate intracellular bacterium, has developed 

several strategies for intracellular survival and to escape from the immune system. One 

of the most attractive mechanisms is the downregulation of MHC II in epithelial cells by 

degradation of the upstream stimulatory factor-1 (USF-1) by the chlamydial protease 

CPAF which is secreted during infection. In WT infected BMDM, where CP does not 

replicate as efficient as in epithelial cells, the surface expression of MHC II is 

downregulated in comparison with the mock control. Addition of IFNγ one day post 

infection did not increase surface MHC II levels compared to IFNγ stimulation alone, 

suggesting that infection with CP blocks the stimulatory ability of this cytokine. 

However, MyD88 -/- infected BMDM did not show any downregulation of MHC II after 

infection, but displayed a substantial upregulation upon infection and subsequent 

IFNγ•stimulation. Surprisingly, heat inactivated CP (HI-CP) behaved similar as CP alive. 

In addition, RT-PCR to detect CPAF RNA showed no presence of the transcript in WT and 

little in MyD88 -/- BMDM, indicating that CPAF may not be involved in MHC II 

downregulation in BMDM during infection, but rather a chlamydial PAMP that signals in a 

MyD88-dependent manner. By contrary, results obtained from infected WT mouse 

embryonic fibroblast (MEFs) showed that HI-CP was not able to impair IFNγ-mediated 

MHC II expression. Also, RT-PCR demonstrated the presence of CPAF RNA after 

infection and USF-1 was degraded in MEFs after CP infection but not after HI-CP 

stimulation. Curiously, USF-1 was upregulated in a MyD88-dependent manner in BMDM 

after CP or HI-CP infection, indicating that induction of USF-1 and increased surface 

expression of MHC II are not correlated in this cell type. In summary, these data show 

that CP prevents IFNγ-induced MHC II upregulation in BMDM via PAMP stimulation 

through MyD88-dependent but CPAF-independent mechanism while in MEFs CPAF is 

involved and therefore an infection is required.

Annabelle Schnaith, Sonja A. Leggio, Martin Krönke, Oleg Krut 

Staphylococcus aureus Subverts Autophagy for Induction of 

Caspase-independent Host Cell Death 

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. 

aureus can invade various types of non-professional phagocytes to produce host cell 

death. We show here that shortly after invasion of HeLa cells S. aureus transit to 

autophagosomes that was characterized by double membranes and co-localization with 

LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient 

atg5 -/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not 

acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. 

aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. 

aureus escape from autophagosomes into the cytoplasm, which results in caspaseindependent 

host cell death. S. aureus strains deficient for agr, a global regulator of S. 

aureus virulence, were not targeted by autophagy and did not produce host cell death. 

Autophagy induction by rapamycin restored both replication and cytotoxicity of agrdeficient 

S. aureus strains, indicating that an agr-regulated factor(s) is required for 

autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction 

of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host 

cell killing.

Stella Eugenie Autenrieth, Dirk Middendorf, Ingo Birger Autenrieth 

Yersinia enterocolitica induces cell death in splenic dendritic 

cells: evidence for YopP dependent and independent 

mechanisms 

The virulence factor YopP of Yersinia enterocolitica (Ye) induces cell death in mouse 

bone marrow-derived dendritic cells (DCs). Here we have investigated whether YopP of 

Ye might induce cell death in DCs in vivo. For this purpose, C57BL/6 mice were infected 

i.v. with Ye wild type (pYV + ) or with the YopP deficient mutant strain (pYV + ΔyopP). 

One, two and three days post infection the number of splenic DCs including 

subpopulations as well as the frequency of apoptotic DCs was analyzed by flow 

cytometry and immunofluorescence microscopy. Infection of mice with the pYV + 

resulted in a higher number of CD11c + cells in the spleen one day post infection 

compared to untreated mice. This effect was not observed in the spleen of mice infected 

with pYV + ΔyopP, indicating that the rapidly increased number of DCs in the spleen one 

day after Yersinia infection is mediated by YopP. Three days post infection the number 

of CD11c + cells was three times lower in the spleen of mice infected with pYV + 

compared to untreated mice. The decrease in CD11c + cells was associated with 

significantly higher numbers of apoptotic and necrotic DCs three days post infection with 

the wild type strain pYV + compared to uninfected mice or to mice infected with the YopP 

deficient mutant strain pYV + ΔyopP as revealed by immunostaining of active caspase 3, 

TUNEL reaction and staining with propidium iodide. However, infection with pYV + ΔyopP 

also resulted in a significant, although lower increase of apoptotic DCs suggesting that 

part of DC death after Yersinia infection in vivo is caused by mechanisms independent 

of YopP. Recent published data suggest, that LPS promotes DC death in bacterial 

infections in vivo. Furthermore, analysis of DC subpopulations revealed that Ye induces 

cell death predominantly in CD4 + CD8α - DCs. Whether and how this affects priming of T 

cells required for the control of Ye infections remains to be shown in future studies. 

Taken together, our data demonstrate YopP dependent and YopP independent induction 

of DC death in vivo suggesting that YopP, possibly in concert with LPS, induces cell 

death in splenic DCs.

Heribert Appelhans, Michael Walther, Andrea Friße, Michael A. Harvey, Stephen A. 

Judice, Brett A. Stillman, Hans Peter Seelig 

A CE-Certified Autoimmune Disease Diagnostic Protein 

Microarray 

CombiChip® Autoimmune is an auto-antigen microarray that enables laboratories to 

measure the presence of 14 different auto-antibodies associated with collagenosis and 

vasculitis-related autoimmune diseases simultaneously from a single serum sample. 

Thirteen protein antigens, double stranded DNA, and an internal calibrator (human IgG) 

are arrayed on each of 16 pads of nitrocellulose on a coated glass microscope slide 

(FAST® Slide). Seventy microliters of diluted patient’s serum is added to each array, 

and the presence of reactive antibodies is detected with DY647-conjugated anti-human 

IgG. The calibrator, a series of dilutions of human IgG arrayed on each pad, is used to 

construct a standard curve to quantify the levels of autoimmune antibodies present 

against each antigen. This microspot immunoassay yields reproducible results which are 

in good agreement with standard methods such as ELISA and Western Blot. It greatly 

reduces assay time and delivers an auto-antibody profile for each patient. This 

diagnostic assay has been validated to ensure compliance with the CE guidelines for invitro 

diagnostics.

Martina Papadopoulos, Frank Momburg 

A complex sequence motif of tapasin stabilizes the TAP2 

protein 

The type I endoplasmic reticulum (ER) glycoprotein tapasin (Tpn) is essential for loading 

of major histocompatibility class I (MHC-I) molecules with an optimal spectrum of 

antigenic peptides and for stable expression of the heterodimeric, polytopic TAP peptide 

transporter. In a detailed mutational analysis the transmembrane domain (TMD) and ERluminal 

connecting peptide (CP) of mouse Tpn were analyzed for their capacity to 

stabilize the TAP2 subunit. Replacement of the TMD of Tpn by TMDs from calnexin or 

the Tpn-related protein, respectively, completely abolished TAP2 stabilization after 

transfection of Tpn-deficient cells, while TMDs derived from distantly related Tpn 

molecules (chicken, fish) were functional. A detailed mutational analysis of the TMD and 

adjacent residues in the ER-luminal CP of mouse Tpn was performed to elucidate amino 

acids that control the stabilization of TAP2. Single amino acid substitutions, including a 

conserved Lys residue in the center of the putative TMD, did not affect TAP2 expression 

levels. Mutation of this Lys plus four additional residues, predicted to be neighbors in an 

assumed α-helical TMD arrangement, abrogated the TAP2-stabilizing capacity of Tpn. In 

the presence of a wild-type TMD, also the substitution of a highly conserved Glu residue 

in the CP of Tpn strongly affected TAP2 stabilization. Defective TAP2 stabilization 

resulted in impaired cell surface expression of MHC-I molecules. This study thus defines 

a novel, spatially arranged motif in the TMD of Tpn essential for stable expression of the 

TAP2 protein and a novel protein interaction mode involving an ER-luminal Glu residue 

close to the membrane.

Andreas Oliver Weinzierl, Dominik Maurer, Florian Altenberend, Nicole Schneiderhan- 

Marra, Karin Klingel, Oliver Schoor, Thomas Joos, Hans-Georg Rammensee, Stefan 

Stevanovic 

A cryptic VEGF T-cell epitope: Identification and 

characterization by mass spectrometry and T-cell assays 

The vascular endothelial growth factor A (VEGF) is involved in various physiological 

processes such as angiogenesis or wound healing but is also crucial in pathological 

events such as tumor growth. Clinical anti-VEGF treatments have been developed which 

could already prove to have enormous beneficial effects for cancer patients. In this 

article we describe a VEGF-derived CD8 T-cell epitope. The natural HLA ligand 

SRFGGAVVR was identified by differential mass spectrometry in two primary renal cell 

carcinomas (RCC), significantly over-presented on both tumor tissues. SRFGGAVVR is 

derived from a cryptic translated region of VEGF presumably by initiation of translation 

at the non-classical start codon CUG499. SRFGGAVVR specific T-cells were generated in 

vitro using peptide loaded dendritic cells or artificial antigen presenting cells. 

SRFGGAVVR specific CD8+ T-cells were identified by HLA tetramer analysis after in vitro 

stimulation. These T-cells were fully functional T-effector cells, which were able to 

secrete IFN-gamma upon stimulation and killed tumor cells in vitro. Additionally, we 

have quantitatively analyzed VEGF mRNA and protein levels in RCC tumor and normal 

tissue samples by gene chip analysis, qRT-PCR, in situ hybridization and bead based 

immuno assay. In the future, T-cells directed against VEGF as a tumor associated 

antigen may represent a possible way of combining peptide-based anti-VEGF 

immunotherapy with already existent anti-VEGF cancer therapies.

Susanne Tartz, Holger Ruessmann, Jana Kamanova, Peter Sebo, Angelika Sturm, Volker 

Heussler, Bernhard Fleischer, Thomas Jacobs 

A heterologous prime/boost immunization using recombinant 

Salmonella and Bordetella adenylate cyclase induces 

complete protection against P. berghei malaria 

Sterile immunity against malaria can be achieved by the induction of cytokine-producing 

CD8+ T cells that target infected hepatocytes presenting epitopes of the 

circumsporozoite protein (CSP). In the present study we evaluate the protective efficacy 

of a heterologous prime/boost immunization protocol based on the delivery of the CD8+ 

epitope of Plasmodium berghei CSP into the MHC class I presentation pathway, by 

either a type III secretion system of live recombinant Salmonella and/or by direct 

translocation of a recombinant Bordetella adenylate cyclase toxoid fusion (ACT-CSP) 

into the cytosol of CD11b-expressing professional antigen-presenting cells (APC). A 

single intraperitoneal application of the recombinant ACT-CSP toxoid, as well as a single 

oral immunization with the Salmonella vaccine, induced a specific CD8+ T cell response 

and the latter could be further enhanced by repeated application of the live vaccine, 

which however conferred only a partial protection on mice against a subsequent 

sporozoite challenge. In contrast, a heterologous prime/boost vaccination with the live 

Salmonella followed by ACT-CSP led to a significant enhancement of the CSP-specific T 

cell response and induced complete protection in all vaccinated mice

Nousheen Zaidi, Timo Herrmann, Daniel Baechle, Sabine Schleicher, Jeannette Gogel, 

Christoph Driessen, Wolfgang Voelter, Hubert Kalbacher 

A new approach for distinguishing cathepsin E and D activity 

in antigen processing organelles 

Cathepsin E (CatE) and D (CatD) are the major aspartic proteinases in the 

endolysosomal pathway. These proteinases exhibit similar specificity and therefore it is 

difficult to distinguish between them since known substrates are not exclusively specific 

for one proteinase. Here we present a substrate-based assay, which is highly relevant 

for immunological investigations since it detects both, CatE and CatD in antigenprocessing 

organelles. Therefore it could be used to study the involvement of these 

proteinases in protein degradation and the processing of invariant chain. An assay 

combining a new monospecific CatE antibody and substrate MOCAc-Gly-Lys-Pro-Ile-Leu- 

Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH2 [where MOCAc is (7-methoxycoumarin-4-yl) 

acetyl and Dnp is dinitrophenyl] is presented. This substrate is digested by both 

proteinases and therefore can be used to detect the total aspartic proteinase activity 

(TAPA) in biological samples. After depletion of CatE by immunoprecipitation, the 

remaining activity is due to CatD and the decrease of activity can be assigned to CatE. 

The activity of CatE and CatD in cytosolic, endosomal and lysosomal fractions of B cells, 

dendritic cells and human keratinocytes was determined. Our data clearly indicate that 

CatE activity is mainly located in endosomal and that of CatD in lysosomal 

compartments. Hence this assay can also be used for characterization of subcellular 

fractions using CatE as an endosomal marker, whereas CatD is a well-known lysosomal 

marker. Moreover, the highest TAPA was detected in dendritic cells and the lowest in B 

cells. The assay presented exhibits a lower detection limit than common 

antibody-based methods without lacking the specificity.

Wiebke Hartmann, Markus M. Simon, Bernhard Fleischer, Simone Korten 

A new role for granzymes: Suppression of defence against 

helminths 

Granzyme (gzm) A and B are major cytotoxic serine proteases of NK cells and cytotoxic 

T cells, known to play a key role in the destruction of tumor cells or infected cells. 

Recent studies show that gzmB is released by regulatory T (Treg) cells suppressing 

effector T cells, antigen-presenting cells, B and NK cells. On the other hand, gzmA and 

B can facilitate leukocyte migration by degrading extracellular matrix proteins. Their 

role in helminth infection has not been examined so far. As CD4+ T cells, NK cells, B 

and Treg cells affect the mixed Th1/Th2 (Th2-biased) immune defence against filariae, 

conferred substantially by granulocytes, we investigated the role of gzmA and B during 

infection of mice with the rodent filaria Litomosoides sigmodontis. Resistant wildtype 

C57BL/6 (wt) mice (infection is finished before worms reach sexual maturity) and 

gzmAxB knock out (ko) mice, generated on this background, were naturally infected via 

mites. GzmAxB ko mice harboured a lower worm load in the pleural cavity than wt mice 

in the early and late phase of infection rendering them hyperresistant. This lower worm 

load in ko mice was associated with early reduction of NK and apoptotic cells and a 

reduction of Th1 specific IgG2b, but increase of pleural IL-5 compared to wt mice. 

Pleural Foxp3+CD4+ Treg cells were detectable early with numbers unaffected by gzmA 

or B deficiency and declining with infection. GzmAxB deficiency did not generally 

influence leukocyte migration, as size and proportions of leukocyte subpopulations were 

unaltered. Therefore, we conclude that a pronounced Th2 response in gzmAxB deficient 

mice induced by a still unclear way leads to hyperresistance in this filarial infection and 

indicates for the first time a defence-suppressive role of granzymes in an infection.

Elisa Kieback, Jehad Charo, Daniel Sommermeyer, Thomas Blankenstein, Wolfgang 

Uckert 

A new safeguard eliminates TCR gene-modified autoimmunereactive 

T cells after adoptive therapy 

The T cell specificity can be redirected by the transfer of TCR genes. The modified T 

cells can elicit effector functions against the new target, and adoptive transfer of 

redirected T lymphocytes into cancer patients has recently led to clinical success. 

However, the adoptive transfer of TCR gene-modified lymphocytes is associated with 

potential risks. First, many tumor antigens are also expressed in normal tissue. 

Recognition of these antigens on non-tumor cells might lead to autoimmunity. Second, 

the introduced TCR chains might form heterodimers with the alpha and beta chains of 

the endogenous TCR. The specificity of these mispaired TCRs cannot be predicted and 

they might have autoimmune capacity. Third, it has been shown that activation of the 

introduced TCR might also trigger signal transduction of the endogenous receptor. 

Therefore, it is essential to have the possibility to eliminate autoreactive T cells in vivo 

in case of severe side effects. 

The strategies developed so far include suicide genes or apoptosis-inducing fusion 

constructs. However, they either do not lead to efficient depletion or require purification 

of gene-modified cells which might hamper their functionality. 

Here, we introduce a novel method to eliminate autoreactive TCR-redirected T cells. By 

introducing a myc-tag into the murine OT-I and P14 TCR or the human gp100 TCR it 

was possible to deplete T cells in vitro and in vivo with a tag-specific antibody. Tagmodified 

TCRs exhibited equal functionality compared to the wild type receptor 

concerning MHC-multimer binding and cytokine secretion. In vivo depletion of 

adoptively transferred cells entirely prevented disease in an autoimmune mouse model. 

Here, OT-I/myc TCR-transduced splenocytes were injected into RIP-mOVA mice which 

express the OT-I specific antigen ovalbumin in the beta-islet cells of the pancreas. 

Destruction of these cells by the adoptively transferred T lymphocytes led to severe 

diabetic disease in untreated control mice. Mice receiving a dose of myc-antibody after 

T cell transfer remained healthy and showed no increase in blood glucose levels.

Matthias Bros, Frank Jährling, Andrea Renzing, Nadine Wiechmann, Ngoc-Anh Dang, 

Arne Sutter, Ralf Ross, Jürgen Knop, Stephan Sudowe, Angelika B. Reske-Kunz 

A newly established murine immature dendritic cell line 

exerts tolerogenic function at its immature state and upon 

alternative activation in the presence of glucocorticoid 

The phenotype and function of murine dendritic cells (DC) is primarily studied using 

bone-marrow-derived dendritic cells (BM-DC), but may be hampered by the 

heterogenous phenotype of BM-DC due to differential state of maturation. We 

characterized a newly established murine DC line (SP37A3) of myeloid origin. During 

maintainance in the presence of GM-CSF and M-CSF, SP37A3 cells resemble immature 

DC characterized by low expression of MHC II and costimulatory molecules and low T 

cell stimulatory capacity. Upon stimulation, SP37A3 cells acquire a mature phenotype 

and activate naive T cells as potently as BM-DC. SP37A3 cells at their immature state as 

well as upon alternative activation by TNF-a and IL-1ß in the presence of 

dexamethasone (DEX) induced regulatory T cells which were anergic upon restimulation 

and suppressed proliferation of naive T cells. Alternatively activated SP37A3 cells 

displayed lower expression levels of costimulatory molecules and proinflammatory 

cytokines as compared with mature cells, as well as upregulated expression of FcgRIIB 

and IL-1RA. SP37A3 cells were responsive to DEX even when applied at later time 

points during activation suggesting functional plasticity. Taken together, the myeloid DC 

line SP37A3 represents a suitable model to study functions of myeloid dendritic cells at 

tolerogenic and immunogenic state, circumventing restrictions associated with the use 

of primary DC and BM-DC.

Iris Watermann, Jeannette Gerspach, Matthias Lehne, Klaus Pfizenmaier, Harald 

Wajant 

A novel CD95L-Prodrug Fusion Protein with tumorselective 

antitumoral activity 

Activation of CD95 induces apoptosis in a variety of tumor cells. The use of soluble 

agonists of CD95 in tumor therapy is precluded due to their high systemic toxicity. To 

circumvent this problem a CD95L-prodrug was developed that strictly induces CD95 

activation in an antigen-dependent manner after processing of tumor-expressed 

proteases. 

In a tumor model, local application of the prodrug reduced the growth of target antigen 

expressing, but not target antigen negative tumor cells, verifying targeted activation 

and anti-tumoral action in vivo. Despite this potent apoptosis-inducing activity in tumor 

cells in vivo, no side effects have been observed when applied systemically. 

Thus, a tumor localized action of the CD95L is ensured by tumor restricted expression of 

both, the target antigen and prodrug processing proteases. Here, a novel drug that 

fulfills the essential criteria of a target-restricted, anti-tumoral action was developed 

that may hold promise for cancer therapies in the future.

Ines Gütgemann, Jasmin Teresa Ney, Qi Zhou, Christian Kurts, Hubert Schorle, 

Andreas Limmer, Martina Berg, Reinhard Büttner 

A novel model of antigen recognition in a hepatic tumor 

microenvironment: c-myc/OVA x LAPtTA transgenic mice 

Murine hepatic tumor models to investigate antigen presentation and antigen specific Tcell 

recognition are hampered either by the lack of a defined cognate antigen or by the 

need to inject tumor cells leading to an artificial change of the local host immune 

response. Here, we report the generation of a novel conditional mouse model system of 

hepatocellular carcinoma (HCC), in which liver specific antigen presentation and 

recognition can be followed. When c-myc/OVA transgenic mice expressing chicken 

ovalbumin (OVA) and the proto-oncogene c-myc are crossed with liver specific inducer 

mice (LAP tTA), the resulting mice (c-myc/OVA x LAPtTA) develop doxicycline 

dependent multinodular tumors, resembling human HCC. Although efficient antigen 

presentation by intratumoral dendritic cells occurs, the antigen specific T-cell response 

is inefficient: Transferred OVA specific OT1 T-cells are rapidly activated, proliferate and 

accumulate in the liver and slightly delayed, in systemic immune organs. However, 

these CD8+ T-cells show a partial downregulation of the T-cell receptor and lack 

Interferon gamma production indicating T-cell tolerance. This model will be useful in 

dissecting antigen-specific and non-specific immune responses during different stages of 

hepatocarcinogenesis and testing of immune modulatory strategies within an organ 

specific tumor microenvironment.

Peggy Riese, Thomas Ebensen, Claudia Link, Kai Schulze, Michael Morr, Carlos Guzmán 

A pegylated derivative of alpha galactosylceramide exhibits 

improved immune stimulatory activities 

The glycolipid alpha galactosylceramide (alpha GalCer) has immune modulatory 

properties, leading to activation of various cell subsets of the innate and adaptive 

immune system. However, its poor solubility makes alpha GalCer a suboptimal 

compound for in vivo applications. A well-known method to improve the solubility of 

chemical compounds in aqueous solutions is the conjugation with polyethylene glycol. A 

pegylated derivative of alpha GalCer, called alpha GalCerMPEG, is characterized, which 

exhibits improved physiological and biological activities. The new compound is watersoluble 

and retains the specificity for the CD1d receptor of alpha GalCer. In vitro studies 

showed the same stimulating activities on different subsets of immune cells (dendritic 

cells, natural killer cells), even when tested at 33-fold lower concentrations. Natural 

killer cells isolated from mice treated with alpha GalCerMPEG have also stronger activity 

on Yac-1 cells than those obtained from animals receiving either alpha GalCer or CpG. 

Intranasal immunisation studies performed in mice showed, that alpha GalCerMPEG 

exerts stronger adjuvant activities than the parental compound alpha GalCer. Coadministration 

of beta-galactosidase with alpha GalCerMPEG resulted not only in high 

titers of antigen-specific antibodies in serum, but also in the stimulation of stronger Th2 

and sIgA responses, both at local and remote mucosal effector sites (i.e., nose, lung, 

vagina). The new synthetic derivative alpha GalCerMPEG represents a promising tool for 

the development of immune interventions against infectious and non-infectious diseases.

Eva Schlosser, Carolina Otero, Benedikt Kessler, Mariola Edelmann, Rene Brunisholz, 

Daniel Legler, Marcus Groettrup 

A prostate carcinoma antigen reveals a new cytosolic class I 

processing pathway for endoplasmic reticulum targeted 

proteins 

Proteins that bear an N-terminal endoplasmic reticulum leader peptide are inserted into 

the lumen of the endoplasmic reticulum followed by cleavage of the signal peptide and 

glycosylation. It has been shown that major histocompatibility complex class I restricted 

T cell epitopes can be generated from these glycoproteins by the proteasome after their 

retrotranslocation from the endoplasmatic reticulum into the cytosol. In this study we 

show that an HLA-A*0201 restricted T cell epitope from prostate stem cell antigen, that 

is recognized by prostate carcinoma specific cytotoxic T lymphocytes, encompasses the 

cleavage site of the endoplasmic reticulum signal peptidase. This cleavage yields 

truncated peptides that fail to bind to HLA-A*0201 and to be recognized by cytotoxic T 

lymphocytes. Since processing of prostate stem cell antigen by signal peptidase occurs 

immediately after cotranslational insertion, the epitope must be synthesized and 

processed from polypeptides that have never reached the endoplasmic reticulum. The 

processing of this epitope relies on the activity of the proteasome and the transporter 

associated with antigen processing. This is the first example of a new pathway of class I 

processing that relies on the failure of endoplasmic reticulum targeted proteins to reach 

their compartment of destiny.

Mathias Konstandin, Guido Wabnitz, Hülya Aksoy, Martin Klemke, Thomas Dengler, 

Yvonne Samstag 

A sensitive assay for the quantification of integrin-mediated 

adhesiveness of human stem cells and leukocyte 

subpopulations in whole blood 

Adhesion of leukocytes is an early step in the formation of adaptive or innate immunity. 

Also in chronic inflammatory pathologies like atherosclerosis regulation of adhesiveness 

is pivotal for the accumulation of leukocytes within the vessel wall. Therefore, the 

quantification of adhesion is crucial for the understanding and monitoring of immune 

responses in patients. However, so far functional analysis of leukocyte adhesion was 

time consuming and required prior purification of cell populations from peripheral blood. 

This reduced the number of samples and cell populations that could be analyzed from 

limited patient material. Here we introduce a novel method that enables a rapid 

quantification of integrin mediated leukocyte adhesion in human whole blood using flow 

cytometry. The quantification relies on soluble multivalent immuncomplexes and is thus 

called “ligand-complex-based adhesion assay” (LC-AA). The LC-AA enables evaluation of 

both integrin affinity and avidity in T-cells, NK-cells and monocytes from as little as 20•l 

whole blood. Furthermore, with the LC-AA for the first time measurement of 

adhesiveness of extremely rare cell populations like CD34+ peripheral blood stem cells 

can be assessed in the absence of prior purification steps. Finally, small blood volumes 

needed for adhesion analysis with the LC-AA allow the evaluation of multiple cell 

subpopulations in large sample collectives, e.g. required in clinical studies.

Heiko Bruns, Frank Stegelmann, Steffen Stenger 

Abl-tyrosine kinase modulates immune responses to 

Mycobacterium tuberculosis 

Tyrosine kinases are checkpoints for activation of cellular immune responses. We 

hypothesized that Mycobacterium tuberculosis (M.tb.) interferes with tyrosine kinase 

function to circumvent killing by the host cell. 

We chose to focus on Abl (Abelson) - tyrosine kinase, because it is known to orchestrate 

immune response to mycobacterial infection like proliferation, adhesion and apoptosis. 

We measured TNF and IFN-gamma release by human PBMC in the presence of the 

specific tyrosine kinase inhibitor STI-571. STI-571 treatment inhibited TNF and IFNgamma 

release induced by mycobacterial antigens up to 90% (n=7). Furthermore 

inhibition of Abl-tyrosine kinase influenced lymphocyte migration. The cell-migration 

towards the chemokines CCL21 and CCL5, both required for the containment of 

Mycobacterium tuberculosis in granulomas, were affected. Specifically, migration of 

PBMC towards CCL21 was enhanced (three-fold), whereas movement towards CCL5 was 

reduced. 

These findings may be clinically relevant, because STI-571 (Imatinib) is a standard 

treatment in chronic myeloid leukaemia. Deviation of cellular immunity as described 

here may increase the risk for the occurrence of infectious diseases during Imatinib 

therapy.

Angelika Stöcklinger 

Ablation of Epidermal Langerhans Cells has no Impact on 

Gene-Gun mediated Immune Responses 

Gene gun immunization, i.e. bombardment of skin with DNA-coated particles, is an 

efficient method for the administration of DNA vaccines. Direct transfection of APC or 

cross-presentation of exogenous antigen acquired from transfected non-immune cells 

enables MHC-I-restricted activation of CD8+ T cells. Additionally, MHC-II-restricted 

presentation of exogenous antigen activates CD4+ T helper cells. Being the principal 

APC in the epidermis Langerhans cells (LC) seem ideal candidates to accomplish these 

functions. However, the dependence on LC of gene gun-induced immune reactions has 

not yet been demonstrated directly. This was primarily hampered by difficulties to 

discriminate the contributions of LC from those of other dermal dendritic cells. To 

address this problem we have employed Lang-DTR knock-in mice that allow for 

selective inducible ablation of LC. LC-deficiency, even over the entire duration of 

experiments, did not affect any of the gene gun-induced immune functions examined, 

including proliferation of CD4+ and CD8+ T cells, interferon-gamma secretion by spleen 

cells, antibody production, cytotoxic T lymphocyte activity, and development of 

protective anti-tumor immunity. Together, our data show that gene gun immunization is 

capable of inducing humoral and cell-mediated immune reactions independently of LC.

Brigitte Santner-Nanan, Nabila Seddiki, Cindy Zhu, Verena Quent, Anthony Kelleher, 

Barbara Fazekas de St. Groth, Ralph Nanan 

Accelerated age-dependent transition of human regulatory T 

cells to 

effector memory phenotype 

We and others recently described a method for isolating viable FoxP3+ 

T regulatory cells (Tregs) by means of the surface phenotype 

CD4+CD127loCD25+. In this study, we used the new strategy to measure 

Treg numbers, phenotype and function at different ages. Mean 

percentages of CD4+CD127loCD25+ Tregs increased only slightly 

throughout life, from 6.10% in cord blood to 7.22 % in PBMC from 

adults between 20 and 25 years and 7.50% in PBMC from adults over the 

age of 60. In all age groups, a higher proportion of Tregs had 

acquired a CD45RA- memory phenotype compared with CD4+Foxp3- 

conventional T cells. This increase was entirely attributable to 

increased Tregs with an effector memory phenotype, whereas central 

memory phenotype cells were equally represented within the Treg and 

conventional CD4+ T cell populations. Expression of CD95 also differed 

between Tregs and conventional CD4+ T cells at all ages. However there 

was no difference in the suppressive capacity of the different naïve 

and memory Treg subsets. These results suggest that, compared with 

their conventional CD4+ T cell counterparts, Tregs undergo 

preferential differentiation from a naïve to an effector memory 

phenotype, driven by their specificity for self rather than foreign 

antigen.

Jasmin Herz, Julian Pardo, Hamid Kashkar, Erik Bos, Reinhard Wallich, Peter J. Peters, 

Elmon Schmelzer, Martin Krönke, Markus M. Simon, Olaf Utermöhlen 

Acid sphingomyelinase is a critical regulator of cytotoxic 

granule secretion by primary T lymphocytes 

Granule-mediated cytotoxicity is the major effector mechanism of cytotoxic CD8 + T 

cells. The phosholipase acid sphingomyelinase (ASMase) hydrolyzes sphingomyelin into 

phospocholine and ceramide and is localized in cytotoxic granules. Therefore, we 

hypothesised that ASMase might be involved in the secretion of cytotoxic granules. 

We report here that CD8 + T cells from ASMase-deficient (ASMase -/- ) mice are defective 

in exocytosis of cytotoxic granules, which results in markedly attenuated cytotoxic 

activity and significantly delayed elimination of the Lymphocytic 

Choriomeningitis (LCM) virus in vivo. Compared to wild type cytotoxic T cells, ASMase -/- 

CD8 + T cells contain equal amounts of granzyme- (gzm) and perforin- specific mRNA 

transcripts and functional protein. Strikingly, T cell receptor-mediated release of 

cytolytic effector molecules like granzyme A and hexosaminidase is more than ten-fold 

reduced in ASMase -/- CD8 + T cells. Cytotoxic granules of ASMase -/- CD8 + T cells are 

correctly directed towards the immunological synapse and fusion pores are readily 

formed that release low molecular weight dextrans yet retain the macromolecular 

complex of serglycin-proteoglycan, granzymes and perforin. Thus ASMase is critically 

involved in regulating the secretion of cytotoxic granules of CD8 + T cells.

Manuela Rossol, Undine Meusch, Holm Häntzschel, Sunna Hauschildt, Ulf Wagner 

Activated CD4 T cells induce TNF production in human 

monocytes via reverse signalling of membrane TNF 

Activated CD4 T cells are able to activate human monocytes by direct cell-cell contact, 

which leads to the production of pro-inflammatory cytokines. This process is believed to 

be important in chronic inflammatory diseases like rheumatoid arthritis (RA). Previously, 

we described membrane TNF expressed on activated T cells and TNF receptor (TNFR) 1 

and 2 expressed on monocytes as ligand/receptor pair responsible for monocyte 

activation. Now we propose a second mechanism participating in T cell-monocyte cell 

contact, namely reverse signalling of membrane TNF in which TNF acts as a signal 

transducing receptor. 

Several lines of evidence indicate the involvement of TNF reverse signalling in T cellmediated 

monocyte activation. (i) Membrane TNF is expressed on monocytes and 

TNFR2 is expressed on activated T cells. (ii) Blockade of TNFR2 on activated CD4 T cells 

by antibodies led to a diminished TNF production and Erk phosphorylation in monocytes. 

(iii) Resting CD4 T cells transfected with a TNFR2 plasmid induced TNF production in 

monocytes. (iv) Knockdown of TNFR2 on CD4 T cells by specific siRNA led to a 

decreased TNF production in monocytes. (v) Crosslinking of membrane TNF on 

monocytes by a plate-bound TNFR2:Ig construct induced a strong production of TNF. 

This TNF production depends on activation of MAP kinase Erk and casein kinase 1, the 

enzyme responsible for TNF phosphorylation. 

In conclusion, we identified reverse signalling of membrane TNF as a second pathway 

leading to TNF production in T cell-mediated monocyte activation. Accordingly, 

monocytes of RA patients treated with an anti-TNF antibody did not respond to 

activated T cells of healthy donors, pointing to a possible blockade of membrane TNF 

reverse signalling due to the anti-TNF antibody.

Milena Josefina Tosiek, Marcus Gereke, Jan Buer, Dunja Bruder 

Activation and regulation of autoreactive CD8 T cells in the 

lung 

The lung mucosa as it is positioned between the body and environment has to keep the 

balance between immunity and tolerance. To better understand mechanisms of 

induction and regulation of CD8 T cell-mediated immune responses in the lung we 

investigate an SPC-HAxCL4 transgenic mouse. This mouse expresses a model antigen 

(viral hemagglutinin, HA) in alveolar epithelial cells type II of the lung simultaneously 

with transgenic CD8 T cells of HA-specificity providing an autoimmune environment in 

the lung. Autoreactive CD8 lymphocytes escape from clonal deletion in the thymus and 

migrate to the lung. Histopathological analysis of SPC-HAxCL4 mice reveals progressing 

with age formation of lymphocytic infiltrations in the lung, resembling inducible 

secondary lymphoid tissue; nevertheless the disease is not lethal suggesting presence 

of some regulatory mechanisms. 

HA-specific lymphocytes of SPC-HAxCL4 mice, regardless of their autoreactive potential 

and infiltration of the lung, show naïve phenotype. In contrast while reisolated, HA- 

specific CD8 T cells respond in vitro to their specific antigen by massive proliferation 

which excludes their anergic phenotype. 

To investigate the acute form of CD8 T cell-mediated autoimmune response, we 

perform adoptive transfers of HA - specific CD8 lymphocytes into SPC-HA mice 

expressing HA in the lung epithelium. 

Surprisingly, CD8 T cells when transferred naïve, fail to proliferate and do not cause 

airway inflammation. In contrast, when cotransferred together with in vitro preactivated 

CD4 T cells of the same antigen specificity, naïve CD8 cells migrate to the 

draining BLN and the lung where they proliferate and trigger inflammation. These data 

suggest the importance of T helper cells in facilitating processes of priming and homing 

of autoreactive CD8 lymphocytes in the lung. 

The next matter of interest is the role of CD4 cells of HA-specificity in maintaining self- 

tolerance in SPC-HAxCL4 mice as well as the conditions under which the tolerance could 

be broken.

Xuefang Ren, Marion Schneider, Ying Wang, Lin Xu, Zhenggang Jiang, Fei Ye 

Activation induced cell death of CD4+CD25+Foxp3+ T cells by 

TCR re-stimulation involves a CD95/CD95L dependent 

mechanism 

In order to better understand how regulatory T cells (Treg) would be modulated, we 

isolated murine CD4+CD25+T cells from naive BALB/c or DO11.10 mice by MACS 

(magnetic cell sorting). Treg were functionally verified by suppressing an 3H cell 

proliferation assay. RT-PCR and intracellular staining were applied to detect the 

expression of Foxp3 in these cells. To induce actvation induced cell death (AICD), Treg 

from BALB/c mice were stimulated with anti-CD3/CD28 antibodies, and those from 

DO11.10 mice were incubated with OVA323-339 peptide plus antigen presenting cells 

(APCs), respectively. SuperArray and Real-time PCR were established to detect the 

apoptosis gene expression pattern in Treg. Flow cytometry was performed to determine 

the apoptosis of Treg. The influence of anti-FasL, anti-TRAIL neutralizing antibody and 

zVAD-fmk on AICD of Treg was evaluated. 

Results: CD4+CD25+Foxp3+ Treg act to suppress activation of the immune system and 

thereby maintain immune system homeostasis and tolerance to self. Management of 

Treg during an immune response is very significant. To augment Treg is advantageous 

in autoimmune disease, allergy and organ transplantation. On the contrary, depletion of 

Treg is beneficial to cancer patients. Activation induced cell death (AICD) is one of the 

main mechanisms for T cell contraction in vivo. We show here CD4+CD25+T cells from 

murine splenocytes could be successfully classified by MACS with 98% purity and 

specifically express Foxp3 which is the ‘master control gene’ for Treg cells and 

demonstrated suppression of proliferation of CD4+CD25-T cells. Treg can be induced to 

AICD by TCR re-stimulation, which may reach up to 39-45• following in vitro 

activationfor 7 days. The results of SuperArray and real-time PCR showed different gene 

expression patterns of TNF family members in freshly isolated Treg and activated Treg. 

TCR re-stimulation of Treg in vitro revealed a CD95/CD95L dependent pathway toward 

AICD. These findings open up to more detailed investigation of Treg modulatory events 

in vivo.

Alexei Gratchev, Julia Kzhyshkowska, Sheila Kannookadan, Miriam Ochsenreiter, Anna 

Popova, Xiaolei Yu, Isabelle Muller-Molinet, LiMing Gooi, Sergij Goerdt 

Activation of a TGF-β-specific multistep gene expression 

program in mature M2 macrophages requires glucocorticoidmediated 

surface expression of TGF-β receptor II 

Alternatively activated (M2) macrophages regulate steady state-, cancer- and 

inflammation-related tissue remodeling. They are induced by Th2-cytokines and 

glucocorticoids (GC). The responsiveness of mature macrophages to transforming 

growth factor (TGF)-β a cytokine involved in inflammation, cancer and atherosclerosis is 

currently controversial. Recently, we demonstrated that interleukin-17 receptor B 

(IL17RB) is up-regulated in human monocyte derived macrophages differentiated in the 

presence of Th2 cytokine IL-4 and TGF-β1. Here we show that mature human 

macrophages differentiated in the presence of IL-4 and dexamethasone (M2IL-4/GC ) 

respond to TGF-β by 5-fold induction of IL17RB mRNA. Further TGF-β induced a gene 

expression program comprising 111 genes in mature human M2IL-4/GC , but not M2IL-4 which includes transcriptional/signalling regulators (ID3, RGS1) as early response 

genes, and immune modulators (ALOX5AP, IL17RB) as well as atherosclerosis-related 

genes (ALOX5AP, ORL1, APOC1, APOC2, APOE) as late response genes. Analysis of 

molecular mechanism underlying GC/TGF-β cooperation revealed that surface 

expression of TGF-βRII was high in M2GC and M2IL-4/GC , but absent from M2IL-4 , while 

the expression of TGF-βRI/II mRNA, TGF-βRII total protein and surface expression of 

TGF-βRIII were unchanged. TGF-βRII surface expression was dependent on GC dose in 

a range of physiological to therapeutic GC concentrations and determined the strength 

and duration of Smad2-phosphorylation-mediated signaling. In summary, mature 

human M2 macrophages made permissive to TGF-β by GC-induced surface expression 

of TGF-βRII activate in response to TGF-β1 a multistep gene expression program 

featuring traits of macrophages found within an atherosclerotic lesion.

Umme Amara, Miriam Kalbitz, Andreas Klos, Markus Huber-Lang 

Activation of Complement by the Coagulation System 

Introduction: Both, the complement and the coagulation system belong to the "first line 

of defence" and undergo massive activation early after severe injury. A dialogue 

between the two cascades has often been proposed but the precise molecular pathways 

of this interaction are still unclear. To elucidate the mechanisms involved, the effects of 

various coagulation factors on generation of the complement activation products C3a 

and C5a was investigated. 

Methods: In vitro cleavage of C5 or C3 in presence or absence of various coagulation 

factors (F) was determined in a time- and dose-dependent manner by ELISA and 

Immunoblotting. Biological activity of the cleavage products was also assessed by C3adependent 

mast cell (HMC-1)- and C5a-dependent neutrophil-chemotactic activity. Oneway 

ANOVA and Tukey multiple comparison test, p

Luisa Cervantes-Barragan, Ulrich Kalinke, Roland Züst, Constantino Lopez-Macias, 

Volker Thiel, Burkhard Ludewig 

Activation of myeloid cells through plasmacytoid dendritic 

cell-derived type I interferon secures control of murine 

coronavirus infection 

The secretion of type I interferons (IFN) is one of the fundamental aspects of the innate 

immune response against viruses. However, the role of type I IFNs in the pathogenesis 

of coronavirus infections has not yet been fully clarified, neither its importance for 

particular coronavirus infected cell populations. We have shown recently that 

plasmacytoid dendritic cell derived type I interferons (IFNs) are of prime importance for 

the initial control of mouse hepatitis virus (MHV) infection (Cervantes-Barragan Blood. 

2007 109:1131-7). In the present study, we have determined the major target cell 

populations of type I IFNs. To this end, we have first generated a series of chimeric 

mice expressing the type I interferon receptor (IFNAR) either on hematopoietic or nonbone 

marrow-derived cells. Early control of MHV depended mainly on IFNAR expression 

on hematopoietic cells. To establish which leukocyte subset responds most efficiently to 

type I IFN, mice conditionally deficient for the type I interferon receptor (IFNAR) on 

different leukocyte subsets were infected with MHV. This genetic analysis revealed that 

IFNAR expression on macrophages/neutrophils and dendritic cells is of prime 

importance for the early containment of MHV within secondary lymphoid organs, while 

IFN activity on B cells or T cells is not required for the clearance of MHV infection. Taken 

together, our results indicate that the protection of macrophages/neutrophils and 

CD11c-positive dendritic cells through type I IFN is essential for the control of murine 

coronavirus infection.

Alexander Donald McLellan, Patrizia Stoitzner, Jacquie Harper, Sarah Saunderson, 

Ralph Jack, Anthea Bouwer 

Activation of natural killer cells by dendritic cells stimulated 

with gram positive bacteria 

Interferon-gamma (IFNγ) release by natural killer (NK) cells plays a key role in the 

resolution of cancer or viral infection. We have previously shown that the cell wall 

components of Gram-positive bacteria stimulate a rapid release of IFNγ from human and 

murine leukocytes. The effect has also been found to be mimicked by the cell wall 

component lipoteichoic acid (LTA) but not by other Toll-like receptor-agonists. NK cell 

activation requires membrane-contact with bacteria-stimulated dendritic cells (DC), but 

is independent of the adhesion molecule LFA-1 and the NK cell activation molecule 

NKG2D. IL-12 and IL-18 expression was found to be essential for NK cell activation. 

Activation of DC/NK with whole bacteria was maintained in TLR2-/- and TLR4-/- 

backgrounds, however the stimulation of DC/NK with the cell wall component LTA was 

lost in the TLR4-/- background, further fuelling the controversy surrounding the role of 

this TLR in the recognition of Gram-positive bacteria. Despite inducing potent cytokine 

secretion in DC/NK cells, Gram-positive cell wall stimulation did not lead to increased 

natural or antibody-dependent (ADCC) cytotoxicity of NK cells against the NK-sensitive 

target YAC-1 or antibody sensitised A20 cells. We are currently investigating the in vivo 

potential of these adjuvants to drive NK cell activation and Th1 polarisation for the 

treatment of tumours or infectious diseases.

Dimitra Kotsougiani, Birgit Prior, Gertrud Maria Hänsch, Christof Wagner 

Activation of T lymphocytes in bacterial infection: production 

of interferon 4gamma, expression of the chemokine receptor 

CXCR6, and up-regulation of Toll-like receptors (TLR) in 

patients with implant-associated posttraumatic osteomyelitis 

Implant-associated posttraumatic osteomyelitis is caused by bacterial biofilms, mostly 

consisting of staphylococci species that colonize devices used for osteosynthesis, such 

plates, nails, or screws, or prosthesis for joint replacement. In most cases, removal of 

the infected implant is required. During surgery, cells having infiltrated the infected site 

can be collected for phenotypical and functional characterization. Predominantly highly 

activated polymorphonuclear neutrophils (PMN) were found as well as T lymphocytes, 

the latter amounting to approximately 20 % of infiltrated cells. The majority of the 

infiltrated T cells expressed CXCR6, a homing receptor for non-lymphoid tissue, while 

CXCR6 expression on peripheral T cells was well below 20 %. 

The infiltrated T cells, particularly the CD8+CD28-, produced 4gamma IFN, while the 

peripheral blood T cells obtained from the same patient did not. Moreover, again 

predominantly on the infiltrated T-cells Toll-like receptors were expressed. TLR1, TLR2 

or TLR4 were found on small, but conspicuous T-cell populations that also expressed 

CD11b+. The data lead to the following conclusion: persistent, localised bacterial 

infections lead to the activation of T cells, and their infiltration into the infected site. It 

is feasible that by producing 4gamma IFN the T cells enhance the efficiency of the local 

effector cells, particularly of the PMN, and thereby support the local host defence.

Eva Distler, Catherine Wölfel, Sylvia Köhler, Marion Nonn, Elke Schnürer, Ralf G. 

Meyer, Christoph Huber, Thomas Wölfel, Udo F. Hartwig, Wolfgang Herr 

Acute myeloid leukemia (AML)-reactive cytotoxic T 

lymphocyte clones rapidly expanded from CD8+ CD62L(high) 

+ T cells of healthy donors prevent AML engraftment in NOD/ 

SCID IL2Rγnull mice 

Current in vitro techniques for isolating acute myeloid leukemia (AML)-reactive cytotoxic 

T lymphocytes (CTLs) from healthy donors are of relatively low efficiency and yield 

responder populations with unknown biological significance. We established an 

allogeneic mini-mixed lymphocyte-leukemia culture (mini-MLLC) approach by 

stimulating donor CD8+ T cells with HLA class I-matched primary AML blasts in 

microtiter plates. Before culture, CD8+ T cells were separated into CD62L(high)+ and 

CD62L(low)+/neg subsets enriched for naive/central memory and effector memory 

cells, respectively. In 8 different related and unrelated donor/AML pairs, numerous CTL 

populations were isolated that specifically lysed myeloid leukemias in association with 

various HLA-A, -B, or -C alleles. These CTLs expressed T-cell receptors of single Vβ 

chain families, indicating their clonal origin. The majority of CTL clones were obtained 

from mini-MLLCs initiated with CD62L(high)+ cells. Using antigen-specific stimulation, 

multiple CTL populations were amplified to 10e8-10e10 cells within 6-8 weeks. Two 

representative CTL clones were capable of completely preventing the engraftment of 

human primary AML blasts in NOD/SCID IL2Rγnull mice. We concluded that the mini- 

MLLC approach allows the efficient in vitro expansion of AML-reactive CTL clones from 

CD8+CD62L(high)+ precursors of healthy donors. These CTLs can inhibit AML 

engraftment in immunodeficient mice, suggesting their potential biological relevance.

Sarvari Velaga, Stephan Halle, Sabrina Dähne, Oliver Pabst 

Adaptation of Solitary Intestinal Lymphoid Tissue in response 

to microbial stimulation 

Besides Peyer´s patches, solitary intestinal lymphoid tissue (SILT) provides a structural 

platform to efficiently initiate immune responses in the murine small intestine. SILT 

consists of dynamic lymphoid aggregates that are heterogeneous in size and 

composition, ranging from small clusters of mostly lineage-negative cells known as 

cryptopatches to larger isolated lymphoid follicles rich in B cells. Here we report a novel 

technique that allows monitoring the dynamic behaviour of individual SILT over time. 

We demonstrate that colonization of germ free mice with commensal bacteria provokes 

an adjustment of the spectrum of SILT to that observed under specific pathogen free 

conditions by the conversion of pre-existing lymphoid structures into larger-sized SILT. 

Further enhanced microbial stimulation by means of oral infection with the 

enteropathogen Salmonella yields SILT that exceed the size spectrum of structures 

observed under pathogen free conditions. Salmonella directly infects SILT triggering a 

vigorous inflammatory response and immunopathology that leads to enlargement and 

morphological destruction of SILT. However, neither colonisation of germ free mice nor 

infections trigger the de novo formation of lymphoid tissue. The functional analysis of 

chemokinereceptor and integrin-deficient mouse strains indicates that the adaptation of 

SILT in response to microbial stimulation is mediated by a unique population of lineage 

negative SILT cells expressing the stem cell factor receptor cKit, chemokinereceptor 

CXCR5 and lymphotoxinα. In conclusion, our findings establish SILT, a lymphoid 

compartment frequently neglected in earlier studies, as a bona fide lymphoid tissue in 

the intestine that acts as a major site for Salmonella invasion and ensuing mucosal 

pathology.

Nancy Brewig, Adrien Kissenpfennig, Bernard Malissen, Alexandra Veit, Bernhard 

Fleischer, Uwe Ritter 

Adaptive immune response to Leishmania parasites is induced 

in the absence of epidermal Langerhans cells 

Epidermal Langerhans cells (LCs) represent a subset of dendritic cells (DCs), which 

appear in the outer compartment of the skin. The biological role of this DC 

subpopulation still remains controversial. In the experimental model of leishmaniasis 

LCs were discussed to be critical for regulation of cutaneous immune reactions. 

However, recently it was documented that dermal-derived DCs rather than epidermal 

LCs might represent the key players in experimental leishmaniasis. These contradictory 

findings might be explained by the overlapping phenotype of epidermal-derived LCs and 

dermal-derived DCs. To answer the question whether LCs are indeed involved in T cell 

mediated immune response against Leishmania (L.) major, knock-in mice expressing a 

diphteria toxin receptor under the control of the langerin (CD207) gene were used to 

conditionally ablate LCs. Following depletion, mice were infected subcutaneously into 

the footpad with L. major parasites and the immune response in the footpad draining 

lymph node (LN) and at the site of infection was analyzed over the time. 

Investigation of i) the course of infection, ii) the production of L. major-specific 

immunoglobulines and iii) the development of a memory T cell response revealed no 

differences between LC-ablated and control mice, indicating a minor role for LCs in the 

immune response against experimental leishmaniasis. To examine the cellular immune 

response in skin draining LNs cells were isolated and restimulated with soluble 

Leishmania antigen. We detected comparable proliferation of antigen specific CD4+ T 

cells in LC-ablated and control mice. In contrast, the proliferation of CD8+ T cells 

occured just up to day 10 after infection and was significantly diminished in LC-ablated 

mice. Therefore, we assume that LCs interact transiently with CD8+ T cells, known to 

play a minor role in the high dose infection model of experimental leishmaniasis, but not 

with CD4+ T cells. 

In conclusion, this is the first study demonstrating that epidermal LCs are dispensable 

for the induction of a protective immune response against Leishmania parasites.

Gaubert Sophie, Zimmermann Andra, Arbach Olga, Rossa Simone, Thiel Andreas, 

Voigt Sebastian, Ebell Wolfram 

Adenovirus-specific T cell therapy in paediatric stem cell 

transplants: Isolation and expansion of donor-derived T 

lymphocytes 

Human adenoviruses rarely cause severe clinical symptoms in healthy children and 

adults. However, they become a critical issue for immunocompromised patients and 

result in significant morbidity and mortality in allogeneic haematopoietic stem cell 

transplant children. Antiviral medication has not been unequivocally effective, 

encouraging new strategies. Virus-specific T cells are described as a key component in 

immunity against adenoviruses. 

In our lab a protocol was developed to generate adenovirus-specific T cell lines for 

paediatric haematopoietic stem cell-transplant patients under Good Manufacturing 

Practices, in order to infuse these cells in the early post transplant period. Starting with 

100 ml whole blood from the designated allogeneic healthy donor we stimulate 

peripheral mononuclear cells with a defined mix of overlapping peptides covering the 

hexon protein primary sequence (PepMix). Reactive cells are selected for interferongamma 

secretion using the CliniMacs device in a class A environment. Positively sortedcells 

are expanded for at least two weeks with autologous feeder cells and a cytokine 

cocktail. When ready, the T cell line is phenotyped for its specificity and cytotoxicity 

using autologous B-LCL, tested for the microbiological and viral status, and 

cryopreserved in three aliquots for prophylactic infusions on day +20, +60 and +100 

post-transplant with a cell dose of 1 x 107/m² body surface of the recipient. Our 

validation runs demonstrate that the final product contains variable mixtures of NKcells, 

CD4+ and CD8+ T-cells with specificity higher than 60 %. Further details of the 

protocol and clinical trials will be discussed.

Iris Bellinghausen, Barbara Häringer, Beatrice Lafargue, Tamara Hilmenyuk, Heinz 

Decker, Joachim Saloga 

Allergological implication of the quaternary hexameric 

structure of the cockroach allergen Per a 3. 

Cockroach allergens play a very important role in allergic diseases, especially asthma. 

The major allergen of the American cockroach (Periplaneta americana), Per a 3, 

naturally occurs as isoforms of hexamers. The aim of this study was to investigate 

whether the hexameric structure of Per a 3 influences its allergenicity and 

immunogenicity. Therefore, we compared the different effects of native hexamers and 

dissociated monomers of cockroach hemolymph extracts, containing almost only Per a 3 

proteins, on proliferation and Th1/Th2 cytokine production of CD4+ T cells in coculture 

with allergen-pulsed monocyte-derived dendritic cells as well as the leukotriene release 

of basophils. In Periplaneta americana-sensitized and non-sensitized donors the 

monomer was internalized faster by immature DC and induced higher proliferation and 

IFN-gamma production than the native hemolymph extracts (hexamers). While in nonsensitized 

donors IL-4 and IL-5 as well as IL-10 production were also increased after 

stimulation with monomeric Per a 3-pulsed DC, Th2 cytokine and IL-10 production were 

only enhanced in Periplaneta americana-sensitized donors using hexameric Per a 3pulsed 

DC. Furthermore, in the leukotriene release assay the monomer was less 

effective than the hexamer. Our data indicate that the quaternary structure can 

influence both allergenicity and immunogenicity, also depending on the sensitization 

status. In addition, the monomeric variant of Per a 3 allergens could be a possible 

candidate for a specific immunotherapy because the IgE-mediated allergic reaction and 

the Th2-inducing capacity are diminished while the Th1-inducing capacity is retained.

Ingrid Schuster, Elfriede Eppinger, Christoph Salat, Bernhard Frankenberger, Joachim 

Ellwart, Elfriede Nößner, Dirk Busch, Angela Krackhardt 

Allorestricted T cells with high specificity for the FMNL1derived 

peptide PP2 show tumor reactivity but also limited 

alloreactivity against other MHC alleles – implications for 

adoptive T cell therapy using allorestricted T cell receptors 

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor 

leukocyte infusions has curative potential, especially in hematological malignancies. 

However, this approach is severely restricted due to graft-versus-host-disease (GvHD). 

Therefore, we aimed to generate HLA-A2-allorestricted T cells with defined specificity 

using the CLL-associated antigen Formin-related protein in leukocytes 1 (FMNL1) as 

target. 

After stimulation with peptide-pulsed T2 cells, we were able to isolate an HLA-A2allorestricted 

effector T cell clone specifically recognizing the peptide FMNL1-PP2 

(RLPERMTTL) derived from FMNL1. Potent lytic effector functions were observed against 

different tumor cell lines and EBV-transformed B cells. The T cells showed also reactivity 

against CLL cells. 

Investigating the specificity of this T cell clone, we observed high peptide specificity 

against T2 cells pulsed with the specific peptide, but not T2 cells pulsed with seven 

different control peptides. Moreover, T2 cells presenting the highly homologous HDAC6derived 

peptide RLAERMTTR in an HLA-A2-restricted manner were not recognized. 

However, the T cell clone was reactive against different HLA-A2 subtypes as well as two 

out of 16 HLA-A2- samples. Peptide pulsing of HLA-A2 subtypes increased target 

recognition in several cases indicating that the selected peptide might be presented by 

these HLA-A2 subtypes. Concerning the two HLA-A2- samples which have been 

recognized, MHC-restriction could be specified in one case to HLA-A*3303 by testing 

A*3303-transfected C1R cells. In this case peptide pulsing did not increase specific 

recognition of target cells suggesting that another peptide may be recognized in the 

context of HLA-A*3303. 

These data indicate that highly peptide-specific allorestricted T cells may harbour 

polyspecificity against different peptides and MHC molecules which can result in 

unpredicted cytotoxicity. This is certainly of special interest for the use of allorestricted 

peptide-specific T cell receptors for adoptive transfer.

Andrey Bogdanov, Tatyana Rybakova, Nikolai Belyaev 

Alpha-fetoprotein impair recovery of cell-mediated immunity 

normal function after tumor ablation 

It was shown tumor successfully grown when it were thought of as having escaped from 

immunosurveillance. Tumor-induced immunosuppression based on changing of immune 

cells functional activity, structure and viability. At that, tumor produces many factors 

that may involve in suppression of anti-tumor immunity. One of prospective factor is 

alpha-fetoprotein (AFP), which induced disorders of certain immunity effector reactions 

in vitro and provides immunosuppression in overproducing transgenic mice. We 

investigated the role of exogenous AFP in immunosuppression maintaining after noncancerous 

tumor ablation. In result, AFP exposures lead to derangement of normal 

function recovery of cell-mediated immunity after tumor nodule ablation. Thus, cytolytic 

activities of NK-, NKT- and •D8+ T cells as well as PHA-induced proliferations of NKT- 

and •D8+ T cells were much more low that in placebo-injected animals. Moreover, 

induced production of IFN-γ and TNF-α from NK cells and •D8+ T cells as well secretions 

of IFN-γ from NKT cells and TNF-β from •D8+ T cells were inhibited compare with 

placebo-injected animals. Dendritic cells from AFP-injected animals showed weak 

antigen-presenting ability and suppressed secretion of IL-12, IL-18 and IL-23. In 

addition, they secreted IL-10, PGE2 and TGF-β1, which not detected in placebo-injected 

animals. Investigation of effector •D4+ T cells demonstrated still higher Th2 cells 

prevalence on Th1 cells in AFP-injected animals whereas Th1 cells quantity was higher 

that Th2 cells in placebo-injected animals. Besides, TGF-β1- and IL-10-produced 

hematopoietic stem cells as well as functionally active of Th3- and Treg1 cells presented 

in spleen of AFP-injected animals. At that, values of all investigated parameters of AFPinjected 

animals were higher or similar with same parameters of tumor-bearing animals’ 

prior tumor rejection. Consequently, AFP maintains and in some cases elevates tumorinduced 

immunosuppression through direct and/or indirect (via activation regulatory T 

cells and hematopoietic stem cells) ways even after tumor ablation.

Connie Schulze, Petra Heyder, Sandra Franz, Kerstin Sarter, Luis Munoz, Stefan 

Pöhlmann, Hanns-Martin Lorenz, Martin Herrmann, Martin Schiller 

Alteration of glycocalyx and exposition of mannose residues 

during apoptosis are essential for the clearance of apoptotic 

marterial 

Apoptosis and removal of apoptotic cells are essential for the maintenance of 

homeostasis in multicellular organisms. Defects in these processes lead to the 

development of autoimmune diseases. To date, a variety of receptors regulating uptake 

of apoptotic cells are known. Recently, alterations of sugar residues on the surface of 

apoptozing cells were observed. Thus, we wanted to characterize changes in the 

glycocalyx of dying lymphocytes. Further, we wanted to analyze whether these changes 

are important for the clearance of apoptotic cells. We analyzed the binding of lectins to 

apoptotic lymphocytes and subcellular fragments shed from these cells. A receptor for 

poly-mannose residues expressed on dendritic cells is DC-SIGN (DCS). To substantiate 

the role of DCS for the engulfment of apoptotic material we used Raji cells stably 

transfected with DCS (Raji-DCS). Engulfment by Raji-DCS and untransfected Raji cells 

of apoptotic material was compared. 

Apoptozing lymphocytes showed an early binding of Maakia amurensis lectin I (MAL I) 

and Sambuccus nigra agglutinine (SNA) (2h after apoptosis induction). These lectins 

recognize sialic acid residues. Further, on we detected a time dependent increase in the 

binding of Narcissus pseudonarcissus lectin (NPn), Griffonia simplificolia lectin II (GSL 

II) and Ulex europaeus agglutinine I (UEA I). NPn recognizes poly-mannose, GSL II is 

specific for N-acetyl-glucosamine, and UEA I for fucose residues. These lectins also 

bound to subcellular fragments shed from apoptozing cells. Co-incubation of subcellular 

fragments with Raji-DCS as well as untransfected Raji cells revealed that the uptake of 

subcellular fragments depends on the presence of DCS. Untransfected Raji cells showed 

a significantly attenuated and delayed engulfment of apoptotic material. 

Taken together we found an alteration of the glycocalyx on the surface of apoptotic cells 

as well as subcellular fragments shed from these cells during apoptosis. The recognition 

of altered glycocalyx especially of mannose residues by DCS is crucial for the 

engulfment of apoptotic material.

Sabine Höpner, Katharina Dickhaut, Jamina Eckhard, Shashank Gupta, Kirsten Falk, 

Olaf Rötzschke 

Amplification of CD4 T cell responses by catalysing antigenloading 

through MHC-loading Enhancer (MLE) 

In previous studies we identified a set of small molecular compounds which have the 

capacity to enhance loading of peptides/proteins onto MHCII molecules. We showed the 

enhancement of antigen-loading on soluble as well as membrane bound MHCII- 

Molecule. In particular adamantanethanole (AdEtOH) strongly accelerates the antigenloading 

rate. This effect is obvious for HLA-DR molecules expressing Glycine at position 

beta 86 of the pocket P1, which is located in the binding site. The dimorphic position 

(Glycine/Valine) determines the depth of the conserved pocket P1 in the MHC-molecule. 

MLEs transiently occupies the P1 and stabilizing the receptive conformation, which leads 

to rapid ligand exchange. 

We are able to show the correlation between the depth of the pocket P1 and allelespecificity. 

Additionally there is evidence for using allele-specific compounds, like 

AdEtOH as molecular tools for amplifying immune reactions in vivo. 

In mouse models we could obtain strongly enhanced specific CD4+- T-cell response 

against tumour associated antigens (TAA) by using MLE as vaccine additive 

(“adjuvant”). However the observation of rapid ligand exchange from outside by MLEs 

could be a risk factor for allergy or autoimmune diseases and gives rise to investigate 

environmental factors, which acts by the mechanism of MLEs. Preliminary data show 

modulation of autoimmune reaction in EAE-models using AdEtOH. So far, it is important 

to investigate the role of MLE in tumour vaccination as well as autoimmunity functions 

of small molecular compounds.

Stefanie Scheu, Philipp Dresing, Richard M. Locksley 

An IFNβ Reporter Mouse Model for the Visualization of the 

Initiation of the Type I Interferon Response in vivo 

Type I interferons (IFNs) were initially identified on the basis of their antiviral activities. 

However, recent studies place these cytokines at the interface between the innate and 

adaptive immune system, with additional important roles in bacterial and parasitic 

infections and antitumoral and immunomodulatory features. Structurally, type I IFNs 

can be grouped into a protein family of multiple IFNα subtypes, encoded by at least 13 

different genes in the mouse, and a single IFNβ subtype. The canonical pathway for 

IFNα/β production is initiated by the IRF-3 mediated expression of IFNβ leading to a 

positive feedback loop via the IFN type I receptor and IRF-7. Although the interferon 

produced first in most situations is IFNβ the celltypes responsible for this initial 

production remain unclear. To reveal the identity of these IFNβ producers in vitro and in 

vivo we created an IFNβ reporter knock in mouse which expresses the enhanced yellow 

fluorescent protein (eYFP) from a bicistronic mRNA linked by an IRES to the endogenous 

IFNβ message. In vitro experiments with bone marrow derived macrophages (BMDM) 

and dendritic cells (BMDDCs) show IFNβ production from distinct cell subpopulations in 

response to defined pathogen compounds. The highest frequencies of IFNβ/YFP + cells 

were observed in BMDMs after poly(I:C) treatment. This response was already 

detectable 4 hours after poly(I:C) stimulation with a peak at 24 hours. A small 

population of GMCSF-grown BMDDCs produced IFNβ after poly(I:C), CpG or LPS 

treatment whereas FLT3-L cultured B220 + pDCs responded mainly to CpG. Initial in vivo 

data show that after poly(I:C) injection IFNβ producing CD11c + cells localized to the 

marginal zone and T cell areas of the spleen whereas in lymph nodes accumulations of 

IFNβ producing cells could be observed in the subcapsular sinus. Additional data will be 

presented validating these IFNβ/YFP reporter mice as a valuable tool for the 

visualization and characterization of IFNβ producing cells in vitro and in vivo after 

challenge with defined pathogen compounds or in infection models.

Frank Schmitz, Antje Heit, Tobias Haas, Hermann Wagner 

An mTOR dependent transport mechanism of cytosolic 

receptors licenses TLR-independent recognition of nucleic 

acids 

Nucleic acid recognition by innate receptors constitutes an integral part of the 

mammalian anti-viral immune responses. The endosomally expressed Toll-like receptors 

(TLR)-3 and -9 recognize natural and synthetic double stranded (ds)RNA and single 

stranded (ss)DNA, respectively. The synthetic mimics of dsRNA, Poly I:C, and ssDNA 

(CpG-ODN) have been reported to display synergistic activation of innate immune cells 

and subsequent enhanced production of type I interferons, ultimative leading to 

increased cytotoxic T cell responses. 

When stimulating myeloid dendritic cells (mDC) with those ligands, we made the 

unexpected observation, that the production of interferon alpha (IFNa), so far 

considered a key feature of plasmacytoid DCs, is independent of TLR-3 and -9. The 

restricted translocation of both ligands to endosomal/lysosomal compartments leads us 

to hypothesize that the non-TLR nucleic acid receptors RIG-I and Mda5 are involved. To 

date, these receptors are described to operate exclusively in the cytosol. To elucidate 

the mechanism allowing cytosolic receptors to respond to endosomally located ligands, 

we investigated the role of autophagy, a process for transport of cytosolic material to 

lysosomes. Indeed, chemical inhibitors of autophagy did significantly reduce the 

observed IFNa production. Further, rapamycin, and inductor of autophagy operating at 

the cellular kinase mTOR was capable to increase the cellular response both to CpG- 

ODN plus Poly I:C and to Poly I:C alone. 

These findings suggest a novel transport mechanism of cytosolic receptors to 

endosomes/lysosomes for the recognition of extracellular nucleic acids. Ultimatively, 

this process may mechanistically explain the immunogeneicity of apoptotic host-cells to 

induce tumor-protective T cell responses.

Linda Sender, Zoe Waibler, Camilla Merten, Roland Hartig, Matthias Gunzer, Peter 

Reichardt, Ulrich Kalinke, Burkhart Schraven 

An unusual signalling signature suggests a molecular basis 

for the adverse side effects of anti-human CD28 

superagonistic antibodies 

Superagonistic CD28 antibodies (CD28SA) activate T-lymphocytes without concomitant 

perturbation of the TCR/CD3-complex. In rats and mice they induce the preferential 

expansion of regulatory T-cells and can be used for the treatment of autoimmune 

diseases. Therefore, it was proposed that CD28SAs might also be applicable for 

humans. However, a phase I clinical trial using the CD28 superagonist TGN1412 showed 

that the reactivity of human T-cells towards CD28SA-mediated stimulation dramatically 

differs from the response of mouse, rat and even monkey T-cells. The molecular basis 

for the severe side effects of CD28SAs in humans is as yet unclear. Here we show that 

two distinct CD28SAs (commercially available ANCD28.1 and original TGN1412) induce 

a delayed and weak but extremely sustained calcium response in human, but not in 

rhesus or cynomolgus monkey T-cells. The sustained Ca++-signal is accompanied by a 

global and extended activation of the major intracellular signalling pathways. Together 

these signals culminate in the synthesis of high amounts of pro-inflammatory cytokines, 

most notably IFNgamma and TNFalpha but also IL-2, -4, -5 and -10. Our findings 

suggest a molecular basis for the dramatic side effects that CD28 superagonists produce 

when administered to humans.

Lorena Martinez Gamboa, Henrik Mei, Karin Reiter, Kristin Kemnitz, Arne Hansen, 

Florian Emmerich, Abdulgabar Salama, Thomas Dörner 

Analysis of B cell subpopulations in patients with autoimmune 

thrombocytopenia: effects of splenectomy and implications 

for therapy 

Background: Evidences of B cell depletion (i.e. by anti-CD20, Rituximab) strongly 

supports an immunopathogenic role of B cells in autoimmune diseases, including RA, 

SLE and autoimmune thrombocytopenia (ITP). Disturbances of B cell homeostasis have 

been identified in patients with SLE and Sjögren’s syndrome. Because Rituximab as well 

as splenectomy are effective treatment options in severe ITP, and autoantibodies 

against platelets surface antigens represent an immunopathogenic key element in this 

entity, B cell abnormalities are expected to be of great importance for the disease. 

Therefore, we analysed distribution and activation of B cell subsets in peripheral blood 

samples from ITP patients and healthy donors (HD). 

Methods: B cell subsets were defined by FACS analysis of surface CD19 and CD27 in 

peripheral blood samples from non-splenectomized ITP patients (ITP) and patients in 

remission after splenectomy (“ITP-spl”), either splenectomized more than two years ago 

(“long-term-spl”) or less than 6 months ago (“recent-spl”), and HD. Activation of cell 

subsets was assessed by measurement of surface expression of CD95, CD38, ICOS-L, 

HLA-DR and the chemokine receptors CXCR3, CXCR4 and CXCR5. Migration capacity of 

B cells was assessed by chemotaxis assays towards CXCR3, CXCR4 and CXCR5 ligands. 

Results: Frequency of memory B cells was significantly decreased in ITP-spl patients 

compared to ITP patients without splenectomy or HD. Remarkably, this effect was more 

pronounced in long-term-spl patients. Differences were confirmed comparing absolute 

numbers of memory B cells between long-term-spl patients and HD, while no significant 

differences were observed for naïve or total B cells. Assessment of activation showed 

elevated number of CD95 expressing cells in ITP patients (either splenectomized or not) 

when compared to HD in all B cell subsets, indicating a steady activation state even 

after splenectomy. Cells expressing CXCR3 were reduced in all B cell subsets, specially 

in long-term-spl patients, while no differences were observed in cells expressing CXCR4 

nor CXCR5. Analysis of migration capacity towards CXCR4 or CXCR5 ligands showed 

also no differences between subsets when compared to HD, while CXCR3+ cells from 

ITP patients showed an increased migration towards CXCR3 ligands. 

Conclusion: Splenectomy apparently favours clinical remission in ITP patients through 

removal of the site of platelets destruction, and also leads to significant reduction of 

memory B cells. Our observations indicate different dynamics and regulation of diverse 

B cell subsets. Identification of splenic survival factors for memory B cells may have the 

potential for new therapeutic approaches targeting specific B cell subsets.

Timo Lischke, Andreas Hutloff, Richard A. Kroczek 

ANALYSIS OF CD4+ T CELL IMMUNITY IN VIVO 

We intend to study the development of CD4+ T cell memory in vivo. In a first step, 

many of the in vitro data on the expression of cell surface communication molecules 

have to be re-derived from in vivo experiments. To establish a baseline for future 

experiments aimed at defining the influence of inflammatory agents on the development 

of CD4+ T cell memory, we have adoptively transferred transgenic, ovalbumin (OVA)specific 

CD4+ T cells into syngeneic murine recipients, which were subcutaneously 

immunized with OVA + lipopolysaccharide. The draining lymph nodes were removed at 

defined time points and the OVA-specific CD4+ T cells were analysed for the expression 

of activation markers such as CD69 and CD25, and for the expression of the 

costimulatory molecules 4-1BB, OX40, ICOS, PD-1, and BTLA. CD69 and CD25 became 

detectable already 6 h after immunization on most OVA-specific CD4+ T cells; 4-1BB 

was hardly expressed at all. Expression of OX40 and ICOS could be seen at 12 h, with 

maxima at 24 h and 48 h, respectively. The kinetics for PD-1 and BTLA, the negative 

regulators of T cell activation, were more extended. Further, we determined the exact 

expansion and contraction kinetics of CD4+ T cells using CSFE labelling. In the late 

phase of the immune response (days 5 – 21), we recorded the development of OVA 

specific CD4+ CD44+ CD62L− effector-memory and CD44+ CD62L+ central-memory T 

cells, but did not observe the appearance of FoxP3+ regulatory T cells. The degree of T- 

B interaction was assessed by measuring the serum levels of OVA-specific IgG1, IgG2a, 

and IgG2b. This comprehensive analysis of the CD4+ T cell response will be 

instrumental for the future dissection of the decision points for memory formation in 

vivo.

Katja Maurer, Ellen Harrer, Andreas Goldwich, Kathrin Eismann, Silke Bergmann, Birgit 

Schätz, Sandra Müller, Thomas Harrer 

Analysis of CTL mediated immune selection in a dominant HLA 

B8-restricted CTL Epitope in Nef 

Objectives: To study the role of CTL escape for disease progression in HIV-1-infection 

we analyzed the CTL response to the dominant HLA B8-restricted CTL epitope 

FLKEKGGL (FL8) in HIV-1 Nef. 

Design: Fifty seven HLA-I-typed patients at different stages of HIV-1 infection were 

included in this study. One subject was used for longitudinal analyses. 

Methods: HIV-1 nef genes derived from plasma or PBMC were analyzed by PCR based 

sequencing. Nef FL8 - specific immune responses were detected by gamma-IFN 

ELISPOT using freshly isolated PBMCs and synthetic peptides. CTL lines were generated 

by peptide stimulation. 

Results: We could detect a significant correlation between mutations in the Nef epitope 

FL8 and the presence of the HLA-B8 allele. Mutations within the FL8 epitope decreased 

CTL recognition depending on the individual CTL line. The longitudinal analysis of a HIV- 

1-infected patient with good control of HIV-1 viremia for several years demonstrated 

the emergence of a K to Q – mutation at position P5 with rising viremia and disease 

progression. 

Conclusion: Our data demonstrate a strong selection pressure on the Nef FL8 epitope by 

CTL. The longitudinal analysis in a patient indicate an important role of CTL escape 

mutations for disease progression.

Simone Wüst, Denise Tischner, Anna Kleimann, Ralf Gold, Jan P. Tuckermann, Holger 

M. Reichardt, Fred Lühder 

Analysis of Glucocorticoid action in 

Experimental Autoimmune Encephalomyelitis (EAE) 

Currently application of high-dose glucocorticoids (GCs) is the standard therapy of acute 

relapses in multiple sclerosis. Despite their widespread use, the mechanisms of GC 

action in the treatment of neuroinflammatory disorders are not yet fully understood. 

This refers to the cell-types that are targets of GCs, the question whether genomic or 

rather non-genomic mechanisms are involved and the processes that are modulated. In 

order to address these questions, we made use of the well-established MOG35-55 

induced EAE model in the C57Bl/6 mouse. 

Therapeutic and preventive administration of dexamethasone ameliorates the disease 

course in a dose-dependent manner. To elucidate the role of the GR in mediating this 

effect we used heterozygous GR knock-out mice and hematopoietic stem cell chimeras. 

These experiments revealed that the cytosolic GR is a prerequisite for most GC actions 

since the therapeutic efficacy of high doses of dexamethasone was very low and 

transient in these models as demonstrated by the clinical scores and histological 

analysis of CNS inflammation. Induction of EAE in different conditional GR knock-out 

mice showed that, at the cellular level, expression of the GR in T cells is essential for 

the beneficial effects of GCs in neuroinflammatory diseases. At present experiments are 

under way that should help to identify the processes and genes that are specifically 

targeted by GCs in T cells. With the new knowledge in hands we anticipate that 

therapeutic approaches for multiple sclerosis may be improved.

Anett Schulz, Manuela Rossol, Matthias Pierer, Sybille Arnold, Holm Häntzschel, 

Christoph Baerwald, Ulf Wagner 

Analysis of polymorphisms in the TNFR2 gene in rheumatoid 

arthritis and possible functional relevance 

The gene for TNF receptor 2 (TNFR2) is located on chromosome 1p36.2, a region, which 

has already been described to be associated with susceptibility to autoimmune diseases. 

To determine a possible association of TNFR2 polymorphisms with rheumatoid arthritis 

(RA), PCRs with sequence specific primers were carried out to identify known 

polymorphisms in RA patients and controls. The frequency of the rare allele of the 1668 

T/G polymorphism, which is located in the 3ÚTR of the TNFR2 gene, was higher in RApatients 

than in healthy controls (4.6% vs. 2.7%, OR 1.77, p=0.029). The 

polymorphism which leads to an amino acid change (position 676, Met/Arg) and the 

other two polymorphisms (1663 G/A, 1690 T/C) in the 3ÚTR were equally distributed 

in RA patients and controls. 

Next we analyzed the expression of TNFR2 on immune cells in donors with the RA 

associated 3ÚTR polymorphism or the 676 T/G polymorphism with a resulting amino 

acid change. The 3ÚTR polymorphism resulted in a decreased expression of TNFR2 on 

CD4 T cells and monocytes in patients with RA, while the 676 T/G polymorphism had no 

influence on the expression of the protein on the surface of these cells. As the 676 T/G 

polymorphism results in an amino acid change, functional studies were carried out for 

this polymorphism. We previously described a TNFR2 dependent cell contact assay, 

which might resemble the pro-inflammatory milieu in RA. Monocytes of donors with and 

without the polymorphism were co-cultured with activated, fixed T cells and cytokine 

production was measured. We observed a decreased production of TNF, IL-8 and 

soluble TNFR2 in monocytes of healthy donors and RA patients with the 676 T/G 

polymorphism. The effects are specific for T cell induced monocyte activation, because 

neither LPS induced nor spontaneous production of TNF, IL-8 and soluble TNFR2 are 

dependent on the polymorphism. 

In summary, a 3ÚTR polymorphism in the TNFR2 gene is associated with RA and leads 

to a diminished cell surface expression of the protein. The functional 676 T/G 

polymorphism results in a decreased cytokine production in an inflammatory setting.

Sonja Kothlow, Benjamin Schusser, Nicola Penski, Georg Kochs, Peter Staeheli, Bernd 

Kaspers 

Analysis of potential antiviral Mx activity in the chicken 

The chicken is a natural host of influenza A virus and the relevance of avian FLUAV 

infections for human health and the poultry industry is highlighted by the ongoing 

pandemic H5N1 outbreak. One of the key players in antiviral defence against FLUAV is 

the type I IFN system and studies in mice identified PKR and Mx proteins as main 

effectors of the IFN-induced antiviral state. Mx proteins are present in all vertebrates 

and belong to the dynamin superfamily of large GTPases. In the chicken, Mx was 

initially described as a cytoplasmic protein that lacks antiviral activity. But recent 

studies revealed that the chicken Mx gene is highly polymorphic and suggested that an 

Asn/Ser polymorphism at amino acid position 631 might determine antiviral activity. 

To examine whether the 631Asn isoform of Mx does mediate resistance against FLUAV 

infection embryo fibroblasts (CEF) from individual chicken embryos with defined 

homozygous Mx 631 genotype were generated. To induce Mx expression these cells 

were preincubated with different concentrations of chIFN&alpha and subsequently 

infected with different avian influenza viruses ChIFN treatment of CEF induced a dose 

dependent antiviral state and was effective for all viruses tested. Mx protein was 

detected in cells of both Mx isoforms but no differences in antiviral activity could be 

observed. To exclude additional polymorphisms in the Mx genes of these cells, identical 

chMx constructs differing only in AS631 were generated and are analysed for their 

antiviral properties. 

Hence further detailed studies will be necessary to finally identify factors which 

influence antiviral Mx activity in the chicken and IFN mediated resistance to FLUAV 

infection.

Andreas Jeron, Susanne Pfoertner, Tanja Toepfer, Jan Buer, Robert Geffers, Andres J 

Schrader 

Analysis of regulatory T cells in renal cell carcinoma 

In many cancer diseases an increased frequency of peripheral CD4+CD25+ regulatory T 

cells (Treg) could be observed correlated with poor prognosis of cancer patients. 

Therefore we characterized human Treg cells from patients with renal cell carcinoma 

(RCC), which is known to be an immunogenic tumor. 

Using magnetic cell separation we isolated highly pure CD4+CD25+ Treg and naïve CD4 

+CD25- T cells from the peripheral blood of 12 RCC patients and 11 healthy donors. In 

FACS analyses we confirmed elevated levels of peripheral CD4+CD25+ Treg cells in RCC 

patients. These cells were further analysed for expression profiles on the Human Treg 

Chip, a self-developed customized oligonucleotide microarray representing 350 Treg 

associated genes. 

Significant differentially expressed genes between CD4+CD25+ and naïve CD4+CD25- 

T cells were conducted to gene ontology resulting in a functional clusterization of the 

corresponding genes. We observed association to apoptosis, cell cycle, proliferation, 

signal transduction and regulation of transcription as well as to categories like 

cytokines, chemokines and surface receptors. Combining gene regulation and the 

functional background of the affected molecules we concluded that Treg cells derived 

from RCC patients might be less responsive to apoptotic stimuli, which might promote 

their accumulation in the periphery and hereby also tumor immune escape. 

Further experiments will compare peripheral Treg cells to tumor surrounding Treg cells 

to identify new targets explaining a potential crosstalk between tumor and Treg cell.

Nicole Warnecke, Burkhart Schraven, Luca Simeoni 

Analysis of TCR-mediated MAPK activation in primary T cells. 

The mitogen-activated protein kinase (MAPK) family includes three major wellcharacterized 

groups of kinases: the extracellular signal-regulated protein kinases 

(ERK), the p38 MAP kinases and the c-Jun NH2-terminal kinases (JNK). In immune 

cells, MAPKs preside to the regulation of lymphocyte development as well as to both the 

innate and the adaptive immune responses. T cells possess multiple possible ways to 

activate MAPKs. Previous data had suggested that RasGRP is required for ERK activation 

and positive selection while Grb2 regulates another branch of the TCR-mediated 

signaling cascade leading to JNK and p38 activation and negative selection in the 

thymus. To assess how MAPKs are activated in peripheral T lymphocytes, we performed 

RNA interference by using short interfering RNA (siRNA). Efficient downregulation of 

RasGRP1, but not of Grb2 or Sos1, resulted in a severe reduction of TCR-mediated Erk 

phosphorylation in human primary T cells. Thus, similar to thymocytes, also peripheral T 

cells activate ERK via a pathway involving RasGRP. Surprisingly, when we suppress 

Grb2 or Sos1 expression we found that ERK phosphorylation was enhanced. Finally, we 

demonstrated that Grb2 and Sos1 are dispensable during TCR-mediated JNK and p38 

phosphorylation in primary human T cells, thus indicating that JNK and p38 activation 

occurs through different mechanisms in mature in comparison to immature T cells. In 

summary, our data suggest that in primary T lymphocytes Grb2/Sos1 are not positive 

regulators of MAPK activation but rather appear to be component of a negative 

regulatory complex required to inhibit T-cell activation.

Eva Billerbeck, Hubert E Blum, Robert Thimme 

Analysis of virus specific FoxP3+ regulatory CD8+ T cells 

Regulatory T cells have been shown to be involved into the suppression of virus specific 

T cell responses. Recent studies focused on the regulatory CD4+CD25+ T cell subset, 

however, very little information is currently available about the role of regulatory CD8+ 

T cells in virus infections. To address this important issue, we analyzed the expression 

of the regulatory T cell specific transcription factor FoxP3 in HCV-, Flu-, EBV- and CMVspecific 

CD8+ T cells from the blood of chronically HCV infected patients and healthy 

controls ex vivo and after peptide-specific stimulation. Of note, we did not see a 

significant FoxP3 expression in virus-specific CD8+ T cells ex vivo. However, in vitro 

stimulation of human PBMC with HCV- or Flu -specific peptides gives rise to two distinct 

antigen-specific T cell populations: FoxP3- and FoxP3+ CD8+ T cells. Of note, the 

expansion of HCV- and Flu specific FoxP3+ CD8+ T cells was blocked by IL-2- and IL-10neutralizing 

antibodies. The HCV- and Flu-specific FoxP3+ CD8+ T cells displayed typical 

surface markers of CD4+ regulatory T cells, such as GITR and CTLA-4 and had a cellcell 

contact dependent suppressive activity. Interestingly, the stimulation of PBMC with 

CMV specific peptides did not induce the generation of virus specific FoxP3+ regulatory 

CD8+ T cells. In sum, our results indicate that stimulation with a defined viral antigen 

leads to the expansion of two different cell populations: FoxP3- memory/ effector as 

well as FoxP3+ regulatory virus-specific CD8+ T cells that may contribute to the 

suppression of virus-specific T cells in a strict antigen-specific manner.

Julia Hoffmann, Ralf-Holger Voss, Ruth Frommolt, Matthias Theobald, Udo Hartwig 

Analyzing GVL-immune responses of p53-specific CD8+ 

cytotoxic T cells to leukemia cell lines and acute myeloid 

leukemia in vitro and in vivo in humanized NOD/SCID/IL- 

2Receptor gamma-chain null mice 

Graft-vs-leukemia (GVL) immune responses are of key interest in allogeneic 

hematopoietic stem cell transplantation and are mediated by donor T-cells recognizing 

hematopoietic minor-histocompatibility-antigens as well as common leukemiaassociated 

antigens (e.g. WT1, p53). Adoptive transfer of leukemia-specific T cells 

would potentially increase GVL-immunity in the absence of graft-versus-host disease 

(GVHD). Using a C57BL/6 HLA-CyA2-Kb transgenic mouse model, we generated HLA-A2 

restricted, human p53- and PRAME-peptide specific CD8+ cytotoxic T lymphocytes 

(CTLs), which are able to recognize p53 peptide epitopes presented by p53-transfected 

tumor cells (e.g., EL4-A2Kb-p53, Saos2/143) as well as various leukemia cell lines 

naturally expressing p53 or PRAME (e.g., HL60, KG-1 and PCI13) as shown by 51Crrelease 

and CFSE-labelling based cytotoxicity assays. In ongoing experiments, p53specific 

CTL are also being tested for cytolytic activity to acute myeloid leukemia (AML) 

blasts, and results demonstrate AML-reactivity to selected samples. 

The potential of p53-specific CTL to mount GVL-immunity in vivo without GVHD is 

currently evaluated in a tumor protection model using NOD/SCID IL2R common gamma 

chain deficient (gamma c null) mice. After co-culture of CTL and p53 expressing tumor 

cells for 24h in vitro followed by adoptive transfer into NOD/SCID/IL2Rgamma c null 

recipients previously engrafted with purified HLA-A2+ CD34+ stem cells, persistence of 

tumor and human B cells as well as GVL-reactivity are examined by genomic PCR and 

flow cytometry, and the results will be presented. 

In summary, our results suggest that HLA-A2 restricted p53 specific CD8+ T cells are a 

promising tool to confer GVL immunity in the absence of GVHD.

Heiko Weyd, Lucie Doerner, Andrea Mahr, Nadine Eberhardt, Dagmar Riess, Bjoern 

Linke, Christine S. Falk, Peter H. Krammer 

Annexin I – an anti-inflammatory signal on the surface of 

apoptotic cells 

In multicellular organisms, apoptotic cell death takes place continuously to remove 

excess cells, maintaining tissue homeostasis. To preclude the release of noxious cellular 

contents and the development of autoimmunity, dying cells are rapidly cleared by 

neighbouring phagocytes, such as macrophages or dendritic cells (DC), which reside in 

peripheral tissues as sentinels of the immune system. Under steady state conditions, 

the uptake of apoptotic cells does not lead to an autoimmune response, even though 

many self-antigens are presented by professional antigen presenting cells. We reasoned 

that apoptotic cells might play an active part in preventing autoimmunity by modulating 

DC activity, possibly leading to the development of “tolerogenic” DC or to the induction 

of regulatory/anergic T cells. In an attempt to define signals on the surface of apoptotic 

cells involved in this process, we raised monoclonal antibodies against apoptotic cells 

and screened for their ability to differentiate between live and apoptotic cells. Using this 

approach, the lipid binding protein annexin I was identified on the surface of early 

apoptotic cells by an antibody which we named DAC5. Blocking experiments with DAC5 

as well as addition of recombinant annexin I confirmed a role of this protein in 

suppression of pro-inflammatory cytokine secretion by DC. Our results indicate that 

annexin I acts as an anti-inflammatory signal suppressing immune responses against 

self antigens, possibly contributing to the induction of peripheral tolerance.

Nicole Gerlach, Cassandra James, Ulf Dittmer 

Anti-retroviral effects of type I interferon subtypes in vivo 

Type I interferons (IFN) play a very important role in the innate immunity against viral 

infections. In mice type I IFN belongs to a multigene family including 14 IFNα subtypes 

and a single IFNβ subtype. IFNα/β is produced very early after infection and induces an 

antiviral state in the cells. Interestingly, individual IFNα subtypes differ in their 

biological activity. However, nothing is known yet about the exact biological function of 

IFNα subtypes in a retroviral infection. In the current study we have used the Friend 

retrovirus model to determine the differences in the antiviral effects of the IFNα 

subtypes (1, 4, 6, and 9) as well as to analyse the mode of action of these subtypes in 

vivo. 

After FV infection of mice only IFNα4 and IFNα9 mRNA transcripts could be detected in 

splenocytes. However, IFNα subtypes 1, 4, 6 and 9 were all biologically active against 

FV in vitro, but differed in their relative biological efficacies. Treatment of FV-infected 

mice with the IFNα subtypes IFNα1, IFNα4 and IFNα9, but not IFNα6 led to a reduction 

to various degrees of the viral load. IFNα subtype therapy regulated the cytokine 

production in the plasma of FV-infected mice and thus may modulate the subsequent 

innate and adaptive immune responses. Along these lines decreased viral load after 

IFNα1 treatment correlated with augmented FV-specific CD8+ T-cell and NK-cell 

responses, whereas the lower viral load in the IFNα4 and IFNα9 treated mice exclusively 

correlated with the activation of NK-cells in the spleen. The current results show that 

treatment of virus infections with selected IFNα subtypes can be an effective antiviral 

therapy. The knowledge about the different antiviral properties of the IFNα subtypes 

against retroviruses may facilitate their clinical use in retroviral infections in the future.

Christina Stöckle, Timo Burster, Thomas Rückrich, Alexander Beck, Christoph 

Driessen, Arthur Melms, Eva Tolosa 

Antigen Processing in the Human Thymus 

The interaction of the TCR of developing thymocytes with peptide-MHC complexes on 

thymic antigen presenting cells (APC) is crucial for T cell development, both for the 

positive selection of “useful” thymocytes as well as the negative selection of 

autoreactive and potentially harmful thymocytes. Therefore, the self-peptide- 

MHC complexes displayed by thymic APC such as dendritic and epithelial cells shape the 

T cell repertoire of each individual. These peptides are generated by intracellular 

proteases such as the cathepsins and AEP (MHC-II). Proteolytic destruction of potential 

T cell epitopes from self-antigens will lead to a lack of presentation and 

consequently, escape of autoreactive T cells recognising these epitopes. To draw a 

picture of the processes that govern generation or destruction of self-epitopes in 

thymic APC we studied the antigen processing machinery and self-antigen processing in 

the human thymus using a combination of approaches including RT-PCR, active site 

labeling and in vitro digests with lysosomes from separated APC subsets. While in 

peripheral dendritic cells the main immunogenic epitope of myelin basic protein is 

destroyed by cathepsin G, thymic DC completely lack CatG activity. Epitope destruction 

in these cells is dominated by cathepsin S and AEP. Thymic DC therefore 

differ remarkably in their antigen processing machinery from their peripheral 

counterparts.

Patrick C. Rämer, Susanne Haemmerling, Mathias H. Konstandin, Thomas Giese, 

Thomas J. Dengler, Sivanandam Vijayshankar 

Antigen-presenting capacity and T cell costimulation of 

endothelial progenitor cells is comparable to monocytes 

Endothelial progenitor cells (EPC) home to sites of vascular repair and therefore have 

potential implications in vascular diseases and in allogenic transplant settings. This 

study aimed to investigate the antigen presenting capacity of EPC and their T cell 

costimulatory capacity compared to HUVEC and monocytes. 

EPCs were isolated from PBMCs by adhesion to fibronectin. Coculture assays of EPC, 

HUVEC and monocytes were performed with allogenic CD4 T cells and were quantified 

by thymidine incorporation. Cytokine production was assessed by qRT-PCR (Light 

Cycler). 

Flow-cytometric analyses revealed an expression of endothelial antigens (e.g. KDR) as 

well as monocytic antigens (e.g. CD14). In PHA-based CD4 costimulation assays, EPC 

induced similar stimulation indices (35) compared to monocytes (30), while HUVECinduced 

proliferation was clearly less (~20). This T cell activation was strongly 

dependent on B7-CD28 interaction, as blocking with CTLA-4 fusionprotein resulted in 

70% decrease of proliferation. In addition, costimulation with EPC resulted in 6-fold 

upregulation of IL-2 mRNA after 8h, comparable to monocytes (7x), but significantly 

stronger than HUVEC (1.5x). 

In contrast to HUVEC, EPC activate naïve CD4/CD45RA T cells. Stimulation of PHAactivated 

naïve T cells with EPC resulted in a stimulation index of 380, similar to 

monocytes (340), while HUVEC failed to induce such strong proliferation (30). 

EPC were moreover able to present antigen in a HLA-DR restricted way. The peptide 85 

B MTB was effectively presented to DR3A3 hybridoma, a cell line specifically recognizing 

this peptide presented via HLA-DR. After preincubation of EPC with 85 B MTB, IL-2 

production of the hybridoma was 20fold increased. 

In conclusion, although EPC exhibit endothelial-like surface markers, functional 

characteristics place these cells more in a monocytic lineage. This is further supported 

by tests of antigen-presentation in which EPC resemble APCs more than actual 

endothelial cells. These findings will pertain to the use of endothelial precursors 

especially in a transplant setting.

Marcela Fajardo-Moser, Christine Hambrecht, Heidrun Moll 

Antigen-pulsed dendritic cell-derived exosomes as cell-free 

vaccines against infection 

Dendritic cells (DC) are the most potent antigen-presenting cells of the immune system 

with the unique capacity to initiate and regulate adaptive immune responses. We 

previously demonstrated that susceptible mice immunized with bone marrow-derived 

DC (BMDC) that had been pulsed ex vivo with Leishmania major antigens and activated 

with CpG were protected against infection. DC secrete a 60-100 nm diameter 

membrane vesicle population of endocytic origin, called exosomes. Besides MHC and 

costimulatory molecules, DC-derived exosomes bear several adhesion proteins, which 

are probably involved in their specific targeting and immunological properties. We 

investigated the capacity of exosomes secreted by antigen-pulsed and CPG-activated 

BMDC to induce protective immunity against leishmaniasis. Our data indicate that 

treatment with exosomes derived from antigen-pulsed and CpG-activated BMDC elicit a 

Th1-biasted Leishmania-specific immune response in vivo and confer long-lasting 

protection against infection. These data encourage us to further investigate the 

mechanisms responsible for the immunostimulatory capacity of DC-derived exosomes in 

vivo and in vitro in order to exploit their full potential as vaccination tools.

Christian Schütz, Andreas Mackensen, Jonathan P. Schneck, Jürgen Schölmerich, 

Mathias Oelke, Martin Fleck 

Antigen-specific CD8+ T cell depletion mediated by apoptosisinducing 

HLA-A2-Ig based artificial APCs 

Different cell based immunotherapy strategies have been developed to specifically 

modulate T cell mediated immune responses. To expand CD8+ T cells in antigenspecific 

fashion, we previously generated an “artificial-Antigen-Presenting-Cell” (aAPC) 

by immobilizing soluble HLA-A2-Ig dimer molecules and anti-CD28-mAb onto microbeads. 

Here we present phenotypical and functional data of aAPC-based “Killer-artificial- 

Antigen-Presenting-Cells” (•aAPC), generated to overcome problems related to current 

cell-based approaches for the treatment of autoimmunity. •aAPC were generated by 

coupling different amounts of an apoptosis-inducing α-Fas(CD95)-mAb onto the 

previously established aAPC. FACS-analysis revealed multiple HLA-A2-Ig based-•aAPC 

phenotypes, which induced Fas-mediated apoptosis in Jurkat T cells at different levels. 

To test, whether •aAPC could also be used to eliminate unwanted cytotoxic T 

lymphocytes (CTL) in an antigen-specific fashion, additional co-culture experiments 

were established with CMVpp65- and Melan-A26-35 specific CTL. High levels of 

apoptosis were only observed in CMVpp65 or Melan-A26-35 specific CTL co-cultured 

with •aAPC loaded with the corresponding peptide. Control •aAPC pulsed with a nonspecific 

peptide did not induce apoptosis in CTL. Elimination of specific CTL by cognate 

peptide-pulsed •aAPC was time and ratio dependent, not due to activation-induced-celldeath 

(AICD), and almost completely abrogated in the presence of a blocking α-FasmAb. 

This study demonstrates for the first time, that HLA-A2-Ig based-•aAPC can be used to 

eliminate unwanted CTL in an antigen-specific fashion through induction of Fasmediated 

apoptosis. Future studies will exploit this novel •aAPC technology to 

determine its potential for the treatment of autoimmune diseases and prevention of 

allograft rejections.

Heike Koehler, Andreas Hombach, Hinrich Abken 

Antigen-specific T cell activation is repressed by TGF-b which 

can be overcome by CD28 costimulation 

Solid tumors are frequently protected from a cellular anti-tumor attack by an 

immunological barrier, the predominant mediators of which are thought to be IL-10 and 

TGF-b. We explored the impact of these cytokines on the individual, tumor-specific T 

cell effector functions upon antigen engagement. Isolated, naïve CD4+ and CD8+ T 

cells were antigen-specifically redirected towards CEA+ tumor cells by expression of a 

recombinant T cell receptor (immunoreceptor) which binds CEA and triggers T cell 

activation via CD3z. Immunoreceptor activated T cells secrete IFN-g, proliferate, and 

lyse CEA+, but not CEA- tumor cells. Whereas IL-10 has no direct effect on 

immunoreceptor triggered effector functions of both CD4+ and CD8+ T cells, TGF-b 

represses proliferation, but neither IFN-g secretion nor specific cytolytic activities. TGFb 

mediated repression of proliferation is reverted by B7-CD28 costimulation. 

Consequently, combined CD28-CD3z signaling via recombinant immunoreceptor 

converts antigen-triggered T cell proliferation largely resistant to TGF-b mediated 

repression. This demonstrates that TGF-b induced suppression of a specific anti-tumor T 

cell attack is not primarily due to repression of cytolytic activities but indirectly to 

repression of T cell proliferation which, however, can be overcome by costimulatory 

CD28-CD3z signaling providing a strategy to overcome TGF-b mediated immunological 

tumor protection.

Nonsikelelo Mpofu, Konstantinos Iordanidis, Matthias Hardtke-Wolenski, Micheal. P 

Manns, Elmar Jaeckel 

Antigen-specific, Foxp3 transduced T cells for therapy of type 

1 diabetes 

In vitro expanded antigen specific CD4+CD25+ regulatory T cells (Tregs), have been 

shown to suppress autoimmune diabetes, suggesting a novel approach to cellular 

immunotherapy for autoimmunity. However to interfere with ongoing disease requires 

at least 10 6 in vitro expanded antigen specific Tregs which are difficult to obtain from a 

polyclonal repertoire. Hence an alternative approach would be to instruct naïve or 

antigen specific CD4+ T cells to obtain regulatory function. Indeed retroviral 

transduction of antigen specific T cells with Foxp3, a Treg lineage specific transcription 

factor, results in the generation of Tregs that can interfere with established 

autoimmunity in a non-lymphopenic nonobese diabetic (NOD) mouse model. However, 

conditions for transduction into NOD T cells have not yet been optimized. Furthermore 

very little is known about the in vivo effect of these Foxp3 transduced antigen specific T 

cells. Here we show that ecotropic pseudotyped viruses are more efficient at 

transducing NOD CD4+ T cells than VSVG pseudotyped viruses. In addition retroviral 

transduction of Foxp3 into NOD CD4+ primary T cells carrying the transgenic TCR from 

an islet Ag-specific T cell clone, BDC2.5, confers a Treg phenotype on these cells as 

they exhibit Treg signature characteristics such as in vitro anergy, suppressive capacity 

and upregulation of CD62L, CD25 and GITR. Adoptive transfer of Foxp3 transduced BDC 

T cells into NOD recipients shows that the cells home and proliferate in an antigen 

specific manner.

Anjana Singh, Miri Blank, Yehuda Shoenfeld, Harald Illges 

Antiphospholipid syndrome patients display reduced titers of 

soluble CD21 in their sera irrespective of circulating anti-beta- 

2-glycoprotein-I autoantibodies. 

B cells have a central role in the development of many autoimmune diseases, relating to 

their mechanism of activation, genetics and the successful application of anti-B-cell 

therapy. The membrane protein complex CD19/CD21 couples innate immune 

recognition by the complement system to the activation of B cells. A soluble form of the 

complement receptor CD21 (sCD21) is shed from the lymphocyte surface. sCD21 is able 

to bind all known ligands such as CD23, sCD23, Epstein-Barr-Virus and C3d in immune 

complexes. The levels of sCD21 in the serum may modulate the immunity. Here, we 

show the serum levels of sCD21 in sera of Antiphospholipid syndrome (APS) patients. 

APS is an autoimmune disorder in which autoantibodies cause heart attack, stroke and 

miscarriage. APS might may appear as primary or in association with Systemic Lupus 

erythromatosus (SLE) and other autoimmune diseases. Here we ask whether APS 

patients have different sCD21 titers compared to healthy persons and whether sCD21 

levels correlate with the presence of anti-ß2-GPI autoantibodies. We show that 

autoimmune APS patients have significantly reduced amounts of sCD21 in their sera, 

irrespective of the presence of anti-ß2-GPI autoantibodies. In our APS patients cohort 

additional SLE, vasculities, DVT (Deep vein thrombosis), fetal loss or thrombosis did not 

correlate to the reduced level of sCD21.

Stefan Lienenklaus, Marcin Lyszkiewicz, Jadwiga Jablonska, Siegfried Weiss 

ANTIVIRAL AND ILLUMINATED 

A NEW MOUSE LINE TO MONITOR β-INTERFERON INDUCTION 

Type I Interferons have been reported to be involved in a wide variety of diseases like 

viral and bacterial infections as well as inflammatory, allergic and autoimmune 

reactions. Within the group of Type I IFNs which consists of more than 10 α-IFNs and a 

single β-IFN, β-IFN has been shown to play a central role. In fibroblasts the complete 

Type I IFN response depends on the presence of this “immediate early” interferon. 

Although this regulatory function has been carefully described, up to now the impact on 

Type I IFN dependent disease is poorly understood. Since spleen cells in contrast to 

fibroblasts can produce α-IFNs independent of β-IFN a key question is which cells are 

the Type I IFN producers in various situations. To adress this questions we generated a 

new mouse line in which the β-IFN gene can be inactivated tissue specifically. The cre 

mediated deletion places a luciferase gene under the β-IFN promoter and thus allows 

the sensitive detection of the β-IFN producing cells. Here we demonstrate the β-IFN 

monitoring capacity after deletion of the floxed β-IFN allele in the whole mouse. The 

heterozygote mice have an intact Type I IFN response and at the same time express 

luciferase if β-IFN is induced. Detecting the light emission we follow the β-IFN producers 

in uninduced as well as in virus infected mice. This new mouse line provides a powerful 

tool to pinpoint the β-IFN producing cells in different diseases and to characterise their 

function in the induction of the Type I IFN response.

Carola Pongratz, Benjamin Yazdanapanah, Hamid Kashkar, Martin Kroenke 

Antiviral implications of a convertible HIV-1 directed siRNA 

library 

RNA interference (RNAi) has been recognized as a powerful gene therapy strategy 

against RNA viruses like human immunodeficiency virus (HIV). However, the efficacy of 

transiently transfected or stably expressed RNAi is limited by the rapid development of 

escape mutant viruses. Here we describe the construction of a genome-wide 

randomized lentiviral RNAi library directed against HIV type-1. Employing appropriate 

selection procedures we have selected approximately 484 short interfering RNA 

sequences. Potent siRNA sequences were subcloned into a lentiviral expression cassette. 

The possible regulatory effects of the shRNA vectors on HIV expression were assessed 

by co-transfection with an HIV-derived pNL4.3-luc expression vector followed by a 

luciferase assay. More than 50% of the lentiviral expression cassettes proved to inhibit 

HIV-driven luciferase expression by greater than 70%. These siRNAs will be scrutinized 

for their binding to conserved or variable regions of HIV, respectively, and tested for 

their suitability of long-term inhibition of wild type HIV isolates.

Cosima Kretz, Bartlomiej Berger, Lucie Dörner, Heiko Weyd, Ingo H. Tarner, Ulf Müller- 

Ladner, Hanns-Martin Lorenz, Peter H. Krammer, Annegret Kuhn 

Apoptosis in Systemic Lupus Erythematosus: 

Influence on Immune Response and Peripheral Tolerance 

Systemic lupus erythematosus (SLE), a human autoimmune disease, is associated with 

abnormal immune responses; however, the pathogenesis of SLE is still not well 

understood. It has been suggested that accumulation of apoptotic cells in the tissue and 

circulation is associated with this disease. Several reports proposed that a complete and 

safe disposal of apoptotic remnants might be crucial for the maintenance of peripheral 

tolerance. Furthermore, it has been demonstrated that dendritic cells (DCs) cocultivated 

with apoptotic cells are suppressed in their production of inflammatory 

signals. The aim of this study was to investigate whether an impaired clearance of 

apoptotic cells or a defect in the apoptotic pathway itself is responsible for the 

manifestation of SLE. DCs derived from peripheral blood mononuclear cell samples were 

analyzed regarding their functionality in clearance of apoptotic cells. Preliminary data 

suggest that DCs of patients with SLE are highly sensitive to inflammatory stimuli. In 

addition, we observed that the activation of patients’ DCs could still be suppressed by 

apoptotic tumor cells. To determine if tolerance can be induced by apoptotic cells of SLE 

patients, we co-cultivated apoptotic neutrophils with U-937, a tumor cell line displaying 

DC qualities. Interestingly, we observed an increase of inflammatory parameters in 

some of the patients in contrast to normal healthy donors when apoptotic neutrophils 

were used. These data support our hypothesis that there is a molecular defect in 

peripheral tolerance in SLE patients, which might be due to impaired apoptosis. Further 

analysis is necessary to understand the mechanism and impact of these findings and to 

allow therapeutic approaches in the future.

Katharina Randers, Telja Pursche, Doreen Finke, Kirsten Jacobsen, Robert Hoerster, 

Christian Brockmann, Holger Hennig, Tony Marion, Sigfried Goerg 

Apoptotic DNA is able to induce in vivo IFN α production 

within the splenic marginal zone via a TLR dependent 

pathway. 

Introduction: Systemic lupus erythematosus is a multigenic autoimmune disease related 

to impaired waste disposal and elevated levels of Interferon alpha. Here we provide 

evidence that targeting of apoptotic DNA towards the splenic marginal zone explains 

TLR-9 pathway dependent elevated levels of Interferon alpha in lupus susceptible 

complement C4 deficient mice. 

Methods: Levels of intravascular DNA of wt mice or C4null mice were determined by 

ELISA and TUNEL dotblot assay. C57/Bl6 wildtype mice were injected with 4 µg 

apoptotic or native DNA with or without prior treatment with chloroquin. Splenic CD11b 

cells were isolated 6h later by magnetic cell sorting, mRNA was purified and IFN α and 

TLR9 were measured by RT-PCR. 

Results: We found that the levels of DNA were significantly higher in serum of C4null 

mice than in wt mice, but TUNEL dotblot assay detected apoptotic DNA in both mice. 

Application of apoptotic DNA showed a significant increase of Interferon alpha mRNA 

and TLR-9 in CD11b+ cells whereas native DNA resulted just in a minimal increase. 

Prior treatment with chloroquine reduced the expression of Interferon alpha and TLR-9 

mRNA in CD11b+ cells. 

Conclusion: Here we demonstrate that untreated C4null mice have elevated levels of 

intravascular DNA, which may be of apoptotic origin and that targeting of apoptotic DNA 

towards the marginal zone increases levels of Interferon mRNA in C4null mice as well as 

in wt mice. Blockade of in vivo TLR signal transduction with chloroquine prevented the 

apoptotic DNA dependent interferon alpha activation, demonstrating that endogenous 

apoptotic DNA plays an important role for the induction of interferon alpha SLE.

Katharina Weibhauser, Bernd Kaspers, Sonja Kothlow 

APPLICATION OF THE RCAS RETROVIRAL VECTOR SYSTEM 

FOR FUNCTIONAL IN VIVO STUDIES OF THE CYTOKINE BAFF 

IN THE CHICKEN 

We recently showed that ChBAFF, the chicken homolog of the mammalian B cell 

activating factor of the TNF family (BAFF), is an essential survival factor for peripheral 

mature chicken B cells. To further analyze the influence of ChBAFF especially on 

immature prebursal and bursal B cells, in vivo studies must be performed. We examined 

whether for this purpose the delivery of selected genes into developing embryos by 

RCAS based retroviral gene transfer provides an alternative to standard methods like 

protein injection. 

We generated RCAS-BP(A) vectors for the overexpression of chBAFF and the in vivo 

neutralization of the cytokine expressing the cross-reacting soluble human decoy 

receptor BCMA. Following the injection of day 3 embryos with infected fibroblasts stable 

expression of the retrovirally induced transgenes was detected both in chicken embryos 

and in birds up to more than two months of age. Immunhistochemical staining for viral 

protein revealed virus replication in all examined tissues giving strong positive signals in 

endothelial cells and the medulla of bursal follicles. To determine the biological activity 

of RCAS expressed proteins, B cell numbers in peripheral blood and spleen were 

analysed by means of flow cytometry and immunohistochemistry. 2 week old BAFF 

transgenic birds showed a more than 2-fold increase in B cell numbers in peripheral B 

cell compartments In contrast, neutralization of BAFF activity by BCMA-Fc expression 

lead to the nearly complete absence of B cells in secondary lymphoid tissues. BAFF 

overexpression also significantly increased amounts of serum immunoglobulins. Effects 

of transgen proteins were detectable for the complete examination period, though at 

two month of age differences decreased, probably due to homeostatic control 

mechanisms. 

These results demonstrate that the RCAS retroviral vector system can be effectively 

applied for the expression of soluble molecules and is a valuable tool to overcome the 

limitations of functional in vivo studies in the chicken. 

This work was supported by the “Elite-Förderung Bayern” and the DFG (GRK 1029).

Johanna Oberlies, Carsten Watzl, Thomas Giese, Claudia Luckner, Stefan Meuer, 

Markus Munder 

Arginine depletion by human granulocyte arginase impairs NK 

cell function 

The arginine-hydrolysing enzyme arginase is constitutively expressed by human 

granulocytes (PMN). Upon PMN cell death arginase is liberated and depletes arginine in 

the microenvironment. This amino acid depletion suppresses T cell proliferation and 

cytokine secretion and emerges as a key mechanism of immunosuppression during 

chronic inflammation. 

Here we show that PMN arginase also severely impairs several functions of primary 

human NK cells as well as lymphokine-activated killer cells (LAK). In arginine-free 

medium or medium depleted of arginine by liberated PMN arginase, NK cell proliferation 

and IL12/IL-18-induced IFN-&gamma secretion are severely diminished. We also see 

impairment of NK cell degranulation in the context of arginine depletion. NK cell viability 

is unaffected by the absence of arginine. The mechanism of NK cell suppression by 

arginine depletion is likely posttranscriptional since mRNA transcript frequency is 

unaffected upon NK cell activation in the absence of arginine. Finally, the arginase 

inhibitor N-&omega-hydroxy-nor-L-arginine is able to restore NK cell functions 

completely in our experimental system. Arginase inhibitors therefore seem to be 

promising pharmacological agents to treat unwanted suppression of the adaptive (T 

cell) as well as innate (NK cell) immune system.

Katrin Drögemüller, Martina Deckert, Ulrike Hellmuth, Monika Sakowicz-Burkiewicz, 

Dirk Reinhold, David Gutmann, Werner Müller, Dirk Schlüter 

Astrocyte gp130-expression is critical for astrocyte survival, 

downregulation of intracerebral immune responses and 

survival of experimental autoimmune encephalomyelitis and 

Toxoplasma encephalitis 

In cerebral inflammatory diseases, there is robust astrocyte activation; however their in 

vivo function is largely unknown. To study their role in experimental autoimmune 

encephalomyelitis (EAE), we generated mice deficient in astrocytic expression of gp130 

(GFAP-Cre gp130fl/fl), the signal transducing receptor for cytokines of the IL-6 family. 

numbers of GFAP+ astrocytes were strongly reduced in experimental autoimmune 

encephalomyelitis (EAE) of GFAP-Cre gp130fl/fl mice. Clinically, GFAP-Cre gp130fl/fl 

mice were characterized by a significantly more severe EAE and finally succumbed to 

chronic EAE, whereas control mice recovered. Histopathologically, inflammation and 

demyelination were more intense, widespread and persisted in GFAP-Cre gp130fl/fl 

mice. In addition, GFAP-Cre gp130fl/fl mice were significantly impaired in control of 

Toxoplasma gondii ultimately dying of a necrotizing Toxoplasma encephalitis (EAE). 

While GFAP+ astrocytes of infected gp130fl/fl control mice were activated and increased 

in number, which was associated with effective parasite control and survival of TE, 

GFAP-Cre gp130fl/fl mice lossed GFAP+ astrocytes during TE. In vitro, survival of T. 

gondii-infected, LPS- and TNF-stimulated astrocytes was also gp130-dependent. Thus, 

astrocytes are of crucial importance in EAE and TE, and gp130 is a critical survival 

factor of astrocytes in inflammatory CNS disorders.

Mihály Józsi, Stefanie Strobel, Hans-Martin Dahse, Wei-shih Liu, Peter F. Hoyer, Martin 

Oppermann, Christine Skerka, Peter F. Zipfel 

Autoantibodies block C-terminus of factor H in atypical 

hemolytic uremic syndrome 

Atypical hemolytic uremic syndrome (aHUS) is a severe renal disease and is associated 

with defective complement regulation. Mutations in several complement regulating 

genes and also factor H (FH)-specific autoantibodies have been described in aHUS 

patients. Our aim was to investigate the role of FH-autoantibodies in aHUS. 

Plasma samples of 51 aHUS patients were screened and FH-autoantibodies were 

identified in five cases, which corresponds to ~10% of the patients. Specificity was 

confirmed by removal of IgG from the plasma, which eliminated FH binding. Isolated 

IgG bound to FH and to its C-terminal fragment SCR19-20 in ELISA, and reacted with 

FH in Western blot assay. Furthermore, FH binding activity was contained in the F(ab’)2 

fragments of isolated patient IgG. For mapping of binding epitopes, recombinant FH 

deletion mutants were used in ELISA. This approach localized for all five autoantibodies 

the binding site to SCR19-20 of FH. C-terminal binding of the five FH-autoantibodies 

was verified in competition assays using recombinant FH fragments and domain mapped 

monoclonal antibodies. 

C3b-binding of FH was reduced by addition of patient IgG in ELISA. In hemolysis 

assays, patients’ plasma caused increased lysis of sheep erythrocytes. In one patient 

the autoantibody titer was followed during treatment. Plasma exchanges were effective 

in reducing the FH-autoantibody titer, which was accompanied by improvement in 

clinical parameters. 

Our data indicate that FH-autoantibodies associated with aHUS block the C-terminus of 

FH, which is essential for binding to C3b and to cellular surfaces. Autoantibody binding 

may also reduce the stability or alter the conformation of FH. These effects may result 

in a reduced FH activity and contribute to the development of aHUS.

Martin Schiller, Isabelle Bekeredjian-Ding, Petra Heyder, Norbert Blank, Klaus Heeg, 

Hanns-Martin Lorenz 

Autoantigens are translocated into apoptotic bodies during 

apoptosis 

Dysregulation of apoptosis and clearance of apoptotic debris contributes to the 

development of autoimmune disease like systemic lupus erythematodes (SLE). A 

hallmark of apoptosis is the formation of membrane vesicles (apoptotic bodies) which 

separate from the dying cell. In the present study we analyzed whether nuclear 

antigens are translocated into apoptotic bodies and whether apoptotic bodies are 

effectively engulfed by monocyte derived phagocytes. We found an accumulation of 

nuclear antigens within apoptotic bodies. In detail, we detected an accumulation of 

DNA, histones and ribonuclear proteins. Interestingly, other cytoplasmic molecules like 

p53, Hsp 70, cytochrome C, prohibitin, Lamin B and NUMA were excluded from 

apoptotic bodies. Purified apoptotic bodies were rapidly engulfed, when coincubated 

with monocyte derived phagocytes. In this context it is important to note that those 

molecules which accumulated in apoptotic bodies are preferred targets of the 

autoimmune response in SLE. Our data suggest that during apoptosis “immunogenic 

molecules” are actively translocated into apoptotic bodies. This is followed by an 

effective engulfement of apoptotic bodies by environmental phagocytes. In autoimmune 

diseases a defective clearance of apoptotic bodies or a dysregulation of apoptotic body 

formation may contribute to the development of autoimmunity and the formation of 

autoantibodies against structures present in apoptotic bodies.

Karin Loser, Sandra Balkow, Kerstin Klimmek, Claus Kerkhoff, Wolfgang Nacken, 

Thomas A. Luger, Stefan Beissert 

Autoreactive CD8+ T cell development in CD40L-mediated 

autoimmunity is controlled by S100A8 and A9 proteins 

CD40-CD40L signaling is involved in the development of autoimmunity and transgenic 

(tg) overexpression of CD40L in basal keratinocytes spontaneously leads to systemic 

autoimmunity as evidenced by autoantibodies, nephritis, proteinuria, and autoimmune 

dermatitis, which can be adoptively transferred by injecting CD8+ T cells into naive 

recipient mice. To identify genes involved in the development of MHC class I-restricted 

autoreactivity gene expression profiling of sorted CD8+ T cells from CD40L tg mice 

before and after onset of disease was performed and surprisingly, an increased 

expression of S100A8 and A9 genes both members of the S100 family of Ca-binding 

proteins was detected. To determine the functional relevance of S100A8/A9 expression 

for the development of autoimmunity in vivo, CD40L tg mice were crossed to S100A8/ 

A9-/- mutants. Interestingly, CD40LxS100A8/A9-/- mice showed significantly reduced 

autoimmune dermatitis and markedly decreased numbers of skin lesion infiltrating 

lymphocytes. Since CD40L tg mice show renal IgG/IgM depositions and 

glomerulonephritis, renal function was analyzed in CD40LxS100A8/A9-/- mice. 

Importantly, CD40LxS100A8/A9-/- mice showed a complete loss of nephritis, renal 

immunoglobulin depositions, and proteinuria indicating normal kidney function. 

Additionally, the activation status of lymph node CD8+ T cells was determined in 

CD40LxS100A8/A9-/- mice demonstrating a decreased expression of cytotoxic/ 

autoreactive markers like CD43 and granzyme B. CD8+ T cells isolated from 

CD40LxS100A8/A9-/- mice produced significantly reduced amounts of IL-17 a cytokine 

which has been suggested to mediate the inflammation associated with several 

autoimmune diseases. Moreover, adoptively transferred CD8+ T cells from 

CD40LXS100A8/A9-/- mice failed to elicit autoimmune dermatitis in recipient mice 

indicating that the expression of S100A8/A9 proteins may be critically involved in the 

pathogenesis of autoreactive MHC class I-restricted T cells in CD40L-induced systemic 

autoimmunity.

Bernd Lepenies, Klaus Pfeffer, Michelle Hurchla, Theresa Murphy, Kenneth Murphy, 

Juliane Oetzel, Bernhard Fleischer, Thomas Jacobs 

B and T lymphocyte attenuator (BTLA) ligation prevents 

cerebral malaria during P. berghei ANKA infection 

BTLA (CD272) is a coinhibitory receptor that is expressed on T and B cells and dampens 

T cell activation. In this study, we analyzed the function of BTLA during infection with P. 

berghei ANKA. This strain provokes a strong activation of T cells that is in C57BL/6 mice 

associated with a pathology that resembles cerebral malaria in humans. During the 

course of infection we found an induction of BTLA in several organs, which was either 

due to up-regulation of BTLA expression on activated T cells in the spleen or due to 

infiltration of BTLA-expressing T cells into several organs. In the brain we observed a 

marked induction of BTLA and its ligand HVEM during cerebral malaria, which was 

accompanied by an accumulation of predominantly CD8+ T cells, but also of CD4+ T 

cells. Application of an anti-BTLA mAb caused a significantly reduced incidence of 

cerebral malaria compared to control Ig. Treatment with this antibody also led to a 

decreased number of T cells that sequestered into the brain of P. berghei ANKA-infected 

mice. Our findings indicate that BTLA/HVEM interactions are functionally involved in 

sequestration of T cells in brain capillaries and that BTLA engagement is of importance 

to prevent immunopathology during infection with P. berghei ANKA. This study suggests 

that BTLA is a potential target for therapeutic interventions in severe malaria and might 

also have implications for the treatment of other inflammatory processes.

Janine Suffner, Kristin Hochweller, Natalio Garbi, Günter Hämmerling 

BAC-transgenic mice for depletion of Foxp3+ regulatory T cells 

It is well-documented that Foxp3+CD4+ CD25+ T cells, so called regulatory T cells 

(Tregs), play a fundamental role for the maintenance of self-tolerance. In order to 

analyse Treg function in vivo, we generated a novel BAC-transgenic mouse line, 

expressing eGFP, the diphtheria toxin receptor, and luciferase under the control of the 

Foxp3 locus. These Foxp3 GDL mice offer unique opportunities to study biology and 

function of Treg cells. Live Foxp3+ (GFP+) cells can be positively selected by flow 

cytometry. After treatment with 30ng DT/g bw for three times, we observed efficient 

depletion of luciferin expressing cells, visible in bioluminescence imaging (IVIS), as well 

as complete depletion of GFP+. 

The transfer of in vivo depleted CD4+ cells from Foxp3-GDL mice into Rag2-/- recipient 

mice resulted in severe colitis, weight loss and death of the recipient mice. This shows 

that DT treatment effectively depleted regulatory T cells necessary for prevention of 

colitis in immunodeficient recipients. 

Although we efficiently deplete all eGFP+ cells, the efficiency of depletion of CD4+ Foxp3 

+ cells varied between different mouse lines. Hence, the BAC does not appear to be 

expressed in all Foxp3+ cells. We observed that continuous depletion of GFP+Foxp3+ 

Tregs leads to homeostatic expansion of the remaining GFP-Foxp3+ Tregs. Foxp3+ cells 

expanded most rapidly in the blood, followed by the spleen. GFP-Foxp3+ cells in 

mesenteric LN and thymus did not expand within 7 days. 

Further investigation includes the thymic involvement in homeostatic expansion of 

Tregs, the expansion of Tregs in different peripheral organs, and assessment of 

proliferative capacity and factors required for homeostatic expansion in the periphery.

Matthias Peiser, Juliana Koeck, Burghardt Wittig, Reinhard Wanner 

Bacterial lipopeptides activate human Langerhans cells via 

Toll-like receptor 2 

Dendritic cells (DCs) are central in linking the innate with the adaptive immune system. 

They are known to be equipped with a plethora of pathogen-detecting Toll-like receptors 

(TLRs). However, the pattern of TLRs on epidermal DCs, the Langerhans cells (LCs) 

remained as yet poorly characterized. As detected by monoclonal antibodies, we show 

that LCs from human skin express TLR1, 2, 5, 6, and 9, the cognate receptors for 

detection of bacterial-derived molecules. Langerin+LCs generated from monocytes and 

sorted by the CD1c molecule (CD1c+MoLCs) acquired a matured phenotype when 

activated by lipopolysaccharide (LPS), peptidoglycan (PGN), macrophage-activating 

lipopeptide 2 (MALP2) and lipopeptide Pam3CSK4. In human epidermal LCs, the TLR2 

ligand PGN, the TLR2/1 ligand Pam3CSK4, and the TLR2/6 ligand Pam2CSK4 enhanced 

the expression of the costimulatory molecules CD86 and CD40. TLR2-triggering 

lipopeptides activated CD1c+MoLCs by inducing IL-1 receptor-associated kinase (IRAK) 

1 phosphorylation, secretion of the proinflammatory cytokines interleukin (IL)-6 and 

tumor necrosis factor (TNF)α and migration in response to chemokines CCL19 and 

CCL21. Upregulation of TNFα and IL-6, nuclear factor-κB (NFκB) activation and 

proliferation of allogeneic CD4+T cells could be blocked by TLR2-specific antibodies in 

TLR2-stimulated CD1c+MoLCs. Coblocking of the TLR2 heterodimeric receptors by anti- 

TLR1/anti-TLR2 and anti-TLR2/anti-TLR6 indicated an exclusive role of TLR2 in IL-6 

induction in human LCs. Collectively, these findings demonstrate that TLR2 expressed 

by LCs mediates inflammatory responses to lipopeptides and plays a central role in 

sensing bacterial pathogens in human skin.

Konrad Bode, Klaus Heeg, Alexander Dalpke 

Bacterial origin histone deacetylase inhibitor butyric acid 

inhibits dendritic cells 

Posttranslational modifications of histones by acetylation are a major mechanism to 

modify chromatin structure and gene expression in eukaryotic DNA. In a recent work we 

could show that histone deacetylase (HDAC) inhibitors like trichostatin A or 

suberoylanilide hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibit 

Toll-like receptor (TLR)-induced cytokine induction of dendritic cells. In the present 

work we investigated whether the HDAC inhibitor butyric acid, a product of the 

metabolism of some anaerobic bacteria, also inhibits dendritic cell activation by TLRs. 

We can show in bone marrow derived dendritic cells that butyric acid strongly inhibits 

the expression of several TLR induced cytokines like IL-12p40, IFNβ and TNFα on 

protein (ELISA and FACS-analysis) and mRNA (real-time RT-PCR) level. Whereas TLR 

stimulation resulted in transient histone H4 acetylation at selected regions of the IL- 

12p40 promoter as shown by chromatin immunoprecipitation, butyric acid induced a 

hyperacetylation of the whole IL-12p40 locus for a prolonged period. HDAC inhibitors 

had no effects on upstream NFκB activation and nuclear translocation as well as on MAP 

kinase signaling (immuno-blot and gel-shift) but recruitment of selected transcription 

factors to the IL-12p40 promoter region was impaired in the presence of butyric acid. 

The results give evidence that butyric acid affects immune cells by inhibiting HDACs. 

This might indicate that butyric acid is involved in the induction of tolerance towards the 

physiological bacterial flora in the mucosa of the intestine.

Marta Rizzi, Ulrich Salzer, Klaus Warnatz, Stefanie Hamm, Sigune Goldacker, Beate 

Fischer, Hans Hartmut Peter, Hermann Eibel 

BAFF Receptor expression in CVID patients 

BAFF Receptor (BAFF-R) is a member of the TNF-R superfamily and is expressed mainly 

on mature B cells. Common variable immune deficiency (CVID) is a primary immune 

deficiency characterized by hypogammaglobulinemia. Several genetic defects 

contributing to this phenotype have been identified (mutations in ICOS gene, TACI 

gene, CD19 gene). Our group recently identified a deletion mutation within the BAFF-R 

gene, removing the transmembrane domain of the protein. The homozygous mutation 

precludes the syntheses of functional BAFF-R. Therefore, B cell development in the 

BAFF-R deficient patient is arrested at the transitional B cell stage. As a result, the 

patient lacks IgG-producing plasmacells and is severely B lymphopenic. 

Searching for additional BAFF-R mutations we screened a subgroup of our cohort of 

CVID patients with very low number of B cells in peripheral blood (

Jenny Dieckmann, Susanne Rehfeld, Bernd Kaspers, Sonja Kothlow 

BAFF regulates the expression of bcl-2 family members in 

chicken B cells 

BAFF (B-cell activating factor of the TNF-family) is a central regulator of B cell 

homeostasis in mammals and birds. In vitro and in vivo studies with recombinant 

ChBAFF protein have shown that in contrast to man and mice in the chicken BAFF is not 

only involved in the regulation of mature B cells but also in the development of very 

early B cells stages in the bursa of Fabricius, the chickens unique primary B cell organ. 

ChBAFF does not induce cell proliferation but rather acts as an anti-apoptotic factor. To 

analyze whether this anti-apoptotic activity is mediated by the regulated expression of 

bcl-2 family members, detailed expression studies for chicken homologues of pro- and 

anti-apoptotic members of this key protein family in apoptosis were performed by 

means of RT-PCR and quantitative RT-PCR. 

ChBAFF is mainly expressed in an autocrine way by B cells in the bursa, where all bcl-2 

family members showed varying but continuous expression during embryogenesis and 

post hatch with significantly increased bok expression around hatch. 

For the characterization of BAFF effects in vivo the retroviral gene transfer system RCAS 

was used to either overexpress the cytokine or generate a functional BAFF knockdown 

by expressing the soluble decoy receptor huBCMA. Though cytokine overexpression did 

significantly increase the peripheral B cell number it did not affect the size of the 

embryonic and post hatch bursal B cell compartment or change the expression levels of 

bcl-2 family members in the bursa. In contrast, BAFF neutralisation significantly 

decreased both peripheral and bursal B cell pools. Strikingly, the few remaining B cells 

in the bursa of BCMA transgenic birds showed enhanced BAFF expression and massively 

increased amounts of anti-apoptotic bcl-2 and Nr13 mRNA. This suggests that in the 

bursa effects of the highly expressed endogenous BAFF are maximal and cannot be 

further enhanced by cytokine overexpression, but if endogenous BAFF signalling is 

blocked only those B cells survive which succeed to produce high amounts of BAFF and 

anti-apoptotic proteins. 

This work was supported by the DFG (GRK 1029).

Astrid Karbach, Evelyn Rossmann, Veronique Kitiratschky, Heidelore Hofmann, Markus 

M. Simon, Peter Kraiczy, Reinhard Wallich 

BbCRASP-1 of the Lyme disease spirochetes induces 

antibodies to nondenatured structural determinants in 

humans 

Pathogenic species of the Borrelia (B.) burgdorferi sensu lato complex express 

complement regulator-acquiring surface proteins (CRASP) that bind serum factor H and 

factor H-like protein 1 (FHL-1). The binding of factor H and FHL-1 to the bacterial 

surface is thought to locally increase the efficiency of C3b cleavage and simultaneously 

downregulate the alternative complement cascade. BbCRASP-1 is the dominant factor H 

and FHL-1 binding protein. Inactivation of BbCRASP-1 increases sensitivity to 

complement in vitro, and the introduction of BbCRASP-1 into serum-sensitive B. garinii 

strains imparts partial serum resistance suggesting that BbCRASP-1 plays an important 

role in immune evasion. Von Lackum et al. demonstrated that spirochetes express 

BbCRASP-1 in the mouse skin at tick bite site for 72 h post infection. However, it is still 

debated whether spirochetes express BbCRASP-1 during infection in humans. The goal 

of this study was to investigate BbCRASP-1 specific antibody responses in Lyme disease 

patients. We have compared the reactivity of serum antibodies derived from patients for 

various B. burgdorferi antigens, including BbCRASP-1, VlsE and OspC. The quality of 

antibodies generated during natural infection was analyzed by using nondenatured and 

denatured forms of the three recombinant outer surface proteins. In summary, the data 

obtained suggest that spirochetes express BbCRASP-1 during infection in humans. 

However, antibodies generated are exclusively directed towards nondenatured 

structural determinants of the protein. These findings may have important implications 

for the analysis of humoral immune responses to proteins in general.

Evelyn Rossmann, Peter Kraiczy, Pia Herzberger, Christine Skerka, Michael Kirschfink, 

Markus M. Simon, Peter F. Zipfel, Reinhard Wallich 

BhCRASP-1 of the relapsing fever spirochete Borrelia hermsii 

is a factor H and plasminogen binding protein 

Borrelia (B.) hermsii is the most frequent tick-borne relapsing fever agent in North 

America and is transmitted to humans through the bites of infected Ornithodoros ticks. 

B. hermsii employs several mechanisms to persist in the blood and evade immune 

response. Antigenic variation is clearly important to escape humoral immunity. 

Complement may be more important in opsonization and phagocytosis. Here we 

identified a novel member of the complement regulator acquiring surface protein 

(CRASP) family expressed by B. hermsii and designated BhCRASP-1. The ability to bind 

the complement regulators, factor H (FH) and factor H related protein 1 (FHR-1) but not 

FH-like protein 1 (FHL-1) has important implications for the host-pathogen interaction. 

In this study, we demonstrate that pathogens that bind FH exploit the regulatory 

activity of this protein, which serves to control C3b deposition and C3 convertase 

activity. BhCRASP-1 specifically interacts with the short consensus repeat 20 of FH. 

Heterologous expression of BhCRASP-1 in the serum-sensitive B. burgdorferi B313 

strain protects transformed B313 cells to some extent from complement-mediated 

killing. Furthermore, we show that FH and plasminogen can bind concurrently to 

BhCRASP-1, however via distinct, non-overlapping domains. Binding of plasminogen to 

BhCRASP-1 in the presence of host-derived urokinase-type plasminogen activator (uPA) 

leads to the formation of active plasmin that degrades high molecular weight 

glycoproteins, such as fibrinogen. Our findings will be helpful to further elucidate the 

molecular basis of B. hermsii interactions with host factors in the pathogenesis of 

relapsing fever.

Xin Ding, Niklas Beyersdorf, Gregor Blank, Fred Lühder, Kevin Dennehy, Ralf Gold, 

Thomas Kerkau, Thomas Hünig 

Blockade of CD28-B7 interactions by anti-CD28 antibodies 

protects from 

immunopathology in vivo 

Co-stimulation through CD28 achieved either by interactions of CD28 with B7 molecules 

or by agonistic anti-CD28 antibodies is critically involved in the initiation of T cell 

responses. 

Here we have studied the in vitro and in vivo properties of a novel mouse anti-mouse 

CD28 antibody, E18, which recognizes an epitope close to the B7 binding site. In vitro, 

both, the intact antibody and its Fab fragment, completely blocked the binding of B7 

molecules to CD28 expressed on mouse thymocytes. E18 Fab also inhibited T cell 

proliferation after stimulation with anti-CD3 or the superantigen staphylococcal 

enterotoxin B (SEB) in vitro, while the intact antibody triggered suboptimal costimulation. 

Application of E18 mAb to normal BALB/c mice in vivo or to newborn 

thymus organ cultures in vitro selectively reduced the generation of CD25+ FoxP3+ 

regulatory T cells (Treg cells). In the periphery, a single injection of 250 •g E18 to adult 

BALB/c mice led to a gradual loss of CD4+ T cells from secondary lymphoid organs, 

while not reducing Treg cell frequencies among CD4 cells. Even after weekly treatment 

with 100 •g E18/ mouse for a period of 26 weeks only a two-fold reduction in Treg cell 

frequencies among CD4+ cells was observed, which thoroughly rebounded after 

cessation of treatment. Preventive application of E18 mAb prior to in vivo stimulation 

with SEB strongly reduced the proliferation of responding Vβ8+ CD4+ T cells. The 

capacity of mAb E18 to inhibit effector T cell responses in vivo translated into protection 

from acute graft versus host disease-associated lethality and provided efficient 

treatment for experimental autoimmune encephalomyelitis. This work was supported by 

grants from the DFG (Hu295/8), the GHS (1.01.1/06/001) and the Wilhelm Sander- 

Stiftung (2005.133.1).

Tea Gogishvili, Beate Geyer, Susanne Grunewald, Thomas Hünig 

Blockade of CD28-mediated co-stimulation ameliorates 

allergic airway inflammation in mice 

Allergic airway inflammation is characterized by a Th2-mediated immune response and 

cytokines, such as IL-4, IL-5 and IL-13 are predominantly responsible for eliciting the 

pathogenic changes. Activation of naïve T-cells involves not only engagement of the 

TCR by antigen, but also requires signals delivered through co-stimulation. Recent 

studies have demonstrated the central role of CD28 in the induction of allergic airway 

inflammation and immunotherapeutic options selectively targeting CD28 have been 

suggested to be desirable. To investigate the requirement for CD28 for functional 

activity of T-cells we examined the effects of an aCD28 mAb, which interferes with 

CD28-B7 interaction, in a murine model of allergic airway inflammation. Treatment with 

mAb was started before or after the allergic state had been established. Blockade of costimulation 

by aCD28 mAb moderately reduced the number of CD4+ T cells. This effect 

was associated with the reduced production of Th2 cytokines to the side of 

inflammation. Interestingly, treatment with aCD28 did not influence IL-10 and IFNgamma 

secretion. Examination of the airways demonstrated that OVA/Alum 

sensitization led to infiltration of eosinophils, which was significantly reduced after 

administration of aCD28. Moreover, total IgE was only slightly reduced, whereas 

synthesis of OVA-specific IgE was significantly inhibited in treated groups. Thus, our 

data demonstrate that blockade of co-stimulation with aCD28 mAb reduces Ag-driven 

immune response in a murine model of allergic airway inflammation.

Mostafa Jarahian, Carsten Watzl, Yasmin Issa, Peter Altevogt, Frank Momburg 

Blockade of natural killer cell-mediated lysis of NCAM140 

expressed on tumor cells 

Expression of the neural cell adhesion molecule (NCAM) on malignant cells of 

neuroendocrine, epithelial and hematopoeitic origin has been reported but its role for 

tumor cell recognition by the immune system remained uncertain so far. We have 

studied the cytotoxicity of the natural killer (NK) cell line NK92 and polyclonal NK cells 

from different donors against NCAM-deficient and NCAM-transfected tumors. While the 

pancreatic carcinoma PANC-1 and the glioblastoma T98G showed no enhanced 

susceptibility to NK lysis after NCAM transfection, de novo NCAM expression in HeLa 

cervical carcinoma, SHEP neuroblastoma and the multiple myeloma lines RPMI-8226 

and LP-1 was associated with significantly decreased lysis by NK cells. Binding of an 

NCAM-specific monoclonal antibody to NCAM-positive target cells was able to reverse 

the reduced lysis susceptibility. Conjugate formation of NCAM-expressing tumor cells 

with NK cells was blocked and could be restored by anti-NCAM. NK cell-expressed NCAM 

molecules which might engage in homotypic cis- or trans-interactions had no apparent 

inhibitory function. The known cis-ligands of NCAM, heparan sulfate proteoglycan and 

L1-CAM, were also not directly involved in NK inhibition. ICAM-1 cell surface expression 

was down-modulated in NCAM-transfected HeLa cells. ICAM-1 is involved in killer cell 

immune synapse formation. Its downmodulation may therefore contribute to the 

reduced lysis of NCAM-expressing target cells. We conclude that aberrant expression of 

NCAM on tumor cells of different histogenetic origin can lead to inhibition of target cell 

recognition and lysis by NK cells.

Nicole Bethke, Matthias Böthe, Siegfried Kohler, Matthias Niedrig, Andreas Thiel 

Bystander activation of recall antigen-specific CD4+ T-cells 

during primary immunization with live attenuated yellow 

fever virus 

Immunization with the attenuated yellow fever virus 17D-204 confers protection in the 

majority of vaccinees. However, the underlying mechanisms for the generation of 

specific cellular immunity have remained unclear so far. To gain insight into vaccinationinduced 

immunological processes we have analyzed absolute and relative frequencies of 

various immunocyte subsets and assessed yellow fever vaccine-specific and bystander 

activated CD4+ T-cells. 

Human blood was obtained from 8 healthy volunteer donors before and at 8 time points 

after vaccination with yellow fever virus 17D-204. Absolute and relative frequencies of 

CD4, CD8, DC, NK and B-cell subsets were analyzed by multicolour flow cytometry. 

Antigen-specific CD154+ CD4+ T-cell were analyzed for expression of IL-2, IFNγ, TNFα, 

IL-4 and IL-10 after stimulation with yellow fever vaccine 17D-204, recall antigens 

(CMVpp65, TT) and SEB. 

Hitherto all vaccinees responded with significant generation of yellow fever vaccinespecific 

CD154+ CD4+ T-cells with maximum responses for IL-2, IFNγ, TNFα and IL-4 

between day 10-14. In 6 out of 8 donors increased frequencies of CMV and/or TTspecific 

CD154+ CD4+ T-cells peaked shortly before or in parallel to the appearance of 

yellow fever vaccine-specific T-cells. 

Our data demonstrate that primary yellow fever immunization induces vaccine-specific 

CD4+ T-cells but also mobilizes recall antigen-specific CD4+ T-cells with polyfunctional 

features.

Olaf Gross, Andreas Gewies, Katrin Finger, Martin Schäfer, Tim Sparwasser, Christian 

Peschel, Irmgard Förster, Jürgen Ruland 

Card9 controls a non-TLR signaling pathway for innate antifungal 

immunity 

Fungal infections are increasing worldwide with the dramatic rise in immunodeficiencies 

including AIDS. However, immune responses to fungi are poorly understood. Dectin-1 is 

the major mammalian pattern recognition receptor for the fungal component zymosan. 

It represents the prototype of innate non-Toll like receptors (TLRs) containing 

immunoreceptor tyrosine-based activation motifs (ITAMs) related to those of adaptive 

antigen receptors. Here we identify Card9 as a key transducer of Dectin-1 signaling. 

While being dispensable for TLR/MyD88 induced responses, Card9 controls Dectin-1/Syk 

mediated myeloid cell activation, cytokine production and innate anti-fungal immunity. 

Card9 couples to Bcl10 and regulates with Bcl10/Malt1 zymosan induced NF-κB 

activation. Yet, Card9 is dispensable for antigen receptor signaling that utilizes Carma1 

to link to Bcl10/Malt1. Thus, our results define a novel innate immune pathway and 

indicate that evolutionary distinct ITAM receptors of the innate and adaptive system 

employ diverse adaptor proteins to selectively engage the conserved Bcl10/Malt1 

module.

Christina Stöckle, Vinod Sommandas, Hubert Kalbacher, Athur Melms, Eleni 

Adamopoulou, Ekkehard Weber, Christoph Driessen, Bernhard Boehm, Eva Tolosa, 

Timo Burster 

Cathepsin distribution in primary human antigen presenting 

cells 

Distinct proteases are required for the processing of antigens in order to make them 

suitable for loading onto major histocompatibility complex (MHC) class II molecules for 

presentation to CD4+ T cells. Several classes of cathepsins, including cysteine and 

aspartate, are involved in this processing. In addition, we recently described a serine 

protease cathepsin G (CatG) as a new member of this antigen processing machinery. 

CatG was detected in primary human B cells, in contrast to B cell lines and active CatG 

was evident in primary human dendritic cells (DC), but not in monocyte-derived DC. In 

vitro experimentation demonstrated that CatG degraded the immunodomminant T cell 

epitope of the multiple sclerosis-associated autoantigen myelin basic protein (MBP). 

Here we directly compared the cathepsin distribution of primary B cells with both DC 

subsets: myeloid DC (mDC) and plasmacytoid DC (pDC). Higher levels of CatG and 

CatD were found in pDC compared to mDC or B cells. During maturation of mDC CatG 

expression was decreased, resulting in suppressed T cell activation when pulsed with 

tetanus toxoid C-fragment (TTC). The expression of other cathepsins was to a large 

extent unchanged. We therefore propose that CatG is crucial in processing of TTC in 

primary human mDC.

Marion Leick, Tanja Hartmann, Susann Ewers, Andrea Diefenbacher, Robert Nibbs, 

Meike Burger 

CCL5 upregulates surface expression of the orphan atypical 

chemokine receptor CRAM-A/B in a heparin sulphate 

dependent manner in pre-B acute lymphoblastoid leukemia 

cells 

Chemokines work as cellular recruitment molecules. Specific combinations of 

chemokines, chemokine receptors, and adhesion molecules determine which subgroups 

of leukocytes migrate and what their destinations are. These migratory processes are 

also regulated by the availability and binding of chemokines on endothelial layers by 

interactions with glycosaminoglycans and atypical chemokine receptors that are 

described as silent or decoy receptors. Here, we report that pre-B acute lymphoblastoid 

leukemia cells Nalm6 and G2 express transcripts of a rare distributed splice variant of 

the atypical orphan receptor CRAM-A. These pre-B cells show high surface expression of 

CRAM in connection with CCL5-mediated functional responses. We investigated these 

responses in detail in Nalm6 cells that do not express any of the known CCL5 receptors 

but still specifically phosphorylate ERK1/2 in response to CCL5 stimulation. CCL5 did not 

induce calcium responses or migration in Nalm6 cells. CRAM-A/B transfected cell lines 

also did not show any migratory responses but a change in actin polymerization 

together with ERK1/2 phosphorylation. In addition, CCL5-induced p44/42 

phosphorylation was not inhibited by pertussis toxin suggesting that CRAM-A/B does not 

signal via Gi proteins. Intriguingly, CRAM surface expression on Nalm6 cells is 

specifically upregulated in response to CCL5 but not to other chemokines. The 

upregulation of surface expression follows distinct time and concentration kinetics and is 

mediated by heparin sulfate binding of CCL5 together with CCL5 binding to CRAM. 

Radioactive ligand binding assays also indicate low affinity CCL5 binding directly to 

CRAM. Our results characterize CRAM-A/B as belonging to the group of atypical 

chemokine receptors that rather play a role in modulating chemokine induced immune 

responses than in triggering direct motility.

Uta Elisabeth Höpken, Susann Winter, Kerstin Krüger, Armin Rehm, Martin Lipp 

CCR7 REGULATES LYMPHOCYTE EGRESS AND RECIRCULATION 

THROUGH BODY CAVITIES 

Continous lymphocyte circulation from the blood into non-lymphoid tissues and from 

there back to local lymphoid organs via afferent lymphatics maintains immune 

surveillance under homeostatic as well as inflammatory conditions. We recently showed 

that the CC chemokine receptor CCR7 controls not only homeostatic B and T cell 

trafficking to and within secondary lymphoid organs, but also homeostatic recirculation 

of lymphocytes through non-lymphoid epithelial tissues. Lack of CCR7 results in a 

massive accumulation of lymphocytes with the development of ectopic lymphoid follicles 

in the gastric mucosa, and age-dependent histomorphological changes with profound 

cystic hyperplasia and an increased rate of mucosal proliferation. 

Here, we demonstrate that CCR7 deficiency also results in disturbed lymphocyte 

reciculation through body cavities. Lack of CCR7 leads to a significant increase of T- and 

B lymphocyte numbers within the peritoneal and pleural cavity. To address the 

molecular basis of lymphocyte accumulation in body cavities, we adoptively transferred 

biotinylated splenocytes derived from either wt or CCR7-/- mice into the peritoneal 

cavities of wt recipients. Twenty four hours after adoptive transfer of biotinylated 

splenocytes derived from either wt or CCR7-/- mice into the peritoneal cavities of wt 

recipients, the amount of recirculated donor lymphocytes was determined in the 

draining parathymic lymph nodes by flow cytometric analysis and immunohistology. 

Thus, peritoneal accumulation of lymphocytes is caused by impaired egress of CCR7 

deficient lymphocytes from the peritoneum via the afferent lymphatics to the draining 

lymph nodes and this process is highly dependent on CCR7 expression. We conclude 

that the homeostatic chemokine receptor CCR7 serves as a central regulator in 

peripheral lymphocyte recirculation and cellular homeostasis at peripheral sites.

Tim Worbs, TR Mempel, J Bölter, UH von Andrian, R Förster 

CCR7-ligands stimulate the intranodal motility of T 

lymphocytes in vivo 

In contrast to lymphocyte homing, little is known about molecular cues controlling the 

motility of these cells within lymphoid organs. Applying two-photon intravital 

microscopy in mice, we demonstrate that signals provided by the chemokine receptor 

CCR7 enhance the intranodal motility of CD4+ T cells. Analyzing motility properties of 

adoptively transferred T cells in the T cell area of wild type recipients we observed a 

33% reduction of the average cell velocity and a 55% reduction of the mean motility 

coefficient of CCR7-deficient CD4+ T cells compared to wild type cells. Both parameters 

were similarly reduced once adoptively transferred wild type T cells were analyzed in 

the T cell area of lymph nodes of plt/plt mice, which lack CCR7-ligands. Importantly, 

systemic application of the CCR7-ligand CCL21 was sufficient to rescue the motility of 

wild type T cells inside the T cell area of plt/plt recipients. Comparing the movement 

behavior of T cells in subcapsular areas, which are devoid of detectable amounts of 

CCR7-ligands even in wild type mice, we failed to reveal any differences in wild type 

and plt/plt recipients. Furthermore, in both wild type and plt/plt recipients subcutaneous 

injection of the CCR7-ligand CCL19 induced the rapid recruitment of highly motile T cells 

into the subcapsular region. Together, these data identify CCR7 and its ligands as 

important chemokinetic factors stimulating the basal motility of CD4+ T cells inside 

lymph nodes in vivo.

Matthias Krusch, Tina Baessler, Katrin Miriam Baltz, Helmut Rainer Salih 

CD137 ligand expression on acute myeloid leukemia cells 

modulates immune surveillance of human NK cells identified 

to express CD137 upon activation 

Studies of haploidentical bone marrow transplantation (BMT) suggest that NK cells play 

an important role in immunosurveillance of leukemia, and acute lymphoblastic 

leukemias (ALL) were shown to be less susceptible to NK-mediated lysis than acute 

myeloid leukemia (AML). NK cell reactivity is governed by a balance of activating and 

inhibitory receptors including various members of the TNF receptor (TNFR) family. The 

TNFR family member CD137/4-1BB has been shown to stimulate proliferation and IFN-γ 

production, but not cytotoxicity of NK cells in mice. Surprisingly, yet nothing is known 

regarding the consequences of CD137-CD137 ligand (CD137L) interaction for NK cell 

reactivity in humans. Here we report that NK cells express CD137 upon stimulation with 

activating cytokines like IL-2 or IL-15. In addition, we demonstrate that primary acute 

myeloid leukemia (AML) cells of patients express CD137L in 14 of 34 (41%) cases. 

CD137L expression in AML was not associated with expression of HLA-class I, CD80/86 

or ligands for the activating immunoreceptor NKG2D. Reverse signaling via CD137L into 

AML cells induced the release of the immunoregulatory cytokines IL-10 and TNF. 

Furthermore, AML-expressed CD137L stimulated both cellular cytotoxicity and IFN-γ 

production of CD137-expressing NK cells, since NK cell reactivity was significantly 

reduced by addition of blocking CD137 antibodies in coculture assays. Taken together, 

our data indicate that CD137 is inducibly expressed and mediates different effects in 

human compared to murine NK cells. Furthermore, CD137L expression substantially 

influences tumor immunoediting by AML cells and modulates NK cell-mediated 

immunosurveillance of leukemia.

Holger Hoff, Zulema Cabail, Karin Knieke, Marion Rudolph, Heike Hirseland, Barbara 

Bröker, Monika Brunner-Weinzierl 

CD152 (CTLA-4) controls CD28-independently cell cycle 

progression and resistance against apoptosis of human T 

lymphocytes 

The activation of T-lymphocytes is tightly controlled by positive and negative costimulatory 

receptors. Primary costimulatory molecules are CD28 and CD152 (CTLA-4) 

of the Ig-superfamily. Both are stimulated by the same ligands B7-1 (CD80) and B7-2 

(CD86) on antigen-presenting cells. While CD28 is mediating positive co-stimulatory 

signals the crosslinking of CD152 leads to cell-cycle arrest and reduced production of 

cytokines, but CD28 as well as CD152-mediated signals induce resistance against 

activation-induced cell death (AICD). A subpopulation of human T-helper cells (CD4+) 

and cytotoxic T cells (CD8+) that has lost the expression of CD28 (CD28null T cells) 

show reduced proliferation and longevity. In this study we could show that despite the 

loss of CD28 expression these cells are able to express CD152 at their cell surface 

shortly after beginning of their stimulation. Their surface CD152 is functional as the 

serological blockade of CD152 leads to enhanced proliferation of CD4 and CD8 CD28null 

T lymphocytes. In addition we were able to show that blockade of CD152 signal 

transduction using CD152 Fab-fragments during stimulation leads to enhanced induction 

of apoptosis. The anti-apoptotic effect of CD152 was confirmed by cross-linking of 

CD152 which leads to reduced activation of caspases. The CD152-mediated resistance 

against apoptosis is mediated by enhanced activation of the anti-apoptotic kinase Akt. 

Thus, surface CD152 expression and CD152-mediated signalling is independent of CD28signalling. 

These data also demonstrate that CD152 initiates CD28-independent 

induction of anti-apoptotic signalling pathways.

J. Kolja Hegel, Pushpa Pandiyan, Paula Kolar, Karin Knieke, Steven L. Reiner, Monika 

Brunner-Weinzierl 

CD152 (CTLA-4) utilizes Eomesodermin, but not T-bet, for 

regulating effector function of individual CD8 T lymphocytes: 

Implication for tumor therapy 

Immune responses of CD8 T lymphocytes are primarily regulated by positive and 

negative costimulatory molecules such as CD28 and CD152 (CTLA-4), respectively. We 

were able to show that surface expression of CD152 is highly up regulated on activated 

CD8 T lymphocytes already during primary immune responses, suggesting a prominent 

regulatory role. Traditionally, CD152 blockade or deficiency was thought to only 

increase CD4 T cell help or enhance the overall frequency of cytotoxic CD8 T cells 

responding an antigenic stimulus. We could now reveal that CD152 enhances effector 

functions (cytotoxicity and cytokine response) of individual CD8 T cells. Mechanistically, 

we could demonstrate that signals induced by CD152 reduce the frequency of IFNgamma 

and GranzymeB expressing CD8 T cells independently of T-bet by selectively 

inhibiting Eomesodermin downstream of the ERK/MAPK pathway. Enhanced cytotoxicity 

of individual CD8 cells primed in the absence of CD152 signaling could be demonstrated 

in vivo. These novel insights could explain the efficacy of anti CD152 treatment for 

tumor rejection.

Karin Knieke, Holger Hoff, Frank Maszyna, Paula Kolar, J. Kolja Hegel, Alf Hamann, 

Gudrun F. Debes, Monika Brunner-Weinzierl 

CD152 (CTLA-4)- signalling promotes homing to secondary 

lymphoid organs 

Migration of antigen-experienced T cells to secondary lymphoid organs is important for 

immune responses. Here, we show that CD152 (CTLA-4), known as a major down 

regulator of T-cell responses, enhanced CD4 T cell migration towards CCL19, a 

chemokine involved in directing lymphocytes to lymph nodes and leaving tissues. 

Paralleling the enhanced chemotaxis, CD152 signalling increased T cell expression of 

CCL19 binding receptor CCR7. Importantly, the CD152-mediated signals amplified the 

migration of Th1 cells to lymph nodes in vivo indicating an unexpected role for CD152 in 

the orchestration of effector T cell recirculation through secondary lymphoid organs.

Baerbel Keller, Mirzokhid Rakhmanov, Sylvia Gutenberger, Sigune Goldacker, Dirk 

Holzinger, Elisabeth Nikolopoulus, Paul Fisch, Hermann Eibel, Hans-Hartmut Peter, 

Klaus Warnatz 

CD21low B Cells Represent a Polyclonally Activated B Cell 

Population Exposed to Type I Interferons 

Most patients with common variable immunodeficiency (CVID) exhibit a severely 

disturbed homeostasis of the circulating B cell pool. About 10% of these patients are 

characterized by the expansion of B cells with an unusually low expression of CD21. 

These CD21lowCD38low B cells exhibit an activated phenotype with increased cell size 

and elevated levels of CD86. Spectrotyping of sorted cells identified a polyclonal origin 

of CD21low B cells. A similar expansion of CD21low B cells in SLE as well as HIV 

infected patients suggested a potential role of type I interferon stimulation. Gene 

expression analysis for MxA, OAS1 and IFIT1 of FACS sorted CD21low B cells versus 

naive B cells of HD and CVID patients confirmed the interferon signature in CD21low B 

cells. Since HHV8 was suggested as a possible pathogen in a subgroup of CVID patients 

we tested our patients with expanded CD21low B cells and granulomatous disease, but 

could not confirm a role of HHV8 in our cohort. Currently we are investigating polyclonal 

activation of B cells via different Toll like receptors as a possible source of CD21low B 

cell differentiation.

Isis Ludwig-Portugall, Emma E. Hamilton-Williams, Christian Kurts 

CD25+ regulatory T cells induce peripheral B cell tolerance 

against non-lymphoid tissue autoantigens 

To study mechanisms of peripheral B cell tolerance against non-lymphoid tissue 

autoantigens we generated transgenic RIP-OVA/HEL (ROH) mice expressing the model 

antigens, OVA and HEL in pancreatic islets. T cells in these mice were not centrally 

tolerant to OVA, because double transgenic ROHxOT-I mice produced OVA specific CD8 

+ T cells (OT-I cells) and became diabetic. When ROH mice were immunized with OVA, 

we observed OVA specific IgG titers at day 21, suggesting that CD4+ cells able to 

switch Ig subclass were present. Nevertheless, titers in ROH mice were much lower than 

in nontransgenic controls, indicating specific tolerance. When we depleted CD25+ cells 

with the PC61 mAb, autoantibody production was completely restored, whereas titers 

against the foreign model antigen ßGal were unaffected. These findings suggested that 

regulatory tolerance suppressed autoantibody production, rather then deletion of 

autoreactive B or T cells. 

To investigate whether this tolerance was induced in the periphery, we injected naïve 

HEL-specific B cells form the transgenic IgHEL donor mice into ROH mice and analysed 

their phenotypical changes. After 3 days we detected neither proliferation, nor changes 

in IgHEL cell numbers, nor B cell receptor (BCR) downregulation. After 22 days numbers 

of IgHEL cells in transgenic recipients were reduced, and surviving cells showed reduced 

expression of the BCR. IgMa produced by IgHEL cells rapidly vanished in transgenic 

mice, even when specific help was provided. Thus, IgHEL cells were peripherally 

tolerized to a non-lymphoid tissue autoantigen. We conclude that peripheral B cell 

tolerance against autoantigens in nonlymphoid tissues can be maintained by CD25+ 

Treg

Christian Becker, Tobias Bopp, Jan Kubach, Franz-Josef Schneider, Edgar Schmitt, 

Helmut Jonuleit 

CD4-mediated, TCR-independent functional activation of 

human CD4+CD25+ regulatory T cells 

CD4 coreceptors exert complex regulatory effects on T cell activation and function. We 

recently showed that engagement of CD4 by specific anti-CD4 antibodies induce 

suppressive activity in human CD25+Foxp3+ regulatory T cells (Tregs). CD4 mediated 

activation of Tregs acts independently of TCR stimulation but elicits comparable 

suppressive activity resulting in efficient suppression of effector T cell proliferation and 

cytokine production. Examination of the proximal CD4 signaling events in Tregs after 

CD4 ligation shows phosphorylation of the zeta-associated protein-70 which relays 

signals to downstream signaling components. Selective inhibition of lck activity during 

anti-CD4 stimulation prevents induction of suppressive function in CD25+ Tregs. In 

contrast to Tregs, CD4 ligation of CD4+CD25- T cells by anti-CD4 cross-linking does not 

induce suppressive activity. 

Here we show that the HIV-derived CD4 binding envelope glycoprotein gp120 is a 

potent functional activator of human Tregs. Upon stimulation with gp120, Tregs 

substantially increase production of cAMP, a second messenger previously shown to be 

essentially involved in Treg-mediated suppression. These results demonstrate that 

suppressive activity in human Tregs can be directly triggered by gp120, suggesting that 

immune dysfunctions associated with HIV infection might in part result from CD4mediated 

Treg activation by the virus.

Alexander Donald McLellan, Sarah Charlotte Saunderson, Petra Schuberth, Lilija 

Miller, Amy Charlotte Dunn, Philippa Anne MacKay, Ralph Wilson Jack 

CD40/IL-4-stimulated B cells release exosomes enriched in 

components of the B cell receptor complex. 

Exosomes are lipid-bound nanovesicles released by a variety of cell types and have 

been proposed to play a role in antigen presentation, immunoregulation and viral 

transmission. Recent work in our lab has shown that exosomes are not present in the 

blood of normal humans or mice. Moreover, primary leukocytes do not release 

exosomes unless subjected to potent activation signals. High level exosome release was 

stimulated when splenic B cells were cultured with activated-T cell membranes and this 

was found to be dependent on IL-4 and CD40L signalling. B cell exosomes expressed 

high levels of MHC-I, MHC-II and B220, as well as components of the B cell receptor 

complex (BCR), namely surface-IgM, CD19 and the tetraspanins CD81 and CD9 (a 

marker of marginal zone B cells). Although CD40/IL-4-stimulated B cells secreted a 

range of other immunoglobulin isotypes (IgA, IgG1, IgG2a/2b & IgG3), IgM was the 

predominant isotype enriched on the surface of exosomes. This suggests that the 

exosome pathway may intersect with antigen uptake and processing mediated by the 

BCR complex. We propose that blood-borne antigen entering the marginal sinus of the 

spleen is processed by antigen-specific marginal zone B cells which then release 

exosomes containing Ig-bound antigen for transfer to other cell types, such as dendritic 

cells.

Rachid Marhaba, Margot Zöller 

CD44 blocking induces apoptosis by uncoupling Ezrin from the 

Ras pathway 

The adhesion molecule CD44 is involved in a large array of functions including 

hematopoeisis, lymphocytes homing, migration, activation and apoptosis. The CD44 

gene comprises 20 exons divided into two groups; standard and variable exons. The 

standard exons are common to all CD44 glycoproteins and lead to the expression of the 

CD44 standard isoform (CD44s). The variable exons are expressed by alternative 

splicing in a cell type- and activation state- dependent manner, and lead to the 

generation of several CD44 variant isoforms (CD44v). We have previously shown that 

CD44 crosslinking provides a co-stimulatory signal for the TCR signaling whereas 

CD44v6 and CD44v7 deliver signals independently of the TCR engagement. In this 

work, we studied the effect of CD44 blocking on T cell proliferation and induction of 

apoptosis and the underlying mechanism. We found that CD44 blocking induces 

apoptosis of T cells. This effect is associated with a strong inhibition of ERK and JNK 

phosphorylation. CD44 blocking correlates with the loss of CD44-associated Ezrin and 

inhibits complex formation between Ezrin and Ras leading to the MAPK activation. These 

results suggest that CD44, via an interaction with an unknown cellular ligand, delivers 

signals leading to cell proliferation and inhibition of apoptosis.

Annalena Bollinger, Zane Orinska, Silvia Bulfone-Paus 

CD8+CD38+ T cells: An newly defined IL-15 dependent 

regulatory T cell population with effects in allergic asthma 

Regulatory T-cells (Tregs) are of essential importance to achieve peripheral tolerance 

toward self and harmless alloantigens. The family of Tregs is composed of so called 

naturally occurring and induced Tregs, wherein the characterisation of CD8+ Tregs just 

has begun. However, CD8+ T-cells are essential players for establishment of an 

adequate immune responses. Impaired Treg function is associated with a breakdown of 

tolerance causing autoimmune diseases or allergy. In this context, IL-15, as an 

interleukin with various activating actions on T-cells, has been shown to exert strong 

effects on the course of allergy via influencing both effector T-cells and Tregs. Based on 

this background we postulated that IL-15 regulated CD8+ Tregs may play an important 

role in prevention of allergy. 

In this study, we characterized a very first IL-15 modulated CD8+ Treg population 

phenotypically and functionally for the C57 BL/6 murin system. We elucidated CD38, a 

transmembrane NAD-glycohydrolase with pleiotropic functions, as an IL-15 sensitive 

molecule. Its surface expression clearly correlates with well defined Treg markers, such 

as CD25, glucocorticoid-induced tumour necrosis factor receptor (GITR), the forkhead/ 

winged helix transcription factor foxp3 and others. Concerning functional aspects, we 

found a suppressive capacity of CD8+CD38+ T-cells toward antigen-specific CD8 and 

CD4 T-cell proliferation, which was additionally associated with a decreased level of IL-2 

and an increase of IL-10. Furthermore, we have established the Ovalbumin/asthma in 

vivo model for the use of C57 BL/6 mice in context with regulatory T-cells and allergy. 

Investigations of the effect mediated by CD8+CD38+ T cells elucidated a clear function 

in down-regulating TH2 associated IL-6 release in the bronchioalveolarlavage (BAL). 

Taken together, we determined an IL-15 sensitive CD8+CD38+ Treg population acting 

as a regulator of allergic asthma. 

Funded by SFB/Transregio 22, A14

Katja Lüthje, Svenja Ehrlich, Bernhard Fleischer, Minka Breloer 

CD83 expression level affects spleenic B cell maturation 

The murine transmembrane glycoprotein CD83 is an important regulator for both: 

thymic T cell maturation and peripheral T cell response. CD83 deficiency leads to a 

block in the thymic maturation of CD4 + T cells and addition of soluble CD83 was shown 

to suppress peripheral T cell responses in vivo and in vitro. Recently we showed that 

CD83 that is expressed by activated B cells, is also involved in the regulation of B cell 

function. CD83 overexpression in CD83 transgenic (tg) mice led to a dramatically 

reduced Ig response to TD and TI model antigens in vivo that was due to CD83 

expression on the B cells themselves. 

Here, we investigate the B cell development in CD83 overexpressing transgenic (tg) 

mice and in CD83 mutant (mut) mice that display dramatically reduced amounts of 

CD83. Analysis of B220 + cells in the bone marrow revealed no difference in the 

percentage of IgM - CD43 - pre B cells. Analysis of the spleenic B cell pool highlighted an 

increase in transitional B cells and a reciprocal decrease in follicular B cells. Employing 

two independent CD83tg mouse strains, we further showed that the intensity of CD83 

expression was positively correlated to accumulation of transitional B cells in the spleen. 

Since CD83tg bone marrow displayed the same impaired maturation upon 

transplantation into irradiated wild-type hosts the accumulation of immature B cells in 

the spleen was not induced by CD83 expression on stromal cells but rather due to CD83 

expression on hematopoetic stem cells. 

While the number of transitional and follicular B cells in CD83 mutant mice was normal 

a decreased maturation of marginal zone B cells was observed. This altered maturation 

of marginal zone B cells was only partially reverted to normal upon transplantation of 

CD83 deficient bone marrow into wild-type hosts and therefore may be due to both: 

CD83 deficiency on somatic and on bone marrow derived cells. 

Taken together we provide evidence that CD83, in addition to its well described role in 

thymic T cell maturation, is also involved in B cell development. In contrast to T cell 

maturation where CD83 is necessary to be present on non hematopoetic thymic 

epithelial cells we show that CD83 expression on hematopoetic cells, presumably the 

transitional B cells themselves, plays a role in the final maturation of follicular B2 cells.

Wiebke Hansen, Simone Reinwald, Astrid M. Westendorf, Carsten Wiethe, Jan Buer 

CD83 is involved in the immunosuppressive function of 

regulatory T cells 

The transmembrane protein CD83 has been initially described as maturation marker for 

dendritic cells (DCs). Moreover, there is increasing evidence that CD83 also regulates B 

cell function, thymic T cell maturation and peripheral T cell activation. Here, we show 

that CD83 plays also a pivotal role in regulatory T cells (Treg) biology. CD83 is upregulated 

in CD4 + CD25 + Treg cells of both murine and human origin in comparison to 

their naïve counterparts. Furthermore, stimulation in vitro leads to a further increase in 

CD83 protein expression on Treg cells. Transduction of naïve CD4 + CD25 - T cells with 

CD83 encoding retroviruses confers suppressive activity in vitro, which is accompanied 

by the induction of Foxp3 expression. Functional analysis of CD83-transduced T cells in 

vivo demonstrate that these CD83 + Foxp3 + T cells are able to interfere with the effector 

phase of a severe contact hypersensitivity reaction of the skin and prevent the paralysis 

associated with experimental autoimmune encephalomyelitis (EAE). In summary, our 

data demonstrate that CD83 seems to be involved in the immunosuppressive function 

of Treg cells and thereby might play a crucial role in the course of immune responses.

Birte Kretschmer, Katja Lüthje, Andreas H. Guse, Svenja Ehrlich, Friedrich Koch- 

Nolte, Friedrich Haag, Bernhard Fleischer, Minka Breloer 

CD83 modulates B cell function in vivo and in vitro 

The murine transmembrane glycoprotein CD83 is an important regulator of both: 

thymic T cell maturation and peripheral T cell responses. Here we provide evidence that 

CD83 is also involved in the regulation of B cell function. We therefore compared the 

function of CD83Tg B cells that overexpress CD83 and CD83 mutant (CD83mu) B cells 

that display a drastically reduced CD83 expression. Correlating with CD83 expression, 

the basic as well as the LPS induced expression of the activation markers CD86 and 

MHC-II was significantly increased on CD83Tg B cells and reciprocally decreased on 

CD83mu B cells. Wild-type B cells rapidly upregulate CD83 within three hours post BCR 

or TLR engagement. The forced premature expression of CD83 on CD83Tg B cells 

resulted in reduced calcium signaling, reduced Ig secretion and a reciprocally increased 

IL-10 production upon in vitro activation. This altered phenotype was mediated by CD83 

expressed on the B cells themselves, since it was observed in purified B cells and 

therefore in the absence of accessory cells. Although reduced CD83 expression on 

CD83mu B cells did not alter the response of whole spleen cell cultures to LPS 

stimulation in vitro, a slight reduction in IL-10 release together with a non-significant 

increase in Ig secretion was observed in purified CD83mu B cells. 

Employing a non-conditional transgenic model we cannot rule out the possibility that 

artificial CD83 expression on B cells during their maturation within the CD83Tg mice 

results in the generation of dysfunctional B cells. Nevertheless we were able to show 

that the treatment of non-Tg C57BL/6 mice with anti-CD83 mAb led to a dose 

dependent 10-fold increase in the antigen-specific IgG1 response to TI immunization 

thus demonstrating a genuine role for naturally induced CD83 in the regulation of 

peripheral B cell function. Although we still have to define the CD83-positive cell 

population(s) targeted by the anti-CD83 mAb in vivo, this finding already demonstrates 

a genuine role for naturally induced CD83 in the regulation of peripheral B cell function.

Thomas Bickert, Andrea Horst, Christoph Wagener, Bernhard Fleischer, Uwe Ritter 

CEACAM1 is crucial for lymphvasculogenesis during 

inflammatory immune responses 

Recently, it was shown that Carcinoembryonic antigen-related cell adhesion molecule 1 

(CEACAM1) plays an important role in angiogenesis. To investigate the possible impact 

of CEACAM1 in lymphvasculogenesis during a local inflammatory response, Ceacam1 

knock out (Ceacam1 -/- ) mice were infected subcutaneously with the obligate 

intracellular protozoan parasite Leishmania (L.) major. Ceacam1 -/- mice developed both 

humoral and cellular immunity against L. major. However, the lack of CEACAM1 resulted 

in delayed wound healing and oedema formation at the site of infection, indicating that 

formation of lymphatic vessels might be affected by the lack of CEACAM1 expression. To 

test this hypothesis, we analyzed the dermal infiltrate caused by the infection with L. 

major. These data revealed that CEACAM1 is highly expressed on infiltrating CD11b + 

cells, which in turn are known to be pivotal in lymphangiogenesis as recently shown in a 

transplantation model of mouse cornea. Furthermore, a subpopulation of these 

infiltrating myeloid CD11b + cells were positive for CEACAM1 and lymphatic vessel 

endothelial hyaluronic acid receptor (LYVE-1). Therefore, it is likely that CD11b + / 

CEACAM-1 + cells might be involved in formation of new lymphatic vessels. Detailed 

FACS analysis of bone marrow and blood exhibited that CD11b + monocytes were 

significantly reduced in Ceacam1 -/- mice leading to the assumption that a diminished 

precursor population was responsible for the failure in lymphvasculogenesis resulting in 

oedema formation. Interestingly, we could not detect reduced numbers of LYVE-1 + / 

CD11b + cells in the infected footpad of Ceacam1 -/- mice. However, histological staining 

of the inflamed footpad for LYVE-1 revealed striking differences. While LYVE-1 + 

lymphatic vessels were found within the inflammation in wild type mice, no 

incorporation of these cells into vessel-like structures was observed in Ceacam1 -/- mice. 

In conclusion, our results show that CEACAM1 is pivotal for the formation of functional 

cutaneous lymphatic vessels under inflammatory conditions caused by L. major.

Ulf Alexander Wenzel, Sonja Möller, Werner Solbach, Tamas Laskay, Ger van 

Zandbergen 

Cell death regulation controls Leishmania disease 

development 

An early marker for apoptosis is the exposure of phosphatidylserine (PS) on the outer 

leaflet of the cell membrane. PS can be detected using annexin A5 (AnxA5) and uptake 

of AnxA5 binding cells by professional phagocytes results in a silencing of the immune 

response. Leishmania major (L. major) is an obligate intracellular parasite elegantly 

misusing the apoptotic cell clearance system for disease development. This parasite 

enters polymorphonuclear neutrophil granulocytes (PMN) in its promastigote form. 

Hiding inside AnxA5-positive PMN the parasite transfers into macrophages (MF), where 

it multiplies in its amastigote form. We found that the presence of AnxA5-binding 

population of promastigotes is responsible for the survival and infectivity of viable 

promastigotes. It has been suggested that to silently infect MF, the amastigote form of 

the parasite uses PS-expression without subsequent cell death as a form of apoptotic 

mimicry. 

In this study we developed a novel in vitro method to culture the amastigote form of L. 

major. We compared cultured amastigotes with MF derived, as well as in vivo derived, 

amastigotes. AnxA5-binding and TUNEL-positivity were used as markers for apoptotic 

like death. Inside MF we found the presence of both TUNEL-positive and -negative 

amastigotes. In addition, we found that purified L. major amastigotes, taken from 8 

week infected BALB/c mice, contained both TUNEL-positive and -negative amastigote 

populations. Finally, we found that amastigote cultures contained an AnxA5-binding 

subpopulation. Moreover we could demonstrate that amastigote cultures contain a 

TUNEL-positive, non viable subpopulation. In contrast to the mimicry hypothesis, this 

data suggests that L. major parasites misuses an apoptotic like death system in at least 

three stages of the disease development. AnxA5-binding promastigotes help viable 

parasites to enter PMN, subsequently apoptotic PMN transfer the promastigote silently 

into MF. In MF the L. major amastigotes develop, again containing both a viable and an 

AnxA5-binding population that could help silencing the next host MF.

Norbert Koch, Alexander McLellan, Jürgen Neumann 

Chaperone Controlled Assembly and Matched Isotype Pairing 

of Major histocompatibility complex Class II Subunits 

The major histocompatibility complex class II (MHCII) region encodes highly 

polymorphic polypeptides, which form a heterodimeric peptide receptor. How is it 

achieved that the great variety of inherited allotype or isotype subunits of MHCII, 

combine to functional peptide receptors? Discovery of a novel molecular mechanism for 

MHCII subunit assembly revealed that pairing of alpha and beta polypeptides is 

controlled by expression of invariant chain (Ii). Assembly of MHCII is initiated by 

sequential binding of alpha and subsequent association of beta chain to Ii. Interaction of 

Ii to the highly conserved MHCII alpha chain may explain the promiscuous binding of Ii 

to the large cohort of polymorphic MHCII heterodimers. The various allo- and isotypic 

beta chains expressed in APC compete for binding to the alpha-Ii matrix, yielding 

isotype matched alpha-beta-Ii complexes. Moreover, Ii controls formation of the nonclassical 

MHCII molecule DM. HLA-DR and DM subunits form mixed complexes that are 

only dissolved when Ii is present. In Ii-gene deficient mice aberrant interaction of I-A 

and H2-M subunits impairs the function of H2-M. The catalytic activity of H2-M is 

rescued by expression of low amounts of Ii.

Johann Poetzl, Catherine Botteron, Daniela Maennel, Anja Lechner 

Characterisation of long-lived CCR6 expressing T H cells in the 

immune response 

The function of T cell subsets in vivo depends on their location. Chemokines are small 

chemotactic cytokines that guide leukocytes through the body. The chemokine CCL20 is 

expressed in mucosal and dermal tissues and its only known receptor is CCR6, which is 

a trait of effector and memory T cells. CCR6 positive T cells are associated with different 

autoimmune processes, i.e. psoriasis, atopic dermatitis and colitis. Yet, its function on T 

cells in infectious diseases seems to be elusive. 

In experimental murine leishmaniasis we could recently show that CCR6 deficiency is 

associated with impaired T helper (TH ) cell function. To further elucidate the function of 

CCR6 for the generation of antigen specific TH cells, we analysed the generation of 

CCR6 positive, activated TH cells after ovalbumin immunisation. Phenotypically, the 

population of CCR6 expressing TH cells does not alter significantly after immunisation. 

In mice, TH cells show co-expression of CCR6 with CD127 and a down-regulation of the 

lymph node homing adhesion molecule CD62L. In this regard the expression profile of 

such cells is characteristic for short-lived effector or long-lived effector memory T cells. 

To dissect these two possibilities we performed in vitro proliferation assays at different 

time points after immunisation. After antigen specific restimulation the population of 

proliferating T cells consisted mainly of CCR6 positive T cells. In addition, the preference 

of CCR6 positive T cells for antigen specific proliferation was obvious over the whole 

observation period from day 2 to day 28 compared to CCR6 negative T cells. 

From this data we conclude that CCR6 positive TH cells seem to represent a long lived, 

stable subset of effector memory TH cells, which are generated early in the immune 

response.

Bernhard Fleischer, Juliane Ladhoff, Elke Effenberger, Cornelia Doebis, Hans-Dieter 

Volk, Martina Seifert 

Characterisation of NK cell attacks towards MHC class I 

deficient rat aortic endothelial cells in vitro 

Background: MHC (major histocompatibility) class I deficient rat aortic endothelial cells 

(RAEC) have been generated in the past by expression of an anti-MHC class I-intrabody. 

The MHC-I-down regulation resulted in a reduced susceptibility of RAEC to humoral and 

CTL-mediated immune attacks. Those cells are interesting tools for generating vascular 

allografts. However, due to their MHC class I deficiency RAEC may become a target of 

Natural Killer (NK) cell lysis. 

Methods: Rat NK cells of the recipient rat strain were isolated from spleen by density 

gradient centrifugation combined with nylon wool adherence and MACS-separation 

technology using an anti-NKR-P1 antibody. Characterization of isolated NK cells was 

performed flow cytometrically. Rat NK cells were cultured for 2 days in IL-2 containing 

medium and then used as killer cells in a Calcein-based cytotoxicity assay. NK cell lysis 

was tested with syngeneic, allogeneic and allogeneic but MHC class I deficient cells. YAC- 

1 tumor cell line was used as a positive control for NK cell killing. 

Results: Rat NK cells could be isolated with a purity of 70-80 %, defined by the coexpression 

of ratNKR-P1 and ratNKp46 and the absence of CD3/T cell receptor (TCR) 

expression. In cytotoxicity assays wild type RAEC of syngeneic as well as allogeneic 

origin were killed to a low degree (10-40 %) by NK cells, whereas the YAC-1 cells were 

almost completely lysed. MHC class I deficient RAEC displayed comparable low specific 

lysis rates similar to that of wild type cells. 

Conclusion: Down regulation of surface MHC class I on RAEC does not increase 

susceptibility to NK cell mediated lysis. This immunological resistance of MHC I deficient 

RAEC would allow their use as an essential component for covering the luminal surface 

of vascular allografts.

Mirjam Peter, Klaus Heeg, Alexander Dalpke 

Characterization of a guanosine-rich suppressive 

oligodesoxynucleotide which inhibits Toll-like receptor-9 

signaling 

Bacterial DNA as well as synthetic oligonucleotides containing unmethylated CpG 

sequences (CpG-ODN) activate immune cells by stimulating Toll-like receptor 9 (TLR9). 

This activation leads to cytokine secretion and up-regulation of costimulatory molecules 

in macrophages and dendritic cells (DCs) as well as B-lymphocyte proliferation. Beside 

these physiological effects TLR9 activation is accused to be responsible for the onset 

and aggravation of autoimmune diseases. Recently, suppressive ODN have been 

described which inhibit TLR9 induced activation of immune cells. Here we describe a 

novel guanosine-rich, suppressive ODN (G-ODN) which inhibits CpG induced activation 

of TLR9 in murine macrophages and DCs as well as in human plasmacytoid DCs (pDCs). 

G-ODN blocked the secretion of TNFα and IL12p40 as well as up-regulation of 

costimulatory molecules. We observed an early block of signaling as activation of MAP- 

Kinases and NF-κB was inhibited but G-ODN did not inerfere with cellular uptake. 

Inhibition was TLR9 specific as it was not observed for stimulation of other TLRs. This 

was confirmed using a TLR9 specific transfection model. G-ODNs even inhibited in a 

molar ratio of 10:1 and were also inhibitory when given up to 6 hours after initial CpG 

stimulation. G-ODNs were also suppressive in an in vivo model of CpG-induced cytokine 

shock. Therefore this class of G-ODN could be of therapeutic use in clinical situations 

with increased TLR9 activation as suggested for certain autoimmune diseases.

Andrea Kiessling, Dagmar Riemann, Susanne Fuessel, Esther Kamphausen, Barbara 

Seliger 

Characterization of expression pattern and regulation of 

ISGylation system in tumors 

Following the identification of ubiquitin as central regulator of proteasomal degradation, 

a number of ubiquitin-like proteins have been identified. Interferon-stimulated gene 15 

(ISG15), a member of this family, was recently described to exert a dual function as 

intracellular modifier of proteins negatively regulating the polyubiquitination and as an 

extracellular cytokine-like modulator of innate immune responses. 

In this study, we analyzed the expression and regulation of ISG15 and of the enzymes 

involved in ISG15 conjugation to proteins (Ube1L, UbcH8, and Herc5) in human tumors. 

By quantitative PCR, all components of the ISGylation system were found in 16/17 renal 

cell carcinoma (RCC), 6/7 squamous cell carcinoma, 37/39 melanoma, and 2/2 prostate 

carcinoma cell lines. However, basal expression levels of the individual components did 

not strictly correlate with each other. The transcript quantification in paired malignant 

and non-malignant tissue samples from RCC patients revealed increased ISG15 and 

UbcH8 expression in the tumors in 9/9 and 6/9 tissue samples, respectively. In 

contrast, Ube1L and Herc5 were heterogeneously expressed in the tumors. In paired 

specimens from prostate cancer patients, ISG15 and UbcH8 were upregulated in 11/14 

tumors and 10/14 tumors, respectively. Furthermore, the tumor-associated expression 

of these genes was more prominent in advanced tumors. 

In order to determine the functional role of ISG15 in tumors, the regulation of 

ISGylation system by various stimuli was analyzed. The ISGylation system was highly 

upregulated by type I IFN in tumor cell lines of distinct origin and by type II IFN 

particularly in melanoma cell lines. According to the presence of several p53 sites in the 

ISG15 promoter, ISG15, but none of the other ISGylation components, was increased 

after exposure to UV light. 

Our results show that ISG15, in contrast to the other components of ISGylation, is 

upregulated in tumors, indicating (i) an IFN-independent, cell stress-mediated 

mechanism of regulation, and (ii) a role of ISG15 in tumors that is independent from its 

function as covalent modifier of proteins.

Christoph Treese, Franziska Lange, Nadja Hilger, Anja Mittag, Attila Tarnok, Andreas 

Lösche, Frank Emmerich, Ulrich Sack 

Characterization of fibroblasts eroding cartilage in arthritis 

INTRODUCTION: The pathology of rheumatoid arthritis (RA) is characterized by 

destruction of articular cartilage and bone. In this process, functionally altered 

fibroblasts play a key role as they secrete distinct patterns of matrix proteinases, 

cytokines, and express variable levels of costimulatory and adhesion molecules. We 

have compared a destructive and a non-destructive fibroblast cell line for functional and 

phenotypic differences. The purpose of this study was to gain a deeper understanding of 

cartilage destruction in arthritis. 

METHODS: The destructive fibroblast line LS48 and the non-destructive 3T3 cells were 

cultured and characterized by slide based and flow cytometry, using antibodies against 

several adhesion molecules, pro- and anti-inflammatory cell surface antigens, and line 

and progenitor specific cell markers. 

RESULTS: Invasive fibroblasts are characterized by significant higher expression of 

adhesion molecules such as CD51 (vitronectin receptor), CD61 (GPIIIa), CD44 (H-Cam), 

and CD147 (EMMPRIN) that are involved in attachment and invasion of these cells. 

Furthermore, destructive cells were shown to express more DR5 (TRAIL-R2), CD95 

(Fas), and CD40 (Bp50) that are responsible for apoptotic and inflammatory processes. 

Finally, CD90 (Thy-1) and CD34 were shown to be strongly depressed. Expression of 

further molecules such as Fc receptors could be induced by culture in the presence of 

added immunoglobulin preparations. 

CONCLUSION: Our results reveal different phenotypic and functional characteristics of 

invasive and non-invasive fibroblasts. The expression is regulated by mediators that are 

typically present in arthritic joints. These findings will help us to gain further knowledge 

about the role of fibroblast in the pathogenesis of RA.

Jan Leipe, Alla Skapenko, Hendrik Schulze-Koops 

Characterization of human Interleukin 17 (IL-17)-producing T 

helper cells (Th17 cells) in healthy individuals and in patients 

with rheumatoid arthritis 

Th17 cells have recently been described as a distinct pro-inflammatory CD4 T cell 

population that contributes to the development of several autoimmune disorders in 

murine disease models. Their role in human autoimmune diseases, however, is less 

clear as their phenotype and effector functions in humans has not been thoroughly 

delineated. We analyzed Th17 cells in healthy human individuals and in patients with 

the prototype autoimmune disease, rheumatoid arthritis. CD4 T cell-derived IL-17 was 

detected by ELISA in the culture supernatants of ex vivo activated freshly isolated 

mononuclear cells from the peripheral circulation of healthy individuals and RA patients 

indicating the presence of Th17 cells in the peripheral circulation of both, controls and 

patients. IL-17 was also produced by CD4 T cells from the synovial fluid of patients with 

RA. Expression of IL-17 could be induced from highly purified memory CD4 T cells by 

TCR stimulation with mAbs to CD3 and further enhanced by costimulation via CD28. Of 

interest, IL-17 production was not modulated by IL-6 or TGF-beta, both of which have 

been implicated in Th17 differentiation in mice. In contrast to memory T cells, naive 

cells did not produce IL-17, even in the presence of IL-23. Together, the data indicate 

the presence of IL-17-producing CD4 T cells in patients with rheumatoid arthritis but 

also in healthy individuals. The precise function of these cells in autoimmune diseases, 

therefore, remains to be shown.

Michael Mihlan, Mario Hebecker, Markus Huber-Lang, Peter F. Zipfel, Mihály Józsi 

Characterization of ligand binding of the human Factor Hrelated 

protein 4 (FHR-4) provides insight into function 

Factor H-related protein 4 (FHR-4) is a unique member of the factor H protein family, as 

two isoforms has been described. FHR-4A has nine short consensus repeat (SCR) 

domains and includes a duplicated region of four domains, where SCR1-4 and SCR5-8 

are closely related. FHR-4B is a short isoform and consists of five SCRs, corresponding 

to SCR1 and SCR6-9 of FHR-4A. 

In order to analyze the function of FHR-4, the two isoforms and fragments of FHR-4A, 

representing SCR1-3, SCR4-9, SCR5-7 and SCR8-9, were recombinantly expressed and 

their binding properties to the central complement component C3b and to the acute 

phase protein C-reactive protein (CRP) were analyzed. Both FHR-4 isoforms as well as 

SCR4-9 and SCR8-9 bound to C3b, suggesting a binding site in the two C-terminal 

domains. In fluid phase cofactor assay, both FHR-4 isoforms enhanced the cofactor 

activity of factor H for C3b cleavage, but the FHR-4 variants alone lacked cofactor 

activity. 

Both FHR-4A and FHR-4B bound to CRP in the presence of Ca2+, independent of ionic 

strength. SCR1-3 but not the other FHR-4A fragments bound to CRP, indicating that 

SCR1 is necessary for CRP binding. CRP-binding was confirmed by surface plasmon 

resonance analysis and by coimmunoprecipitation. In addition, complexes of FHR-4A 

and CRP were isolated from serum of sepsis patients by immunoprecipitation, thus 

demonstrating in vivo interaction between the two proteins. 

In summary, we identify complement C3b and CRP binding domains of FHR-4A. The 

binding characteristics of the protein fragments give further insight into the function of 

the FHR-4 proteins, as the separation of binding sites allows simultaneous binding of 

FHR-4A to C3b and CRP. Thus, our data indicate a role of FHR-4 in innate immune 

response and inflammation.

Sandra Klein, Cosima Kretz, Nina Oberle, Martin Hartmann, Alexander Enk, Peter H. 

Krammer, Elisabeth Suri-Payer, Annegret Kuhn 

Characterization of regulatory T cells in human autoimmune 

diseases with skin manifestations 

A dysfunction of CD4 + CD25 + regulatory T cells (Treg) is known to be involved in a 

variety of human autoimmune diseases. Recently, our group demonstrated that the 

number of Treg in skin lesions of patients with lupus erythematosus (LE) was 

significantly reduced compared to other chronic inflammatory skin diseases. 

Furthermore, a significant reduction in the number of peripheral Treg was only found in 

patients with an active flare of systemic organ manifestation of the disease. We also 

analyzed the frequency and phenotype of Treg in different forms of scleroderma, 

another complex autoimmune disease with skin and systemic organ involvement. Thus, 

the number of Treg in skin specimens of patients with scleroderma was investigated 

using anti-Foxp3 and anti-CD4 monoclonal antibodies for immunohistochemistry. In 

addition, characterization of peripheral Treg from patients with different forms of the 

disease was carried out by flow cytometry. Quantitative analysis of CD4 + T cells in skin 

lesions of patients with scleroderma revealed a significantly reduced number compared 

to LE and other chronic inflammatory skin diseases such as psoriasis and atopic 

dermatitis. Moreover, the frequency of Treg in the skin expressed as percentages of 

Foxp3 + cells from CD4 + cells in the inflammatory infiltrate of scleroderma was similar to 

the frequency found in LE but significantly reduced compared to the other control 

diseases. In peripheral blood, preliminary data show a higher number of total Foxp3 + 

Treg in patients with scleroderma compared to normal healthy donors while the number 

of CD4 + CD25 high Foxp3 + Treg seems to be normal. In conclusion, our results suggest a 

significant reduction of Treg in the skin of patients with different forms of scleroderma. 

Further investigations will evaluate if there is also a peripheral dysfunction of Treg in 

patients with this autoimmune disease.

Björn Kolbe, Ulrike Kolrep, Roland Wagner, Jürgen Schmitz, Ian C.D. Johnston 

Characterization of the natural ligand of the plasmacytoid 

dendritic cell-specific marker CD303 

CD303, the human Blood Dendritic Cell Antigen (BDCA)-2, belongs to the Type II C- 

Type Lectin family and is specifically expressed on Plasmacytoid Dendritic Cells (PDC). 

The Type I IFN secretion of PDC plays a major role in inflammatory autoimmune 

diseases such as Systemic Lupus Erythematosus. The natural ligand of CD303 may 

prove to be a key molecule in understanding these diseases, as it has been shown that 

cross linking of CD303 with the monoclonal antibody AC144 inhibits PDC IFN production. 

In order to identify the CD303 ligand (CD303L), we generated a CD303-Fc fusion 

protein containing the extracellular domain of CD303, an HA-tag and a human IgG1-Fc 

domain for purification and detection of the protein. CD303 protein purified by ProteinA 

chromatography can be detected by a panel of CD303 antibodies in ELISA studies and 

inhibits the staining of PDC with AC144 antibody. However, as high titers of protein 

were required in these assays, a second affinity purification step using AC144-coupled 

Sepharose was introduced. CD303 purified via this 2-step protocol showed an at least 

10x better binding to AC144 in ELISA than the ProteinA purified protein, indicating that 

only a fraction of the original protein was in fact correctly processed and fully functional. 

Many human and non-human cell lines express CD303L (determined by FACS analysis) 

and the highly purified CD303 also stained these cells 200x more effectively than only 

ProteinA purified protein. Therefore, high affinity binding of CD303 to AC144 correlates 

with binding to the natural CD303L. 

This protein will now be used to isolate the CD303L by immunoprecipitation from L+ 

cells, and as lectins are known to recognize pathogen carbohydrate structures (eg. DC- 

SIGN), glycan binding studies will also be performed.

Stefanie Gross, Uwe Trefzer, Wolfram Sterry, Peter Walden 

Characterization of tumor-specific versus virus-specific tumorinfiltrating 

and peripheral blood T cells from melanoma 

patients 

Successful tumor immunotherapy depends on two aspects. First, the generation of 

sufficient numbers of tumor-specific T cells and, second, that these cells finally „do their 

job“. In numerous studies it has been shown, that the first part can be achieved now 

quite well. Following vaccination, vaccine-specific T cells can be monitored in the blood 

of the patients, but the clinical outcome still remains poor. In a few cases T cells have 

been monitored within the tumor as well. These cells are different in phenotype and 

functional capacity compared to those from the blood. The changes T cells are 

undergoing within the tumor microenvironment are still poorly understood, but remain 

one of the obstacles for successful immunotherapy. 

To address the question, whether or not, the changes T cells are undergoing within the 

tumor microenvironment depend on the specificity of the T cells, we evaluated the 

phenotypes of tumor-specific tumor-infiltrating T cells (TIL) and peripheral T cells in 

melanoma patients in comparison to T cells specific for immunodominant viral antigens. 

We used 12 color flow cytometry to analyze Dextramer stained cells ex vivo. In total 76 

samples (54 tumors and 22 PBMC) of 49 patiens and 3 healthy donors were analyzed. 

Tumor specific (Tyrosinase, Telomerase and gp100) Dextramer positive cells were 

compared to virus specific (CMV and EBV) Dextramer positive cells. These cells were 

phenotyped for the expression of markers that are indicators for activation, 

differentiation and negative regulation. 

In the tumor, we found both, tumor-specific and virus-specific T cells. The tumorspecific 

T cells are enriched in the tumor compared to the peripheral blood, but still it 

seems that non-tumor-specific T cells represent the majority of T cells within the tumor. 

However, the tumor specific T cells showed a different expression pattern for the 

activation markers CD25 and CD69 and for the negative regulatory markers PD-1 and 

CTLA-4 than the virus specific T cells. 

From our results it can be concluded that the infiltration of T cells into the tumor does 

not depend on their specificity. T cells which do not recognize tumor cells do enter the 

tumor and are affected by the tumor associated cytokine milieu. 

Nonetheless, some of the phenotypic changes T cells undergo within the tumor 

microenvironment seem to depend on the recognition of their cognate epitope on the 

tumor cells.

Marco Frentsch, Regina Starke, Sarah Meier, Gebhardt Friedemann, Dirk Busch, 

Chiara Romagnani, Andreas Thiel 

Characterization of versatile CD8 memory T-cells with potent 

APC helper function 

A classical paradigm in T-cell immunity is that CD8+ T-cells exert cytolytic cellular 

immunity while CD4+ T-cells provide helper and inducer functions for a variety of cells 

involved in the humoral as well as in cellular immune responses. Based on our recently 

established methods to assess antigen-induced CD40L (CD154) activated CD4+ T-cells 

we have identified a new subset of memory CD8+ T-cells in healthy humans capable of 

CD40L expression after activation. 

Most CD8+ TT40L-cells display a characteristic central memory T-cell phenotype 

expressing CD27, CD28, CD62L and CD127 but lacking surface markers characteristic 

for effector T-cells such as CD56. Upon activation CD8+ TT40L-cells secrete mainly IL-2, 

TNFα, MIP1β and partly IFNγ or IL-4 but lack expression of perforin or GranB. CD8+ 

TT40L-cells express a chemokine receptor profile predisposing them to migrate to 

peripheral-epithelium and to recirculate through secondary lymphoid organs. In vitro 

CD8+ TT40L-cells exert potent helper function such as induction of dendritic cell 

maturation and B-cell activation. 

CD8+ TT40L-cells might represent essential cells in protective immunity particularly in 

situations when classical CD4+ Th-cells are absent or their appropriate function is 

altered. According to their unique functional CD8+ TT40L-cells may become potent 

candidate cells for qualitative immune monitoring and adoptive T-cell therapies in 

infection and cancer.

Shipra Gupta, Sebastian Rieder, Sylvia Escher, Aleksandra Heitland, Wolf-Georg 

Forssmann, Jörn Elsner, Ulf Forssmann 

Chemokine receptor-mediated intravascular inactivation of 

leukocytes by a non-glycosaminoglycan(GAG)-binding variant 

of NNY-CCL14. 

Chemokines play an important role in the selective recruitment of leukocytes during 

inflammation and immunosurveillance. Glycosaminoglycan (GAG) binding has been 

shown to be essential for the in vivo activity of certain chemokines. NNY-CCL14 has 

been proposed as a potential therapeutic compound acting as a CD26-resistant, 

agonistic inactivator of CCR1, CCR3 and CCR5. In this study, a non-GAG-binding variant 

of NNY-CCL14 was generated by mutating basic (B) amino acids to glycine or alanine 

within the identified 49BBXB52 GAG-binding motif. This variant, NNY-CCL14(G50,A51), 

does not bind to heparin in contrast to its precursor NNY-CCL14. Its biological activity 

was tested on CCR1, CCR3 and CCR5 using stably transfected cell lines or leukocytes in 

comparison with NNY-CCL14. NNY-CCL14(G50,A51) demonstrated a two-fold reduced 

ability to activate CCR1, but internalization of CCR1 was not affected. Surprisingly, the 

activity on human CCR3 was strongly reduced. The biological effects on human CCR5 

remained totally unaffected. There was a marked decrease in chemotaxis of eosinophils 

and monocytes in response to NNY-CCL14(G50,A51). In mice treated i.v. with NNY- 

CCL14(G50, A51), a sustained in vivo down-modulation of CCR5 was observed over a 

3h period. 

This non-GAG-binding mutant NNY-CCL14(G50,A51) retains the potential to induce 

receptor desensitization and internalization, but cannot be presented on the 

endothelium via heparin binding sites. Thus, after injection, it is available in the 

circulation distant from otherwise physiologically presented GAG-bound chemokines. 

These properties suggest that, when administered systemically, this receptor agonist is 

able to modulate leukocyte functions (i.e. receptor internalization) prior to their 

interaction with other endothelium-bound chemokines expressed under pathophysiological 

conditions such as allergic inflammation. 

Financial support: DFG grant FO77/10-1

Silvia Capellino, Peter Angele, Werner Falk, Maurizio Cutolo, Rainer H. Straub 

Chromaffin-like cells and catecholamine production: role on 

the inflammatory response in rheumatoid arthritis (RA) 

patients 

Introduction: It is well known that norepinephrine (NE) influence the immune response. 

We already demonstrated that in RA synovial tissue there is a loss of sympathetic nerve 

fibers compared to osteoarthritis (OA). However, there is no difference in NE release 

from synovial tissue in both groups. 

Aim: To identify cells positive for tyrosine hydroxylase (TH) and PGP9.5 (neuronal 

marker) and to understand the role of the local catecholamine production during the 

inflammatory response in RA patients. 

Materials and methods: Synovial samples were obtained from OA and RA patients who 

underwent knee joint replacement surgery. Tissue samples were frozen and than 

analyzed for TH, PGP9.5 and synovial cell markers by doublestain-immunofluorescence. 

From the same patients, synovial cells were isolated by tissue enzymatic digestion, and 

cultured with Reserpine (10 -6 to 10 -8 M) or medium (control) for 24 hours. 

Determination of TNFα, IL-10, IL-8 and IL-6 was performed by Luminex technique and 

ELISA. 

Results: Only in RA we found cells expressing PGP9.5. These cells did not doublestain 

with markers for fibroblasts, macrophages, T-cells and B-cells but also express TH. The 

blockade of catecholamine release by Reserpine 10 -6 M caused a reduction of TNFα by 

60% compared to control cells in RA patients. In OA there was a reduced TNFα 

production in Reserpine-treated cells, but the effects were lower than in RA. The 

production of IL -6 and IL -8 tended to be lower in Reserpine-treated RA cells, but not in 

OA. 

Conclusions: We hypothesize that chromaffin-like cells are present in RA synovial tissue, 

producing NE and other catecholamines, and acting on local inflammation. Our future 

goal is to investigate the effects of the different catecholamines on the inflammatory 

reaction.

Thomas Bollinger, Monika Bajtus, Annalena Bollinger, Stojan Dimitrov, Werner Solbach 

Circadian Rhythm of Regulatory T Cell distribution and 

function 

Vaccination is a powerful tool for the prevention of infectious diseases. Relatively little is 

known about the impact of the time point of vaccination on the ensuing immune 

response. Previous studies showed that vaccination in the afternoon compared to that 

applied in the morning hours caused a highly significant approx. fourfold higher mean 

antibody titer. No information is available on the underlying mechanisms. One 

possibility could be modulation of anti-vaccine immune responses by regulatory T cells 

(Treg). Thus, it seems reasonable that number and/or function of Treg might have a 

circadian rhythm. We investigated 12 healthy young men for 24 hours and monitored 

various parameters in the peripheral blood bi-hourly. We analysed the absolute 

numbers of Tregs by FACS using CD4-FITC- and CD25-PE-antibodies . Cosinor analysis 

revealed significant circadian rhythm in this population with highest levels during night 

time (approximately 95 Treg/µl blood) and lowest levels in the day (approximately 55 

Treg/µl blood) . In a second study we addressed the analysis of the functional circadian 

rhythm of Treg by performing proliferation and suppression assays for the measurement 

of the suppressive capacity of Treg at different time points during a 24 hour period. The 

first data provide evidence for circadian rhythm of regulatory T cell function with the 

lowest suppressive capacity of Treg at 7:00 am. At 3:00 pm for example we measured a 

significant higher suppressive capacity (p

Nicholas Schwab, Christoph Leder, Chi Wang Ip, Antje Kroner, Klaus-Armin Nave, 

Klaus Dornmair, Rudolf Martini, Heinz Wiendl 

Clonal expansions of pathogenic CD8+ effector T cells in the 

CNS of myelin mutant mice 

The causal interrelation of CNS degeneration and adaptive immunity is assumed as a 

critical pathogenic element influencing the clinical phenotype in neuroinflammatory as 

well as in neurodegenerative disorders. We have recently shown that primary 

oligodendrocytic damage (by overexpression of PLP) leads to a low-grade inflammatory 

reaction of high pathological impact in the CNS, probably reflecting pathogenic 

principles in some forms of multiple sclerosis. As a completely novel finding, these 

reactions were mediated by CD8+ T cells whereas CD4+ cells were not necessary for 

the pathological effects of the immune system. 

We here present a detailed immunological characterization of systemic and CNS-specific 

CD8+ T cell responses in PLP transgenic mice and aged wild type mice. We provide 

evidence that T effector cells are surveying the CNS of PLP transgenic and wild-type 

mice. These cells accumulate with age and show a higher level of activation in PLP 

transgenic mice. Exclusively in PLP transgenic mice, T cells show clonal expansions, as 

demonstrated by T cell receptor CDR3-spectratype analysis. Although we could not find 

specific CD8+ T cell responses against myelin-antigen-derived peptides, Vβ/Jβ 

similarities strongly suggest specificity against a common antigen. 

Our data thus demonstrate that a primary degenerative CNS disorder can be associated 

with secondary expansions of pathogenically relevant CD8+ T cells in the CNS. These 

findings have implications for our understanding of the role of secondary adaptive 

immune reactions in the context of neurodegenerative and neuroinflammatory diseases.

Andreas Grahnert, Steffi Richter, Sunna Hauschildt 

Cloning and characterization of an enzymatically active ADPribosyltransferase 

4 (ART4) from chicken 

ADP-ribosylation is a posttranslational protein modification, by which the ADP-ribose 

moiety of NAD + is transferred to an amino acid of the target protein. Enzymes 

catalyzing this reaction are called Mono-ADP-ribosyltransferases (ARTs). In mammals 

five ARTs have been identified. Except for human ART3 and ART4 their catalytic cleft 

contains an R-S-EXE motif and they are enzymatically active. By database searches a 

predicted chicken ART4 (chART4) gene was found. Using 5’ and 3’ RACE-PCR as 

molecular cloning procedures (based on the predicted nucleic acid sequence) we 

obtained the cDNA of this chART. Compared to the other human ARTs, the derived 

amino acids show the highest degree of identity with human ART4 (hART4, 42 %). 

Furthermore, the chART and the hART4 map to regions of conserved linkage synteny. 

Therefore the cloned chART could represent an ortholog of the human ART4. We 

detected the mRNA of this protein in bone marrow cells and yolk sack. In contrast to 

human and mouse ART4 the chART4 contains the R-S-EXE motif, characteristic for 

arginine-specific ARTs. In transfection experiments we could show, that the chART4 is 

indeed enzymatically active. We confirmed the arginine specificity and showed, that the 

chART4 is an about 40 kDa, GPI-linked and glycosylated protein. Like human ART4 the 

mRNA of chART4 was found in an erythroblast cell line. We would assume that the 

chART4 may also play a role in other cells originating from myeloid stem cells like 

human monocytes, in which we already detected ART4 mRNA.

Jens Wuerfel, Alina Smorodchenko, Elena Pohl, Johannes Vogt, Eva Tysiak, Robert 

Glumm, Sven Hendrix, Robert Nitsch, Frauke Zipp, Carmen Infante-Duarte 

CNS-irrelevant T cells enter the brain, cause blood-brain 

barrier disruption but no cellular neuropathology 

Invasion of autoreactive T cells and alterations of the blood-brain barrier (BBB) 

represent early pathological manifestations of multiple sclerosis (MS) and its animal 

model experimental autoimmune encephalomyelitis (EAE). Non-CNS-specific T cells are 

also capable of entering the CNS. But studies investigating the spatial pattern of BBB 

alterations as well as the exact localization and neuropathological consequences of 

transferred non-CNS-specific cells have been thus far lacking. Here, we used magnetic 

resonance imaging (MRI) and multiphoton microscopy (MPM) as well as histochemical 

and high-precision unbiased stereological analyses to compare T cell transmigration, 

persistence, relation to BBB disruption, and subsequent effects on CNS tissue in a model 

of T-cell transfer of ovalbumin (OVA)- and proteolipid protein (PLP)-specific T cells. BBB 

alterations were present in both EAE-mice and mice transferred with OVA-specific T 

cells. In the latter case, BBB alterations were less pronounced, but the pattern of initial 

cell migration into the CNS was similar for both PLP- and OVA-specific cells. Increased 

microglia density and astrogliosis were, however, observed exclusively in the brain of 

EAE mice. While mice transferred with non-neural-specific cells showed similar levels of 

rhodamine-dextran extravasation in susceptible brain regions, EAE mice presented huge 

BBB disruption in brain stem and moderate leakage in cerebellum. This suggests that 

antigen-specificity and not the absolute number of infiltrating cells determine the 

magnitude of BBB disruption and cellular pathology.

Maria Lawrenz, Alexander Visekruna, Thorsten Joeris, Nicole Schmidt, Anjo Kroesen, 

Stefan H.E. Kaufmann, Ulrich Steinhoff 

Coherence or Coincidence ? - ERK activation and 

Immunoproteasomes in Crohn Disease 

It is a common hypothesis, that Inflammatory Bowel Disease (IBD), namely Crohn 

Disease (CD) and Ulcerative Colitis (UC), results from inappropriate immune response 

to normal mucosal microflora. LPS signals via Toll Like Receptor 4 (TLR4) and activates 

the TPL2/ERK pathway, a MAPK pathway, that is linked to NF-kB and leads to 

production of proinflammatory signals. In this study, we investigated the role of ERK 

activation in IBD. Inflamed colonic mucosa from 25 patients with either CD or UC was 

analysed by immunohistochemistry and Western Blot. We observed highly activated 

ERK in the inflamed colon of patients with CD but not in patients with UC. Furthermore, 

in vitro experiments with macrophages from proteasome mutant LMP2 and LMP7 KO 

mice revealed a correlation between ERK activation and proteasomal activity. These 

findings demonstrate, that high amounts of immunoproteasomes present in inflamed 

mucosa of CD patients contribute to activation of the proinflammatory TPL2/ERK 

pathway. We suggest that blocking of the immunoproteasome activity by specific 

inhibitors is a promising therapeutic intervention strategy in treatment of CD patients.

Marcus Gereke, Jan Buer, Dunja Bruder 

Collaboration of the innate and adaptive immune system 

leads to immunological tolerance in the lung 

The lung with its exposed position has many tolerogenic properties to avoid an 

immunological collapse due to infection and inflammation. However, little is known 

about synergisms of innate and adaptive immunity in inducing peripheral tolerance in 

the lung. To get further immunological and molecular insights in the mechanisms 

underlying peripheral tolerance induction in the lung, CD4+ T cell reactivity to a lungspecific 

antigen was studied by transgenic expression of hemagglutinin (HA) in alveolar 

type II epithelial cells. Concomitant expression of HA and a MHC class II-restricted T cell 

receptor specific for HA in double transgenic mice resulted in a severe immunemediated 

interstitial lung disease. Despite aggressive and rapid progression of 

inflammation in lungs of young mice, pulmonary inflammation reached a plateau state 

in elder mice, suggesting the induction of peripheral tolerance mechanisms. Intensive 

characterization of self-reactive CD4+ T cells isolated from the inflamed lung suggested 

the induction of functional regulatory T cells at the site of inflammation. Epithelial cells 

represent a part of the innate immune system and play an important role supporting 

immune responses. Isolation of antigen-expressing alveolar type II epithelial cells (AEC 

II) enabled us to directly demonstrate that these particular cells are capable to induce 

antigen-specific T cell proliferation. Moreover, AEC II from diseased mice have a 

reduced capacity to stimulate naïve self-reactive CD4+ T cells and exhibit broad 

changes in their gene expression profile compared to AEC II derived from healthy 

control mice. Furthermore, AEC II from the inflamed lung secrete soluble factors that 

down modulate T cell proliferation. Data obtained by extensive functional and molecular 

characterization suggest an important role of alveolar type II epithelial cells for the 

induction of T cell tolerance and immune regulation in the lung and point at a close 

collaboration between the innate and adaptive immune system.

Jan Hendrik Niess, Frank Leithauser, Guido Adler, Jörg Reimann 

Commensal-driven local TH17 responses trigger inflammatory 

bowel disease 

The gastrointestinal immune system has evolved to respond to a vast array of stimuli 

for the innate and adaptive immune system derived from the enteric microflora. To 

determine if the development of TH17 cells in the colonic lamina propria (cLP) depends 

on the enteric microflora CD4 T cells that can be induced to secrete IL-17A were 

isolated from the lamina propria of B6 mice housed under standard pathogen free (SPF) 

or syngeneic (age- and sex-matched) germ-free (GF) mice. Increased numbers of CD4 

TH17 cells were found in the lamina propria of the ileum and colon but not the 

duodenum, jejunum, mesenteric lymph nodes, spleen or liver of SPF mice. In syngeneic 

(age- and sex-matched) GF mice, the absence of the commensal bacteria in the gut 

correlated with low numbers of cLP CD4 TH17 cells demonstrating that the microflora is 

required for the accumulation of IL-17-producing CD4 T cells in the cLP. Studies in 

genetically deficient, congenic SPF mice indicated that (type I and type II) interferons 

suppress but IL-4 and IL-12p40-containing cytokines support the accumulation of CD4 

TH17 cells in the cLP. cLP CD4 TH17 cells produce IL-17 but not IFNγ, IL-4 or IL-10. 

Colitis can be readily induced by adoptive CD4 T cell transfer into congenic, 

immunodeficient SPF hosts characterized by progressively increasing numbers of CD4 

TH17 cells that ‘spontaneously’ produce abundant amounts of IL-17 which can be 

further accelerated by transfer of IFNγ-deficient CD4 T cells into immunocompromised 

SPF recipients. In absence of the enteric flora naïve CD4 T cells failed to proliferate in 

transplanted GF but not in SPF hosts. Increased IL-17 levels were only found in sera of 

SPF but not GF hosts and thus immunocompromised GF recipients lacking the enteric 

flora are protected from the development of colitis. A deregulated, commensal bacteriadriven 

local expansion of CD4 TH17 cells is a key feature of the inflammatory bowel 

disease in this model and the increased numbers of TH17 cells in the cLP may restrict 

the potential harmful action of the enteric flora to the local microenvironment.

Maria Diedrichs-Möhring, Georges M.G.M. Verjans, Seerp G. Baarsma, Gerhild 

Wildner 

Comparison of antigen-specific cytokine secretion and 

proliferation of T lymphocytes in vitro 

Response of T lymphocytes following stimulation with antigen or mitogen can be 

determined either by proliferation or by secretion of distinct cytokines. Due to limited 

material it is often not possible to establish cultures for early and lately secreted 

cytokines as well as to detect proliferation. Therefore, we combined both methods. 

Lymphocytes were stimulated in 96-well-plates with different antigens or mitogen. 

Culture supernatants were collected after 24 h, 48 h, 72 h and 96 h and frozen until 

use. The remaining cultures were used to determine proliferation by 3H-thymidine 

uptake. Supernatants from the various time points were pooled to analyze the secreted 

cytokines by multiplex bead immunoassay. To verify this approach peripheral blood 

lymphocytes from two donors were stimulated in the same way and cytokine 

concentrations were determined from samples of single time points (24 h, 48 h, 72 h 

and 96 h) as well as from pooled supernatants. 

Using this approach we were able to detect early as well as lately secreted cytokines in 

the supernatants of stimulated T lymphocytes. Interestingly, the peak of distinct 

cytokine secretion in response to a certain antigen varied between different individuals. 

Comparing cytokine secretion and proliferation of T lymphocytes we found no 

correlation: high amounts of TNF-α and / or IFN-γ were found in weakly proliferating 

cultures and vice versa. These results indicate that cytokine secretion and proliferation 

are two different aspects of lymphocyte response following stimulation and these 

parameters are not replaceable by each other. 

Supported by Deutsche Forschungsgemeinschaft, SFB 571

Vera Jakobi, Swen Wagner, Michael Loos, Franz Petry 

Complement activation of Cryptosporidium parvum 

(Apicomplexa, Protozoa) via the classical and lectin pathways 

C. parvum causes self-limiting diarrhoea in the immunocompetent and chronic and lifethreatening 

disease in immunocompromised individuals. In order to overcome the 

infection a specific CD4+-T-cell response is essential. Infection is mainly restricted to 

the epithelium of the small intestine and no systemic infections have been reported. We 

investigated if complement contributes to the host defence against cryptosporidiosis. 

We could show that both, human and mouse C1q bind to the extracellular stages of C. 

parvum, the sporozoites and oocysts, independently of specific antibodies. Binding was 

demonstrated by incubation of the parasites with normal sera or C1q-deficient sera 

supplemented with purified C1q. After intensive washing parasites were lysed in SDS 

sample buffer, electrophoresed and blotted onto nitrocellulose. C1q was detected by 

immunoblotting. The binding of C1q was dependent on the ionic strength and 

temperature. Affinity of purified C1q to the parasite was higher than serum C1/C1q. 

Similar experiments were performed to demonstrate human and mouse MBL binding to 

C. parvum. MBL binding was Ca++ dependent and could be competed with mannose 

during incubation. Activation of complement was demonstrated by deposition of C3b/ 

iC3b on oocysts in an immunofluorescence test. Despite the in vitro activation of the 

classical and lectin pathway mice deficient in C1q or MBL-A and MBL-C were not more 

susceptible to infection than wild type controls as judged by parasite shedding in the 

faeces. However, RT-PCR amplification of parasite mRNA from ilea of infected mice 

suggested slightly higher infection levels than C57BL/6 controls. From these data we 

conclude that the classical and lectin pathways may contribute to the restriction of the 

infection to epithelia of the host.

Feng Guo, Debra Weih, Elke Meier, Falk Weih 

Constitutive alternative NF-?B signaling promotes marginal 

zone B cell development but disrupts the marginal sinus and 

induces HEV-like structures in the spleen 

Nuclear factor (NF)-?B plays a crucial role in B cell and lymphoid organ development. 

Here, we studied the consequences of constitutive, signal-independent activation of the 

alternative NF-?B pathway for the splenic marginal zone (MZ). In contrast to nfkb2-/- 

mice, which lack both p100 and p52, mice that lack only the inhibitory p100 precursor 

(p100-/-), but still express the p52 subunit of NF-?B2, had markedly elevated MZ B cell 

numbers. Both cell intrinsic mechanisms and increased stromal expression of vascular 

cell adhesion molecule-1 (VCAM-1) contributed to the accumulation of MZ B cells in 

p100-/- spleen. While migration of p100-/- MZ B cells towards the lysophospholipid 

sphingosine-1 phosphate (S1P) was not affected, CXCL13-stimulated chemotaxis was 

impaired, correlating with reduced migration of MZ B cells into follicles in response to 

LPS. Strikingly, p100 deficiency resulted in the absence of a continuous marginal sinus 

but strongly induced expression of mucosal addressin cell adhesion molecule-1 

(MAdCAM-1) and glycosylated cell adhesion molecule-1 (GlyCAM-1) and the formation 

of ectopic high endothelial venule (HEV)-like structures in the splenic red pulp. Thus, 

constitutive activation of the alternative NF-?B pathway favors MZ B cell development 

and accumulation but leads to a disorganized microarchitecture of the splenic MZ.

Christian Koble, Jens Derbinski, Bruno Kyewski 

CONSTITUTIVE CROSS-PRESENTATION OF ENDOGENOUS 

SELF- ANTIGENS BY THYMIC DENDRITIC CELLS 

The role of central tolerance induction has recently been revised following the discovery 

of promiscuous expression of tissue-restricted self-antigens in the thymus. The extent 

of tissue representation afforded by this mechanism and its cellular and molecular 

regulation are barely defined. We have previously shown that medullary thymic 

epithelial cells (mTECs) are specialized to express a highly diverse set of genes 

representing essentially all tissues of the body thus mimicking the transcriptome of 

multiple peripheral tissues and maximizing the scope of central self-tolerance. It is, 

however, less clear whether mTECs and/or thymic dendritic cells (DCs) are responsible 

for presentation of these tissue-restricted self-antigens to developing thymocytes. Here, 

we analyzed ex vivo presentation of MHC class I- and II-restricted, endogenous selfantigens 

by purified mTECs and thymic DC. Although expression of all tested selfantigens 

at the RNA level was largely confined to mTECs, we consistently found crosspresentation 

by thymic DC. As a result of this constitutive cross-presentation within the 

thymic medulla, the frequency of APCs potentially tolerizing for a specific self-antigen is 

larger than previously estimated, when self-antigen expression had been detected at 

the mRNA or protein level [1, 2]. In conjunction with extensive scanning of medullary 

stromal cells by thymocytes, spreading of self-antigens from mTECs to hemopoietic 

APCs should enhance the efficacy of self-tolerance induction. 

[1] Klein, L. et al.: Sampling of complementing self-antigen pools by thymic stromal 

cells maximizes the scope of central T cell tolerance, Eur. J. Immunol. 2001 Aug; 31(8): 

2476-86 

[2] Derbinski, J. et al.: Promiscuous gene expression in medullary thymic epithelial cells 

mirrors the peripheral self, Nat. Immunol. 2001 Nov; 2(11): 1032-9

Katja Sabel, Oleg Krut, Martin Krönke, Alexander Klimka 

Construction of a recombinant intrabody library to select for 

inhibitors of intracellular pathogens 

Intracellular interactions between pathogen and host cell factors during infection are 

very complex and offer a wide range of therapeutic targets. Single chain fragment 

antibodies (scFv) can be functionally expressed inside the cell (intrabodies) and 

specifically bind cellular or pathogenic molecules thus inhibiting microbial pathogenicity. 

To reveal the anti-infectious potential of intrabodies experimentally, we have 

constructed a lentiviral scFv library which was stably expressed in mammalian target 

cells. To ensure that diversity of the scFv library was maintained during library cloning, 

randomized clones have been analysed by DNA fingerprint and sequencing and 

intrabody expressing cells were selected by negative selection using cytotoxic agents. 

As a proof of concept an HSV-1 thymidine kinase (TK) expressing cell line has been 

transduced with the intrabody library and subsequently treated with the cytotoxic base 

analog ganciclovir, which resulted in intrabody expressing, gancyclovir-resistant cell 

lines. As to the infection model, scFv-transduced HeLa cells were infected with the 

cytotoxic Staphylococcus aureus strain MW2. After four rounds of infection a significant 

reduction of pathogenicity was observed. Surviving HeLa cell clones are currently 

analysed to identify and characterize the specificity of intrabodies inhibiting S. aureus 

cytotoxicity. The findings of our study demonstrate the potential of intrabodies to both 

investigate and target eukaryotic as well as microbial factors determining host-pathogen 

interactions.

Vladimir Kocoski, Norbert Tautz, Eberhard Burkhardt 

CONSTRUCTION OF A STABLE TRANSFECTED, PERMANENTLY 

SECRETING BHK Tet-On CELL LINE CARRYING THE SINGLE- 

CHAIN CANINE IL-12 FOR APPLICATION IN THE TUMOR 

IMMUNOTHERAPY IN DOG 

INTRODUCTION: Besides being the most important tumor patient in veterinary 

medicine, the dog is a promising model for tumor immunotherapy in man. As one of the 

approaches in tumor immunotherapy, the cytokine stimulation of Natural Killer and 

cytotoxic T cells represents a promising way to specifically fight the tumor cells. Among 

the cytokines showing potent anti-tumor activities the heterodimeric interleukin-12 (IL- 

12) plays a significant role. However, in contrast to mice and human, detailed studies 

on this area in dogs are lacking. In order to provide reliable sources for future 

investigations on the field of tumor immunotherapy in dogs, we (1) constructed a singlechain 

canine IL-12 sequence, (2) stably transfected it into a baby hamster kidney (BHK)- 

Tet-On cell line, which would serve as a constant and inducible (Doxycycline) protein 

source and (3) demonstrated the biological activities of the constructed protein. 

METHODS: Both sequences coding for the canine IL-12, p35 and p40, were amplified by 

PCR, ligated as a single chain in a pTRE/luciferase vector and afterwards stably 

transfected into the BHK-Tet-On cell line. Using canine interferon-gamma (IFN- 

&gamma) ELISA and cytotoxicity test (Rose Bengal Assay), the bioactivity of the IL-12 

containing supernatants was investigated. 

RESULTS: Canine IL-2 blasts incubated with the supernatant from the transfected clone 

showed significantly increased IFN-&gamma production, which was completely blocked 

by an anti-canine IL-12 neutralizing antibody. The supernatant also showed increased 

cytolytic activity of the IL-2 blasts against the canine thyroid adenocarcinoma (CTAC) 

target cells at 50:1 ratio. 

CONCLUSIONS: The transfected clone produces the single-chain canine IL-12 in the 

supernatant, which shows the full bioactivity like the commercially available IL-12. 

Perspectively, this cell clone can be used for further investigation of the IL-12 induced 

anti-tumor effects of canine lymphocytes, especially NK cells. Moreover, the nucleotide 

sequence of the single-chain IL-12 could be used in gene delivery studies in dogs.

Alexey Popov, Julia Driesen, Zeinab Abdullah, Claudia Wickenhauser, Tomo Saric, 

Svenja Debey-Pascher, Trinad Chakraborty, Martin Krönke, Olaf Utermöhlen, Joachim L. 

Schultze 

Containment of pathogens and induction of the local immune 

privilege by IDO + denritic cells in granulomatous infections 

and its implication for human disease 

In the first decades following the discovery of the tryptophan-catabolizing enzyme 

indoleamine 2,3-dioxygenase (IDO), the molecule was thought to be mostly involved in 

the repression of bacterial and viral infections. Recently, however, evidence has 

emerged that this enzyme is also capable of promoting local immune privilege. 

However, these opposite activities of IDO could hardly be imagined within one 

pathological condition. Here, we attempted to link two converse sides of IDO inhibitory 

function – bacteriostasis and immune inhibition – in one in vivo situation. We could 

show for the first time in human listeriosis, but also in granulomatous infections, caused 

by other intracellular pathogens (i.e. tuberculosis or cat-scratch disease), that IDO + 

dendritic cells (DC) and macrophages prevent the spreading of the pathogens by 

formation of granuloma, which encapsulate the bacteria and “keep the bug at bay”. At 

the same time, massive enrichment of IDO-competent APC inhibits effector immune 

cells from entering the granulomas, thereby preventing these granulomas from 

destruction and avoiding pathogen dissemination, a dangerous situation detrimental to 

the host’s survival. Infection of human DC by Listeria monocytogenes in vitro results in 

significant induction of IDO both on transcriptional and protein levels. Moreover, 

enzymatic function of IDO in listeria-infected DC is tightly controlled by TNFα, which 

might explain exacerbation of granulomatous infections during anti-TNFα therapy. 

These findings clearly demonstrate that IDO can play a dual role during life-threatening 

infections, inducing local immune privilege and pathogen containment at the same time. 

It will be of great interest to map out if IDO plays a similar role in other infectious and 

non-infectious diseases.

Kathrin Westphal, Sara Leschner, Holger Loessner, Siegfried Weiss 

Containment of tumor colonizing bacteria by host neutrophils 

Systemic administration of several facultative anaerobic bacteria into tumor bearing 

mice leads to a preferential accumulation and proliferation of the microorganisms inside 

the solid tumor. Up to now, all known tumor-targeting bacteria show a poor 

dissemination inside solid tumors as they accumulate exclusively inside large necrotic 

areas and spare a rim of viable tumor cells. Interestingly, this bacteria-containing 

necrosis is separated from the viable tumor cells by a barrier of host neutrophils that 

have migrated into the tumor tissue. We report here that depletion of host neutrophils 

results in enhanced dissemination of tumor-targeting bacteria. Besides a noticeable 

higher total number of bacteria inside the tumor, we found an increase of the size of 

necrosis and a migration of bacteria into the vital tumor tissue. The border of host 

neutrophils that obviously trap the bacteria inside the necrosis had vanished and the 

few neutrophils left were bypassed by the bacteria. In addition, neutrophil-depletion 

might positively influence the therapeutic effects of tumor-colonizing bacteria. Thus, 

depletion of neutrophils should be considered as additional measure during bacterial 

tumor therapy.

Martin Schlee, Michael Bscheider, Veit Hornung, Andrea Ablasser, Stefan Endres, 

Gunther Hartmann 

Contrasting roles of p38 in TLR signaling 

Toll-like receptors 7 and 8 are activated by the synthetic compound Resiquimod (R848). 

According to previous studies, TLR7/8 stimulation involves NF?B, IRF and MAP kinase 

activation, which leads to secretion of proinflammatory cytokines in myeloid cells as well 

as type I IFN release in plasmacytoid dendritic cells (PDC). 

To further evaluate the role of different MAP kinases, we incubated peripheral blood 

mononuclear cells (PBMCs) or murine bone marrow-derived dendritic cells obtained 

from a Flt3-ligand (Flt3–L) culture with synthetic inhibitors and monitored cytokine 

secretion induced by TLR stimulation. 

Here we present data that suggest a negative influence of p38 MAP kinase on IL12 

secretion after stimulation with R848 but not LPS. Using the well-characterized selective 

chemical p38 inhibitor SB203580 from the family of the pyridinyl imidazoles, we could 

dose-dependently increase the amount of IL12 secreted by human monocytes. This 

increased cytokine secretion was independent of the Th1-inhibitory cytokine IL10. In 

contrast, inhibition of p38 almost completely abolished IFNa production after R848 

stimulation of PDCs. Similar results were gained regarding the signaling of murine 

myeloid bone marrow-derived dendritic cells (mDC) obtained from a Flt3–L culture. An 

increase of IL12 production in the presence of SB203580 after R848 and CpG 

stimulation was observed, while IFN alpha secretion by murine Flt3–L PDCs was 

strongly inhibited by SB203580. Surprisingly, IL12 secretion by murine Flt3–L PDCs was 

not increased but slightly decreased by SB203580, pointing towards a cell type-specific 

role of p38 in TLR signaling rather than a target gene-specific signaling.

Bishnudeo Roy, Oliver Pabst, Swati Shukla, Sandra Düber, Siegfried Weiss 

Contribution of B-1 cells to gut associated humoral immunity 

The relative contribution and repertoire of B-1 and B-2 cell derived IgA in the gut 

immune system is still a controversial issue. The lack of an exclusive B-1 cell marker 

has made it difficult to delineate the B-1 and B-2 cell derived IgA producing plasma 

cells. To circumvent these problems, we have used the L2 mouse line which is 

transgenic for λ2315 light chain and virtually consists of B-1 cells exclusively. A 

particular feature of these mice is the presence of a few B-1 cell derived specificities at 

dominating frequencies in the peritoneum. In this work, these specificities, detectable 

as VH sequences, were used as markers to investigate the participation of B-1 cells in 

IgA production at the intestinal mucosa. 

Analysis of IgM VH sequences derived from Peyer´s patches (PP) B cells of L2 mice 

showed that 10% of these IgM VH sequences were identical to IgM VH sequences 

derived from peritoneal cavity (PEC) B-1 cells of these mice. Also, some commonality 

amongst the IgM VH sequences derived from the lamina propria (LP) B cells, and PEC B- 

1 cell associated sequences from L2 mice could be observed. 

On the other hand, analysis of IgA VH sequences derived from the LP and PP B cells 

showed no match with B-1 cell associated Ig VH sequences. Additionally, IgA VH sequences derived from the LP associated plasma B cells was rather heterogeneous and 

showed N/P nucleotide addition in the CDR3 region and somatic hypermutation through 

out the VH chain. Histology done for the intestine showed a decreased number of IgA+ 

plasma cells in the LP of L2 mice in comparison to NT control mice. Consistent with this, 

there was a decrease in the levels of secretory IgA in the intestinal lumen of L2 mice. 

In addition, IgA expressing B cells, majority of which was B-1 cells (IgA+CD43+) could 

be observed in the PEC of L2 as well as NT mice. Analysis of PEC B-1b cell derived IgA 

VH sequences of normal mice showed the presence of nucleotide exchanges through out 

the VH sequences at a high frequency. 

Altogether, these data suggest that IgA producing B cells might be a highly selected 

population and PEC B-1 cells might diversify due to antigen driven selection in the 

GALT.

Susanne Kirschnek, Robert Paul, Bianca Obermaier, Georg Häcker, Uwe Koedel 

Contribution of cell death in phagocytes and resident cells to 

the outcome of pneumococcal meningitis in mice 

Efficient uptake and elimination of bacteria by neutrophil granulocytes and macrophages 

is a crucial step in the defence against bacterial infections. We observed phagocytosisassociated 

apoptosis of macrophages and granulocytes after uptake and digestion of 

bacteria, which proceeds via the mitochondrial pathway and is at least in part 

dependent on the BH3-only protein Bim. To investigate the significance of bacteriainduced 

phagocyte apoptosis for infection, a mouse model of pneumococcal meningitis 

was used. Phagocyte and/or resident tissue apoptosis was inhibited by Bcl-2 

overexpression or Bim deficiency. In this model, neutrophil granulocyte function is 

known to be crucial for efficient defence against bacterial infection. Mice overexpressing 

Bcl-2 in the hematopoietic compartment had more severe symptoms and higher 

lethality than wild type littermates. We observed a similar recruitment of leukocytes into 

the brain at early time points during infection but a significantly higher leukocyte count 

in the cerebrospinal fluid at later stages. This was accompagnied by a loss of bloodbrain-barrier 

function and impaired clearance of bacteria in the blood in Bcl-2transgenic 

mice. In addition, more pronounced histopathological alterations, indicative 

of enhanced inflammation, were observed. Experiments with bim-/- mice further 

indicate that the loss of the pro-apoptotic Bcl-2 family protein Bim leads to a different 

phenotype where higher leukocyte counts in the cerebrospinal fluid are correlated with 

lower bacterial load in the brain and better maintenance of the blood-brain-barrier. 

Thus, in contrast to Bcl-2 overexpression (which was restricted to the hematopoetic 

compartment), Bim deficiency in all cell types conferred enhanced resistance to 

pneumococcal meningitis. These results indicate a physiological function for phagocyte 

apoptosis in the control of bacterial infections and suggest that cell death in nonhematopoetic 

cells contributes to the outcome of pneumococcal meningitis.

Gabriele Weintz, Michael Hammer, Ilona Moßbrugger, Leticia Quintanilla-Martinez, 

Christian Stemberger, Dirk H. Busch, Roland Lang 

Control of inflammation and host resistance during Listeria 

infection by the MAPK-Phosphatase DUSP1 

Innate immune cell activation via pattern recognition receptors induces the release of 

inflammatory cytokines, chemokines and mediators that are essential for the control 

and elimination of pathogens. To avoid excessive inflammation and tissue damage, the 

intensity and duration of this response are tightly regulated by multiple mechanisms. 

We and others have recently identified the MAPK phosphatase DUSP1 as an essential 

negative regulator of the inflammatory response to TLR activation. In the absence of 

DUSP1, LPS causes deregulated p38 activation in macrophages in vitro and high 

lethality in vivo due to excessive production of inflammatory cytokines. Infection with 

Listeria monocytogenes activates p38 MAPK and has been shown to induce expression 

of DUSP1 in macrophages. Here, we ask whether DUSP1 controls the type and intensity 

of the host response to Listeria infection, in terms of inflammation and control of 

bacterial replication. Our data show that DUSP1-deficient mice have reduced bacterial 

loads in the spleen on day 3 after intravenous infection compared to wildtype mice. At 

the histological level, the development of necrotic lesions in the spleen and liver 

appeared to be reduced. However, contrary to our expectation, the better protection 

against Listeria was not accompanied by enhanced cytokine production, neither 3 days 

nor 2 hours after infection, when bacterial loads were equal in DUSP1-deficient and 

wildtype mice. We are currently determining the efficiency of bacterial killing, the 

production of nitric oxide, and the kinetics of p38 MAPK activation of in vitro infected 

DUSP1-deficient macrophages to elucidate the mechanism(s) responsible for the 

increased resistance to Listeria infection.

Caspar Ohnmacht, Nico van Rooijen, David Voehringer 

Cooperation between innate and adaptive immunity during 

type 2 immune responses in vivo. 

Type 2 immune responses are associated with allergic diseases and parasite infections. 

They are mediated by IL-4/IL-13 expressing effector cells of the innate and adaptive 

immune system. Using sensitive IL-4/eGFP reporter mice (4get mice) we could identify 

eosinophils and basophils as the main IL-4 expressing cell types of the innate immune 

system recruited to the lung during infection with the helminth parasite Nippostrongylus 

brasiliensis or OVA-induced allergic inflammation. Reconstitution of T cell-deficient mice 

with IL-4/IL-13-deficient CD4+ T cells was sufficient to mediate parasite expulsion from 

the intestine and induction of effector cell recruitment and airway hyperreactivity in the 

lung. Therefore, innate IL-4/IL-13 expressing cells play a crucial role during the effector 

phase of type 2 immune responses. 

We analyzed development, migration and turnover of eosinophils and basophils in vivo. 

Both cell types could be identified in the fetal liver. Staining for the surface markers 

FIRE, CCR3, Siglec-F and CD62L revealed a gradual maturation process of eosinophils 

and enabled us to determine the activation status of eosinophils in different tissues. 

Eosinophils showed an activated phenotype in lymph nodes, thymus and Peyers’ 

patches indicating that they had entered these tissues via the afferent lymph from sites 

of inflammation. In contrast to basophils, eosinophils actively migrated to the peritoneal 

cavity, where they survived for several days and from where they could recirculate to 

other organs.

Kathrin Schönberg, Gesine Kögler, Johannes Fischer, Markus Uhrberg 

Correlation of KIR expression and presence of HLA-C ligands 

in adult but not neonatal NK cells: transition from a naïve to 

an adult NK cell repertoire 

The functional repertoire of human natural killer (NK) cells is shaped by interaction of 

killer cell inhibitory receptors (KIR) and HLA class I ligands. It is so far unknown, how 

HLA class I-dependent education of NK cells is achieved. Here, a comparative analysis 

of NK cell repertoires was performed in peripheral blood (PB, n=154) and cord blood 

(CB, n=100) of a mainly Caucasoid cohort. A 6-color flow cytometric protocol was set 

up to differentiate between the four HLA class I-specific KIR encoded on group A 

haplotypes in combination with NKG2A. Similar to a recent study in a Japanese cohort, 

the presence of HLA-C ligands strongly correlated with an increased frequency of NK 

cells from PB expressing the respective cognate KIR. In contrast, no influence of HLA-C 

on the KIR repertoire was detected in NK cells from CB. Generally, significantly more 

KIR-expressing NK cells are present in PB than in CB. Nonetheless, the percentage of 

NK cells that are lacking inhibitory receptors for self-HLA class I is significantly lower in 

CB. This is due to a much higher frequency of NKG2A-expressing NK cells in CB. The 

data suggest that the NK cell repertoire goes through a transition from birth to 

adulthood, which is characterized by a marked decrease of NKG2A and an increase of 

self-HLA class I-specific KIR. The repertoire transition appears to be completed at 

adulthood as no significant changes of NKG2A and KIR frequencies beyond age 20 were 

detectable in the cohort. The possible consequences regarding change in NK cell 

function and the clinical use of cord blood as stem cell source will be discussed.

Zoe Waibler, Martina Anzaghe, Abdo Konur, Shizuo Akira, Werner Müller, Ulrich Kalinke 

CpG 1668 treatment stimulates an anti-inflammatory 

environment that abrogates CpG 2216 induced type I IFN 

responses by pDC 

Upon incubation with a wide range of concentrations of the synthetic CpG 

oligodeoxynucleotide 2216 (CpG 2216), mouse bone marrow derived plasmacytoid 

dendritic cells (BM-pDC) are stimulated to produce interferon (IFN)-alpha, whereas only 

intermediate but not high doses of CpG 1668 induce IFN-alpha responses. To address 

why high dose CpG 1668 does not induce type I IFN, we co-incubated BM-pDC with high 

doses of CpG 2216 and CpG 1668 that resulted in the shut off of CpG 2216 induced IFNalpha 

responses. Incubation with supernatant of high dose CpG 1668 treated BM-pDC 

caused more than 90% inhibition of CpG 2216 induced IFN-alpha responses indicating 

that secreted inhibitor(s) plaid a major role. Among cytokines found in the inhibitory 

supernatant, IL-10 turned out to be one important negative regulator. In line with this, 

supernatant of IL-10 deficient BM-pDC stimulated with high dose CpG 1668 did not 

significantly inhibit IFN-alpha production, whereas addition of IL-10 neutralizing 

antibodies dramatically reduced the inhibitory capacities of wild-type supernatants. 

Moreover, recombinant IL-10 inhibited CpG 2216 induced IFN-alpha production. 

Interestingly, also IFN-alpha secretion upon stimulation with the DNA-encoded mouse 

cytomegalovirus (MCMV) was significantly diminished upon co-incubation with high dose 

CpG 1668. Thus, depending on the concentration used, CpG 1668 can be stimulatory or 

regulatory by inducing IL-10 that affects signaling by synthetic and/or natural Toll-like 

receptor (TLR) 9 ligands. 

Mechanisms described here probably reflect a physiological system to protect the 

organism from hyperactivation of dendritic cells that could lead to autoimmune 

reactions.

Viktor Kölzer, David Anz, Michaela Golic, Cornelia Wurzenberger, Stefan Endres, 

Carole Bourquin 

CpG Oligonucleotide Treatment Alters the Morphological 

Distribution and Phenotype of Regulatory T Cells 

Regulatory T cells (Treg) contribute to the maintenance of immunological homeostasis 

and limit autoimmune reactions. However, Treg also suppress anti-tumor immunity and 

may reduce the efficacy of cancer immunotherapy. Synthetic oligonucleotides containing 

immunostimulatory CpG motifs (CpG) that signal via Toll-like receptor 9 evoke Th1-type 

responses and pro-inflammatory cytokine production. They effectively induce anti-tumor 

immune responses in many experimental cancer models and are currently tested in 

phase III clinical studies. Despite this promising application in cancer therapy, little is 

known about their influence on Treg in vivo. 

In healthy, CpG-treated mice, we evaluated Foxp3-positive Treg populations in 

secondary lymphoid organs by flow cytometry and immunohistology. The influence of 

CpG-based cancer therapy on Treg was investigated in mice carrying subcutaneous 

tumors derived from a C26 colon carcinoma. 

We show that CpG treatment leads to a shift in the CD25low/CD25high Treg ratio in the 

spleen and lymph nodes, with an increase of CD25low expressing Treg phenotypes. Our 

analysis also demonstrates that regulatory T cells accumulate in the spleen during CpG 

treatment and display an altered morphological distribution. At the same time, CpG 

treatment decreases the fraction of CD25low-expressing Treg within PBMC and reduces 

the accumulation of Treg in peripheral blood of tumor-bearing mice. CpG application 

also slightly reduces the Treg population within tumor-draining lymph nodes. 

In summary, we show that CpG-based immunotherapy leads to a shift in the balance 

between CD25low and CD25high Treg phenotypes and to their accumulation in the 

spleen. We suggest that the described enhancement of anti-tumor immunity through 

CpG is unlikely to result from a general reduction of the Treg population. Instead, a 

redistribution of Treg during immunotherapy could support anti-tumor immune 

responses. Further investigation of the influence of immunotherapy on Treg may 

support the development of improved therapeutic regimens.

Wolfgang Kastenmüller, Georg Gasteiger, Ingo Drexler 

Cross-competition of CD8+ T cells shapes the 

immunodominance hierarchy during recall vaccination 

CD8+ T cell responses directed against multiple pathogen-derived epitopes are 

characterized by defined immunodominance hierarchy patterns. One possible 

explanation for this phenomenon is that CD8+ T cells of different specificities compete 

for the access to epitopes on antigen presenting cells and that the outcome of this so 

called cross-competition reflects the number of induced T cells. In our study using a 

vaccinia virus infection model, we found that T cell cross-competition is highly relevant 

during boost vaccination, thereby shaping the immunodominance hierarchy in the recall. 

We demonstrate that competition was of no importance during priming and unaffected 

by the applied route of immunization. It strongly depended on the timing of viral 

antigen expression in infected APC and was characterized by poor proliferation of T cells 

recognizing epitopes derived from late viral proteins. To our knowledge, this is the first 

demonstration of functional importance of T cell cross-competition during a viral 

infection. Our findings provide a basis for novel strategies of how boost vaccination to 

defined antigens can be selectively improved. They give important new insights into the 

design of more efficient pox-viral vectors for immunotherapy.

Nanette von Oppen, Linda Diehl, Rene Tolba, Percy Knolle 

CROSS-PRESENTING LIVER SINUSOIDAL ENDOTHELIAL CELLS 

ESTABLISH ANTIGEN-SPECIFIC ADHESION OF NAÏVE CD8 T 

CELLS LEADING TO T CELL TOLERANCE IN VIVO 

Presentation of antigen in the liver leads to induction of T cell tolerance rather than 

immunity. The ability of liver sinusoidal endothelial cells (LSEC) to cross-present 

exogenous antigens on MHC I molecules to naïve CD8 T cells and to induce CD8 T cell 

tolerance contributes to the hepatic immune regulatory function. However, the 

mechanisms underlying recruitment of naïve CD8 T cells to the liver remained unclear. 

We provide evidence that organ-resident cross-presenting liver sinusoidal endothelial 

cells recruit naïve CD8 T cells antigen-specifically to the liver. Antigen-specific hepatic 

recruitment of naïve CD8 T cells occurred rapidly within 4 hours after antigen-challenge 

and was exclusively observed in the liver but not in spleen, lymph nodes or lung. 

Expression of CD54 supported antigen-specific T cell adhesion but was not essential. 

Antigen-specific adhesion was accompanied by rapid stimulation of T cells, as shown by 

up-regulation of CD69, which was also predominantly observed in the liver. Experiments 

employing bone marrow chimeric mice unequivocally demonstrated that organ-resident 

but not myeloid APC were responsible for antigen-specific naïve T cell adhesion. 

Importantly adhesion of naïve CD8 T cells to cross-presenting LSEC in bone marrow 

chimeric mice resulted in tolerance of these cells. Our data attribute a novel function to 

LSEC, i.e. antigen-specific retention of naïve CD8 T cells, which is not observed for 

other organs. Delivery of a flow-stop signal by tolerogenic LSEC to passenger naïve CD8 

T cells in the hepatic circulation provides a mechanistic insight into the early steps of 

induction of CD8 T cell tolerance in the liver.

Heinke Conrad, Kerstin Gebhard, Julia Neudorfer, Christian Peschel, Helga Bernhard 

Cross-reactivity of HER2p369-377 reactive CTL clones against 

other HER family members 

Immunotherapies against tumor associated antigens are a promising field in oncology. 

Until now several vaccination studies against HER2 which is overamplified in about 25% 

of breast cancer were performed but the objective response rate achieved was only 

2.6%. The goal of our studies is the adoptive transfer of in vitro stimulated and 

expanded tumor-reactive, HER2-specific cytotoxic T cells (CTL) for patients with HER2overexpressing 

breast cancer. 

(1) CD8 T cells derived from an HLA-A2 donor were stimulated with autologous dendritic 

cells (DC) transfected with HER2-mRNA. (2) CD8 T cells isolated from an HLA-A2 

negative donor were stimulated with allogeneic HLA-A2 positive DC pulsed with the 

peptide HER2p369-377 known to be naturally presented with HLA-A2. HER2p369-377 

specific T cells were screened via ELISpot and FACS sorting, respectively, cloned and 

further tested in 51CRA and ELISA using HER3 and HER4 peptides corresponding to 

HER2p369-377 pulsed as well as HER2-, HER3- and HER4 overexpressing cell lines. 

Additionally, cell lines were transfected with either of the HER cDNA and a HER2 and 

HER3 overexpressing tumor cell line was transfected with the respective siRNA. 

The HER2-reactive CTL derived from both stimulation methods recognized the peptide 

HER2p369-377 and lysed the same panel of HLA-A2-matched, HER2-overexpressing cell 

lines as determined by lytic activity and cytokine secretion. Interestingly, the HER2p369- 

377 specific CTL clones were not only able to recognize the synthetic HER3 and/or HER4 

peptides corresponding to HER2p369-377 but also a cell line expressing either HER2, 

HER3 or HER4. Using downregulation of the respective HERs we could show that HER2 

and HER3 both contribute to the recognition by the CTL clones. 

As HER2 and HER3 both contribute to the malignant phenotype of HER2/HER3overexpressing 

breast cancer cells, the parallel targeting of HER2 and HER3 by crossspecific 

CTLs may inhibit the selective outgrowth of escape variants. These results will 

contribute to the design of T cell-based therapies for the treatment of breast cancer 

patients.

Mandy Pierau, Engelmann Swen, Thomas Drewes, Thabo Lapp, Dirk Reinhold, 

Burkhart Schraven, Ursula Bommhardt 

Cross-talk between PKB/Akt and TGFβ signalling in T cell 

activation 

The TGFβ family of cytokines are important regulators of cellular proliferation, apoptosis 

and differentiation. TGFβ is a key regulator of inflammatory responses and has been 

linked to the pathogenesis of experimental autoimmune encephalomyelitis (EAE). 

Binding of TGFβ to type II and type I serine-threonine kinase receptors leads to 

phosphorylation of the intracellular signal mediators, the Smad proteins. Activated 

Smad2 and Smad3 bind Smad4 and as heteromeric Smad complexes, once translocated 

to the nucleus, cooperate with other nuclear co-factors to induce the transcription of 

target genes. Several Smad regulatory proteins control the subcellular localization, 

phosphorylation and the binding of transcriptional partners of the Smads. Interestingly, 

a cross-talk between TGFβ signalling and the PI3K/PDK1/PKB pathway has been 

suggested from findings in cell lines that Smad proteins can physically interact with PKB 

or PDK1. We previously showed that T cells from transgenic (tg) mice expressing a 

constitutively active form of PKBα (myrPKB) are hyperreactive and show enhanced 

survival. Surprisingly, MOG-peptide induced EAE in myrPKB tg mice showed a milder 

disease progression compared to wild type mice. Since the amelioration of EAE 

pathology in myrPKB tg mice could be coupled to differences in TGFβ-mediated 

signalling events, we studied the response of T cells to TGFβ. In contrast to wild type 

CD4+ T cells TCR/CD3 induced proliferation of myrPKB tg T cells proceeded in the 

presence of TGFβ. Active PKB thus counteracts the inhibitory signals of TGFβ, although 

nuclear translocation of activated Smad2/3 proteins was normal. Data on candidate 

proteins that are affected by myrPKB and likely contribute to the "resistance" to TGFβ 

will be presented and discussed.

Sven Burgdorf, Christian Kurts 

Current models and mechanisms of antigen crosspresentation 

After internalization and processing of extracellular antigens, dendritic cells can induce 

an adaptive immune response by presenting antigenic epitopes on both MHC II 

molecules (to activate CD4+ T helper cells) and MHC I molecules (to activate CD8+ 

cytotoxic T killer cells). The latter process has been termed cross-presentation. 

Increasing evidence supports an important role of cross-presentation in various 

biological processes. Nevertheless, the molecular mechanisms regulating intracellular 

processing of internalized antigens and loading of the derived peptides on MHC class I 

molecules remain largely unknown. 

At present, several models have been proposed to explain how endocytosed antigens 

might reach the MHC I presentation pathway. Most of them point out a decisive role of 

the ER associated degradation machinery (ERAD) and the cytoplasmic proteasome. Our 

recent findings support a further mechanism in which distinct endocytosis mechanisms 

selectively introduce soluble antigens into an organelle dedicated to cross-presentation 

and distinct from classical lysosomes. In this presentation, I will review the current cellbiological 

models of cross-presentation, focussing on their similarities and distinctions 

and on the unanswered questions pertaining to its molecular regulation.

Jörg Rossbacher, Frank Wilde, Gerd Müller, Martin Lipp 

CXCR5 as a therapeutic target in Non Hodgkin lymphomas 

and autoimmune disease 

Homeostatic chemokine receptors and their ligands control the trafficking of 

lymphocytes to and within secondary lymphoid organs. The receptor CXCR5 is 

expressed on mature recirculating B cells, a subset of memory T cells and follicular B 

helper cells (TFH). In cooperation with its ligand, CXCL13, which is expressed on 

stromal and follicular dendritic cells in the B cell follicle, CXCR5 is responsible for B/T 

separation in lymphoid tissues. It was shown that CXCR5 plays a major role in the 

development of secondary lymphoid structures like Peyers Patches and certain lymph 

nodes and therefore is suspected to be essential in ectopic follicle formation in chronic 

inflammatory diseases like Rheumatoid Arthritis, Sjogren's syndrome and Helicobacter 

pylori-induced chronic gastritis.Together with the fact that 90% of B cell non Hodgkin 

lymphomas (NHL) express significant levels of CXCR5 on their surface we propose that 

CXCR5 is a suitable therapeutic target in B cell NHL and chronic autoimmune disease. 

We therefore generated a hybrid hybridoma producing the trifunctional bispecific 

antibody bsCXCR5xCD3 to perform CXCR5 positive cell depletion. Bispecificity should 

enhance the lytic ability by bringing target and effector cells into close proximity and 

the intact Fc portion could attract FcR bearing effector cells or even initiate complement 

lysis. We tested the antibody on CXCR5 expressing NHL cell lines and observed 

increased cell lysis at various conditions and time points. In addition we tested primay B 

cells obtained from healthy human donors and observed efficient cell lysis at 

comparable concentrations and time points. These results show that the bispecific 

antibody is able to deplete target cells “in vitro”, no matter if they are cancer cell lines 

or primary human cells.

Sven Hartmann, Antje M. Wengner, Uta E. Hoepken, Peter K. Petrow, Uta Schurigt, 

Rolf Braeuer, Martin Lipp 

CXCR5- and CCR7-dependent lymphoid neo-genesis in a 

chronic model of antigen-induced arthritis (AIA) 

Rheumatoid arthritis (RA) is a common autoimmune disease affecting about 1% of the 

adult population and is mainly characterized by chronic, polyarticular, synovial 

inflammation, which can lead to long-term joint damage resulting in chronic pain and 

disability. The molecular and cellular pathogenic mechanisms, which lead to RA and 

maintain the chronicity of the disease, are still poorly understood. Characteristically for 

RA is the infiltration into the synovial tissue by granulocytes and large numbers of 

mononuclear cells, such as T and B lymphocytes, monocytes, neutrophils and 

macrophages. We have developed a novel model of antigen-induced arthritis in mice 

resembling the chronic phase of the human disease including frequent formation of 

ectopic follicular structures, a hallmark of human RA. In our model formation of ectopic 

follicles with segregated B and T cell areas can be regularly induced by intra-articular 

injection of mBSA into the knee joints of pre-immunized C57BL/6 and BALB/c mice. In 

CXCR5- and CCR7-deficient mice the formation and organization of these ectopic 

structures are severely impaired in proving that both chemokine receptors play an 

important role in lymphoid neo-organogenesis during chronic inflammatory conditions 

as in RA. Remarkably, most follicles show topologically segregated T and B cell areas 

with presence of CD8 and CD4 T cells, the formation of active germinal centers (GC) 

and generation of antigen-specific CD138+ plasma cells. Moreover, formation of ectopic 

follicles is entirely dependent on the presence of the chemokine receptors CXCR5 or 

CCR7. Our results suggest that continuous inflammatory stimuli, e.g. by autoantigens, 

are sufficient to induce lymphoid neo-organogenesis at extra-nodal sites, which in turn 

allows local antigen-dependent interaction of memory/effector B and T lymphocytes 

resulting in aberrant chronic autoreactive immune responses.

Tanja Nicole Hartmann, Bretton Summers, Valentin Grabovsky, Eilon Woolf, Ziv 

Shulman, Eike Buss, Tom Schall, Marcus Thelen, Ronen Alon 

CXCR7 blockage inhibits in human hematopoietic progenitor 

cells and T cells a CXCR4 subset specialized in integrin 

activation by CXCL12 under shear stress conditions 

CXCR7 (RDC1) is a novel CXCL12-binding chemokine receptor. Our earlier results 

suggested a crucial role for the major CXCL12 binding GPCR, CXCR4, in motility and 

integrin activation of human HPCs and T lymphocytes. We investigated the function of 

CXCR7 in CXCL12-mediated motility and integrin activation processes in these cells. We 

report that CXCR7 modulates the function of a small CXCR4 subset, which is specialized 

in CXCL12-stimulated integrin activation under shear flow. Inhibition of CXCR7 by 

blocking antibodies and a small molecular weight compound specifically interfered with 

CXCL12-dependent LFA-1 and VLA-4 activation and T cell adhesiveness to surfaceimmobilized 

anti-CXCR4 under shear flow. However, CXCR7 blockage did not interfere 

with CXCR4 surface expression nor with CXCL12-triggered T cell motility or chemotaxis 

in shear free and integrin independent assays, in contrast with previous reports. 

Strikingly, CXCR7 was not found on the surface at detectable amounts, but highly 

expressed in the cytoplasm. We suggest that both the inhibitory antibodies and the 

compound enter the cell via the small recycling pool and inhibit CXCR4-mediated 

integrin activation. This is a first demonstration that inhibition of an intracellular GPCR 

with undetectable expression on the cell surface can effectively interfere with a 

physiological key function of this GPCR.

Tobias Bopp, Christian Becker, Matthias Klein, Stefan Klein-Heßling, Alois 

Palmetshofer, Edgar Serfling, Marc Becker, Jan Kubach, Schmitt Steffen, Sabine Stoll, 

Hansjoerg Schild, Martin Staege, Michael Stassen, Helmut Jonuleit, Edgar Schmitt 

Cyclic AMP: The decicive component of naturally occuring 

regulatory T cell-mediated suppression 

Naturally occurring regulatory T cells (nTregs) are crucial for the maintenance of 

peripheral tolerance by suppressing auto-reactive T cells via cell contact-dependent 

mechanisms. Moreover, by limiting the magnitude of all adaptive immune responses, 

nTregs also play a pivotal role in adequately controlling immune pathologies. Although 

the underlying mechanisms of nTreg-mediated suppression are still elusive, a hallmark 

of this process is the inhibition of IL-2 production in the responder T cells. We found 

that nTregs harbour high levels of cyclic AMP (cAMP). This second messenger is known 

to be a potent inhibitor of IL-2 production and subsequent proliferation of conventional 

T cells. Upon co-culture with nTregs the cAMP content of the conventional T cells 

strongly increases. Furthermore, we demonstrate that nTregs and conventional T cells 

communicate via cell-contact dependent gap junction formation. The suppressive 

activity of nTregs is abolished by a cAMP-specific antagonist as well as by a gap junction 

inhibitor which blocks the cell contact-dependent transfer of cAMP to responder T cells. 

Hence, our results demonstrate that cAMP is crucial for nTreg-mediated cell contactdependent 

suppression by traversing membranes via gap junctions.

Bianca Paul, Linda Diehl, Alexander Knorre, Percy Knolle, Marc Beyer, Waldemar 

Kolanus 

Cytohesin-3 Links B7H1 mediated Shut-down of the PI3 

Kinase Pathway to the Repression of IL-2 Synthesis in 

Anergic T cells 

T cell anergy, defined by the repression of cytokine production in the presence of 

cognate antigen, is a hallmark of peripheral tolerance. This process is controlled by 

poorly defined signaling through co-inhibitory molecules, such as PD-1 on T cells and its 

ligand B7H1 on antigen presenting cells. Here we show that the guanine nucleotide 

exchange factor cytohesin-3 is an important, conserved repressor of IL-2 transcription 

in anergic T cells. B7H1-mediated inhibition of PI3-kinase activation in T cells results in 

a strong induction of cytohesin-3 expression. Furthermore, cytohesin-3, which lacks a 

carboxy-terminal serine phosphorylation site present in other cytohesins, is shown to 

mediate selective inhibition of NFkB- and ERK1/2-, but not of NFAT-dependent signal 

transduction pathways. We propose that cytohesin-3, an endogenous antagonist of 

phospho-cytohesin-1 signaling, is an essential component of a novel type of inhibitory 

circuitry leading to the attenuation of immune effector functions.

Sabrina Hoffmann, Michael Winkler, Marcus Gutscher, Helmut Fickenscher, Carsten 

Watzl 

Cytomegalovirus infected fibroblasts downregulate ligands 

for the Natural Cytotoxicity receptors NKp30 and NKp44 

Activation of Natural Killer (NK) cells is controlled by an elaborate system of activating 

and inhibiting receptors. The family of Natural Cytotoxicity Receptors (NCR) plays an 

important role in NK cell activation. The members of this family, including NKp30 and 

NKp44, have been shown to be involved for NK cell activation during tumor clearance 

and the lysis of virally infected cells. Although the cellular ligands to these receptors still 

remain elusive to date, we are able to detect these ligands using novel trimeric NCR 

fusion proteins. 

Human Cytomegalovirus (hCMV) is known to introduce a vast number of changes within 

the host cell machinery upon infection. Here we examine the role of NCR ligands during 

CMV infection of primary human foreskin fibroblasts (HFF). Non-infected fibroblasts 

readily express the ligands for NKp30 and NKp44 on their surface. Upon infection HFF 

show a clear decrease in staining intensity for both ligands. Killing of HFF by human NKcells 

is partly dependent on NKp30. Interestingly, the lysis of CMV infected HFF is no 

longer dependent on NKp30. UV-inactivation of viral particles and inhibition of 

expression of CMV early genes abrogates this effect suggesting that a specific CMV gene 

product is responsible. These results demonstrate that CMV infection causes a downmodulation 

of the ligands for NKp30 and NKp44, leading to reduced activation of NK 

cells. The down-regulation of NCR ligands might therefore constitute a new strategy of 

hCMV to escape the attac of NK cells.

Doris Urlaub, Sven Mesecke, Hauke Busch, Roland Eils, Carsten Watzl 

Decision making in NK cells 

The effector functions of NK cells are controlled by a balance of positive and negative 

signals that are transmitted via various kinds of surface receptors. To date our 

understanding about the integration of positive and negative signals and the decision 

making process inside NK cells remains poor. 

With the help of bioinformatic modelling we try to understand how NK cells first 

integrate antagonising signals and then compute a reliable killing decision. Gradual 

signal input through activating and inhibitory receptors is integrated to come to a "yes 

or no" decision by the NK cell to kill an attached target cell. Triggering of activating 

receptors leads to Src kinase activation and Vav-1 phosphorylation, whereas inhibitory 

receptors dephosphorylate Vav-1 via the phosphatase SHP-1. Therefore, we proposed in 

a first hypothesis, that Vav-1 is the decision making point in the signal transduction 

network. With this hypothesis we created a simplified model describing NK cell 

activation upon various stimuli and compared the results with experimental data. A 

Lattice Gas Monte Carlo Simulation showed that increased clustering of activating 

receptors already leads to a rapid switch-like increase in Src kinase phosphorylation. We 

confirmed this experimentally by showing that increasing the amount of activating 

receptor stimulation already behaves switch-like on the level of Vav-1 phosphorylation. 

Similarly, also the engagement of inhibitory receptors leads to a switch-like 

dephosphorylation of Vav-1. 

We are currently refining our mechanistic model and are testing predictions derived 

from this experimentally. This model of NK cell regulation enables a novel insight into 

the decision making process during lymphocyte activation.

Jochen Maul, Susanne Pförtner, Robert Geffers, Kerstin Kapp, Jan Buer, Martin Zeitz, 

Rainer Duchmann 

Decreased expression of CCR4 on CD4+CD25 high regulatory T 

cells as a possible mechanism for impaired migration to 

inflamed mucosa in Crohn´s disease 

Background: Regulatory T cells (Treg) prevent and treat established colitis in animal 

models. Although previous findings show that CD4+CD25 high FOXP3+ Treg from patients 

with Crohn´s disease (CD) display normal suppressive function to allogeneic antigens in 

vitro, Treg function may be impaired in vivo. 

Aims: To elucidate possible mechanisms for impaired Treg function in CD. 

Materials and Methods: Treg and naive T cells were isolated from peripheral blood of 

patients with active (aCD; n=3) and inactive CD (iCD; n=3) and healthy controls (HC; 

n=11) using MACS. Hybridisation to a self-developed microarray (Human TReg Chip) 

enabling simultaneous expression measurement of 350 genes selected for their 

potential implication in Treg biology was performed and differential expression was 

analysed by SAM. Regulation of candidate genes was confirmed by FACS analysis with a 

different group of HC (n=5) and CD patients (n=9). 

Results: 39 genes are significantly up-regulated when comparing Treg from HC to aCD 

and 25 genes comparing HC to iCD. In both groups, 10 genes were up-regulated more 

than 5fold. Transcripts for chemokine receptor 4 (CCR4) were among the strongest 

expressed. FACS analysis showed no significant difference in the percentage of CCR4+ 

Treg comparing HC to CD (57.9±11.3 vs. 54.4±12.7), but comparison of normalized 

mean fluorescence intensity (nMFI; MFI CCR4+/MFI CCR4-) showed a significant 

decrease of nMFI in CD (15.8±3.7 vs 10.4±3.4; p=0.019). 

Conclusions: CCR4 is a possible target gene for Treg pathobiology in CD. Since the 

chemokines TARC and MDC – both ligands for CCR4 - are expressed in inflamed 

mucosa, the decreased expression of CCR4 points to an impaired mucosal Treg 

migration in CD.

Mahmoud Sadeghi, Gerhard Opelz, Volker Daniel, Cord Naujokat, Rainer 

Zimmermann, Angela Huth-Kühne, Caner Süsal 

Decreasing Soluble CD30 and Increasing IFN-γ Plasma Levels 

are Indicators of Effective Highly Active Antiretroviral 

Therapy. 

Abstract 

It was previously reported that, without highly active antiretroviral therapy (HAART), 

secretion of Th1 cytokines and antiviral IFN-γ in HIV-infected patients is decreased, 

whereas the production of Th2 cytokines, proinflammatory cytokines, and TNF-α is 

increased. We studied the effect of HAART on Th1-, Th2-, and monocyte-derived 

cytokines, and on the Th2-type immune response marker soluble (s)CD30 in HIV-1infected 

hemophilia patients. Viral Load (VL), CD4+ lymphocyte counts, and plasma 

levels of sIL-1RA, IL-2, sIL-2R, IL-3, IL-4, IL-6, sIL-6R, IL-7, IL-10, TNF-α, TGF-β2, IFNγ, 

and sCD30 were measured in 18 patients who received HAART. Nine patients were 

initially treatment-naïve and were monitored after the initiation of HAART. sCD30 

median levels were significantly higher in treatment-naïve patients than in patients who 

were on HAART (77 vs. 30U/ml, p=0.005). A strong association was observed between 

sCD30 and VL (r=0.85, p=0.004). After the initiation of HAART, sCD30 levels decreased 

and remained low (at 1 year: 38; at 2 years: 41U/ml; p=0.012 and p=0.021, 

respectively, as compared to baseline level) and this was accompanied by a decrease in 

VL and monocyte-derived IL-6 and an increase in CD4+ lymphocyte counts and Th1derived 

IFN-γ. One year after the initiation of HAART a strong inverse correlation was 

observed between IFN-γ and VL (r=-0.83, p=0.006). In contrast to sCD30 and IFN-γ, 

CD4 counts and plasma IL-6 did not correlate with VL at any time. Our data suggest 

that decreasing sCD30 and increasing IFN-γ plasma levels are indicators of effective 

HAART treatment and CD4 Th1 cell recovery in HIV-infected patients.

Nadine Voelxen, Sylvia Gutenberger, Hans-Hartmut Peter, Hermann Eibel, Klaus 

Warnatz 

Defective activation of B cells in persistent polyclonal B cell 

lymphocytosis (PPBL)? 

Persistent polyclonal B cell lymphocytosis (PPBL) is a disorder which mainly affects 

female smokers in their 4.-6. decade. The origin of this polyclonal lymphocytosis of 

atypical B cells often associated with a polyclonal rise of serum IgM is not understood. 

We therefore examined B cells of 6 PPBL patients (all female, age: 35-63 years) 

phenotypically by FACS as well as by activation in vitro. 

The characterization of the B cell subpopulations showed a polyclonal expansion of CD27 

+IgD+IgM+ memory type B cells up to 88.8% of CD19+ B cells (normal range: 7.8 – 

36%) as well as an increased number of CD21low B cells up to 30% of CD19+ B cells 

(normal range: 1.1-6.9%). There were no aberrations within the T cell compartment 

detectable. 

Functional analysis revealed a reduced proliferation especially after stimulation with anti- 

CD40, a decreased expression of CD86 on the surface of cells(17.98-70.43% with a 

mean of 37,91% compared to 53.54-73.76% with a mean of 62,59% in healthy donor), 

as well as enhanced apoptosis revealed by staining for annexin (21.69-55.81% with a 

mean of 39,76% versus 5.82-19.68% with a mean of 15,43% in healthy donor), 

despite an expansion of these cells in vivo. 

Based on our findings we currently investigate early and late signalling events 

downstream of CD40 and the B cell receptor in isolated B cells via western blotting and 

FACS to identify potential defects in these pathways.

Christine Skerka, Nadine Lauer, Claudia N Keilhauer, Lars Fritsche, Bernhard H.F. 

Weber, Peter F. Zipfel 

Defective Binding of Factor H (Y402H) and FHL-1 to CRP and 

Collagen in Age Related Macular Degeneration 

The common Y402H variant in the human complement Factor H (CFH) is linked to agerelated 

macular degeneration (AMD), a prevalent disorder leading to visual impairment 

and irreversible blindness in elderly patients. At present it is unclear how the variant of 

CFH contributes to the occurrence of drusen and the progression to AMD. In order to 

define a molecular role of CFH in AMD we purified CFH from plasma of genotyped AMD 

patients and control persons which are homozygous HH402, homozygous YY402 and 

heterozygous H/Y402. In addition we recombinantly expressed FHL-1, the alternative 

splice product of CFH, with the risk H402 and the protective Y402 variant. Functional 

tests were performed to compare these CFH and FHL-1 subtypes. The risk variants 

402H of both proteins CFH and FHL-1 showed reduced binding to C reactive protein 

(CRP). Using extracellular matrix protein array analysis reduced binding of the risk 

variants was identified to collagen 1, a major component of drusen. This reduced 

binding may cause inefficient complement regulation at the cell surface, particularly 

under conditions of inflammation, when CRP is recruited to injured sites and tissue. CFH 

and FHL-1 may act in concert and in the eye the reduced surface binding may result in 

inappropriate local complement control, which leads to inflammation, disturbance of 

local physiological homeostasis and progression to cell damage. As a consequence, 

these processes may lead to AMD pathogenesis.

Florian Börncke, Beatrix Pollok-Kopp, Mladen V. Tzvetkov, Martin Oppermann 

Defective Binding to C-Reactive Protein and Impaired 

Cofactor Activity of the Allotypic Y402H Variant of 

Complement Factor H 

Common polymorphisms in the human complement Factor H (FH) gene have been 

associated with either enhanced or reduced risk of developing age-related macular 

degeneration. Using FH preparations which were isolated from the sera of genotyped 

individuals we show that allotypic variation of two different amino acids (Y402H and 

I62V) affects binding of native FH to C-reactive protein (CRP), but not to C3b, heparin 

or retinal pigment epithelial cells. Variant-specific monoclonal antibodies which 

recognize distinct SCR7 epitopes showed that amino acid 402 not directly interacts with 

CRP, but probably indirectly affects SCR7 conformation. While both polymorphic FH 

forms showed similar cofactor activities for the Factor I-mediated C3b cleavage, the 

resulting iC3b fragment was much less efficiently converted to C3dg in the presence of 

the FH H402 risk variant compared to FH Y402. Reduced binding of FH H402 via CRP to 

cellular surfaces and sustained expression of iC3b, the major ligand for complement 

receptors CR3 and CR4 on macrophages, could result in enhanced inflammation in the 

subretinal space of AMD patients.

Frank Guenther, Gertud Maria Hänsch, Christof Wagner 

DEFENCE AGAINST BACTERIAL BIOFILMS: ROLE OF 

POLYMORPHONUCLEAR NEUTROPHILS (PMN) 

The formation of bacterial biofilms is increasingly recognised as the leading cause of 

chronic infections, particularly in patients with implanted devices, such as catheters, 

artificial heart valves or orthopaedic prostheses. It is generally assumed that the 

infection persists because bacteria organised as biofilms escape the host defence 

mechanisms. On the other hand we observed massive infiltration of leukocytes, 

predominantly of PMN, into the site of infection. To examine the question how PMN 

interact with bacterial biofilms, Staphylococcus aureus were cultivated under conditions 

allowing biofilm formation. The biofilms were then opsonised with normal human serum 

(NHS), immunoglobulin-depleted NHS, or complement-inactivated NHS and incubated 

with PMN derived from healthy donors. Interactions were observed by time lapse video 

microscopy, and by confocal laser scan microscopy. PMN adhered to, migrated on and 

into the biofilm. The PMN aggregated considerably, and eventually the formation of 

large cells clusters was observed. The clusters contained also extracellular PMN-derived 

DNA, consistent with the formation of neutrophil extracellular traps (NETs). Bacteriafree 

zones appeared around the PMN, and phagocytosis, apparent as uptake of bacteria 

into the PMN, was also observed. Depletion of bacteria and efficiency of phagocytosis 

depended on the opsonisation of the biofilm and of its maturation in vitro. Taken 

together, our data provide evidence that bacteria in biofilms are not entirely protected 

against host defence but that phagocytosis is still possible. Whether NET and cluster 

formation contributes to bacteria killing in biofilms cannot be decided as yet, but 

remains an attractive alternative.

Tobias Schulze, Katrin Räbel, Sven Golfier, Martin Lipp 

Deficiency in Sphingosine-1-phosphate receptor 4 (S1P4) 

results in deviated humoral immune responses 

The receptors S1P1 and S1P4 represent the major populations of Sphingosine-1phosphate 

receptors expressed on lymphocytes. Besides various functions in other cell 

types of the immune system, S1P1 is required for egress of lymphocytes from 

peripheral lymph nodes and thymus as well as B-cell positioning within the spleen. The 

role of S1P4 in lymphocyte homeostasis has not been well established yet. In order to 

assess the biological function of the later receptor, we have generated a murine S1P4-/model. 

Overall B and T cell distribution in secondary lymphoid organs were not significantly 

different in S1P4-/- and wild type (WT) mice. However, analysis of immunglobuline 

isotypes showed significant differences concerning IgG, IgA and IgE levels in the plasma 

and serum. Interestingly, IgA levels were similar in brocheal lavage fluids of S1P4-/- 

and WT mice. Epicutaneous challenge with FITC of previously sensitised mice revealed 

increased reactivity in the S1P4-/-mice. In contrast, the type IV hypersensitivity 

response were significantly decreased in S1P4-/- mice. 

Our findings in the S1P4-/- mice indicate that, although there are no imbalances in 

overall T- and B cell distribution, S1P4 is implicated in the polarisation of the immune 

response. Interestingly, it has been reported that S1P1 bias the immune response 

towards an TH2 phenotype. The possibility of a potential interaction between S1P1 and 

S1P4 in the polarisation of the immune response is currently assessed using lentiviral 

based shRNA mediated S1P1 knock down in S1P4-/- mice.

Anja Erika Hauser, Tobias Junt, Thorsten R. Mempel, Michael W. Sneddon, Steven H. 

Kleinstein, Sarah E. Henrickson, Ulrich H. von Andrian, Mark J. Shlomchik, Ann M. 

Haberman 

Definition of Germinal Center B Cell Migration In Vivo Reveals 

Predominant Intra-zonal Circulation Patterns 

The formation of germinal center (GC) derived high affinity memory B cells and plasma 

cells constitutes an important element of an effective adaptive immune response. 

Proliferation, mutation and selection in the GC are thought to occur in distinct 

microanatomical compartments—the dark zone (DZ) and the light zone (LZ). The DZ 

compartment is located more proximal to the T cell zone. It is primarily comprised of 

blasting B cells and represents the site where somatic hypermutation occurs. The LZ is 

characterized by the presence of follicular dendritic cells (FDCs), which are able to trap 

antigen (Ag) in the form of immune complexes on their surface. Affinity maturation has 

been posited to require frequent trafficking between zones: newly generated GC B cells 

have to migrate from the DZ to the LZ in order to test their BCR affinity against the Ag 

on the FDCs. After positive selection, they migrate back to the DZ for further 

proliferation. 

Here, we report the use of multi-photon in vivo microscopy to determine migration 

patterns of GC B cells. Analysis of time-resolved images revealed unexpected patterns 

of movement as well as GC B cell morphology. In contrast to an anticipated frequent 

movement between the DZ and LZ, few cells were observed to cross the DZ/LZ 

interface. Moreover, cell track trajectories indicated that cell movement in this region is 

predominantly parallel to the interface, suggesting that B cells circulate within individual 

LZ and DZ compartments. The results suggest a revision to our views of B cell 

circulation within the GC and the functional relationship of its two major compartments.

Christine Skerka, Mihály Józsi, Stefanie Strobel, Stefan Heinen, Matthew Edey, Svante 

L. H. Zipfel, Judith A Goodship, Timothy H.J. Goodship, Christoph Licht, Peter F. Zipfel 

Deletion of CFHR1 and CFHR3 correlates with presence of 

Factor H autoantibodies in hemolytic uremic syndrome 

Atypical Hemolytic Uremic Syndrome (aHUS) is a severe renal disease that is caused by 

defective complement regulation and multiple factors predispose to the disease. Disease 

associated mutations have been described in the genes encoding the complement 

regulators factor H (CFH), membrane cofactor protein (MCP), factor I (IF)) and factor B 

(FB). We showed in two independent cohorts of aHUS patients that a chromosomal 

deletion of 84 kb in the RCA gene cluster results in the loss of the genes coding for 

factor H related proteins CFHR1 and CFHR3 and increases the risk for aHUS. Here we 

identify in the same cohort of 121 aHUS patients 16 juvenile individuals (i.e. 13%) who 

are positive for CFH autoantibodies. Interestingly 13 of these individuals showed also 

homozygous CFHR1 and CFHR3 deficiency. Family studies showed a strong correlation 

of the presence of autoantibodies and CFHR1/CFHR3 deficiency with HUS and that the 

absence of CFHR1/CFHR3 alone represents a risk factor for HUS. Using a novel protein 

nanoarray with domain mapped Factor H monoclonal antibodies (mAb) the binding 

epitope of all analyzed autoantibodies was localized to the C-terminus of Factor H. This 

domain harbors the cell binding region of the complement inhibitor and represents a hot 

spot for HUS associated mutations. In hemolytic assays two C-terminally binding mAbs 

C02 and C18 which compete for autoantibody binding and serum from autoantibody 

positive patients showed enhanced lysis of unsensitized sheep erythrocytes. Thus we 

conclude that CFHR1/CFHR3 deficiency is a risk factor to develop Factor H 

autoantibodies that are associated with HUS.

Thorsten Feyerabend, Annette Tietz, Herve Luche, Freddy Radtke, Hans Joerg 

Fehling, Hans Reimer Rodewald 

DELETION OF NOTCH-1 IN KIT+ PRO-T CELLS BLOCKS T CELL 

DEVELOPMENT, BUT DOES NOT CONVERT T CELL 

PROGENITORS INTO THYMIC B CELLS 

Loss of Notch-1 in bone marrow cells, or interference in the thymus with Notch-1-Notch 

ligand interactions is associated with a block in T cell development in favor of aberrant B 

cell development. It is, therefore, widely accepted that Notch-1 signaling determines 

the developmental choice within single uncommitted progenitors between the two major 

lymphocyte lineages. To ‘fate map’ hematopoietic pathways in vivo, we have generated 

a Cre recombinase mouse (Mc-cpa Cre ) in which Cre expression is controlled by a 

myeloid gene locus (mast cell-carboxypeptidase A [Mc-cpa]). Surprisingly, when 

crossed to a Cre-dependent red fluorescent protein (Rosa-RFP flox ) indicator mouse, Mccpa 

Cre labels ~90% of T but only ~10% of B lineage cells. Labeling along the T cell 

pathway takes place very early in the thymus in a major fraction of Kit + pro-T cells. We 

have taken advantage of this unprecedented and T cell-biased onset of Cre-expression 

to delete Notch-1 in pro-T cells while simultaneously turning on RFP. Cre-mediated 

Notch-1 deletion recapitulates the thymic Notch-1 deficiency phenotype, i.e. a block in T 

cell development at the apparent expense of B cell development. However, the fraction 

of RFP-labeled thymic B cells is not increased under conditions of Notch-1 deletion in 

pro-T cells. We conclude that loss of Notch-1 in Kit + pro-T cells blocks T cell 

development but does not convert T cell progenitors into B cells. The abundant 

immature thymic B cells that arise under Notch-1 deficiency represent T-lineage 

independent B cells that might capitalize on the free niches in the thymus made 

available by fading Notch-1 -/- pro-T cells.

Carsten Wiethe, Alexander Steinkasserer, Manfred Lutz, Andre Gessner 

Dendritic cell differentiation state and their interaction with 

NKT cells determines Th1/Th2 differentiation in the murine 

model of Leishmania major infection 

Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial 

signals differently, redefining their classical subdivision into an immature endocytotic 

and a mature antigen-presenting differentiation stage. While both signals induce DC 

maturation by upregulating MHC II and costimulatory molecules, only Toll-like receptor 

signals are able to trigger proinflammatory cytokine secretion by DC, including Th1polarising 

IL-12. 

Here, we explored the murine Leishmania major infection model to examine the CD4+ T 

cell response induced by differentially matured DC. Initial experiments showed that 

pulsing DC with L. major lysate did not affect DC maturation with TNF&alpha (TNF-DC) 

or LPS+anti-CD40 (LPS+CD40-DC). When partially matured TNF-DC were injected into 

Balb/c mice prior to L. major infection, the mice failed to control infection and 

developed a Th2 response which was dependent on IL-4R&alpha signaling. In contrast, 

injections of fully matured LPS+CD40-DC induced a Th1 response controlling the 

infection. Furthermore, when the expression of different Notch ligands on DC was 

analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DC whereas 

LPS+CD40-DC upregulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 

polarization induced by TNF-DC required interaction with CD1d-restricted NKT cells. 

However, NKT cell activation by L. major lysate-pulsed DC was not affected by blockade 

of the endogenous glycolipid suggesting exchange with exogenous parasite-derived CD1 

glycolipid antigen. 

In sum, the differentiation stage of DC as well as their interaction with NKT cell 

determines Th1/Th2 differentiation. These results have generic implications for the 

understanding of DC instructed Th cell responses and the development of improved DC 

vaccines against leishmaniasis.

Stefan A. Kaden, Juergen Schmitz, Gregor Winkels 

Dendritic Cell immuno-activating receptor 1 – 

Characterization of a novel member of the C-type Lectin family 

C-type lectins (CLECs) are involved in a variety of immune interactions, in which they 

have been shown to act as both, pathogen recognition as well as adhesion receptors. 

The recently described murine dendritic cell (DC) immuno-receptor (DCIR)/DC immunoactivating 

receptor (DCAR) family consists of six type II CLECs clustered on 

chromosome 6. Beside the already characterised mDCIR, mDCIR2 (33D1) and mDCAR, 

little is known about mDCAR1, mDCIR3 and mDCIR4. 

To study phenotype and function of these gene products, we generated monoclonal 

antibodies (mAbs) against mDCAR1. In contrast to the previously published gene 

expression profile of DCIR/DCAR family genes, in which mDCAR1 was found to be 

restricted to NK cells (NK), we identify the receptor in bone marrow on subsets of F4/80 

+ macrophages and Ly6G + granulocytes, but not on NK or DC. Noteworthy, in thymus, 

expression of mDCAR1 is restricted to CD11c high DC. mDCAR1 + thymocytes comprise a 

typical myeloid DC phenotype characterised by the expression of CD8α and CD205, as 

well as the absence of CD11b. These mature DC express high amounts of MHCII and 

the costimulatory molecules CD40, CD80, CD86. mDCAR1 + thymic DC are positive for 

CD54, CD90, the stem cell and progenitor markers Sca-1 and c-kit (CD117), as well as 

the thymic specific DC marker BP-1. 

The successful generation of mAbs allowed us to identify the expression of mDCAR1, 

being the first step to uncover a presumably interesting function of this representative 

of the CLEC receptor family in the immune system.

Marcin •yszkiewicz, Natalia Zi•tara, Manfred Rohde, Kurt Dittmar, Jadwiga 

Jab•o•ska, Siegfried Weiss 

Dendritic cell like function of ER-TR9 + marginal zone 

macrophages 

ER-RT9 + marginal zone macrophages (MZM)) are found in the spleen at a strategic 

position for capturing blood borne antigen and pathogens. They are located where the 

central arteriole opens into the splenic sinus and form a tight network by extending long 

dendroid like protrusions. This DC like property prompted us to investigate the antigen 

presenting capacity of such cells. ER-TR9 + MZM were able to present peptide and 

soluble antigen to naïve CD4 T cells in vitro. Interestingly, such cells were also able to 

efficiently present peptide to major histocompatibility complex MHC class I restricted 

naïve CD8 T cells. More importantly, ER-TR9+ MZM cells were able to efficiently 

processe native OVA and cross-present it to OT-I T cells in vitro while other splenic 

macrophages (MF), like metalophillic MF and red pulp MF were unable to do so. This 

cross-presenting ability of ER-TR9 + MZM was found for soluble OVA as well as for latex 

bead-associated OVA. ER-TR9 + MZM, in addition to CD8a + DC and plasmacytoid DC, are 

effective in phagocytosis of Listeria monocytogenes that invade the spleen. By ex-vivo 

studies, we could show that both ER-TR9+ MZM and CD8α + DC were able to efficiently 

cross-present antigen secreted by L. monocytogenes to CD8 T cells. Our data identified 

ER-TR9+ MZM as professional splenic APCs with DC like properties. They are able to 

cross-present antigen and to activate naïve CD4 and CD8 T cells.


Dendritic cells control awareness of T lymphocytes for antigen 

In the body, dendritic cells (DCs) are in continuous cointact with T lymphocytes. In 

order to investigate potential consequences of this contact, we generated genetically 

modified mice that allow long-term depletion of DC. In the respective BAC CD11c.DTR 

transgenic mice, designated CD11c.DOG, the human diphtheria toxin receptor (DTR) is 

expressed in CD11c-positive DC. Depletion with diphtheria toxin (DT) eliminated more 

than 90-95% of DC. However, the DCs were regenerated within 3-4 days so that for 

long-term ablation repetitive injections of DT were required. 

CD4 T cells isolated from mice lacking DC exhibited a drastic decline in basal TCR 

signalling as indicated by decreased phosphorylation of the TCR ζ chain. No differences 

were observed in CD4 or TCR β surface expression. The reduced DC - T cell contact had 

profound functional consequences for the response of T cells to antigen. Naive CD4 T 

cells from DT-treated CD11c.DOG x 2D2 mice, which harbour a transgenic TCR reactive 

against the Ab/MOG35-55 determinant, failed to respond to MOG35-55 peptide 

presented by irradiated splenocytes. Likewise, T cells from DC-deficient mice showed a 

drastically reduced response to SEA superantigen presented by functional DC. 

The dramatic effect of the absence of DC on basal T cell sensitivity adds another major 

role to the wide range of functions of DC. Thus, in addition to being crucial for the 

balance between T cell immunity and tolerance, DCs provide continuous stimulation for 

naive T cells, thereby keeping T cells in a state of awareness that allows them to 

effectively respond to foreign antigens.

Mathias Lucas, William Schachterle, Karin Oberle, Peter Aichele, Andreas Diefenbach 

Dendritic Cells Prime Natural Killer Cells 

by trans-Presenting Interleukin 15 

Natural killer (NK) cells are lymphocytes of the rapidly acting innate immune system 

that play an essential role in the recognition and eradication of virally infected cells and 

tumors. Recent in vitro data suggested that dendritic cells (DC)- or macrophage-derived 

cytokines are able to enhance NK cell functions. However, it is unknown if NK cell 

effector responses in vivo depend on the NK cells’ interaction with myeloid cells. Using a 

mouse model for the inducible ablation of DC, we show that the in vivo priming of NK 

cell responses to viral and bacterial pathogens depended on the presence of CD11c high 

DCs. After peripheral Toll-like receptor (TLR) stimulation, priming of NK cells required 

their recruitment to local lymph nodes and interaction with DCs resulting in the 

emergence of effector NK cells in the periphery. NK cell priming was dependent on the 

recognition of type I IFN signals by DCs and the subsequent production and transpresentation 

of IL-15 by DCs to resting NK cells. CD11c high DC-derived IL-15 was 

necessary and sufficient for the priming of NK cells. Our data define a unique in vivo 

role of DC for the priming of NK cells, revealing a striking and previously unappreciated 

homology to T lymphocytes of the adaptive immune system. These results have 

important implications for the development of immunotherapeutic strategies aiming to 

boost NK cell effector

Anja Saalbach, Claudia Klein, Ulf Anderegg, Jan C. Simon 

Dermal fibroblasts induce maturation of dendritic cells 

To trigger an effective T cell-mediated immune response in the skin, dendritic cells (DC) 

migrate into locally-draining lymph nodes where they present antigen to naive T cells. 

During their migration to secondary lymphoid organs, DC have to travel through the 

stromal microenvironment comprised of the extracellular matrix and stromal cells such 

as fibroblasts, macrophages and endothelial cells. Little is known about the interaction 

of DC with these various cellular microenvironments. Here, we show that DC are located 

in close proximity to stromal fibroblasts in inflamed skin. In accordance, a Thy-1- and 

ICAM-1-dependent adhesion of DC to fibroblast could be detected in vitro. Co-culture 

experiments demonstrate that fibroblasts are effective in inducing both phenotypic and 

functional maturation of DC in a TNF alpha dependent manner. The resulting fibroblastmatured 

DC are able to support T cell driven immune responses reflected by CD25 

expression and enhanced T cell proliferation [1]. Together these data demonstrate that 

dermal fibroblast which DC can encounter during their trafficking from skin to lymph 

node may act as potent regulators of DC differentiation and function, and thus may 

actively participate in the regulation and outcome of DC-driven cutaneous immune 

responses. 

[1]Saalbach et al.; J Immunol, 2007, 178: 4966–4974.

Elke Pogge von Strandmmann, Boris Böll, Daniel Re, Andreas Engert, Venkateswara 

Simhadri 

Detection of HLA-B associated transcript 3 (BAT3) in sera 

from Hodgkin Lymphoma patients and its release from 

Hodgkin lymphoma cells 

Major triggering NK cell receptors such as NKG2D and the Natural Cytotoxicity 

Receptors Nkp30, 44 and 46 are critically involved in tumor cell recognition and 

surveillance, since a decrease of the corresponding ligands on tumor cells correlates 

with impaired NK cell-dependent killing and tumor progression. However, sustained 

expression and the release of soluble ligands for the NKG2D receptor negatively 

imprints the local and systemic immune response and correlates with a poor prognosis 

for haematological and epithelial malignancies. 

Hodgkin Lymphoma (HL) patients have impaired NK cell activity in the peripheral blood 

and the level of NK anergy correlates with a bad prognosis. Since spleen derived NK 

cells exhibit normal or increased activity, a serum-derived factor is probably involved in 

NK cell inhibition. In order to asses the role of HLA-B-associated transcript 3 (BAT3), we 

screened the sera of healthy donors and early and late stage HL patients using a BAT3 

specific sandwich-ELISA. BAT3, recently characterized in our lab, is a tumor-released 

ligand, that engages the triggering Natural Cytotoxicity Receptor NKp30. The BAT3 

serum level was significantly elevated in HL patients in comparison to healthy donors 

(p=0.0002). Interestingly, the early stage patients had a more pronounced increase 

compared to the advanced stage patients (p=0.024). We next analyzed whether cellular 

stress signals, such as HDAC inhibition and proteasomes inhibition could modulate the 

expression/release of BAT3 from Hodkin lymphoma-derived cell lines. A panel of cell 

lines were incubated with subtoxic concentrations of valproate and bortezomib and the 

supernatants were collected for a BAT3-specific ELISA. A significant increase of BAT3 in 

response to both substances was detected, that was irrespective of the IkB-a mutation 

status of the cell lines. The analysis of released BAT3 using fractionation and Western 

blotting revealed that different BAT3 isoforms were secreted, that may exhibit distinct 

modulation of NK cell-activity.

Leander Grode, Hans-Heinrich Henneick v. Zepelin, Albrecht Laeufer, Bernd Eisele 

Developing a TB vaccine for human use 

Vakzine Projekt Management GmbH (VPM) acquires promising vaccine candidates from 

academia, develops them with a consortium of partners and commercializes the results. 

With this goal VPM acquired a Tuberculosis vaccine candidate (VPM1002) from Max- 

Planck Institute of Infection Biology and develops this candidate through all necessary 

stages onto clinical Phase I and II. The project is divided into the three parts technology 

transfer & production, preclinic & pharm/tox and clinical Phase I and II. 

VPM1002 is a recombinant bacterial vaccine candidate for the prevention of tuberculosis 

for residents in endemic areas and persons at risk in non-endemic areas. 

It is well known that the existing BCG vaccine offers only little, if any, protection against 

pulmonary tuberculosis in adults. VPM1002’s strength is its ability to induce a CD8 T cell 

response which is crucial in immunity to M. tuberculosis. 

Recombinant M. bovis BCG expressing Listeriolysin (Hly) is able to induce poreformation 

in the phagosomal membrane and to leave the mycobacterial phagosome. 

The lysis by the Hly can be further enhanced by lowering the pH. Therefore the urease 

activity was knocked out by destruction of the ureC gene. Hly promotes antigen 

translocation into the cytoplasm and apoptosis of infected target cells, thus promoting a 

more profound immune response comprising both antigen-specific CD4 and CD8 T cells. 

VPM1002 is manufactured by a novel submerse fermentation in minimal medium. The 

final product is a lyophilised cake of live bacteria. Establishment of a GMP process is 

completed. The process has been designed to offer full scalability. The GLP 

pharmacology is ongoing. The clinical phase I is planned for 2007.

Stefan Porubsky, Anneliese O. Speak, Bruno Luckow, Vincenzo Cerundolo, Frances M. 

Platt, Hermann-Josef Gröne 

Development and function of invariant natural killer T cells in 

mice with isoglobotrihexosylceramide (iGb3) deficiency 

Invariant natural killer T (iNKT) cells represent a distinct lymphocyte population which 

co-express natural killer (NK) surface markers such as NK1.1 (CD161) and a T cell 

receptor (TCR), which is composed of an invariant TCR-chain encoded by Vα14-Jα18 

gene segments in mice and Vα24-Jα18 in humans. iNKT cells play an essential role in 

immunoregulatory processes, such as tolerance, host defense and tumor surveillance. 

iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4+/ 

CD8+ cortical thymocytes, but the identity of the endogenous lipid(s) responsible for 

positive selection of iNKT cells remains unclear. One candidate lipid proposed to be 

responsible for the positive selection is isoglobotrihexosylceramide (iGb3). To directly 

investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in 

iGb3 synthase (iGb3S, also known as α1-3galactosyltransferase 2, A3galt2). These mice 

developed, grew and reproduced normally and exhibited no overt behavioral 

abnormalities. Consistent with the notion that iGb3 is only synthesized by iGb3S, we 

demonstrate the lack of iGb3 in iGb3S-/-, as compared to iGb3S+/- mice. iGb3S-/- mice 

showed normal numbers of iNKT cells in the thymus, spleen and liver with selected TCR 

Vβ chains identical to controls. Upon administration of α-galactosylceramide, activation 

of iNKT and dendritic cells was similar in iGb3S-/- and iGb3S+/- mice, as measured by 

up-regulation of CD69 as well as intracellular IL-4 and IFN-γ in iNKT cells, up-regulation 

of CD86 on dendritic cells and rise in serum concentrations of IL-4, IL-6, IL-10, IL- 

12p70, IFN-γ, TNF-α, Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to 

be an endogenous CD1d lipid ligand determining thymic iNKT selection.

Nina Wantia, Tanja Ertl, Christine Cirl, Nuria Rodriguez, Hermann Wagner, Thomas 

Miethke 

Development of a protein and CpG-based vaccination against 

Chlamydophila pneumoniae 

Chlamydophila pneumoniae induces a pneumonia in C57/BL6 mice after nasal 

application of 1,75x10 6 IFU (infectious units). Six days after infection the mice were 

sacrificed, and chlamydial burden was measured in cell culture and by real-time PCR. 

First we investigated whether protection is achievable, hence mice were low-dose 

(0,5x10 6 IFU) infected three weeks before standard infection with 1,75x10 6 IFU. They 

hardly lost weight and showed no clinical signs of infection. Chlamydial burden was 

reduced to 0,2% in these mice compared to control mice. When we examined lung T 

cells of these mice, we could show that after restimulation with infected dendritic cells 

17,2% of CD4 + cells were IFN-γ positive (mock-infected mice: 0,055%). IFN-γ 

producing T cells are known to be protective against chlamydial infection. 

To generate a protective immunity against Cp. pneumoniae we tested several different 

chlamydial proteins. Membrane proteins of Cp. pneumoniae are accessible to the 

immune system and polymorphic membrane proteins (PMP) have been of 

immunological interest recently. Although the exact function of this new protein family 

is still unknown, it is known that they are able to induce neutralizing antibodies in 

Chlamydia trachomatis and are promising candidates for vaccination, especially PMP21 

and PMP10 and 11 are auspicious for protective vaccination. 

Protein and CpG 1826 were injected subcutaneously, followed by the infectious 

challenge three weeks later. Vaccination with OMP2 reduced chlamyidal burden to 

10,0% compared to correspondig control mice. PMP21 could even decrease chlamydial 

burden to 4,6%, whereas weight loss and lung weight was just slightly decreased in 

both cases. To induce better protection including less weight loss and lung weight we 

trapped the proteins in a organic polymer of Poly-DL-lactide-co-glycolide. This polymer 

was shown to release the antigen slowly into the cell and induces robust T cell 

responses.

Augustin J Kerkdijk, Gerhard Held, Antje Mueller, Wolfgang L Gross, Michael 

Pfreundschuh, Jan Voswinkel 

Development of a System to Test for Specificity of B-cell 

Receptors found in Granulomatous Lesions of Wegeners 

Granulomatosis patients 

Central in the pathogenesis of Wegeners granulomatosis (WG) is the PR3-ANCA, an 

antibody with specificity against proteinase-3 (PR3), a serine protease which is 

expressed on primed neutrophilic granulocytes. The ANCA-antigen-interaction leads to 

vasculitis. The biphasic nature of WG starting with a localised granulomatous 

inflammation followed by systemic necrotising small-vessel vasculitis seems to have a 

pathophysiological background in that the granulomatous disease gives rise to the 

systemic vasculitis by ANCA-priming. 

The granulomatous lesions of WG patients have been examined and germ-like centers 

have been discovered with B-cells and plasma cells in the vicinity of PR3-protein. We 

hypothesized that these germinal-center-like structures function as a tertiary lymphoid 

structure in which the plasma cells which produce the PR3-ANCAs are formed and that 

PR3 might be the antigen upon which affinity maturation takes place. The goal of this 

study was to develop a system which allows us to investigate the specificity of the Bcells 

found in the granulomatous lesions of WG patients. We characterised the antibody 

coding DNA derived from individual B-cells found in the granulomatous lesions and 

cloned this DNA into a phagemid vector which allowed us to produce fab-fragments, 

which are antibodies lacking the Fc-fragment. These fab-fragments are analogous to the 

B-cell receptor from which the DNA was derived. In this way we investigated six B-cells 

from granulomatous lesions of WG patients for their specificity against PR3 using ELISAs 

and Western blots. Furthermore we panned the fab-fragments against a phage randompeptide 

library to find other possible epitopes. We could not find fab-fragment binding 

against PR3 in ELISA or western blot analysis and we could not characterize a currently 

known peptide as a target fo the fabs in the random peptide library. This might be due 

to conformational differences between B-cell receptors and fab-fragments which render 

the latter dysfunctional or to low affinity of ANCA- binding.

Nadja Hilger, Rico Hiemann, Jörg Michel, Ursula Anderer, Martin Weigert, Ulrich Sack 

Development of an completely automatized system for image 

aquisition and detection of HEp-2 immunofluorescence 

patterns 

The standard screening test for the diagnosis of autoimmune diseases is the detection 

of autoantibodies in serum of patients by indirect immunofluorescence (IIF) based on 

HEp-2 cells. Manual evaluation of this test is very subjective, slow and there are no 

objective parameters as guidelines available. Interlaboratory tests show occasionally 

large deviations in the test evaluation resulting in a high variance of results. The aim of 

this project is the fast, objective, safe and economical automatic analysis of HEp-2 IIF 

patterns. Images of IIF patterns were completely automatically captured by use of an 

inverse motorized fluorescence microscope. Thereby, device-specific parameters were 

controlled automatically, too. For fast analysis of IIF patterns new algorithms of image 

processing were developed. Artifacts were recognized and excluded from analysis by the 

developed software. Analysis of more than 80.000 images clearly demonstrated full 

automatization and fast processing of IIF patterns. Additionally serum-specific 

fluorescence could be easily distinguished from background. Even very weak but 

positive patterns can be recognized and used for diagnosis. A detailed separation into 

different basic patterns is possible. 

Objective, fast and disease-related economical analysis of HEp-2 immunofluorescence 

patterns is feasible. The implemented software algorithms allows a mathematically way 

of describing IIF patterns and can therefore be a useful tool for the needed 

standardisation process.

Anne Brüstle, Sylvia Heink, Magdalena Huber, Christine Rosenplänter, Christine 

Stadelmann, Philipp Yu, Enrico Arpaia, Tak W. Mak, Thomas Kamradt, Michael Lohoff 

Development of inflammatory Th17 cells requires interferon 

regulatory factor 4 

The transcription factor IRF4 is essential for T helper 2 (Th2) development. We show 

here that IRF4 is also critical for generating Th17 cells, pro-inflammatory cytokineproducing 

cells associated with autoimmune diseases like experimental autoimmune 

encephalomyelitis (EAE). IRF4-deficient (IRF4-/-) mice did not develop EAE and IRF4-/- 

Th cells failed to differentiate into Th17 cells. Transfer of IRF4+/+ Th cells rendered 

IRF4-/- mice susceptible to EAE. IRF4-/- Th cells showed reduced expression of RORgt 

and increased expression of Foxp3, transcription factors important for Th17 and 

regulatory T cell differentiation, respectively. The dysregulation of both factors 

contributed to the phenotype of IRF4-/- Th cells . Our data position IRF4 at the center 

of Th development, influencing not only Th2 but also Th17 differentiation.

Christian Menge, Evelyn A. Nystrom 

Dexamethasone depletes γδT cells and alters the activation 

state and responsiveness of bovine peripheral blood 

lymphocyte subpopulations 

Administration of dexamethasone (DEX) to cattle is commonly used in models of stressinduced 

effects on the host defense. Even though the effects of DEX on the bovine 

adaptive immune are not fully elucidated yet. Previous studies showed that lymphocyte 

subsets are differentially affected by DEX. The objective of the present study was to 

characterize subsets of circulating lymphocytes in calves prior to and 48 h after the 

onset of parenteral DEX treatment. Treatment significantly reduced the overall 

percentage of circulating lymphocytes, and disproportionately depleted the population of 

γδTCR+/CD8α- cells. Analysis within the CD8α+ population of T cells further revealed 

that DEX treatment also reduced the CD8αlow subset of γδT cells coexpressing the 

activation marker ACT-2+. By contrast, DEX treatment did not affect the percentage of 

CD8αlow/CD25+ cells, indicating that cells with a special activation state were affected. 

Despite a sharp increase in the number of CD25+ PBMC from DEX-treated calves, only 

marginal differences were noted in the percentages and the proliferative capacity of the 

major lymphocyte subsets. Transcription of several Th-prototype cytokines (IL-2, IFN-γ, 

IL-4, TGF-β) was reduced in short term-PBMC cultures from DEX-treated calves, sparing 

il-10. The results of this study extend the evidence that DEX treatment does not 

generally suppress the bovine immune system but has a number of different effects on 

different lymphocyte subpopulations. This information must be considered when utilizing 

DEX treatment to improve bovine infection models.

Christian Menge, William C. Stoffregen, Joachim F.L. Pohlenz, Evelyn A. Nystrom 

Dexamethasone differentially down-regulates L-Selectin 

(CD62L) expression by bovine lymphocyte subsets in vivo and 

depletes the intestinal mucosa of intraepithelial γδT cells 

Intraepithelial lymphocytes (IEL) mainly are non-proliferating cells and less prone to the 

anti-proliferative effect of dexamethasone (DEX). Since DEX also alters the adhesion 

molecule expression, we hypothesized that DEX treatment of cattle affects IEL by 

interfering with lymphocyte homing. The objectives of the study were to investigate the 

effects of parenteral DEX treatment of calves (1) on the expression of CD62L - a 

prototype adhesion molecule - by peripheral lymphocyte subsets and (2) on the 

composition of intestinal IEL. Quantitation of CD62L on peripheral lymphocytes showed 

that DEX treatment down-regulated CD62L 48 h after first treatment. Consistent with a 

principal reduction of CD62L expression on γδT cells, the effect mainly pertained to CD4- 

and to CD8αlow cells. CD8αhigh lymphocytes were not affected. Analysis of IEL 

preparations from ileum, cecum, and distal colon 96 h after onset of treatment showed 

that DEX depleted intraepithelial γδT cells, some of which coexpressed CD8α. Variable 

effects of DEX treatment on other subsets and expression of activation markers by γδTCR 

+ and CD8α+ T cells were noted in ileum, colon, and cecum. Cytokine profiling (mRNA 

of mucosal tissue scrapings) showed that DEX treatment lowered the amounts of il-2 

and il-4 but increased il-8 and ifn-γ transcripts. Despite the variable effects of DEX 

treatment on IEL composition, changes in cytokine transcription were similar throughout 

the intestine. The study provides first evidence that differential effects of DEX on 

adhesion molecule expression by lymphocyte subsets alters the composition of intestinal 

IEL in cattle.

Eva Rieser, Monika Braun, Barbara Simm, Barbara Mosetter, Christine S. Falk 

Differences between cytotoxicity and cytokine expression are 

due to different phosphorylation patterns 

Introduction: The plasticity of human natural killer cells is reflected by the individual 

expression pattern of activating as well as inhibitory receptors. Besides this receptor 

expression, the amplitude of activity is determined by the intracellular signaling status. 

Previous studies have shown that NK cells expressing a similar receptor repertoire do 

not necessarily resemble identical activity regarding cytotoxicity and cytokine 

expression. Therefore, the differences in the intracellular signaling status of each NK cell 

may influence its reactivity to receptor-mediated triggering. Presuming that 

dysfunctions in different signaling cascades might explain the hyporeactivity of certain 

NK cells, we analyzed the intracellular phosphorylation patterns in response to various 

stimuli. We used several NK lines that differ in cytotoxicity and cytokine secretion, 

respectively. 

Materials and methods: Three NK lines were compared regarding cytotoxicity (CD107a 

degranulation) and cytokine secretion (multiplex technology). Phosphorylation patterns 

following PMA/ionomycin and receptor-mediated stimulation were determined by 

phosphoplex analysis quantifying total amounts of more than 10 kinases and their 

phosphorylated proportions. 

Results: Substantial differences were observed in their individual cytolytic activity and 

cytokine secretion pattern. In addition to these functional differences, the 

phophorylation status and the overall amount of signaling molecules also varied 

substantially between these NK lines. Our results implicate a correlation between low 

cytotoxic activity a special set of cytokine production and less phosphorylation for most 

of the tested signaling proteins. Therefore, receptor expression alone is not sufficient for 

NK function because the kinase compositions and the phosphorylation cascades 

determine the individual reactivity and, thus, plasticity of NK cells.

Marcel Andre Krüger, Kathrin Kopplin, Nadine Unterwalder, Christian Meisel, Hans- 

Dieter Volk, Gerald Grütz 

Differences in Lipopolysaccharide and Lipid A desensitisation 

Despite of great efforts, sepsis is still one of the major causes of death in intensive care 

units. It is characterised by an overproduction of pro-inflammatory cytokines in the 

early phase. This can lead to a systemic inflammatory response syndrome (SIRS). In 

prolonged sepsis the overproduction of anti-inflammatory cytokines is becoming more 

and more important due to a counter-regulation. This can develop into a compensatory 

anti-inflammatory response syndrome (CARS), also called immunoparalysis. During 

CARS the antigen-presentation and TNF-alpha production of monocytes is down 

regulated systemically, while the production of anti-inflammatory mediators like IL-10 

and IL-1RA is high. Due to this, the immune function is very low. 

A common in vitro model for CARS is LPS-desensitisation. For this, monocytes are 

incubated with LPS for 24 h. After rechallenge with LPS, the TNF-alpha-production of 

the cells is strongly decreased. This phenotype is similar to that of monocytes from 

patients with immunoparalysis. 

It is commonly believed, that Lipid A is the immunological relevant part of LPS, while 

the sugar residues only play a minor role. In contradiction to this common opinion, we 

demonstrate that desensitised monocytes that are restimulated with Lipid A behave 

different to monocytes restimulated with LPS, concerning TNF-alpha-production. We 

could show that in LPS-desensitisation IL-10 plays a major role when the cells are 

rechallenged with LPS, since desensitisation can be prevented by neutralising anti-IL-10antibodies. 

In contrast to this, neutralising anti-IL-10-antibodies have no effect when 

the cells are rechallenged with Lipid A. 

In summary our results raise new interesting questions on how LPS really interacts with 

its receptors on the surface of the cell.

Jan Diekmann, Olaf Beck, Georg Rauser, Hansjörg Schild, Hermann Einsele, Max S. 

Topp 

Different mechanisms contribute to the immune evasion of 

Epstein-Barr virus latent membrane protein 1 

Epstein-Barr virus (EBV) infection is associated with several pathogenic conditions such 

as nasopharyngeal carcinoma, NK and T cell lymphomas and Hodgkin`s disease, which 

all express latency type II proteins including the latent membrane proteins 1 and 2 

(LMP1/2). Both LMP1 and 2 have been suggested to represent targets for 

immunotherapy with antigen-specific CD8+ T cells. After generation of HLA-A*0201 

restricted CD8+ T cells specific for the YLLEMLWRL and CLGGLLTMV epitopes from the 

LMP1 and LMP2, respectively, we could show, that only the LMP2-specific CD8+ T cells 

were able to lyse antigen-expressing target cells. 

In order to investigate the mechanism responsible for the observed unresponsiveness of 

the LMP1-specific CD8+ T cells against LMP1-expressing target cells, the targets were 

treated with the proteasomal inhibitors MG132 and Epoxomicin. Both inhibitors could 

restore the immunogenicity of the target cells towards the LMP1-specific CD8+ T cells, 

suggesting, that blocking of the proteasome prevents destruction of the epitope and the 

same time enables processing through other proteases. Blocking of the proteasome by 

siRNA targeting of POMP greatly enhanced epitope-specific activation of the T cells 

emphasising, that prevention of proteasomal processing of LMP1 allows sufficient 

generation of viral epitopes. 

Alternatively, genetic truncation of the cytosolic part and the first four transmembrane 

regions of LMP1 could also restore good target recognition, stressing that LMP1 may 

contain sequences for selective inhibition of cytosolic proteases such as tripeptidyl 

peptidase II (TPPII) or thimet oligopeptidase (ThOP). 

These findings indicate, that the LMP1 epitope YLLEMLWRL is prevented from MHC-I 

presentation by distruction through the proteasome and inhibitory influences of the Nterminal 

transmembrane domains of the protein. We propose that this novel function 

may represent an additional immune evasion strategy employed by Epstein-Barr virus.

Diana Dudziak, Alice O'Kamphorst, Gordon F. Heidkamp, Veit R. Buchholz, Christine 

Trumpfheller, Chae Gyu Park, Ralph M. Steinman, Michel C. Nussenzweig 

Differential Antigen Processing and Presentation by Dendritic 

Cell Subsets in vivo 

Dendritic cells (DCs) process and present self and foreign antigens to induce tolerance 

or immunity. In vitro models suggest that induction of immunity is controlled by 

regulating the presentation of antigen, but little is known about how murine DCs control 

antigen presentation in vivo. To evaluate regulation of antigen processing and T cell 

activation by CD11c+/CD8+ and CD11c+CD8- DCs in vivo we delivered antigens to the 

DCs by using chimeric anti DEC205-Ova or 33D1-Ova antibodies. We could show that 

DCs targeted with anti DEC205-OVA or 33D1-OVA in vivo are distinct in their ability to 

present antigen on MHCI and MHCII, in that DEC205 expressing CD8+ DCs are 

specialized for cross-presentation on MHC class I, and CD8- DCs for presentation on 

MHC class II. By using transgenic mice that express human DEC205 under the CD11c 

promoter we could demonstrate that the difference in antigen processing is intrinsic to 

the DC subsets and independend of the receptor that was targeted. Moreover, we found 

an increased expression of proteins involved in MHC class I processing in CD11c+/CD8+ 

DCs and in MHC class II processing in CD11c+CD8- DCs. This specialization may have 

important implications for understanding the initiation of T cell responses in vivo. 

This work was supported by the German Research Foundation (D.D., DU548/1-1)

Seray Cetin, Niels Kruse, Andrew Chan, Ralf Gold, Fred Lühder 

Differential expression of BDNF mRNA splice variants in 

mouse brain and immune cells 

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family and 

plays a key role in neuronal survival, differentiation and plasticity. Its neuroprotective 

character was pointed out by therapeutic application of BDNF. Neurons are the main 

cellular source of this neurotrophin, but it was also demonstrated that immune cells, 

namely T cells, B cells and monocytes express bioactive BDNF. The regulation of BDNF 

gene expression is not fully understood yet, but several mRNA splice variants of BDNF 

were recently described, each producing the same protein. In this study we analyzed 

the expression of mBDNF mRNA splice variants in cells of the immune system in 

comparison to cells of the central nervous system (CNS). We find that all splice variants 

are expressed in the CNS, whereas only mBDNF3 mRNA is expressed in lymphoid 

organs, immune cells and microglia. Activation of purified T cells lead to an increased 

expression of only mBDNF3, while activation of macrophages and microglia results in a 

decreased expression of this splice variant. These results show that mBDNF mRNA is 

differentially regulated in the CNS and the immune system by presumable different 

signalling networks. It additionally offers the chance to manipulate mBDNF expression 

in immune cells without affecting BDNF expression in the CNS.

Ildiko Boross, Christine Lux, Hans-Anton Lehr 

Differential expression of IL-17F and IL-17A in the lung of 

patients affected with bronchioalveolar cell carcinoma 

It is now widely accepted that IL-17 plays a pivotal role in mediating autoimmunity and 

inflammation however its role on the immuno-pathogenesis of tumor has not been 

clarified yet. The IL-17 cytokine family has many members, among them IL-17F and A 

have been shown the highest homology and functional properties. Different studies 

have been reported showing that both TGF-b and IL-6 are required for the 

differentiation of the TH17 clone producing both IL-17A and F. We thus first analysed 

the IL-17A and IL-17F expression in a group of normal subjects (n=9 ) as well as in 

patients with bronchioalveolar cell carcinoma (n=12). We found that although IL-17A 

expression did not differ between subjects with or without this lung tumor, IL-17F 

protein expression was downregulated in lung from patients affected by 

bronchioalveolar cell carcinoma as assessed both by western blot analysis and 

immunohistochemistry. We then investigated TGF-beta and IL-6 protein expression in 

these human lung samples. We found that both TGF-beta and IL-6 were upregulated in 

the lung samples fom subjects affected by bronchioalveolar cell carcinoma. Thus the 

defect we found in IL-17F production in the lung of patients with this tumour must map 

downstream of TGF-beta and IL-6. 

This work is funded by the Graduiertenkolleg 1043 and Immuno-intervention Cluster of 

Excellence (ICE, Mainz, Germany).

Markus Kleinewietfeld, Giovanna Borsellino, Adamo Diamantini, Alexander Sternjak, 

Luca Battistini, Olaf Rötzschke, Kirsten Falk 

Differential expression of VLA-4 by functional Treg and 

effector CD4+ T cells 

Migration and homing of lymphocytes is largely controlled by chemokine receptors and 

adhesion molecules. Since no chemokine receptor is expressed exclusively by Foxp3+ 

CD25+ regulatory T cells (Treg), it remains the question how the trafficking of Treg can 

be selectively controlled. We therefore compared the expression of various integrins on 

human regulatory CD25high T cells and on CD25low effector CD4+ T cells. A striking 

difference was only observed for CD49d, the alpha-chain of the integrin VLA-4 

(alpha4beta1). The effect was evident only on effector/memory-like cells (CD45RO 

+CCR6+), where alpha4 was down-regulated on most of the Treg cells but up-regulated 

on the effector T cells. Loss of VLA-4 on Treg cells was compensated by the expression 

of CLA (cutaneous lymphocyte antigen), which is regulated in opposite to CD49d. 

Therefore, CCR6+ Treg cells are equipped to efficiently bind E-selectin via CLA, whereas 

CD4+ effector T cells preferentially adhere to the VLA-4 ligand VCAM-1. Notably, CD49d- 

CCR6+ Treg cells show the highest Foxp3 expression among all CD25high Treg and only 

these cells express the ectonucleotidase CD39 (ENTPD1), we have recently shown to be 

crucial for Treg mediated immune suppression. In line with the phenotype, the CD49d- 

CCR6+ Treg exhibit the strongest suppressive capacity among human Treg cells. Thus, 

the differential expression of VLA-4 and CLA provides a mechanism for the selective 

recruitment of either CCR6 expressing effector/memory-like Treg (TREM ) or ´ 

conventional ´ effector/memory cells (TEM ). Moreover the absence of CD49d can be 

used together with CCR6 and CD39 as a key-marker to distinguish functional from other 

´ inactive ´ peripheral Treg cells in humans.

Florian Reißfelder, Jutta Schröder-Braunstein, Thomas Giese, Carolin Reiser, Stefan 

C. Meuer, Bernd Sido 

Differential inhibition of human intestinal lamina propria Tlymphocyte 

activation versus peripheral blood T cells by the 

gold-compound auranofin 

The oxidoreductase Thioredoxin (TRX) potentiates cytokine production in and 

proliferation of lymphocytes. Human intestinal lamina propria T-cells (LPT) constitutively 

contain high amounts of TRX, produce large quantities of cytokines and proliferate 

vigorously upon CD2 stimulation as compared to peripheral blood T-cells (PBT). To 

become immunologically active, oxidized TRX needs to be reduced by TRX reductase. 

We, therefore, aimed to inhibit the immune response of LPT in vitro through Auranofin 

(AF), a potent inhibitor of TRX reductase. 

Isolated LPT from fresh surgical specimens of normal colon mucosa and autologous PBT 

were stimulated via CD2 using a combination of mitogenic mAb. AF (0,5•M) inhibited 

proliferation of LPT to nearly background levels, whereas it was enhanced in PBT. 

Correspondingly, the vigorous cytokine mRNA expression in LPT following CD2 

stimulation (IL2, TNFα, TNFβ, IFNγ, GMCSF) was nearly completely abolished by AF in 

contrast to PBT, in which it was enhanced twofold. The CD2 immune response of LPT 

was paralleled by a high-level expression of the antioxidative fraction of TRX as 

determined by redox Western-blot analysis. This fraction was partly oxidized in LPT in 

the presence of AF, whereas it was further increased in PBT. The differential 

immunomodulatory activity of AF in LPT versus PBT was not due to differences in TRX 

reductase expression. However, Annexin V and Propidium Iodide staining revealed that 

LPT are constitutively more sensitive to spontaneous apoptosis than PBT, which is 

further enhanced by AF during CD2 stimulation. Inflammatory bowel disease (IBD) is 

characterized by hyperreactivity of LPT along with resistence to apoptosis as compared 

to LPT from normal gut. AF may, thus, represent an innovative immunomodulatory 

strategy in the therapy of IBD.

Svetlana Karakhanova, Karsten Mahnke, Alexander Enk 

Differential modulation of B7-H1 expression in pDCs and 

mDCs upon maturation of dendritic cells (DCs). 

Expression of regulatory molecules of the B7-H family by DCs plays an important role in 

the regulation of immune responses. However, their function(s) as well as regulation of 

their expression during DC maturation is not completely understood. To test how 

different types of DC maturation affect expression of these molecules, we stimulated in 

vitro prepared monocyte derived DCs (MoDCs) as well as genuine DCs, isolated from 

peripheral blood of healthy donors. Stimulation of MoDCs with a cytokine cocktail 

resulted in increased stimulatory capacity as well as increased expression of CD80, 

CD83, CD86 molecules. In parallel we observed upregulation of B7H1. Similar results 

were observed using genuine DCs. This means that cytokine cocktail maturated genuine 

DCs showed enhanced stimulatory capacity and upregulation of B7H1 expression. While 

MoDC represent a homogenous population of myeloid origin, genuine DCs consist of a 

mixed (mDC and pDCs) population with a different repertoire of TLR receptors. Total 

genuine DC populations were stimulated with various TLR ligands and assessed by FACS 

and functional assays to determine whether the surface expression of B7H1 molecules is 

affected. LPS as well as cytokines enhance expression of B7H1 preferentially in mDC, 

while Poly IC induced B7H1 expression in pDC. We furthermore show that stimulation 

activates the MAPK kinase pathway in MoDCs and blocking of ERK/MAPK 

phosphorylation with a specific inhibitor reduced increased B7H1 expression. This 

indicates that the expression of B7H1, at least in part, is regulated by the MAPK kinase 

pathway. Additional assays are going to be performed to identify supplementary 

signalling events responsible for B7H1 upregulation and to dissect the initial receptor/ 

receptors responsible for activation.

Daniel Engel, Ulrich Dobrindt, Juliane Maurer, Frank Tacke, Christian Kurts 

Differential role of CCR2 on Gr1HI and Gr1LO monocyte 

migration in response to bacterial infection 

Monocytes are crucial immune effectors in bacterial infection, but the mechanisms 

governing their migration are not completely understood. The chemokine receptor 2 

(CCR2) regulates migration of monocytes, which is important for the defense against 

several pathogens. It has been shown recently that CCR2 is required for bone marrow 

(BM) emigration of monocyte precursors expressing high levels of the Gr1 (GR1HI) 

molecules. A differential effect of CCR2 on the migration of Gr1HI and Gr1LO monocytes 

in response to bacterial infection remains to be clarified. 

We have established a murine model of urinary tract infection (UTI) by injection of 

uropathogenic E.coli (UPEC) into the bladder of C57/BL6 mice. The numbers of 

macrophages in infected bladders of CCR2-deficient mice were reduced significantly. 

Using a recently published monocyte tracking method, we found that CCR2 was 

dispensable for immigration of both Gr1LO and Gr1HI monocytes into the infected 

bladder. Analysis of the blood of CCR2-deficient mice revealed a striking reduction of 

circulating monocytes, whereas no changes were seen in the BM. Mixed BM-chimeric 

mice, reconstituted with CD45.1 CCR2-competent and CD45.2 CCR2-deficient BM 

showed, that expression of CCR2 did not affect monocyte numbers in the BM, whereas 

the number of CD45.2 CCR2-deficient donor cells in the blood were severely decreased. 

These data indicated that CCR2 mediated emigration of monocyte precursors out of the 

BM, but neither immigration of mature monocytes into the BM, nor into the infected 

bladder in UTI.

Maik Moermann, Mareike Thederan, Christof Wagner, Inaam Nakchbandi, Gertrud 

Maria Hänsch 

Differentiation of the promonocytic cell-line U 937 to 

osteoclasts by bacterial lipopolysaccharides: a link between 

infection and pathological bone resorption. 

Bacterial biofilms formed on orthopaedic implants elicit persistent inflammation with 

massive tissue destruction and osteolysis. To establish a link between infection and 

osteolysis, we explored the possibility that bacterial infections promote the generation 

of bone resorbing osteoclasts. In that context, the effect of bacterial lipopolysaccharides 

(LPS) on the generation of osteoclasts was tested in vitro. Osteoclasts are derived from 

haematopoietic cells, and differentiate to osteoclasts under the influence of signals 

derived from stromal cells and/or T-lymphocytes. To exclude the effect of those cells, 

the promonocytic cell line U937 was used. U937 were differentiated to monocytes by 

use of phorbol ester, and then cultivated further with LPS. During culture U937 acquired 

the LPS receptor CD14. As the culture progressed, expression of numerous monocytetypical 

receptors, including CD11b, MHC class II, and CD 86 was induced, as were 

osteoclast specific proteins like the tartrate resistant acid phosphatase (TRAP) and 

cathepsin K. Furthermore, between days 4-6 by light microscopy a massive aggregation 

of the cells was observed followed by cell fusion. By day 15 multinuclear cells with the 

morphological characteristics of osteoclasts appeared. These cells were TRAP-positive 

and able to degrade calcium phosphate coated on cover slips and ivory as well. Thus, 

LPS as a single stimulus induces the differentiation of monocytes to cells with the 

phenotypical and functional characteristics of osteoclasts generated by established 

protocols. In conclusion, we propose that local bacterial infections could create a 

microenvironment that promotes the generation of bone resorbing cells, which, in turn, 

could contribute to the infection-associated osteolysis.

Kristine Kohl, Sylvia Schnautz, Elisabeth Klein, Thomas Bieber, Susanne Koch 

DIFFERENTIATION SIGNALS FOR HUMAN LANGERHANS CELL 

PRECURSORS IDENTIFIED BY SEQUENTIAL MIGRATION OF 

MONOCYTES 

Precursors of dendritic cells (DC) are myeloid cells transported via the blood stream, 

which migrate into tissues, where they differentiate to DC, most probably following 

locally released chemotactic signals. Using human peripheral blood monocytes, we 

investigated the impact of trans-endothelial migration on phenotype and function of 

Langerhans cell (LC) precursors, which are paradigmatic DC from the epithelial layer in 

the skin. On their way from the blood vessels to the epidermis, LC precursors need to 

cross the endothelial layers. They are guided by chemoattractants from dermal 

fibroblasts and from epidermal keratinocytes. In an in vitro transwell system, 

monocytes migrated towards medium conditioned by fibroblasts. Only CD14 low cells, 

which had migrated towards fibroblast conditioned medium, began to express CD1a, 

additionally a population of the CD14 low cells became CCR6 positive after migration and 

incubation. 14 hours later, in a second migration assay, only monocytes having 

migrated towards fibroblast-conditioned medium could migrate towards keratinocyteconditioned 

medium. Thus, trans-endothelial migration of human peripheral blood 

monocytes towards fibroblast-conditioned medium enabled them to subsequently 

migrate towards keratinocyte-conditioned medium. This would be a feature expected 

from LC precursors. The investigation of chemokine receptors of freshly isolated 

monocytes and after their various migration steps revealed that their trans-endothelial 

migration towards fibroblast-conditioned medium rescued the expression of chemokine 

receptor CCR2, which was otherwise rapidly down regulated. We therefore hypothesize 

that trans-endothelial migration of monocytes is a critical step for their differentiation to 

Langerhans cells.

Tim Meyer, Susann Beetz, Daniela Wesch, Ina Martens, Dieter Kabelitz 

Direct Costimulatory Effect of TLR3 Ligand Poly(I:C) on T cells 

We and others have recently shown that human αβ- as well as γδ- T lymphocytes 

express Toll-like receptor 3 (TLR3). As a classical pattern recognition receptor, TLR3 

binds double-stranded viral RNA and a synthetic analog, polyinosinic-polycytidylic acid 

[poly(I:C)]. Our earlier results indicated that poly(I:C) alone does not activate γδ- T 

cells but strongly increases the T cell receptor (TCR) stimulated interferon-γ secretion 

and expression of activation markers. Using primary human CD4+ αβ- T cells and Jurkat 

cells, we now also observed enhanced surface expression of CD69 and CD25 as well as 

IL-2 production in CD4+ αβ- T cells stimulated via the TCR in combination with poly(I:C) 

but not by poly(I:C) stimulation alone. To elicit the molecular basis of a potential crosstalk 

between TLR3 and TCR signalling, we performed luciferase assays. We observed 

that costimulation via TLR3 in comparison to TCR stimulation alone enhanced NF-κB but 

not NFAT activation. This tendency was underlined by the fact that the expression of the 

strictly NF-κB-dependent gene A20 was enhanced in Jurkat cells upon costimulation. 

These results indicate that TLR3 signalling modulates TCR-driven responses in different 

T cell populations.

Chiara Massa, Christiane Kellert, Esther Kamphausen, Barbara Seliger 

Disparate modulation of antigen processing components 

during maturation of the different human DC subsets. 

Dendritic cells (DC) are the most potent antigen presenting cells (APC) of the immune 

system and have thus been employed in the cell-based approach to tumor 

immunotherapy. After the modest successes obtained using DC pulsed with single 

tumor-derived epitopes, the latest strategies aim at broadening the repertoire of 

antigens provided by the vaccine in order to activate a more efficient immune response. 

To this purpose DC vaccines have been loaded with the entire tumor antigenic 

repertoire either in the form of RNA or proteins. In this therapeutic setting the final 

pattern of epitopes presented to the immune system is shaped by the antigen 

processing machinery of the DC and can thus be different from the one produced by the 

tumor itself. Indeed, some tumor epitopes can be destroyed by the immunoproteasome 

of professional APC. Aim of this study was to characterise the different subsets of 

human DC for the constitutive expression of the cyosolic and reticular peptidases 

involved in the antigen processing pathway and the modulation of these enzymes 

during the maturation process. To this purpose, myeloid CD1c+ and plasmacytoid 

BDCA4+DC were purified from the blood of healthy donors and stimulated with the 

appropriate TLR ligand. CD14+ monocytes were differentiated into DC either using the 

classical protocol of 7 days culture with GM-CSF and IL4 or the proposed amelioration 

using IL15 for the generation of Langerhans-like cells or a shorter 2 days protocol. The 

characterisation of the mRNA expression patterns of these enzymes revealed an 

opposite behaviour among DC types: a prevalent down-regulation in the expression 

pattern of peptidases was found in the plasmacytoid DC, whereas in myeloid DC the 

enzymes are up-regulated, in particularly in response to poly IC. Functional experiments 

will be performed to confirm whether the different expression patterns identified have 

any consequences on the epitope repertoire presented by the DC types, thus providing 

useful information for the establishment of DC-based vaccines that process tumor 

antigens in the most tumor-like fashion.

Andreas Hombach, Markus Chmielewski, Tobias Riet, Caroline Kopecky, Patrick Schmidt, 

Nadin Fein, Claudia Ederer, Anja Hombach, Heike Koehler, Hinrich Abken 

Dissecting and modulating a redirected anti-tumor T-cell 

response for adoptive immunotherapy: second generation of 

recombinant immunoreceptors. 

During the last years we have shown that naive T cells can be redirected towards 

defined target antigen by retroviral expression of a recombinant immunoreceptor that 

mediates both binding to antigen via an antibody domain and induction of cellular 

activation via the intracellular CD3z domain. The design of the immunoreceptor has a 

number of advantages: (i) the molecule is modularily composed allowing de novo 

composition of signaling and binding properties; (ii) receptor binding is independent of 

MHC presentation of antigen allowing T cell targeting towards unconvential T cell 

targets, i.e., carbohydrates or lipids; (iii) the receptor molecule triggers T cell effector 

functions upon binding. We demonstrated the feasibility of the concept using primary 

tumor cells and T cells from the same tumor patient in order to break tumor tolerance 

and to eliminate the autologous tumor cells, e.g primary melanoma cells, colon 

carcinoma cells or chronic lymphocytic leukemia cells. The concept, moreover, has the 

power to modulate the induced T cell response in a predictive way. By introducing 

costimulatory domains of the CD28 family, i.e., CD28, OX40 or 4-1-BB, we demonstrate 

that the anti-tumor response of T cells is specifically modulated with respect to induced 

secretion of IFN-g and IL-2, cytolytic activity and resistance to activation induced cell 

death. By introducing the CD28 costimulatory domain, moreover, T cells are rendered 

resistant to TGF-b mediated immune suppression as mediated by the tumor itself. By 

altering the epitope specificity and/or the binding affinity of the immunoreceptor, the 

efficacy of antigen-mediated T cell activation is dramatically modulated. By altering the 

structural prerequisites of the immunoreceptor molecule, interactions with the 

endogenous TCR of the engineered T cells are minimized in order to avoid generation of 

unwanted specificities. These newly identified parameters were integrated into a second 

generation of recombinant immunoreceptors which are expected to display an increased 

anti-tumor efficiency upon adoptive transfer.

Marc A. Blank, Olaf Utermohlen, Holger M. Reichardt, Marco J. Herold 

Dissecting the apoptotic pathways induced by Glucocorticoids 

in T-cells 

Glucocorticoids (GC) induce apoptosis in many cell types, but the mechanisms are not 

well understood. Recently it was reported that the proapoptotic Bcl-2 family member 

Bim and an upregulation of ceramides by a caspase-activated acidic sphingomyelinase 

(aSMase) play important roles. In order to study their involvement in GC- induced 

apoptosis we took advantage of the GC-sensitive murine T-cell lymphoma line WEHI 

7.15a. Overexpression of retrovirally expressed shRNAs against Bim or aSMase had no 

influence, whereas knockdown of the GC receptor (GR) itself rendered these cells 

resistant to Dexamethasone. In addition, various pharmacological inhibitors of ceramide 

production had no influence on GC-induced apoptosis. Moreover, thymocytes and 

peripheral T-cells from aSMase knockout mice were equally sensitive to GC induced 

apoptosis as wildtype cells. Furthermore, we confirmed the in vitro knock down results 

of Bim and the GR by introducing shRNAs into hematopoietic stem cells that were used 

to reconstitute lethally irradiated mice. While Bim inactivation did not impact ex vivo GCinduced 

apoptosis, loss of GR expression prevented cell death. Therefore we conclude 

that the involvement of Bim and aSMase is negligible in GC-induced apoptosis of T-cells.

Annelies Verbrugge, Adelheid Cerwenka 

Dissecting the molecular mechanisms involved in the synergy 

of TREM-1 with TLRs 

The innate immune system uses a broad spectrum of receptors to detect invading 

pathogens. A major class of these receptors are formed by the Toll-Like Receptor (TLR) 

s, which directly recognize pathogen-associated molecules and trigger the production of 

pro-inflammatory cytokines. In addition, several receptors can act together to enhance 

the immune response. One receptor that has been shown to cooperate with TLRs to 

induce cytokine production is Triggering Receptor Expressed on Myeloid cells (TREM)-1. 

TREM-1 is expressed on neutrophils and monocytes and is upregulated upon stimulation 

with LPS. Triggering of TREM-1 results in the production of pro-inflammatory cytokines, 

including TNFα. More importantly, TREM-1 synergizes with TLRs in cytokine production. 

The goal of our study is to dissect the molecular mechanisms leading to this synergy. 

Using quantitative RT-PCR, we found that LPS induced TNFα mRNA in primary human 

monocytes. Triggering of TREM-1 did not lead to detectable transcription of TNFα. 

However, when TREM-1 was triggered in the presence of LPS a substantial increase in 

TNFα mRNA was observed, compared to stimulation with LPS alone. The increase in 

TNFα mRNA occurred within the first two hours after stimulation, preceding LPS-induced 

upregulation of TREM-1 cell surface expression. Importantly, we found that preengagement 

of TREM-1 enhanced TNFα production upon subsequent stimulation with 

LPS. In current studies we aim to identify genes involved in the synergy of TREM-1 with 

TLRs using microarray-based gene expression profiling. Insight in the molecular 

mechanisms leading to synergy will enhance our understanding of the inflammatory 

response during sepsis.

Sven Burgdorf, Andreas Kautz, Volker Böhnert, Percy Knolle, Christian Kurts 

Distinct antigen uptake and intracellular routing mechanisms 

for activation of CD4+ and CD8+ T cells 

Adaptive immunity requires activation of T lymphocytes by antigen-presenting cells, 

which present the processed antigens on their MHC molecules. Intracellularly 

synthesised antigens, for example of viral or tumour origin, are presented by MHC I 

molecules and activate CD8+ cytotoxic T cells. Extracellular antigens can be presented 

on both MHC II molecules (to activate CD4+ T helper cells) and on MHC I molecules. 

The latter process has been termed cross-presentation. Current mechanistic models of 

cross-presentation assume that endocytosed antigens must be rescued from lysosomal 

degradation by diversion from a common pool of endosomes towards the cytoplasmic 

proteasome, but the underlying mechanisms remain unresolved and controversial. We 

investigated the influence of antigen uptake mechanisms on the intracellular routing 

and presentation on MHC I vs. MHC II molecules. We could show that if the model 

antigen, soluble ovalbumin, was taken up by pinocytosis or scavenger receptormediated 

endocytosis, it was targeted rapidly into lysosomes, where it was loaded 

selectively on MHC II molecules. Concurrently, ovalbumin was endocytosed by the 

mannose receptor into a distinct early-endosomal compartment, resulting in exclusive 

loading on MHC I molecules. We conclude that the endocytosis mechanism determines 

the intracellular destination of antigen, and thereby its processing and presentation to 

CD4+ or CD8+ T cells. Furthermore, this model implies that soluble antigen intended 

for cross-presentation does not have to be intracellularly diverted from lysosomes, but 

can enter a distinct endosomal compartment already during endocytosis. These findings 

pertain to our general understanding of antigen presentation and may provide a 

mechanistic basis for improving vaccination strategies.

Astrid Menning, Uta Hoepken, Kerstin Siegmund, Martin Lipp, Alf Hamann, Jochen 

Huehn 

Distinctive role of CCR7 in migration and functional activity of 

naïve- and effector/memory-like Treg subsets 

Foxp3+CD25+CD4+ Tregs play a fundamental role in the maintenance of self-tolerance 

and the control of inflammatory reactions. Previous data demonstrated a division of 

labor between naïve- and effector/memory-like Treg subsets, which is largely based on 

their lymph node-recirculating and inflammation-seeking migration behavior, 

respectively. The chemokine receptor CCR7 is expressed on both types of Treg subsets, 

albeit at different levels. Whether it fulfills similar or distinct roles in these subsets has 

not been studied so far. We here show that the recirculation of naïve-like Tregs through 

LNs and, to some extent, the gut is dependent on CCR7. Lack of CCR7 not only 

prevents recirculation but also almost completely abolishes the ability of naïve-like 

Tregs to control the priming phase of an immune response. In contrast, CCR7 deficiency 

in effector/memory-like Tregs promotes their accumulation in inflamed sites, compatible 

with a role of CCR7 for exit from the tissue. Local Treg accumulation was accompanied 

by an enhanced suppression of inflammation. Together, our findings provide conclusive 

evidence that CCR7 expression on Tregs differentially controls in vivo function of the 

naïve- and effector/memory-like subsets.

Julia Polansky, Jennifer Freyer, Stefan Floess, Karsten Kretschmer, Harald von 

Boehmer, Alf Hamann, Jochen Huehn 

DNA methylation controls foxp3 gene expression 

The forkhead box transcription factor Foxp3 is a key regulator for Treg function and 

lineage identity. To date, despite a recent initial characterization of the human FOXP3 

promoter, molecular mechanisms controlling and stabilizing Foxp3 expression are only 

poorly understood. Recently, we have described the Treg specific demethylated region 

(TSDR), an evolutionary conserved element upstream of exon -1 in the foxp3 locus that 

in Tregs showed a selectively demethylated state on all its numerous CpG motifs when 

compared to non-regulatory T cells subsets. However, murine Foxp3+ induced Tregs, 

which were generated in vitro from CD4+CD25-Foxp3- T cells by TCR-mediated 

stimulation in the presence of TGF-ß, displayed only weak and partial demethylation of 

TSDR. Upon restimulation in the absence of TGF-ß, most of these cells lost Foxp3 

expression, indicating that complete demethylation of TSDR might be critical for stable 

Foxp3 expression. Repetitive restimulation and TGF-ß signaling was not sufficient to 

stabilize expression of Foxp3 in induced Tregs. Yet, interference with the DNAmethylation 

status by treatment with 5-aza-2‘-deoxycytidine led to an induction of 

Foxp3 in CD4+CD25- T cells and stabilized Foxp3 expression in TGF-ß-induced Tregs. 

Hence, our data show that DNA methylation is critically involved in the maintenance of 

Foxp3 expression, suggesting that epigenetic modifications allow heritable and stable 

expression of this key transcription factor in Tregs.

Manije Sabet, Maja Frankuski, Anja Reutzel-Selke, Andreas Pascher, Peter Neuhaus, 

Johann Pratschke, Katja Kotsch 

Donor pretreatment with Simvastatin reduces graft 

immunogenicity following prolonged cold ischemia in an 

experimental model of kidney transplantation 

The protective effects of 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors 

(HMG-CoARIs, statins) have been demonstrated in numerous cerebral, cardiac and 

renal ischemia models. The inhibition of free radicals substantially contribute to the 

beneficial effects of statins, however the exact modulatory mechanisms remain unclear. 

Based on their anti-inflammatory and anti-oxidative properties, we investigated the 

potential beneficial effect of donor pretreatment with statins on ischemia/reperfusion 

injury in a rat model of kidney transplantation (Tx). F-344 donor rats were pretreated 

with Simvastatin for 3 days prior to transplantation (10 mg/kg/day). Kidneys were 

grafted into Lewis recipients following a prolonged cold ischemia of 24h. Grafts and 

spleens of recipients were harvested 24h and 14 days post transplantation (n=6/group). 

Frequencies of cell populations were analyzed by flow cytometry and the mRNA 

expression of relevant candidate genes (CD3, IL-12, MHC class II, CD80, CCR7, CCL19 

and CCL21) was evaluated by real-time RT-PCR. After 24h and 14 days post Tx number 

of CD4+ cells were slightly reduced in spleens of recipients following donor 

pretreatment with Simvastatin (Simvastatin vs. control, 24h: 43.9±2.3% vs. 49.2 

±3.7%, p=0.027; d14: 44.3±2.4% vs. 49.8±1.8%, p=0.07). Interestingly, the 

frequency of CD3-CD4+ monocytes was significantly reduced in the Simvastatin group 

(Simvastatin vs. control, 24h: 6.8±2.1% vs. 12.1±2.6%, p=0.007; d14: 20.3±2.9 vs. 

25.0±1.0%, p=0.018), whereas the number of CD3+CD4+ T cells was comparable in 

both groups. In the spleen reduced mRNA expression level of CD3 as a marker of T cell 

infiltration as well reduced mRNA expression of IL-12 and CD80 were detected. 

Furthermore the chemokine receptor CCR7 was markedly reduced in the spleen as well 

as its ligands CCL19 and CCL21 displayed enhanced mRNA expression in the graft. 

Additionally we revealed a notable decrease of MHC class II in both kidney and spleen. 

Our data suggest that donor pretreatment with Simvastatin following prolonged cold 

ischemia reduces graft immunogenicity by modulating potential antigen presenting cells 

and their homing to lymphoid organs. Donor pretreatment with Simvastatin may 

therefore represent an attractive tool to preserve renal function after ischemia/ 

reperfusion injury.

Xiaoqian Wang, Luca Simeoni, Jonathan A. Lindquist, Julio Saez-Rodriguez, Ernst D. 

Gilles, Stefanie Kliche, Burkhart Schraven 

Dynamics of proximal signaling events after TCR/CD8mediated 

induction of 

proliferation or apoptosis in mature CD8+ T-cells 

Engagement of the T-cell antigen receptor (TCR) can induce different functional 

outcomes such as activation, proliferation, survival or apoptosis. How the TCR-mediated 

signaling cascades generating these distinct cellular responses are organized on the 

molecular level is so far not completely understood. To obtain insight into this question, 

we analysed TCR/CD8-mediated signaling events in mature OT-I TCR transgenic T-cells 

under conditions of stimulation that either lead to proliferation or to apoptosis. These 

experiments revealed major differences in the phosphorylation dynamics of ZAP-70, LAT 

and PLC-y1. Moreover, input signals leading to apoptosis induce a strong, but transient 

activation of ERK1/2 mainly at sites of TCR-engagement, whereas stimuli promoting 

survival/proliferation generate a low and sustained activation of ERK1/2 in distinct 

subcellular compartments. The transient activation of ERK1/2 under pro-apoptotic 

conditions of stimulation is at least partially due to the rapid poly-ubiquitination and 

subsequent degradation of ZAP-70 whereas the sustained activation of ERK1/2 under 

survival promoting conditions is paralleled by the induction/phosphorylation of antiapoptotic 

molecules such as PKB and Bcl-xL. Collectively, our data provide signaling 

signatures that are associated with proliferation or apoptosis of T-cells.

Börge Arndt, Burkhart Schraven, Luca Simeoni 

Dynamics of TCR signaling events leading to human T-cell 

proliferation 

The use of cross-linked soluble antibodies (xsAbs) directed against the TCR/CD3 

complex and costimulatory molecules is the standard procedure to study intracellular 

biochemical cascades during T-cell activation. Despite the fact that stimulation of T cells 

via xsAbs results in the activation of several signaling molecules downstream of the 

TCR, it does not result in T-cell proliferation. Instead, it appears that xsAbs induce other 

cellular outcomes like apoptosis or unresponsivness. To investigate the dynamics of the 

signaling events that lead to T-cell proliferation we developed a method to stimulate T 

cells with antibodies immobilized on small particles (iAbs) to mimic an antigen 

presenting cell. We though that under these experimental conditions T cells would 

receive a more physiological stimulation that ultimately would lead to proliferation. 

Indeed, we found that stimulation with CD3 and CD28 iAbs induced a strong 

proliferation of peripheral human T cells, whereas CD3 and CD28 xsAbs induced T-cell 

unresponsiveness. The analysis of TCR-mediated signalling pathways revealed that 

xsAbs induced a very strong, rapid and transient phosphorylation (less than 15 

minutes) of a variety of signalling molecules including ZAP-70, LAT and MAPKs. 

Conversely, stimulation with iAbs results in a very weak but sustained activation of TCR 

proximal signalling molecules (ie, ZAP70, LAT and PLCγ1). Surprisingly, we found that 

the activation of more downstream signalling molecules such as MAPKs or transcription 

factors was very strong and sustained for more than 2 hours in cells stimulated with 

iAbs. In summary, our data demonstrate that the dynamics of signalling pathways that 

lead to T-cell proliferation do not correlate with those induced by crosslinked soluble 

antibodies.

Stephan Meinke, Philipp Eissmann, Carsten Watzl 

Early events in NTB-A signaling 

The activating receptor NTB-A belongs to the family of SLAM-related receptors and is 

expressed on NK, T and B-cells. Engagement of NTB-A on human NK cells by homophilic 

interaction with NTB-A expressing target cells can trigger cytotoxicity, cytokine 

production and proliferation. 

The crystal structure of the NTB-A homodimer has been solved recently. To confirm the 

functional relevance of the interactions seen in the crystal, selected residues in the 

binding region have been mutated. Cells transfected with the mutated receptors have 

been used as targets of NK cells expressing wild type NTB-A in cytotoxicity assays. 

Preliminary data show that the mutation of Histidine 54 or Serine 90 to Alanine reduces 

the susceptibility to wt NTB-A mediated lysis, while changing Glutamine 88 to Alanine 

does not. 

Signal transduction via NTB-A is mediated by two immunoreceptor tyrosine-based 

switch motifs (ITSM) in its cytoplasmic tail, which can bind the adapter molecules SLAMassociated 

protein (SAP) and EWS-activated transcript 2 (EAT-2). To define the role of 

these adapters in NTB-A signaling a stable knock down of SAP has been established in 

NK cell lines. While 2B4 function was abolished in these cells, cytotoxicity against NTB-A 

expressing target cells was not impaired upon SAP knock down. In agreement with the 

functional data, western blotting experiments reveal no difference in NTB-A 

phosphorylation between SAP knock down and control cells before and after receptor 

engagement. These data suggest that the phosphorylation and function of NTB-A are 

independent of SAP expression and imply a novel role of EAT-2 in the signal 

transduction of NTB-A. To confirm these observations in a more physiological setting, 

we are currently knocking down SAP expression in primary human NK cells.

Claudia N. Detje, Hauke Schmidt, Thomas Meyer, Marco Prinz, Ulrich Kalinke 

Early type I interferon responses exclude nerotropic viruses 

from central nervous system 

Mice infected with vesicular stomatitis virus (VSV) mount a protective anti-viral immune 

response. It is generally believed that if clearance of the pathogen in the periphery fails, 

neurotropic virus can enter the central nervous system (CNS) to replicate and cause 

severe brain damage that ultimately leads to death. This concept is supported by the 

observation that type I interferon receptor deficient mice (IFNAR-/-) that usually 

succumb to VSV infection within 2-3 days are protected if they have been immunized 

with UV-inactivated VSV before challenge. 

To investigate whether in IFNAR-/- mice viral infection of the CNS was primarily 

associated with the failure of the peripheral immune system to control the virus or with 

the failure of the interferon system to protect neurons against virus infection, we are 

studying the pathogenesis of VSV infection in mice with a cell type-specific IFNAR 

deletion in neurons of the CNS (NesCre+/-IFNARflox/flox). In recent experiments we 

verified the specificity and efficiency of IFNAR deletion in NesCre+/-IFNARflox/flox mice. 

Unlike IFNAR-/- mice that died within 3 days after VSV infection, NesCre+/-IFNARflox/ 

flox mice did not show signs of disease before 5-6 days after infection. Then, NesCre+/- 

IFNARflox/flox mice became hemiplegic, started to move around in circles and 

eventually died. In contrast, wild type mice did not show any sign of disease and 

survived VSV infection. The different courses of disease observed correlated with a 

severe infection of basically any type of tissue in IFNAR-/- mice, whereas in NesCre+/- 

IFNARflox/flox mice only CNS was infected. Terminally diseased NesCre+/-IFNARflox/ 

flox mice showed approximately 10 to 100-fold higher virus loads in the brain than 

IFNAR-/- mice, whereas only very little or no virus was found in liver, kidney and 

spleen. In contrast IFNAR-/- mice showed high virus titers in all tissues analyzed. 

In conclusion, the data collected so far suggest that early type I interferon responses 

play a crucial role in excluding virus from CNS. It will be a matter of future research to 

determine whether early type I interferon crosses the blood brain barrier or whether it 

is produced within the CNS.

Andrey Bogdanov, Tatyana Rybakova, Anna Nizkorodova, Nikolay Belyaev 

Effect of alpha-fetoprotein-activated hematopoietic stem cells 

on monocytes functional activity 

I was shown, alpha-fetoprotein (AFP) disturbs certain function of monocytes in vitro and 

in vivo models. In our previous work, we discovered AFP as an inductor of mice bone 

marrow hematopoietic stem cells (HSCs) suppression activity in vitro. Whether 

possibility of AFP-activated HSCs modulates monocytes activity and amplifies direct 

effects of AFP is unknown. We investigated effect of AFP-activated mice bone marrow 

HSCs on monocytes functions. Our results show that AFP-activated HSCs suppressed 

the spontaneous expression as well as inhibited LPS-induced up-regulation of •••I, CR3, 

CR4, and especially CD80, CD86, ICAM-1, LFA-1 and •••II. AFP-activated HSCs almost 

completely suppressed synthesis of IL-1β, IL-6, IL-12, IL-18 and TNF-α from other site, 

they increased IL-10 and TGF-β1 production in monocytes. AFP-activated HSCs 

decreased their capacity to secret chemokines as IL-8 and MCP-1 as well as eicosanoids 

as leukotriene C4 and prostaglandin D2. However, some HSCs subpopulations increase 

prostaglandin E2 production. Analysis of monocytes effector functions presented 

number of considerable impairments. The first, HSCs-treated monocytes showed weak 

phagocytosis and NO production abilities. The second, both oxygen-dependent and 

oxygen-independent potentials of HSCs-treated monocytes were significantly 

suppressed. The third, HSCs-treated monocytes inhibited cytotoxic activity of naive NK 

cells and CTL, and were not able to stimulate IFN-γ and TNF-α production in them. The 

fourth, HSCs-treated monocytes had a weak antigen-presenting capacity for naive CD8 

+ T cells. In the end, HSCs-treatment monocytes induced differentiation of naive CD4+ 

• cells and Th1 cells into Th2 cells. Thus, effect of AFP-activated HSCs on monocytes 

contributes to their immunosuppression profile.

Pablo Ariel Casalis, Martin Griebenow, Maria Laura Zenclussen, Ana Claudia 

Zenclussen, Hans-Dieter Volk, Christian Woiciechowsky, Ulrich-Wilhelm Thomale 

Effect of injury severity on the local and systemic cytokine 

expression in a rat model of traumatic brain injury 

Traumatic brain injury (TBI) is a major health problem worldwide, which is often 

associated with immunological complications. Previous works from our group showed 

that patients suffering from brain injury demonstrated a systemic anti-inflammatory 

response without preceding signs of systemic inflammation. The main aim of this work 

was to analyze whether there is a correlation between brain trauma severity and the 

generated local and systemic cytokine profiles. Different groups of two brain trauma 

severities and different endpoints after TBI (4h, 24h, 96h) as well as sham operated 

animals (rats subjected to all surgical procedures except brain injury) and unaffected 

control animals were included in the study. In those animals receiving trauma, a left 

cortical contusion was induced on the exposed brain using a controlled cortical impact 

injury model and trauma severity was adjusted by differing deformation depth of the 

impactor between 2 mm (moderate) and 2.5 mm (severe). The expression of cytokines 

was determined in liver and brain at the respective endpoints using real time RT PCR. 

Locally, augmented levels of IL-6, IL-1β, TNF-α and IL 12 were found 4h after TBI that 

then decreased to normal levels. Surprisingly, IL-10 was also locally augmented 4h 

after TBI. Additionally, immune cell infiltration into the contusion started already at 4h 

and was significantly augmented 24h after trauma, confirming a local inflammatory 

response. Interestingly, a systemic augmentation of the IL 10/TNF-α ratio was observed 

24h after TBI in both groups. Nevertheless, no significant differences between trauma 

severities were found, indicating that in this model if the trauma exceeds a moderate 

level there is no correlation between trauma severity and the intensity of the 

immunosuppression.

Michael Schramm, Ulrike Karow, Albert Haas, Martin Krönke, Olaf Utermöhlen 

Effects of Acid SphingoMyelinase on listeriocidal activiy of 

macrophages 

Deficiency in the enzyme acid sphingomyelinase (ASMase) impairs the capacity of 

macrophages to kill intracellular bacteria, e.g. Listeria monocytogenes. Here, we 

analyzed the impact of deficiency in ASMase on antibacterial effector mechanisms in 

macrophages. 

The classical antibacterial effector mechanisms, i.e. generation of antimicrobial reactive 

oxygen and nitrogen intermediates, was not altered in ASMase-deficient (ASMase -/- ) 

compared to wild type macrophages. In contrast, maturation of phagosomes containing 

either vital Listeria monocytogenes or heat killed L. monocytogenes (HKLM) was 

significantly impaired as indicated by reduced colocalization of bacteria with the 

lysosomal marker protein lamp1 in ASMase -/- macrophages. Both vital L. 

monocytogenes and HKLM showed a significantly prolonged colocalization with the late 

phagosomal marker protein mannose-6-phosphate receptor. Thus, maturation of late 

phagosomes containing L. monocytogenes into bactericidal phagolysosomes is impaired 

in ASMase -/- macrophages. Consistently, the transfer of lysosomal fluid phase markers 

into phagosomes containing HKLM was significantly reduced in ASMase -/- macrophages. 

The transfer of lysosomal cargo of high molecluar weight was more impaired than that 

of low molecular weight. Transfer of cathepsin D, a lysosomal protease involved in 

bacterial degradation, from lysosomes to phagosomes containing L. monocytogenes was 

markedly reduced in ASMase -/- compared to wild type cells. The reduced transfer of 

lysosomal contents into phagosomes containing L. monocytogenes suggests that 

ASMase regulates the fusion of phagosomes with lysosomes which is essentially 

required for elimination of bacteria.

Markus Janke, Jens Poth, Thomas Giese, Gunther Hartmann 

Effects of immunostimulatory RNA on human granulocyte 

populations 

Immunostimulatory RNA (isRNA) is known to stimulate plasmacytoid dendritic cells 

(PDC) and other immune cells via TLR7 or TLR8, which then produce large amounts of 

type I interferon and other cytokines, followed by induction of immune responses of 

potential therapeutical relevance. The expression of TLR7 and TLR8 on granulocytes is 

not doubtlessly clarified and the response to isRNA is still unclear. In this study we 

analyzed the effect of isRNA 9.2s on neutrophils and eosinophils as the largest fraction 

of white blood cells. 

To analyze potential activation of neutrophils and eosinophils we studied upregulation of 

CD11b and downregulation of CD62L as granulocyte activation markers in whole blood 

assays after stimulation with isRNA 9.2s by FACS. TLR expression profile and direct 

effects were analyzed on highly purified neutrophils. Quantification of expressed 

cytokines and chemokines after stimulation was performed by cytometric bead array 

(CBA). For functional analysis of purified neutrophils we tested the impact of isRNA 

stimulation on degranulation and respiratory burst activity. 

We found that neutrophils but not eosinophils are activated after isRNA stimulation in 

human whole blood. This stimulatory effect of isRNA 9.2s was shown to be indirect and 

TLR7 dependent. The only ribonucleic acid recognizing TLR expressed on neutrophils 

was TLR8, which showed functional activity on purified cells. Thus direct neutrophil 

activation via isRNA 9.2s should be restricted to TLR8 but not TLR7 but purified 

neutrophils responded with cytokine and chemokine release, respiratory burst and 

degranulation only after stimulation with synthetic TLR-8 agonists. IsRNA 9.2s did not 

induce any direct effects. 

In conclusion this study clearly shows that neutrophils express only TLR8 but not TLR7 

for nucleic acid recognition. IsRNA 9.2s had no direct influence on neutrophil or 

eosinophil activation. Thus side effects that could emanate from direct activation of 

granulocytes seem to be improbable when isRNA 9.2s is used in prospective 

therapeutical approaches.

Özen Sercan, Günter J. Hämmerling, Bernd Arnold, Thomas Schüler 

EFFECTS OF INTERFERONγ ON CD8 + T CELL HOMEOSTASIS 

Interferon-γ (IFNγ) is an inflammatory cytokine, which contributes to host defenses 

against microbial infections. Besides its anti-microbial activity, IFNγ also regulates 

antigen specific CD8 + T cell homeostasis. However, whether cell types other than 

activated T cells contribute to this process has not been clarified. We addressed this 

question by adoptively transferring T cell receptor transgenic CD8 + T cells into IFNγ- 

and IFNγ receptor (IFNγ)- deficient mice. Our findings suggest that IFNγ promotes 

antigen specific CD8 + T cell expansion but also limits memory T cell formation via its 

actions on host cells. Furthermore, we showed, that in response to IFNγR signaling, 

CD11b + macrophage-like cells are sufficient to normalize expansion and memory 

formation of CD8 + T cells. Macrophage-derived nitric oxide, which suppresses T cell 

expansion in various experimental models, is not responsible for the suppression of T 

cell responses in our system. In conclusion, our experiments demonstrate that IFNγ is 

necessary for efficient T cell expansion and contributes to the regulation of memory T 

cell formation.

Besir Okur, Rainer Glauben, Arvind Batra, Thorsten Stroh, Inka Fedke, Jeannette 

Pietsch, Martin Zeitz, Britta Siegmund 

Effects of Leptin on T helper cell polarisation 

Leptin is an adipokine, initially described as regulator of food intake. Recent data 

suggest an additional regulatory function in the immune system, for instance 

stimulation of naïve T cells in the presence of leptin enhances polarisation to a Th1 

phenotype. Aim of the present study was to characterize the effect of leptin on T cell 

polarisation applying antigen-specific and unspecific systems. Naive CD4 + T cells from 

Balb/c, leptin-deficient ob/ob and ovalbumine-TCR transgenic DO11.10 mice were 

stimulated in vitro under polarising conditions either specifically in the presence of 

antigen presenting cells (APC) and the specific antigen or unspecifically via anti-CD3 

anti-CD28. As described previously stimulation of naive T-cells under non-polarising 

conditions in the presence of leptin did increase the amount of Th1 cells. Furthermore, 

under polarizing conditions in the antigen-unspecific system leptin not only increased 

the polarization of Th1 cells, but furthermore induced a significant decrease of the 

amount of Th2 cells. However, when applying the antigen-specific system under 

polarising conditions, effects of leptin were more diverse and varied depending on the 

antigen and APC chosen: In vitro stimulation of naive DO11.10 T-cells in the presence 

of leptin did not affect the amount of Th1 cells and restimulation even decreased the 

percentage of Th1 polarised cells. This effect was equally evident using WT or ob/ob 

APCs. Similarly, polarisation to Th2 cells was not affected by leptin and restimulation in 

the presence of WT APCs with ovalbumine resulted in increased, but restimulation with 

OVA-peptide led to decreased Th2 polarization. In summary, our data further provide 

strong evidence that the adipokine leptin does affect T helper cell polarisation. However, 

this effect strongly depends on the type of antigen chosen, suggesting that in vivo leptin 

effects might be far more intricately balanced than prior reports based on unspecific 

studies in vivo may have suggested.

Stephan Paxian, Markus P. Kummer, Lars Tatenhorst, Klaus Pfeffer, Frank Kirchhoff, 

Roland Schmid, Michael T. Heneka 

Effects of neuronal and microglial disrupted RelA(p65) in the 

CNS during neuroinflammatory disorders 

Proteins of the NF-κB/Rel family of transcription factors are ubiquitously expressed and 

play a key role in a variety of physiological processes as acute phase, stress- and 

immune response, cell cycle and apoptosis. In mammals, the NF-κB/Rel family 

comprises five members, RelA (p65), RelB, c-Rel, NF-κB1 (p105/p50) and NF-κB2 

(p100/p52). Its role in the immune system and host defence has been well defined over 

the last decades. In contrast, our understanding of the function of this transcription 

factors in the nervous system (NS) is only emerging. Recent reports decipher the 

diverse functions of NF-κB in NS development and activity, which range from the control 

of cell growth, survival and inflammatory response to synaptic plasticity, behavior and 

cognition. Particular attention is given to the specific roles of NF-κB in the various cells 

of the NS, e.g. neurons and glia as NF-κB participates to several neurodegenerative 

disorders, such as Alzheimer´s, Parkinson´s and Multiple Sklerosis. To determine the 

specific role of the NF-κB subunit RelA(p65) in the NS we generated conditional RelA 

knockout mice using the Cre/lox-system to bypass the embryonic letality of mice with a 

conventional inactivated RelA. Intercrossing these mouse line with mice expressing 

either a neuronal (NEXC) or microglial (F4/80) specific Cre generates mice exhibiting a 

specific deletion of RelA in the referring tissue. Current inspections determine the 

efficiacy of the Cre specified RelA deletion in the CNS. In addition, histological analysis 

show a clear tissue defined effect of a Cre induced RelA inactivation. Ongoing 

experiments allocate the specific expression profiles and protein patterns of NF-κB/RelA 

target genes of isolated primary neurons (NEXC) or microglia (F4/80) from mice with a 

disrupted RelA in these cells. In addition, mouse models of neuroinflammatory disorders 

as e.g. MPTP (Parkinson), intercrossed APP/PS1 mice (Alzheimer), EAE (experimental 

autoimmune encephalomyelitis) and stroke should be established, as well as behavioral 

and cognitive (Morris water maze, open field, active avoidance) changes in these mice.

Fanny Edele, Cindy Reinhold, Stefan F. Martin 

Efficiency of T cell defence against melanoma depends on the 

DC immunization route 

We have recently shown that different routes of DC immunization induce effector/ 

memory CD8+ T cells expressing different arrays of tissue-specific homing receptors. 

This polarization is driven in vivo by tissue-specific DC. In our current study, we 

investigate the role of homing receptor polarization by DC vaccination for the efficiency 

of anti-tumor immunity in the mouse model of B16.F10 melanoma. We use the parental 

tumor as well as B16.F10GP33 that expresses a T cell epitope from the glycoprotein of 

lymphocytic choriomeningitis virus (LCMV). 

We examined the role of different DC immunization routes for the homing of T cell 

receptor transgenic P14 effector/memory T cells and wild type T cells that recognize 

GP33 and its correlation with the ability to control tumor growth. 

We adoptively transferred naive P14 T cells and activated them in vivo with GP33pulsed 

DC via the intracutaneous (i.c.), i.v. or i.p. route. After subcutaneous inoculation 

of B16.F10 or B16.F10GP33 melanoma cells into the left and right flanks of C57BL/6 

mice, respectively, tumor size was measured every 2 days. DC-GP33 prevented the 

growth of B16.F10GP33 tumor in all groups independent of the immunization route. 

Thus, induction of skin-specific homing receptors upon i.c. DC injection or the lack of a 

tissue-specific polarization or flexible reprogramming when DC are injected i.v. or i.p., 

respectively, allow efficient tumor defence in this setting. However, a different outcome 

was observed in recipient mice when no P14 T cells were transferred. In this case, longterm 

protective immunity against B16.F10GP33 melanoma required the induction of 

skin-specific CD8+ T cells by immunization with DC-GP33 via the i.c. route. Thus, tissuetargeting 

of tumor antigen-specific effector T cells by the appropriate DC vaccination 

route determines the efficiency of the anti-tumor immune response. 

Our findings are relevant for the biological therapy of human cancers.

Prajeeth Chittappen Kandiyil, Thomas Ebensen, Carlos Guzmàn, Reinhold Schmidt, 

Georg Behrens 

Efficient cross-priming induced by the toll-like receptor 2/6 

agonist MALP-2 

Introduction: MALP-2, a synthetic derivative of a lipopeptide from Mycoplasma 

fermentas which acts via the TLR 2/6 heterodimer has been widely exploited as 

adjuvant. MALP-2 also binds to CD36, a selective sensor of microbial diacylglycerides, 

being this association partially essential for its TLR signalling. Given that CD36 is 

exclusively expressed on CD8 + DC we hypothesized that MALP-2 can also exert a potent 

adjuvant activity for cross-priming against co-administered antigens. 

Methods: DC subpopulations from the spleen were enriched for antigen presentation 

assays in vitro and in vivo using ovalbumin (OVA) as a model antigen. The efficacy of 

MALP-2 in generating effector CTLs by cross-priming was further tested by in vivo killing 

assays. 

Results: Systemic administration of MALP-2 to mice led to upregulation of MHC-II, 

CD80 and CD86 on CD8 + and CD8 - DC in the spleen. Cross-presentation of OVA was 

enhanced in mice receiving MALP-2, as determined by increased proliferation of OVAspecific 

CD8+ T-cells (OT-I) in vivo and in vitro. MALP-2 also increased the antigen 

presentation of spleen-derived CD4 + DC to OVA-specific CD4 + T-cells (OT-II) in vitro. 

Importantly, co-administration of MALP-2 and cellular OVA resulted in generation of an 

effective CTL response, as assessed by an in vivo killing assay. Further, the inability to 

induce effective CTL response in MHC class II deficient mice suggests that MALP-2 aided 

cross-priming is dependent on CD4 + T cell help. In contrast to other TLR ligands, preactivation 

of DC with MALP-2 neither inhibited antigen uptake nor antigen presentation. 

Conclusion: Our data provide evidence that MALP-2 is a potent stimulus to generate 

CTLs via cross-priming and this activity is CD4 + T cell help dependent. Therefore, MALP- 

2 represents a promising tool for the establishment of immune interventions.

Julius Hafalla, Ana Rodriguez, Fidel Zavala 

Efficient development of Plasmodium liver stage-specific 

memory CD8+ T cells during the course of blood stage 

malaria infection 

Immunity to Plasmodium liver stages in individuals in endemic areas is inextricably 

linked to concomitant blood stage parasitemia. While Plasmodium sporozoite infection 

induces measurable CD8+ T cell responses, memory T cell development during active 

erythrocytic infection remains uncharacterized. Using transgenic T cells, we assessed 

antigen-specific CD8+ T cell effector responses induced by normal (NorSpz) and 

radiation-attenuated (IrrSpz) P. yoelii sporozoites. The magnitude, phenotypic 

activation and differentiation pathway of CD8+ T cells were similarly induced by NorSpz 

and IrrSpz. Moreover, in normal mice, memory T cells elicited after priming with NorSpz 

and IrrSpz generated identical recall responses following a heterologous boost strategy. 

Furthermore, these recall responses exhibited comparable in vivo anti-parasite activity. 

Our results indicate that sporozoites which retain their infective capacity induce memory 

CD8+ T cells that are robustly recalled by secondary immunization. Thus, erythrocytic 

infection does not preclude establishment of memory CD8+ T cell responses to malaria 

liver stages.

Stefanie Hoyer, Katrin Birkholz, Verena Wellner, Ina Müller, Erwin Schultz, Gerold 

Schuler, Niels Schaft, Jan Dörrie 

Electroporation of TCR-encoding RNA into CD4+ T cells in 

order to provide T-cell help 

Cancer immunotherapy has mainly focused on CD8+ cytotoxic T cells, even though CD4 

+-mediated T-cell help is required for an efficient anti-tumor response. Therefore, it 

would be beneficial to provide tumor-specific CD4+ T cells either for transfer together 

with tumor-specific CTL or to support tumor vaccination. Up to now, efficient T-cellreceptor 

(TCR) transfer required stable retroviral transduction. However, this method 

includes many risks like insertional mutagenesis, further unwanted effects of viruses, 

and genetic alteration, which could result into long lasting autoimmunity. Thus, we used 

an optimized RNA transfection protocol, to transiently introduce T-cell-receptor (TCR) 

alpha and beta chains, with a known antigen/MHC-specificity. Electroporation of CD4+ T 

cells with EGFP-RNA resulted in a high transfection efficiency (>85%). Furthermore, 

electroporation of CD4+ T cells with RNA coding for either a MAGE-3/HLA-DP4-specific 

TCR or a gp100/HLA-A2-specific TCR resulted in antigen-specific pro-inflammatory 

cytokine production. After antigen-specific stimulation with peptide-loaded DC 

particularly the Th1 cytokines IL-2, TNF, and IFNgamma were produced, but also lower 

amounts of IL-4, IL-6 and IL-10. Moreover, surface-antigen-profiles on T cells and DC 

were determined by FACS. These experiments also showed effects in an antigendependent 

manner; i.e. an increase in CD25, CD80 and CD86 expression on DC and of 

CD25 on CD4+ T cell. These data indicate that TCR-transfected CD4+ T cells can induce 

DC maturation and can therefore be used to provide T-cell help. Accordingly, this 

method for transient TCR transfer using RNA electroporation into CD4+ T cells can form 

a new strategy to induce more efficient CD8+ T-cell responses for the immunotherapy 

of cancer.

Stefanie Helm, Patrick Pankert, Stefanie Eikelmeier, Edward Shang, Hans Ulrich 

Weltzien, Martina Schnoelzer, Hermann-Josef Thierse 

Elements of the innate immune barrier: Proteomic 

identification of allergen-protein interactions in the human 

epidermis 

Background: 

Innate and adaptive molecular events underlying the most common contact 

hypersensitivity towards heavy metal nickel (Ni) are still incompletely understood. 

Aiming to identify primary allergen-protein interactions in the human epidermis, we 

have chosen a specific immunoproteomic approach. Keratinocyte derived Ni-interacting 

proteins were identified by mass spectrometric analysis and compared to previously 

generated data of human antigen presenting cells. 

Results: 

To investigate molecular effects of Ni ions on human keratinocytes immobilized metal 

ion affinity chromatography (IMAC) was used to isolate Ni-interacting proteins from 

primary cell lysates. After 2-dimensional gel electrophoresis (2D-PAGE), laser scanning 

of fluorescent protein gels (Fujifilm Europe, FLA 5100) followed by spot picking (Bruker 

Daltonics, Proteineer II), trypsin digestion and mass spectrometric analysis (Bruker 

Reflex II) more than 15 Ni-interacting epithelial proteins were identified in isolated 

human keratinocytes. Comparative analysis from previous studies revealed differential 

as well as similar Ni-interacting molecules in human B-cells, in-vitro generated DCs and 

primary human keratinocytes. Among others, several heat shock proteins were 

detected, which may be involved in initial cellular stress responses towards heavy metal 

Ni. 

Conclusion: 

Immunoproteomic identification of Ni-interacting proteins in primary keratinocytes is an 

important step in increasing the understanding of molecular mechanisms involved in the 

development and the pathophysiology of human nickel allergy. Results indicate a pivotal 

role for heat shock proteins and are discussed in light of Polly Matzinger's danger 

theory. 

(This work was supported in part by the Landesstiftung Baden-Wuerttemberg, 

Germany, Forschungsprogramm “Allergologie” by grant P-LS-AL/26, and the European 

Union, as part of the project Novel Testing Strategies for In Vitro Assessment of 

Allergens (Sens-it-iv), LSHB-CT-2005 – 018681, www.sens-it-iv.eu).

Benjamin Frey, Luis E. Munoz, Friederike Pausch, Ernst Pöschl, Klaus von der Mark, 

Martin Herrmann, Udo S. Gaipl 

Endogenous AnnexinA5 is Involved in the Immune Reaction 

Against Allogeneic Cells 

The exposition of phosphatidylserine (PS) on the outer leaflet of the cellular membrane 

is a hallmark of apoptotic cells. The high affinity binding of labelled annexinA5 (AxA5) is 

extensively used for the detection of apoptotic cells. In general, AxA5 is a highly specific 

ligand for PS. The latter provides a swift recognition and uptake of dying cells by 

professional phagocytes. Therefore AxA5 may interfere with the immunosuppressive 

effects of apoptotic cells. 

To study the effect of endogenous AxA5 on the immune reaction against allogeneic cells 

we are using AxA5-deficient mice. Interestingly, in contrast to WT mice, the AxA5deficient 

mice showed no reaction against allogeneic cells necrotized by mechanical 

stress. Furthermore, the delayed type hypersensitivity reaction in WT animals was 

significantly higher after immunization with late apoptotic cells. Additional, 

macrophages of AxA5-deficient animals bear a higher immunosuppressive potential. To 

further investigate immunomodulatory effects of AxA5 in the alloreaction, we 

subcutaneously injected viable, allogeneic tumour cells. In WT animals a significantly 

faster decline in size of the injected tumour cells was to be observed. 

As shown previously by us and others, a disturbed PS-dependent clearance by 

macrophages of apoptotic cells leads to the accumulation of the latter and to the 

occurrence of late apoptotic/secondary necrotic cells. This consequently leads to the 

release of danger signals and to a pro-inflammatory microenvironment. We conclude 

that the immunity against allogeneic cells is increased in the presence of endogenous 

AxA5.

Christina Hartwig, Miriam Mazzega, Thomas Tschernig, Detlef Neumann 

Endogenous IL-18 in experimentally induced asthma affects 

cytokine serum levels but is irrelevant for clinical symptoms 

T cells and T cell derived cytokines are involved in the complex pathogenesis of asthma. 

The role of the cytokine IL-18 however, is not clearly defined so far. On the one hand 

side IL-18 induces Th1-type cytokines and thereby might counter-regulate Th2mediated 

allergic asthma. On the other hand IL-18 also bears proinflammatory effects 

possibly enhancing experimental asthma. In order to elucidate the role of IL-18 in 

allergic pulmonary inflammation typical symptoms were compared after induction of 

experimental asthma in IL-18 -/- and in wt mice. Asthma was induced using ovalbumin 

(OVA) as allergen for sensitization and challenge. Sham sensitized and OVA challenged 

mice served as controls. BAL-fluid cytology, leukocyte infiltration in lung tissues, serum 

levels of OVA specific IgE and cytokines, and lung function were analyzed. Clear 

differences could be observed between control and asthma mice in wild type and IL- 

18 -/- animals. Surprisingly, no differences were found between asthmatic wild type and 

IL-18 -/- mice. Thus, in contrast to conflicting data in the literature IL-18 did not 

suppress or enhance the pulmonary allergic immune response in a murine experimental 

model of asthma.

Niklas Engels, Jürgen Wienands 

Enhanced signaling of the IgG-BCR is accomplished by 

tyrosine-phosphorylation of the cytoplasmic mIgG tail 

Depending on the developmental stage, B lymphocytes express on their cell surface 

distinct antigen receptor (BCR) classes, which are defined by the isotype of the 

immunoglobulin (Ig) subunit. All BCR classes utilize Ig-associated Igα and Igβ proteins 

as common signaling subunits. Yet, the BCR of the IgM class on naïve B cells triggers 

activation less potently than the IgG-BCR on class-switched memory B cells during 

secondary immune responses. We now demonstrate that increased signaling by the IgG- 

BCR is accomplished by inducible phosphorylation of an evolutionary conserved tyrosine 

residue in the cytoplasmic tail of the membrane-bound IgG (mIgG) heavy chain. 

Tyrosine-phosphorylated mIgG binds the adaptor molecule growth factor receptorbound 

protein 2 (Grb2) and triggers prolonged activation of protein tyrosine kinases as 

well as intracellular Ca 2+ mobilization. Hence, the Ig component of the IgG-BCR not 

only functions as antigen recognition device but actively improves the immune response 

of memory B cells, which is a fundamental requisite for successful vaccination against 

pathogens.

Fanny Edele, Rosalie Molenaar, Cindy Reinhold, Dominique Gütle, Jan C. Dudda, Reina 

Mebius, Mathias Hornef, Stefan F. Martin 

Environmental instruction of dendritic cells for T cell homing 

receptor imprinting 

Imprinting of tissue-specific homing receptors on T cells is induced by dendritic cells 

(DC) in lymph nodes draining e.g. the skin or the small intestine. However, it is yet 

unclear whether this process is governed by lymph node resident DC or rather by DC 

immigrating from the peripheral tissue. Our recent demonstration that Langerhans cells 

efficiently imprint skin homing receptors on T cells led us to the hypothesis that the 

peripheral tissue microenvironment licenses dendritic cells for homing receptor 

imprinting upon T cell priming in the draining lymph nodes. To analyse the role of nonhematopoietic 

cells in the microenvironment of the skin and the small intestine in the 

imprinting of the DC itself, we used co-cultures of bone marrow-derived (BM-) DC 

pulsed with the T cell epitope GP33 from the LCMV glycoprotein, TCR transgenic P14 

splenocytes and either dermal fibroblasts or small intestinal epithelial cells (SIEC). After 

co-culture of peptide pulsed BM-DC, P14 cells and dermal fibroblasts we observed an upregulation 

of the skin homing receptor E-selectin ligand on the CD8+ P14 T cells. In 

contrast, in co-cultures with SIEC, we observed an up-regulation of the gut homing 

receptors α4β7 integrin and CCR9 on the P14 T cells. 

DC re-isolated from co-culture with SIEC had only marginally up-regulated CD103 (αE 

integrin), but showed a clear expression of retinal dehydrogenases (RALDH). Thus, the 

BM-DC had acquired features of gut-specific DC. 

In summary, our results show that the peripheral tissue microenvironment induces 

tissue-specific characteristics in DC and endows them with the capacity to imprint the 

homing receptor profile specific for their tissue of origin on T cells in the draining lymph 

nodes.

Jana Zeitvogel, Thomas Werfel, Miriam Wittmann 

Epidermal stem cells differ in their response to IFN&gamma 

from other proliferative keratinocytes 

The epidermis has a pool of adult stem cells (ESC). Although the localisation of ESC is 

well described, we lack a clear understanding of their role in perturbed conditions such 

as inflammation. One of the most important mediators in inflammatory skin diseases 

acting on keratinocytes is IFN&gamma. The assumption that ESC might generate a 

protected niche prompted us to investigate their response to the pro-inflammatory 

cytokine IFN&gamma. In this study we isolated two populations of keratinocytes 

according to their adherence ability. ESC enriched by adherence showed a higher CD29 

and CD49f expression compared to other keratinocytes. Surprisingly, surface expression 

of CD54 was more inducible upon IFN&gamma stimulation in the ESC subpopulation. In 

contrary to that, a markedly lower induction of IL-18 and reduced basal production of 

CCL2 were observable in ESC. No differences in IFN&gamma induced IL-10, CXCL10, 

CCL22 or TGF&beta1 secretion was detectable between the two keratinocyte 

subpopulations. These results suggest that ESC respond to IFN&gamma with an 

"restricted" pattern of pro-inflammatory cytokines, and do not build up an antiinflammatory 

microenvironment by means of TGF&beta or IL-10. Activated ESC possess 

the capability to interact with infiltrating lymphocytes via CD54. In conclusion, the ESC 

compartment might actively contribute to the immunological properties of the skin 

organ.

Ellen Andresen, Joern Bullwinkel, Christoph Lange, Holger Heine 

Epigenetic regulation of defensin gene expression in lung 

epithelial cells and COPD 

Chronic obstructive pulmonary disease (COPD) includes emphysema and chronic 

bronchitis and is one of the most common diseases worldwide. Although inflammatory 

processes are thought to play an important role in the pathogenesis of COPD, the exact 

role of the innate immune system of the bronchial system and in particular the mode of 

action of epithelial cells in COPD remains poorly understood. Preliminary results of a 

recently started study with COPD patients of different clinical stages indicated an 

increased expression of human β-defensin (hBD)-1, but not hBD-2, compared with 

healthy controls. However, the mechanism by which the chronic inflammation of 

bronchial epithelial cells in COPD patients leads to a persisting overexpression of 

constitutively expressed antimicrobial peptides is not very well understood. Whereas 

active promoters generally contain histone H3/H4 hyperacetylation and tri-methylation 

at H3 lysine 4, the histone H3 lysine 9 methylation (H3K9me3) has been extensively 

correlated with repression. Our first results with lung epithelial cell lines show the 

absence of active histone modifications under non-induced conditions around the 

transcription start, while H3K9me3 is present throughout the hBD-1 and -2 loci. The 

modulation of defensin gene expression in COPD might be, in part, a consequence of 

epigenetic alternations in the chromatin structure of the defensin genes. The 

understanding of the basic mechanisms that mediate epigenetic regulation of defensin 

gene expression may lead to a better understanding of the pathogenesis of COPD, thus 

providing an unique platform for the development of new therapeutic strategies 

(supported by DFG, SFB617, project A23).

Anke Schütz, Hongqi Lue, Jürgen Bernhagen 

ERK1/2-MAPK signaling induced by macrophage migration 

inhibitory factor (MIF) is influenced by its CXXC motif 

MIF is a pleiotropic inflammatory cytokine that plays a pivotal role in a variety of acute 

and chronic inflammatory conditions such as septic shock, rheumatoid arthritis and 

atherosclerosis. MIF activates the canonical extracellular signal-regulated mitogenactivated 

protein kinase (ERK1/2-MAPK) pathway in a sustained fashion and MIFmediated 

ERK signaling has been suggested to contribute to the pro-inflammatory 

function of MIF in the above diseases. MIF-induced ERK signaling involves CD74, the 

cell surface form of invariant chain, and Src kinase activation, but the structure-function 

relationships of MIF underlying its ability to activate MAPK signaling have been 

unknown. We recently found that MIF activates transient ERK signaling and observed 

that the anti-apoptotic activity of MIF was dependent on its Cys-Ala-Leu-Cys (CXXC) 

redox sequence motif (Lue et al., Cell. Signal. 2006; Nguyen et al., J. Immunol. 2003). 

Here, we wished to examine the role of CXXC on MIF-mediated ERK1/2 signaling in 

fibroblasts. We first compared the effects of recombinant C60SMIF, a mutant in which 

the critical Cys60 residue of CXXC is mutated to serine, with the effect of wildtype MIF 

on ERK1/2 activity in immortalized and primary MIF -/- -mouse embryonic fibroblasts 

(MEFs). Surprisingly, C60SMIF was found to be a potent agonist of the MIF-stimulated 

ERK signaling effect. To confirm this observation and to test for autocrine effects, 

endogenous C60SMIF, which was derived from the supernatants of stimulated MEFs 

isolated from transgenic C60S/C60S-MIF mice (MIF cs/cs ), was analyzed. Compared to 

wtMEF supernatants, MIF cs/cs supernatants led to a marked upregulation of phospho- 

ERK1/2 levels, confirming the critical role that the CXXC motif appears to have for MIFstimulated 

ERK-MAPK signaling.

Winfried Barchet, Vera Wimmenauer, Leonid Gitlin, Susan Gilfillan, Marina Cella, 

Marco Colonna, Gunther Hartmann 

Essential Role of MDA-5 in Type I IFN Responses 

to Poly (I:C) and Encephalomyocarditis Picornavirus 

The cytosolic helicases RIG-I and MDA-5 detect conserved nu- 

cleic acid structures present during RNA virus replication. How- 

ever, the viral RNA ligand for MDA-5 is currently unknown. We 

generated MDA-5 deficient mice and have recently shown that 

MDA-5, not TLR3, is the dominant receptor mediating type I IFN 

secretion in response to the synthetic dsRNA analogue poly- 

riboinosinic:polyribocytidylic acid (poly I:C) in vitro and in vivo. 

Moreover, recognition of the encephalomyocarditis picornavirus 

was also entirely dependent on MDA-5. Consequently MDA-5 

deficient mice exhibit a selectively impaired antiviral response to 

this virus. We are currently evaluating whether a common MDA- 

5 stimulatory motif can be derived and incorporated into short 

RNA oligonucleotides. This would substantially widen the range 

of cell types receptive to immunostimulatory oligonucleotides, to 

include also non-immune cells.

Roman Karwot, Joachim Maxeiner, Steffen Schmitt, Petra Scholtes, Michael Hausding, 

Ildiko Boross, Hans Lehr, Susetta Finotto 

Essential role of NFATc2 in CD8+ cells in a murine model of 

allergic sensitization 

The development of allergic immune responses is mediated by CD4+ effector T cells 

producing T helper 2 cytokines. However the role of IFN-γ in asthma/allergy is not fully 

understood. We demonstrate that mice lacking nuclear factor of activated T cells-2 

(NFATc2) developed increased airway hyperresponsiveness. This AHR was associated 

both with activated, hyperproliferative lung CD4+ Th2 cells and with CD8+ T cells 

defective in IFN-γ production. Moreover, lung effector CD8+ T cells from NFATc2 (-/-) 

and IFN-γ (-/-) mice contained increased numbers of CD8+ CD122+ (IL-2Rβ chain) 

double positive T regulatory (Treg) cells resulting in increased interleukin-10 (IL-10) 

release. Adoptive transfer of ovalbumin (OVA) specific CD8+NFATc2 (-/-) T cells 

enhanced AHR generated by NFATc2 (-/-) CD4+T cells in immunodeficient mice. 

Interestingly, depletion of CD8+CD122+ cells abrogated the increased AHR present in 

CD8+NFATc2 (-/-) T cell-reconstituted SCID mice, by inducing IFN-γ in the airways. 

Thus, NFATc2 deficiency in CD8+ T cells results in IFN-γ downregulation and a 

concomitant increase in AHR. Taken together, our results identify NFATc2 as a key 

transcription factor that governs the function of CD8+ T cells in allergic asthma. 

This work is supportet by DFG FI-187.

Andre Tittel, Daniel Engel, Ulrich Dobrindt, Christian Kurts 

Establishing a murine model system to investigate the 

adaptive immune response against urinary tract infection 

Urinary tract infections (UTI), such as cystitis and pyelonephritis, are among the most 

common infection diseases worldwide. They are usually caused by uropathogenic gramnegative 

Escherichia coli bacteria (UPEC). The mechanisms underlying adaptive 

immunity against UPEC are unknown. 

To address this question, we established a murine model of UTI by inoculating UPEC of 

the E. coli strain 536, which had been isolated from a patient with chronic cystitis, into 

the bladders of C57/BL6 mice. This resulted in bladder infection of 20 % of the recipient 

mice, as detected by counting colony forming units arising from homogenized tissue on 

LB culture plates. In 10 % of the mice, bacterial ascension to the kidney was observed. 

Sporadically, death by urosepsis occurred. When mice were infected a second time after 

3 hours, the proportion of mice with UPEC ascending to the kidney increased to 90-100 

%, enabling studies also on pyelonephritis in our system. 

To study in vivo presentation of UPEC antigens, we transformed E. coli 536 with the 

pnir15ova plasmid containing the ovalbumin gene, and verified expression by western 

blot. In the next step, we injected mice with CFSE-labelled OT-I and/or OT-II cells as in 

vivo probes for presentation of bacterial antigen. After infecting mice with OVAtransfected 

UPEC, we observed specific proliferation of both OT-I and OT-II cells in the 

bladder and kidney draining LNs. Activation may have occurred by presentation of OVA 

associated with phagocytosed bacteria, or of OVA secreted from transfected UPEC. 

Future studies in this model will address the mechanisms of antigen transport from 

infected organs to draining LNs, the identity of the antigen presenting cell that activated 

T cells, and the immune effector functions resulting from such activation.

Marion Nonn, Shamsul A. Khan, Eva Distler, Ralf G. Meyer, Leonard D. Shultz, Rupert 

Handgretinger, Christoph Huber, Wolfgang Herr, Udo F. Hartwig 

Establishment of a NOD/SCID/IL2Rγc null hematopoietic stem 

cell transplantation model to study graft-vs-host and graft-vsleukemia 

immune responses of ex vivo modified human T 

lymphocyte grafts. 

Donor lymphocyte graft engineering to abrogate graft-vs-host (GVH) reactivity while 

improving graft-vs-leukemia (GVL) immunity is of key interest in allogeneic 

hematopoietic stem cell transplantation (HSCT). We established a HSCT model using 

NOD/SCID/IL2R common γ-chain null (γc null ) mice to evaluate GVH and GVL immunity of 

human leukemia-reactive T cell lines in vivo. These T cells were generated by in vitro 

stimulation of donor lymphocytes against primary acute myeloid leukemia (AML) blasts 

followed by CD137-mediated immunodepletion of alloreactivity using allogeneic 

fibroblasts (FB).>sup>1>/sup> 

Firstly, GVH responses of T cell lines were tested by subcutaneously implanting skin 

substitutes composed of primary dermal or bone marrow stromal FB embedded in a 

collagen matrix into NOD/SCID/γc>sup>null mice. Following adoptive transfer of 

undepleted human haploidentical CD3 + T cells, up to 23% of alloreactive T cells 

specifically migrated into viable and vascularized substitutes explanted 21d post 

adoptive transfer. In addition, selected T cell subsets elicited xenoreactivity induced by 

residual murine antigen-presenting cells. In contrast, no allo- and xenoreactivity was 

observed upon transfer of donor T cells enriched for leukemia reactivity. 

Secondly, to study GVL immunity, we examined protection to engraftment of human 

primary AML blasts in NOD/SCID/γc null mice by AML-reactive cytotoxic T cells (CTL) 

expanded from CD8 + donor lymphocytes in short term cultures. After 24h coculture of 

anti-leukemia CTL with AML blasts in vitro, both T cells and leukemia were transferred 

into NOD/SCID/γc null recipients. In contrast to controls, AML-reactive CTL were capable 

of completely preventing AML engraftment in these mice. 

We conclude that the NOD/SCID/γc null HSCT model may be a valuable tool for 

evaluating GVH and GVL immunity in vivo following donor T lymphocyte graft 

modifications in vitro. 

1 Wehler T and Nonn M, et al. Blood 2007;109:365-373

Rainer Wurth, Angelika Bold, Thomas Keller, Ulrike Trahorsch, Peter Voigt, Stefan 

Schubert, Ulrich Sack 

Evaluation and validation of a manual low-cost assay for the 

monitoring of CD4 counts in HIV-infected individuals in non- 

OECD countries 

Background: The CD4+ T-cell count is considered to be the best surrogate marker for 

monitoring the clinical course of infection with HIV. Flow cytometry, as the standard 

reference method for the enumeration of CD4+ T-cells, requires expensive equipment 

and well-trained technicians, thus preventing its widespread use in developing 

countries. 

Objective: In order to make this important surrogate marker available to more patients 

in non-OECD countries, we have modified a commercially available density-based cell 

preparation assay to make it applicable for low-cost cell enumeration. 

Methods: 1.) For evaluation (step 1), whole venous blood taken from 25 HIV-patients 

as well as 29 healthy blood donors was incubated with a cocktail of bi-specific 

tetrameric antibody complexes which crosslink unwanted nucleated cells (NC) to red 

blood cells (RBC) by forming RBC rosettes around targeted NC. After the following 

gradient centrifugation over a density medium, the enriched CD4+ T-cells were 

harvested and then counted on a haemocytometre using a light optical microscope. In 

parallel, the CD4 count of each sample was assessed by flow cytometry. 2.) For 

validation (step 2), this method was performed in blind quintuplicates on 12 HIV+ blood 

samples according to the FDA guidelines ICH-Q2A and ICH-Q2B. 

Results: Association of the introduced method (modified negative selection, MNS) with 

the reference method (flow cytometry, FCM) is given by regression models for both 

steps: 

Step 1: slope = 1.091, intercept = -46.5; (95% confidence intervall [CI] = 0.948 to 

1.240). Step 2: slope = 1.074, intercept = -38.3 (CI = 0.971 to 1.200 (step 2). The 

imprecision of MNS assessed during step 2 was 21.2% (intra-serial) and 18.8% (interserial). 

The cost of the examination of one blood sample is $0.30 - $0.50 per sample. 

Conclusion: The results suggest that the MNS-method, at is capable of providing an 

approximate CD4 count. This would enable a physician attending to an HIV+ patient to 

decide whether or not to start HAART or OI prophylaxis. At a cost of $0.30 - $0.50, it is 

affordable to patients living in resource-restrained areas. The technique has the 

potential to deliver an accurate, precise, low-cost test to monitor the status of HIV+ 

patients, especially in small laboratories in non-OECD countries. 

Keywords: CD4 count; HIV-infection; HIV-monitoring; RosetteSepTM; low-cost 

assay; non-OECD countries

Mathias Fousse, Robert Dinser, Urban Sester, Katinka Albrecht, Mahavir Singh, Hans 

Köhler, Ulf Müller-Ladner, Martina Sester 

Evaluation of latent tuberculosis infection in patients with 

inflammatory arthropathies before treatment with tumour 

necrosis factor-α blocking drugs using a novel flowcytometric 

interferon-γ release assay 

In this study, we compared the efficacy of the conventional skin test and a novel flow 

cytometric whole blood assay in the diagnosis of latent tuberculosis infection (LTBI) in 

patients with rheumatologic diseases evaluated for treatment with TNFα-blocking 

agents. 

97 patients were prospectively assessed for the presence of LTBI through clinical 

history, skin testing, and chest x-ray. In addition, T-cell reactivity towards tuberculin 

(PPD) and the M. tuberculosis specific proteins ESAT-6 and CFP-10 was determined ex 

vivo using a flow cytometric whole blood assay. 

After standard screening, 15% of patients receiving TNFα-blocking therapy were 

pretreated with isoniazide (INH), another 5% of patients did not receive TNFα-blocking 

therapy because of LTBI. PPD-reactivity in the skin was observed in 14% of patients 

compared to 39% with the whole blood test. Analysis of the M. tuberculosis specific 

response to ESAT-6 and CFP-10 revealed positive results in 16% of patients. Using a 

decision tree incorporating history, chest x-ray and either skin-test or ESAT-6/CFP-10 

results, 18 or 22% of patients, respectively, were classified as latently infected with M. 

tuberculosis. Four patients treated with INH because of a positive skin reaction did not 

show reactivity to ESAT-6/CFP-10 in the whole blood assay. Another six patients not 

pretreated with INH because of negative skin tests would have received INH, had the 

results of the whole blood assay been taken into account. 

In conclusion, the skin test has a low sensitivity and specificity for the diagnosis of LTBI 

in our cohort, resulting in both over- and undertreatment with prophylactic INH. The 

flow cytometric analysis of T-cell reactivity to proteins specific to M. tuberculosis is a 

promising approach for refining diagnostic testing.

Katherina Sewald, Maja Henjakovic, Simone Switalla, Norbert Krug, Armin Braun 

Ex vivo Immunomodulatory Testing using Precision Cut Lung 

Slices: Focus on Dendritic Cells 

Introduction: Precision cut lung slices (PCLS) offer the distinctive opportunity to gain 

insight into lung morphology and physiology under in vitro cell culture conditions. 

Objective of this work is the assessment of the immune modulating potency of 

substances on PCLS, especially on dendritic cells (DCs). DC activation and maturation 

are characterized by changes in cell surface marker expression (e.g. MHCII, CD86, 

CD40) and cytokine production. 

Methods: Extraction of lung tissue (mouse) was performed directly post mortem to 

conserve vitality of the tissue. Lungs were filled and cut with a special microtome. After 

preparation of PCLS with a thickness of approx. 220 µm slices were cultivated at 37 °C 

under cell culture conditions. Vitality of the lung slices was either controlled by 

measurement of LDH enzyme activity or live/dead staining with calcein AM/ethidium 

homodimer-1 using confocal laser scanning microscopy (CLSM). Cytokines and 

chemokines were detected with Luminex technology. Dendritic cell markers CD11c, MHC 

class II, CD40 and CD86 were investigated in living mouse PCLS in situ using CLSM. 

Results: Ex vivo incubation of PCLS with LPS induced both changes in cytokine profile 

and in expression of cell surface markers on DCs. LPS induced and dexamethasone 

prevented LPS induced release of cytokines and chemokines such as IL-5 (180 % for 

LPS to 0 % for LPS/dexamethasone), IL-1 (1550 % to 325 %), TNF-alpha (5340 % to 

470 %), IL-12 (723 % to 170 %) and Rantes (1060 % to 120 %) in PCLS. Expression of 

MHC class II, CD40 and CD11c but not CD86 could be observed in naive untreated 

PCLS. After incubation of PCLS with LPS exclusively a strong enhancement of MHC class 

II was found. In contrast, coculture of PCLS with ovalbumin-specific T-cells and 

activation with ovalbumine resulted in an upregulated expression of MHC class II and 

CD86 which was inhibited by dexamethasone. Thus, the method of PCLS might provide 

an in vitro technique to predict immune modulating potencies of inhaled substances on 

dendritic cells.

Ingo Irmler, Mieczyslaw Gajda, Rolf Bräuer 

Exacerbation of Antigen induced Arthritis in IFN-γ-deficient 

Mice as a Result of Unrestricted IL-17 Response 

In rheumatoid arthritis (RA), Th cells are supposed to be involved in induction and 

perpetuation of autoimmune disease with a dominance of the pro-inflammatory Th1 

response. However, the role of IFN-γ, the major cytokine produced by Th1 cells, is still 

incompletely defined. We investigated antigen-induced arthritis, an animal model of RA, 

in IFN-γ-deficient and wild-type C57Bl/6. 

Inflammatory response in the acute stage was strikingly increased in IFN-γ-deficient 

mice, demonstrated by exacerbated joint swelling, DTH reaction and histopathological 

assessment of arthritis. Intraarticular administration of exogenous IFN-γ significantly 

suppressed inflammation in IFN-γ-deficient as well as in wild-type mice. 

Increased production of IL-2, IL-4, IL-5, IL-6 and in particular IL-17 upon stimulation of 

lymph node and spleen cells from IFN-γ-deficient mice was associated with a decreased 

humoral immune response with low serum levels of total and antigen specific 

immunoglobulins (IgG, IgG1, IgG2a, IgG2b, IgG3). The lack of endogenous IFN-γ 

resulted in large numbers of neutrophil granulocytes infiltrating acute inflamed knee 

joints. Monoclonal antibodies neutralising IL-17 diminished acute inflammation. In vitro, 

we found that Th cell expansion and production of IL-17 upon re-stimulation was 

effectively inhibited by IFN-γ in a dose-dependent manner. These results clearly 

demonstrate in vivo a dominance of anti-inflammatory properties of IFN-γ during the 

initial phase of AIA and suggest disease promoting effects of IFN-γ-deficiency acting via 

IL-17 modulated pathways.

Gasteiger Georg, Kastenmuller Wolfgang, Sutter Gerd, Drexler Ingo 

Exclusive cross-priming of cytotoxic T-cells dictates antigen 

requisites for MVA vector vaccines 

Viral vectors are evaluated as recombinant vaccines but little is known about the 

antigen-presentation pathways that are important to induce efficient T-cell immunity 

with these vectors. Many viruses can infect pAPCs resulting in direct presentation of 

antigenic determinants. MHC-I-restricted cross-presentation of exogenous antigen, 

however, can contribute to antiviral immunity. We confirm metabolic stability as a 

critical factor for access of antigenic substrates for antigen-presenting pathways. We 

analyzed vaccines based on the replication-deficient vaccinia virus MVA in two distinct 

mouse models. Infection with MVA expressing the model antigens tyrosinase or 

ovalbumin either targeted for rapid proteasomal degradation or expressed as minigenes 

enhanced MHC-class-I/peptide presentation on DC as well as non-pAPC in vitro and in 

vivo, but dramatically impaired CD8+ T-cell (TCD8+)priming. Similar results were 

obtained when antigen presentation was restricted to cross-presentation in vivo 

indicating that direct priming plays a minor role for TCD8+ induction. Additionally, 

downregulation of cross-presentation by CpG-induced maturation of DC prior to 

immunization abrogated TCD8+ priming, but did not affect infectivity rate, antigen 

processing or direct priming of TCD8+ in vivo. 

Our results show that MVA vaccines exclusively depend on TCD8+ cross-priming. This is 

surprising, as we show that MVA easily infects DC and allows strong antigen 

presentation by these pAPC in vivo. The data reveal new aspects concerning biological 

properties of antigens that can be selected to induce strong T-cell immunity. Moreover, 

we show that it is possible and necessary to adjust the target antigens to the intrinsic 

requirements of the delivering vector with direct implications for the design of optimized 

vaccines targeting TCD8+ responses in infectious disease and cancer. Recent clinical 

studies suggest that our findings might also apply to humans.

daomin gong 

Expression of human GITRL on myeloid dendritic cells 

enhances their immunostimulatory function but does not 

abrogate the suppressive effect of CD4+CD25+ regulatory T 

cells 

CD4(+)CD25(+) regulatory T cells (Treg) have been described as an important hurdle 

for immunotherapy. Engagement of glucocorticoid-induced TNF receptor-related protein 

(GITR) has emerged recently as an important mechanism to control the suppression of 

CD4(+)CD25(+) Treg. Furthermore, it has been documented extensively that GITR 

ligation is costimulatory for naive and activated T cells in the murine setting. However, 

little is known about the role of the human GITR ligand (huGITRL). We wanted to 

explore whether huGITRL could enhance antigen-specific T cell priming by dendritic cells 

(DC). First, we confirmed the endogenous expression of GITRL on HUVEC. We also 

detected GITRL expression on EBV-B cell lines, whereas no GITRL expression was 

observed on human monocyte-derived DC. Electroporation of GITRL mRNA in monocytederived 

DC resulted in a strong and long-lasting surface expression of GITRL. In 

contrast to data obtained in mice, no significant abrogation of Treg suppression by 

GITRL-expressing human DC was observed. Consistent with our mouse data, we 

showed that huGITRL is costimulatory for responder T cells. Furthermore, we found that 

GITRL-expressing DC primed increased numbers of Melan-A-specific CD8(+) T cells. We 

conclude that although huGITRL is not capable of alleviating Treg suppression of 

responder T cells, huGITRL overexpression on monocyte-derived DC enhances their 

capacity to induce antigen-specific T cell responses. Thus, GITRL incorporation in DC 

might improve the antitumor immune response after vaccination.

Katjana Klages, Anja Stirnweiss, Jörg Reimann, Hansjörg Hauser, Andrea Kröger 

EXPRESSION OF INTERFEON REGULATORY FACTOR-1 IN CT26 

COLON CARCINOMA CELLS INDUCES ANTI-TUMOR ACTIVITY 

The transcription factor Interferon regulatory factor-1 (IRF-1) was originally identified 

as a protein mediating the effects of the interferon system. Besides its regulatory 

functions of the cellular response in host defense, e.g. establishing the antiviral state, 

IRF-1 was found to be a tumor suppressor. To study the tumor suppressive effects of 

IRF-1 in vitro and in vivo the colon carcinoma cell line CT26 was transduced by lentiviral 

vectors to express the IRF-1 gene under control of the tetracycline inducible promoter. 

The tumor cells showed a significant growth arrest after induction of IRF-1. Furthermore 

the reversion of the transformed phenotype could be demonstrated by decrease of 

anchorage independent growth in soft agar assays. Upon expression of IRF-1 an 

increase of MHC class I expression was detected and these tumor cells showed a 

significantly higher recognition and elimination rate by specific cytotoxic T cells. In 

addition, the induction of chemokines like Cxcl10 or Ccl19 was shown and transwell 

assays indicated that IRF-1 expression in tumor cells leads to an increased attraction of 

lymphocytes. Besides these direct effects on tumor cells IRF-1 is capable of mediating 

its anti-tumor effects by inducing a tumor specific immune response in the host. To 

evaluate whether intrinsic IRF-1 effects are sufficient to reject tumors in vivo the tumor 

cells were subcutaneously injected in nude mice. Tumor growth was delayed in nude 

mice, but complete tumor rejection was observed in immune competent syngenic mice. 

Tumor specific cytotoxic T cells were detected in mice rejected the tumor upon IRF-1 

induction. Further experiments revealed that also established tumors were rejected 

upon IRF-1 expression. These results demonstrate that IRF-1 expression in tumor cells 

leads to an induction of a tumor specific immune response, which can be responsible for 

a complete tumor rejection.

Thorsten Stroh, Arvind Batra, Rainer Glauben, Inka Fedke, Stephen Girardin, Martin 

Zeitz, Britta Siegmund 

Expression of NOD2 but not NOD1 is modulated by proinflammatory 

cytokines in murine preadipocytes 

Preadipocytes are linked to the innate immune system indicated by their expression of 

functional Toll-like receptors (TLR) and ability to phagocytosis. Recent studies of our 

group revealed the expression of members of the nucleotide-binding site and leucinerich 

repeat (NBS-LRR) proteins. These proteins belong to the family of intracellular 

pattern recognition receptors which act as sensors for motifs found in the 

peptidoglycans (PG) from bacteria. NOD1 binds to Lactyl-Tetra-DAP a peptidoglycan 

motif mainly found in PG from Gram-negative bacteria, NOD2 detects muramyl 

dipeptide (MDP) a motif contained in PG derived from Gram-positive bacteria. 

This study aimed to investigate the expression of NOD1 and NOD2 and their 

functionality in murine preadipocytes. Changes in expression were analysed after ligandspecific 

stimulation and stimulation with pro-inflammatory cytokines using quantitative 

real-time RT-PCR. Functionality of these receptors was tested by ligand-specific 

stimulation, and the subsequent response was evaluated by IL-6 production and 

activation of the NF-κB pathway. Specificity of stimulation was confirmed by RNA 

interference (RNAi) strategies. NOD1 mRNA was detectable in all samples. The 

expression was not altered after ligand–specific stimulation or stimulation with the 

proinflammatory cytokines IL-1β and TNFα. In contrast, NOD2 was slightly increased by 

NOD1-specific stimulation but could significantly be upregulated by stimulation with IL- 

1β. The strongest upregulation was observed after stimulation with LPS but different 

from stimulation with IL-1β or TNFα, LPS-induced upregulation was only present after 

4h of stimulation. Specific stimulation of NOD1 by LT-DAP resulted in a strong activation 

of the NF-κB pathway and a significant IL-6 production could be observed. 

Our data provide evidence that functional NBS-LRR proteins are expressed in 

preadipocytes and that expression of NOD2 can be modulated by pro-inflammatory 

cytokines. Thus underlining the position of preadipocytes in the innate immune system.

Cemil Korcan Ayata, Cinthia Farina, Markus Krumbholz, Florian Weisel, Thomas 

Winkler, Andreas Rosenwald, Reinhard Hohlfeld, Edgar Meinl 

Expression of p75 neurotrophin receptor (p75NTR) and brain 

derived neurotrophic factor (BDNF) in germinal centers 

The neurotrophin receptor (p75NTR) is a pan receptor for all neurotrophins (NGF, BDNF, 

NT3, NT4/5). Neurotrophins have mainly been studied because of their effect on 

survival of neuronal populations. P75NTR is classified as a member of TNF receptors, 

but no TNF ligand and no function in the immune system has been identified so far. 

We report that full length transcripts of p75NTR were expressed in immune organs and 

some peripheral blood mononuclear cells. Adult CNS tissue and immune organs 

transcribed similar levels of p75NTR as seen by TaqMan PCR. A subset of B cells in 

adenoids/tonsils, but not in blood expressed p75NTR as seen by both FACS and TaqMan 

PCR. Double stainings showed p75NTR expression on follicular dendritic cells. A 

potential ligand of p75NTR, BDNF, was seen in germinal centers by immunostaining and 

confirmed by TaqMan PCR of LASER capture dissected material. P75NTR was also highly 

expressed in extranodal follicles in Hashimoto Thyroiditis and follicular B cell lymphoma. 

The abundant expression of p75NTR and one of it’s ligands in germinal centers in 

normal lymphatic tissue and in extranonal follicles suggests a role of this TNF receptor 

in Ig production and in immunopathology.

Matthias M Gaida, Frank Guenther, Martin Loos, Christof Wagner, Gertrud Maria 

Hänsch, Helmut Friess, Nathalia Giese, Wente Moritz 

Expression of the chemokine receptor CXCR6 on 

polymorphonuclear neutrophils (PMN) in pancreatic tumour 

specimen and in acute, localised bacterial infections 

The chemokine receptor (CXCR) 6 has been described on lymphoid cells, and is thought 

to participate in the homing of activated T-cells to non-lymphoid tissue. We now provide 

evidence that CXCR6 is also expressed by activated polymorphonuclear neutrophils 

(PMN) in vivo: Examination of biopsies derived from patients with pancreatic cancer by 

confocal laser scan microscopy revealed a massive infiltration of PMN that also 

expressed CXCR6. PMN of the peripheral blood of these patients, in contrast, did not. To 

answer the question whether CXCR6 expression is a property of infiltrated PMN, 

leukocytes were collected from patients with localised soft tissue infections in the course 

of the wound debridement. By cytofluorometry, the majority of these cells were 

identified as PMN. Up to 50 % of these PMN were also positive for CXCR6. Again, PMN 

from the peripheral blood of these patients were negative for CXCR6, as were PMN of 

healthy donors. In a series of in vitro experiments, up-regulation of CXCR6 on PMN of 

healthy donors by a variety of cytokines was tested. So far, a minor, although 

reproducible effect of tumour-necrosis factor α was seen: brief exposure of PMN to lowdoses 

of TNF α induced surface expression of CXCR6 on 18.5 ± 9 % of the PMN (mean 

± SD, n=6). In summary, our data provide evidence that CXCR6 is not constitutively 

expressed on PMN, but is up-regulated under inflammatory conditions.

Eric Keil, Nana Ueffing, Linda Clayton, Ellis Reinherz, Klaus Schulze-Osthoff, Ingo 

Schmitz 

Expression profiling identifies Gadd45β as a novel mediator of 

negative selection 

Apoptosis is essential for the development of multicellular organisms and its 

deregulation may lead to various diseases. In the immune system, reduced apoptosis 

results in the accumulation of autoreactive T cells leading to impaired tolerance and 

autoimmunity. Autoimmune diseases like type I diabetes or multiple sclerosis arise from 

defects in peripheral tolerance, while the autoimmune polyglandular syndrome type 1 

(APS-1 or APECED) is due to impaired central tolerance, i.e. negative selection. To get 

further insights into the process of negative selection we have analyzed gene induction 

during peptide-induced negative selection in vivo by oligonucleotide arrays. The most 

strongly induced gene was Gadd45β, which is implicated in differentiation and 

apoptosis. Upregulation of Gadd45β was verified on the mRNA and protein level by 

quantitative PCR and Western blotting, respectively. Importantly, Gadd45β-expression 

in vivo was stimulated by peptides inducing negative selection but not by those inducing 

positive selection. In situ hybridization supported a role for Gadd45β in negative 

selection as its mRNA was specifically expressed in the medulla. Finally, overexpression 

of Gadd45β induced apoptosis in CD4+CD8+ double positive T cells. Thus, Gadd45β 

may induce a yet unrecognized pathway for clonal deletion in the thymus next to the 

established once induced by Bim, Nur77 and FasL/CD95L.

Silke Meister, Kirsten Neubert, Kai Herrmann, Renate Burger, Martin Gramatzki, 

Sabine Hahn, Sandra Schreiber, Ulrich Schubert, Hans-Martin Jäck, Reinhard Voll 

Extensive immunoglobulin production sensitizes myeloma 

cells for proteasome inhibition 

Multiple myeloma (MM), an incurable plasma cell neoplasia, is characterized by 

overproduction of monoclonal immunoglobulins (Ig). The recently clinically approved 

proteasome inhibitor bortezomib (Bz) acts directly on MM cells to cause cell death, 

supposedly by blocking the antiapoptotic factor NF-κB. However, the exact mechanism 

by which Bz acts is still under investigation. Extensive synthesis of Ig in MM cells results 

also in defective ribosomal products (DRiPs) and unfolded proteins degraded by the 

proteasome. Therefore, we hypothesized that the proapoptotic effect of Bz is due to the 

accumulation of unfolded proteins and DRiPs along with inhibition of NF-κB. Using the 

human MM IgG-secreting cell line JK-6L and murine •H-chain-transfected Ag8.H 

myeloma cells, proteasome inhibitor treatment induced markedly more apoptotic cell 

death in subclones producing high compared to low amounts of Ig. Unexpectedly, 

bortezomib did not markedly alter NF-κB activity. In contrast, Ig positive MM cells 

showed a highly induced AP-1 DNA binding activity upon Bz treatment. Importantly, in 

Ig-high MM cells Bz triggered production of reactive oxygen species (ROS) as well as 

strong activation of endoplasmic reticulum (ER) stress components involved in apoptosis 

such as CHOP and caspases. Additionally, cells harbouring Ig showed enhanced 

expression of the proapoptotic factor Bax and reduced expression of the antiapoptotic 

protein Bcl-2 due to Bz treatment. Moreover, proteasome inhibition results in formation 

and accumulation of IgG-derived DRiPs. Hence, we conclude that proteasome inhibition 

preferentially affects cells producing high amounts of proteins. Bz-induced cell death in 

MM cells is most likely mediated by ER stress induced through accumulation of unfolded 

proteins/DRiPs.

Carina Klein, Anja Grahnert, Sunna Hauschildt 

Extracellular NAD+ triggers transient [Ca2+]i changes in LPSactivated 

human monocytes via P2Y receptors 

In previous studies we have shown, that stimulating freshly isolated human monocytes 

with NAD + rapidly causes a transient concentration-dependent elevation of [Ca2+ ] i . This 

increase seems to be performed by Ca2+ influx via ligand-gated P2X receptors. Since 

calcium signals play an important role in triggering monocyte functions such as 

chemotaxis, phagocytosis and cytokine production, NAD + can provide a powerful mean 

to interfere with such functions. Here we asked whether extracellular NAD + also affects 

the [Ca2+ ] i in LPS-activated monocytes. We found that cultivating the cells for 16h 

resulted in a complete loss of the calcium response but this lack of response was 

prevented when LPS was present during the 16h incubation time. The NAD + -induced 

LPS-dependent increase is composed of a release of Ca2+ from intracellular stores and a 

calcium release activated influx of Ca2+ from the extracellular medium. The Ca2+ release from intracellular stores suggests that NAD + seems to exert its effects by 

interacting with G-protein-coupled P2Y members of the P2 receptor family which 

mobilize stored Ca2+ via PLC-β activation and IP3 production. By the use of agonists 

and specific antagonists of P2Y receptor subtypes we identified the P2Y11 receptor as 

one of the main receptors to be involved in the calcium response.

Anja Grahnert, Erik Schilling, Carina Klein, Sunna Hauschildt 

Extracellular NAD + triggers transient [Ca 2+ ] i changes in 

human monocytes 

via P2X-receptors 

Recently extracellular NAD + has been identified as a stimulus capable of inducing a rise 

in [Ca 2+ ] i in different cell types by different mechanism. In this study we could show 

that NAD + leads to a transient increase in [Ca 2+ ] i in human monocytes mediated by an 

influx of [Ca 2+ ] i from the extracellular medium. As NAD + does not exert its effect by 

entering the cells and as ATP prevents the NAD + induced effect we investigated the 

involvement of ATP receptors in the Ca2+ -response. ATP receptors are P2 purinoceptors 

classified into ionotropic P2X and metabotropic, G-protein-coupled P2Y receptors. P2Y 

receptors can be excluded, because NAD + only leads to an influx of extracellular Ca2+ . 

Of the seven P2X receptors P2X1 , P2X4 , and P2X7 were found to be expressed on 

human monocytes. The involvement of P2X1 as a receptor subtype mediating the effect 

of NAD + is supported by the finding that the increase in [Ca2+ ] i is inhibited by the 

specific P2X1 antagonists NF449 and suramin. Furthermore P2X7 plays a role by the NAD 

+ -induced increase in [Ca2+ ]i . The P2X7 antagonist KN62 and an anti- P2X7-Ab block 

the NAD + effect. 

Surprisingly the binding of the P2X7 receptor did not lead to a pore formation. These 

data show for the first time that extracellular NAD + acts as a ligand at P2X receptors of 

human monocytes.


Feed-back control of dendritic cell homeostasis 

Dendritic cells (DCs) are pivotal coordinators of the immune system, governing the 

choice between T cell immunity and tolerance. Despite their importance, the 

mechanisms regulating the size of the DC compartment are largely unknown. In order 

to investigate DC homeostasis we have developed a BAC transgenic mouse strain in 

which CD11c+ DCs express the diphtheria toxin receptor (DTR). In this CD11c-DOG 

strain, about 95% of DCs are depleted by a single DT application. Due to their fast turnover, 

continuous DC depletion requires daily DT application. 

Here we show that a residual fraction of DC progenitors can be induced to repopulate 

the DC compartment using wild-type and CD11c-DOG mixed bone marrow chimeras in 

which most of the DCs derive from CD11c-DOG bone marrow and, thus, can be 

depleted by DT. These results indicate that there exist active homeostatic mechanisms 

controlling the DC pool size. To investigated whether DC progenitors respond to the size 

of the DC compartment by modulating their differentiation potential, we performed 

competitive cotransfers of splenic DC progenitors obtained from mice with normal or 

reduced DC numbers. About two thirds of the donor-derived DCs differentiated from 

progenitors obtained from DC-deficient mice, indicating that a reduced number of DCs 

leads to an increased activity of their precursors. The increased progenitor activity is 

likely due to an increased frequency of DC progenitors in the spleen of DC-deplete mice 

as suggested by colony forming assays in vitro in response to GM-CSF. Furthermore, a 

reduced DC number also appears to increase the differentiation activity of DC 

progenitors on a per-cell basis because the number of DCs recovered after transfer of 

splenic DC progenitors from untreated mice into DC-deficient mice is ten times higher 

than that obtained when the same precursors are transferred into mice with normal DC 

numbers. 

Thus, these results demonstrate that there are active homeostatic mechanisms 

controlling the size of the DC compartment via a feedback loop whereby the number of 

DCs controls the activity of their progenitors.

Karsten Kretschmer, Alexander Marson, Garrett M. Frampton, Julia Polansky, Richard 

A. Young, Harald von Boehmer 

Foxp3-dependent gene regulation requires T cell activation 

Foxp3-expressing CD4+CD25+ regulatory T cells play an essential role in the regulation 

of immune responses under physiological conditions. While the function of Foxp3 in Treg 

lineage specification has been firmly established, the molecular pathways involved in 

the Foxp3-specified developmental and suppressor program are still poorly understood. 

Here we report on new evidence that T cell receptor signaling strength greatly 

influences the induction of Foxp3 expression in initially naïve CD4+Foxp3- T cells. 

Consistent with in vivo experiments, lack of co-stimulation (e.g. via CD28) or inhibition 

of T cell receptor-emanating signals during the conversion process enhances TGF-beta 

dependent induction of Foxp3 expression in vitro. In addition, global gene expression 

profiling in conjunction with genome-wide DNA based location analysis (ChIP-on-Chip) 

using custom-spotted tiling arrays, which interrogated Foxp3 binding to 5’ promoters of 

approximately 16000 annotated mouse genes, indicated that Foxp3 had very little effect 

on gene expression in un-stimulated T cells. In contrast, stimulated Foxp3- and Foxp3+ 

T cells exhibited significant differences in expression of almost 1% of mouse genes. 

Although we found that Foxp3 could also function to activate gene expression, the 

predominant effect of Foxp3 promoter occupancy was to suppress the NFAT-dependent 

activation of target genes upon T cell stimulation. Consistently, Foxp3-bound promoters 

were enriched for NFAT binding sites.

Thomas Quast, Barbara Tappertzhofen, Cora Schild, Waldemar Kolanus 

Function of CD81 in dendritic cell migration 

CD81 belongs to the Tetraspanin superfamily of evolutionary conserved membrane 

proteins. The most distinctive feature of the Tetraspanins is their ability to form lateral 

associations with one another and with numerous other molecules including integrins, 

cell surface receptors and signaling molecules, creating multi-molecular signaling 

complexes at the cell surface. Hence, Tetraspanins have been implicated in the 

coordination of signal transduction, antigen-presentation, activation, cell adhesion and 

migration, cell-cell interactions and cell fusion. 

Dendritic cells are the most important antigen presenting cells at the interface of innate 

and adaptive immunity. Upon capturing antigens in context of an inflammatory 

situation, dendritic cells undergo a phenotypic and functional maturation process, which 

includes the increase of cell motility. The capacity of dendritic cells to activate naive T 

cells depends on their ability to migrate to T-cell areas in the lymph nodes. 

Using siRNA techniques, we could show that comparable to Rac1-deficient cells CD81deficient 

human mature monocyte-derived dendritic cells show significantly reduced cell 

migration, which is accompanied with strong deficiencies in lamellipodia formation. 

Furthermore, CD81 is required for the phosphorylation of members of the ezrin/radixin/ 

moesin (ERM) family of proteins, which function as cytoskeleton linkers that are 

involved in membrane protrusion formation and cell motility. As Tetraspanins recruit 

intracellular-signaling enzymes, including protein kinase C (PKC), and organize them in 

the “Tetraspanin-Web” and the phosphorylation of ERM proteins is thought to be 

downstream of PKC, we propose that CD81 is a crucial component in the signaling 

network coordinating dendritic cell migration.

Sebastian Dütting, Wolfgang Schuh, Kai Hermann, Christiane Lang, Hans-Martin Jäck, 

Dirk Mielenz 

Function of Swiprosin-2/EFhd1 during B cell development 

Functional peripheral B cell subsets are produced from hematopoietic stem cells. An 

important early checkpoint of this process is the transient expression of the pre-B cell 

receptor (pre-BCR) at the transition from the late progenitor B cell (pro-B) to the large 

precursor B cell (pre-B) stage. The pre-BCR consists of a membrane-anchored HC of the 

• isotype (•HC) associated with λ and VpreB, and drives pre B cells into a clonal 

expansion phase of two to five cell divisions. 

Swiprosin-2 (also: EFhd1/Mitocalcin) is a close homologue of Swiprosin-1/Efhd2, an 

adaptor protein we showed recently to be involved in proximal B cell receptor signaling. 

In contrast to Swiprosin-1 that was expressed in all murine B cell lines and primary B 

cells examined, albeit at different levels, Swiprosin-2 protein and mRNA were only 

expressed in a transformed pro-B-cell line (38B9) and in primary pro-B cells. We thus 

hypothesized that Swiprosin-2 is down-regulated by the pre-BCR. Using a transgenic tetoff 

system that allows inducible pre-BCR-expression in primary cells, we demonstrated 

pre-BCR-dependent down-regulation of Swiprosin-2 on mRNA and protein level. We 

corroborated this result with IL-7 dependent R5B pro-B cells and transformed 38B9 pro- 

B cells either lacking or expressing a functional surface pre-BCR. A dysfunctional, 

cytoplasmic •HC did not regulate Swiprosin-2 expression, demonstrating that only 

signals from the surface pre-BCR induce Swiprosin-2 down-regulation. We hypothesize 

that pre-BCR-induced down-regulation of murine Swiprosin-2 is required for pre-B cell 

differentiation and/or proliferation. To test this hypothesis we are currently infecting 

primary pro-B cells with a Swiprosin-2-encoding retrovirus that overcomes downregulation 

of Swiprosin-2. In the future, we want to identify the function of Swiprosin-2 

during B cell development and determine the mechanisms by which Swiprosin-2 

potentially affects B cell development.

Cornelia Rosner, Lutz Walter 

Functional analysis of MHC class I genes in the rhesus 

monkey (Macaca mulatta) 

The rhesus macaque represents a widely used animal model for the study of infectious 

diseases, organ transplantation, and in development of vaccines. Recently, the major 

histocompatibility complex (MHC) of the rhesus macaque (Mamu) was sequenced 

completely (Daza-Vamenta et al., 2004), revealing extensive duplications in the class I 

gene subregions. The sequenced haplotype displayed 2 and 19 loci, which show close 

relationship to HLA-A and HLA-B, respectively. The functional significance of this 

plethora of class I genes is, however, unknown so far. 

In order to study the function of particular class I molecules, we cloned representatives 

of Mamu-A and Mamu-B genes in an expression vector that contains AcGFP. These 

constructs were transfected in the MHC class I-negative cell line K562. Surface staining 

with the human MHC antibody W6/32 revealed variable expression patterns. Natural 

killer (NK) cell activity against different Mamu-A and Mamu-B expressing transfectants 

was tested by degranulation assays with freshly isolated rhesus monkey PBMCs. Some 

transfectants exhibit higher inhibitory capacity, as exemplified by lower cell surface 

expression of the CD107a molecule (degranulation marker). Currently, we are 

investigating the specific binding of KIRs and MHC class I ligands by biochemical means. 

Daza-Vamenta R, Glusman G, Rowen L, Guthrie B, Geraghty DE (2004) Genetic 

divergence of the rhesus macaque major histocompatibility complex. Genome Res 14: 

1501-1515

Simone Abel, Jan Buer, Wiebke Hansen 

Functional analysis of Neuropilin1 in regulatory T cells 

Recently, we could identify Neuropilin1 (Nrp1) as a useful surface marker molecule for 

regulatory T cells (Treg). Nrp1 has been initially described as semaphorin III and VEGF 

receptor, being essential for axonal guidance and vascularisation, but little is known 

about the function of Nrp1 in immune regulation. To gain further insights into the role of 

Nrp1 on Treg cells, we have constructed retroviral vectors encoding the full length Nrp1 

cDNA and have optimized the retroviral transduction protocol to overexpress Nrp1 in 

freshly isolated CD4 + CD25 - naïve T cells. Using this approach we are able to analyse 

the impact of Nrp1 on the proliferative and suppressive capacity of Treg cells. 

Furthermore, we will analyse the expression of the Treg specific transcriptional regulator 

Foxp3 and other molecules known to be involved in Treg cell function as well as the 

cytokine profile of Nrp1-transduced T cells in comparison to control vector transduced T 

cells. These results may contribute to a better understanding in the molecular 

mechanism of Treg cell function.

Gamze Kabalak, Torsten Matthias, Reinhold E. Schmidt, Torsten Witte 

FUNCTIONAL CHARACTERISATION OF ILT6 AS GENETIC RISK 

FACTOR FOR MULTIPLE SCLEROSOS AND SJÖGREN’S 

SYNDROME 

Introduction: Autoimmune diseases Multiple Sclerosis (MS) and Sjögren’s syndrome 

(SS) are polygenic disorders and appear to be triggered by EBV infection. Risk genes for 

Multiple sclerosis (MS) are localized on chromosome 6 and the chromosomal region 

19q13.4, the later harboring the genes of the Immunoglobulin-like transcripts (ILTs). 

ILTs are expressed on antigen presenting cells, T and NK cells and play a role in the 

tolerance induction of the immune system by interacting with regulatory T cells. A 

homozygous deficiency of ILT6 was shown to be associated with MS and SS. As ILT6 is 

lacking a transmembrane domain, it is likely to be secreted and may act as a soluble 

mediator. To address the question of the role of ILT6 the recombinant protein was 

expressed and functional studies were performed. 

Methods: Adhesion tests with MCF-7 cells and ILT6 were performed. These cells express 

MHC class I on there surface. Therefore T cell clones expressing either CD8 alpha or 

beta were incubated together with MCF-7 cells in the presence or absence of ILT6. 

Further mixed lymphocyte reaction (MLR) as well as cytotoxicity assays were performed 

with changing concentrations of ILT6. 

Results: These experiments revealed a concentration dependent blockade of the 

interaction of CD8 α and MHC I by ILT6. Furthermore the proliferation of T cells in the 

MLR was increased by addition of ILT6. On the other hand cytotoxicity assays showed 

no influence of ILT6 on NK cells. 

Conclusion: ILT6 may act as a natural immunomodulator with profound effect on T cells. 

Supported by Gemeinnuetzige Hertie Stiftung, Sicca-Forschungsfoerderung and BMBF 

KN Rheuma C2.12

Kathrin Gube, Inga Gebuhr, Katrin Vogt, Erik Kwidzinski, Christine Brandt, Hans-Dieter 

Volk, Birgit Sawitzki 

FUNCTIONAL CHARACTERISATION OF THE TOLERANCE 

ASSOCIATED GENE (TOAG-1) 

Backround: The tolerance associated gene 1 (TOAG-1) has been identified to be highly 

expressed in graft infiltrating cells of tolerance developing treated recipients in several 

experimental transplant models (Sawitzki et. al. AJT). TOAG-1 is highly expressed in 

DC’s and CD4+ T cells. The amino acid sequences of rat, mouse and human isoforms 

contain no homology to known proteins but they contain a cleavage site for 

mitochondrial presequences. Aim: Here we analysed the subcellular localisation of TOAG- 

1, its transcriptional regulation as well as effects of TOAG-1 over expression in murine 

CD4+ T cells. Methods and Results: Transfection of mouse fibroblast 3T3 cells with Nterminal 

fusion protein of TOAG-1 and EGFP lead to accumulation of green fluorescence 

in organelle like structures. Costaining of TOAG-1-EGFP transfectants with fluorescent 

probes or antibodies (e.g. anti-Hsp60) either accumulating within mitochondria 

(Mitotracker) or lysosomes (Lysotracker) revealed an exclusive expression of TOAG-1 in 

mitochondria. To analyse transcriptional regulation of TOAG-1, CD4+ T cells from naïve 

C57BL/6 mice were stimulated with allogeneic (BALB/c) splenocytes which resulted in a 

10 fold down regulation of TOAG-1 transcription. This down-regulation could be 

inhibited by a treatment with the non-depleting anti-CD4 antibody (YTS177) and 

completely prevented by Cyclosporin A. Overexpression of TOAG-1 in murine CD4+ T 

cells via retroviral gene transfer resulted in a lower mitochondrial membrane potential 

(FACS: TMRM) and a higher predisposition to undergo apoptosis (FACS: Annexin) after 

allo-activation in vitro. Conclusions: TOAG-1 is exclusively localised within mitochondria 

and its transcription tightly controlled after T cell activation in vitro and in vivo. By 

lowering the mitochondrial potential and predisposing T cells to apoptosis TOAG-1 may 

be involved in controlling and terminating T cell activation. Further studies clarifying its 

function are ongoing.

Susanne Stutte, Sabine Brauer, Irmgard Förster 

FUNCTIONAL ROLE OF CCL17 IN ALLERGIC IMMUNE 

REACTIONS OF THE SKIN 

Professional antigen presenting cells of the skin, namely Langerhans cells (LC) and 

dermal dendritic cells (DC), are continuously exposed to environmental stimuli and are 

crucial for the induction of different skin immune reactions via presentation of the 

antigen in draining lymph nodes (LN) and secretion of cytokines and chemokines. The 

chemokine CCL17, which binds to the receptor CCR4, mediates the attraction of 

activated/memory T cells to dendritic cells (DC). We generated CCL17/EGFP knockin 

mice that express the enhanced green fluorescent protein (EGFP) under the control of 

the endogenous ccl17 promoter. These mice can be used as heterozygous reporter mice 

with normal CCL17 production or as homozygous CCL17 knockout mice which also 

express the EGFP reporter. We demonstrated that the EGFP+ cells represent almost 

exclusively CD11c+ DC, including activated LC and dermal DC. Functionally, the 

deficiency of CCL17 leads to reduced contact hypersensitivity responses to the haptens 

DNFB and FITC. Presently, we analyze the role of CCL17 expressing DC in allergic 

reactions of the skin using a mouse model for atopic dermatitis (AD). Since CCL17 is 

consistently upregulated in the skin of AD patients, thus chemokine may play an 

important function in the pathogenesis of AD. First results indicate an essential role for 

CCL17 secreting DC in the induction of the humoral immune response during chronic 

skin sensitization, as CCL17 deficient mice show a decreased antigen specific IgE, IgG1 

and IgG2a production. Surprisingly, in the absence of CCL17 dermal DC and LC exhibit 

a reduced migratory behaviour to the skin draining LN. In addition, a lower number of 

IL-4 and IL-5 secreting CD4+ T cells could be detected in skin draining LN of CCL17 

knockout mice. Thus, CCL17 appears to be critically involved in the immunopathology of 

atopic dermatitis.

Daniel Reim, Kay Westenfelder, Simone Kaiser-Moore, Sylvia Schlautkötter, Bernhard 

Holzmann, Heike Weighardt 

Functional role of T cells during mixed bacterial peritonitis 

The general concept of immune defense against microbial pathogens supports the idea 

that early defense mechanisms are controlled by cells of the innate immune system, 

whereas the adaptive immune system is critically engaged in the immune response at 

later time points. During sepsis the innate immune system is crucial for the induction of 

immune reactions against the pathogen, but, in addition, lymphocytes are involved in 

the immune response early at the onset of septic peritonitis. Mice deficient for RAG-1, 

which lack mature B and T cells, show enhanced susceptibility to acute mixed bacterial 

peritonitis as compared to wildtype mice. Whereas B cell-deficient µMT mice show no 

significant difference in the survival rate after peritonitis induction, T cell-deficient nude 

mice (balb/c nu/nu) exhibit enhanced susceptibility to septic peritonitis as compared to 

controls. 

Analysis of cytokine production in either RAG-1-deficient or T cell-deficient nude mice 

indicated attenuated sepsis-induced IL-12 and IL-10 production both systemically and 

locally in spleens of septic mice as compared to wildtype controls. To further analyse 

the contribution of T cells to the immunopathology of severe sepsis we analysed the 

capacity of T cells to contribute to cytokine production during sepsis. Purified splenic T 

cells of septic mice were capable to produce IL-10 and IFNg. These results imply that T 

cells are not only important for adaptive immune processes but are also able to 

contribute to the immunopathology of severe infections via their ability to produce 

immune mediators.

Jan Kubach, Petra Lutter, Tobias Bopp, Sabine Stoll, Christian Becker, Jürgen Knop, 

Stefan Müllner, John Wijdenes, Edgar Schmitt, Helmut Jonuleit 

Galectin-10, a previously unnoted protein essential for the 

functional activity of human CD4+CD25+ regulatory T cells 

CD4+CD25+Foxp3+ regulatory T cells (CD25+ Tregs) comprise a separate lineage of T 

cells that are essential for maintaining immunological tolerance to self. However, the 

molecules mediating the anergic state and regulatory function of CD25+ Tregs are still 

elusive. 

Here we report the identification and functional characterization of galectin-10, a protein 

found to be predominantly expressed by CD25+ Tregs, which is essential for the anergic 

phenotype and suppressive function of these cells. Using differential proteomics, we 

found three isoforms of a 16kDa protein, identified as galectin-10 being constitutively 

expressed in human CD25+ Tregs and nearly absent in conventional CD4+ T cells. 

Expression was up to 40 times higher in CD25+ Tregs and two of the isoforms were 

previously unknown. In contrast, other members of the galectin family like galectin-1 

showed only slight expression differences in this comparison or were upregulated in 

conventional T cells upon activation (e.g. galectin-8). So far, galectin-10 has only been 

described in granulocytes and its expression is restricted to humans. Predominant 

expression of galectin-10 in CD25+ Tregs was also confirmed on mRNA level. Single cell 

staining and flow cytometry showed a strictly intracellular expression of galectin-10 in 

CD25+ Tregs. 

Most notably, specific inhibition of galectin-10 restored the proliferative capacity of CD25 

+ Tregs and abrogated their suppressive function. Thus, expression of galectin-10 on 

mRNA and protein level seems to be essential for the functional activity of human CD25 

+ Tregs.

Kerstin Sarter, Connie Schulze, Sandra Franz, Benjamin Frey, Luis Munoz, Udo Gaipl, 

Martin Herrmann 

Galectins contribute to the recognition and clearance of 

apoptotic cells 

Galectins are _-galactoside-binding proteins. A characteristic of galectins is the 

presence of one or two carbohydrate recognition domains (CRD) of about 135 amino 

acids with an affinity for _-galactosides. Various functions were ascribed to Galectins, 

which have been detected in numerous cells and tissues. Galectins are present in T 

cells, B cells, macrophages, eosinophils, basophils, and thymic epithelial cells. 

Moreover, galectin expression is modulated by various inflammatory stimuli. Galectins 

display pleiotropic effects. Galectin-1 induces apoptosis of thymocytes and of activated 

T lymphocytes. Galectin-3 induces superoxide production of neutrophils, regulation of T 

cell apoptosis, and is involved in actin remodelations in macrophages during 

phagocytosis. Galectin-9 causes thymocyte apoptosis and serves as chemoattractant for 

eosinophils. 

We show that galectins serve as ligands for apoptotic and necrotic cells. Most 

importantly, in contrast to other ligands for dying or dead cells, galectins show a 

different binding to ageing apoptotic granulocytes and irradiated apoptotic lymphocytes, 

respectively. Furthermore, the binding of the several galectin family members differed 

considerably. In phagocytosis assays we observed that several Galectins significantly 

increase the phagocytic clearance of apoptotic lymphocytes. Furthermore, Galectins 

seem to influence the anti-inflammatory potency of apoptotic cells during phagocytosis 

as demonstrated by the release of TNF_ by macrophages during phagocytosis of e.g. 

Galectin-3,-4, and -5 -opsonised cells. 

We conclude that Galectins play an important role as opsonising molecules for apoptotic 

cells. Furthermore, they may serve as adaptor molecules during phagocytosis. They are 

therefore important molecules in the clearance synapse.

Nadja Hilger, Frank Emmrich, Ulrich Sack 

Gene expression in microdissected invasive fibroblast isolated 

from arthritic joints from patients with RA 

The etiology of Rheumatoid Arthritis remains incompletely understood. Inflammatory 

fibroblasts (FB) are necessary for joint destruction and the propagation of inflammation 

while cytokines and their receptors play an important role in the pathogenesis, namely 

in the intercourse of FB with inflammatory and lymphoid cells and in the destruction of 

cartilage and bone. 

Studies of invasive FB isolated from homogenized synovial membrane failed to provide 

detailed insight into the characteristics of erosive FB at the site of cartilage and bone 

destruction. By modifying the latest microdissection technologies, we have analysed 

small cell groups from destructive regions in biopsies of patients and compared them 

with FB from non-arthritic regions. In this way, differential expression of cytokines and 

further candidate genes can be investigated. So we investigated to what extent an 

investigation of mRNA from few cells is possible and whether RNA amplification should 

be attached. 

Cells were isolated by laser capture microdissection. High laser intensity and specialized 

slides were found to be pre-requisites for successful isolation of small cellular units. 

After optimisation of the different methods, RNA of sequential cell numbers starting with 

1000 cells was isolated. One part was reverse transcribed and the other fraction was 

amplified. In order to examine differences, expression of different genes was examined 

and compared in both preparations. 

We were able to dissect small cell groups out of erosive sites and non-inflammed parts 

of synovial tissues from RA patients. We showed that RNA isolation from very small cell 

numbers with following expression analysis is possible. The house keeping gene and a 

FB-specific genes could be detected in all samples without amplification. The 

quantification of cytokine expression was limited. 

We could show that through our approach an optimized investigation of RNA from few 

cells derived from RA synovial tissue is possible. By identifying function-specific 

markers, erosive FB will be better accessible in future and can be investigated more 

easily.

Alexander Gerbaulet, Julia Scholten, Thomas Krieg, Karin Hartmann, Axel Roers 

Generation of a Mouse Model for Mastocytosis 

Mastocytosis is characterized by infiltration of abnormal numbers of mast cells into skin 

and internal organs. The clinical spectrum is broad ranging from mild increases in 

cutaneous mast cell numbers to severe forms of mast cell leukaemia. The disease is 

associated with activating somatic point mutations of the receptor tyrosine kinase Kit, 

however, the relevance of these mutations for the different forms of the disease is 

unclear. We generated a transgenic mouse model based on a 200 kb BAC containing the 

entire kit gene. Two modifications were introduced: 1) the activating point mutation 

D814V and 2) a floxed “stop” cassette, allowing Cre/LoxP-mediated control of transgene 

expression. The construct was pronucleus-injected and founder animals transmitted the 

transgene in the germline. Kit D814Vflox transgenic mice were bred to the hCMV (deleter)- 

Cre-line for ubiquitous deletion of the stop element, which should result in expression of 

the mutated kit under the control of the kit promotor elements contained in the 

transgene. Kit D814Vflox deleter-Cre double transgenic mice were born in numbers 

significantly lower than expected indicating a high frequency of lethality during 

gestation. The few animals born alive displayed a diverse spectrum of phenotypes. 

While some mice showed cutaneous mastocytosis of variable intensity, others 

developed various additional neoplasms including malignant mast cell tumors. We are 

presently investigating the cause of embryonic death in the majority of Kit D814Vflox 

deleter-Cre double transgenic embryos, the effect of Kit D814V expression in mature mast 

cells of Kit D814Vflox Mcpt5-Cre double transgenic mice and the induction of Kit D814V 

expression in adult Kit D814Vflox Mx-Cre double transgenic mice.

Dafne Müller, Bettina Meißburger, Katharina Frey, Anette Karle, Ines Höfig, Roland 

Stork, Roland E. Kontermann 

Generation of an improved recombinant bispecific antibody 

molecule and B7 fusion proteins for targeted cancer 

immunotherapy 

The recombinant bispecific antibody format single-chain diabody (scDb) has shown to 

be able to retarget T lymphocytes to tumor cells, leading to their destruction. However, 

therapeutic efficacy is hampered by the short serum half-life of this small molecule (55 

kDa). Thus, improvement of the pharmacokinetic properties of small bispecific antibody 

formats is required to enhance efficacy in vivo. We have generated a fusion protein of 

single chain diabody and human serum albumin (scDb-HSA) and analyzed this molecule 

for biological activity and pharmacokinetic properties. The scDb-HSA, which is directed 

against the tumor antigen carcinoembryonic antigen (CEA) and the T cell receptor 

complex molecule CD3, retained full binding capacity to both antigens and showed 

strong increase in circulation time compared to the unfused scDb molecule. In order to 

provide a tumor target specific costimulatory signal, fusion proteins of the extracellular 

domain of B7.2 (CD86) and single chain Fv or diabody against CEA were generated. 

This constructs showed specific binding to CEA and CD28/CTLA-4. Costimulatory 

properties were assayed in combination with the scDb (providing the first stimulatory 

signal) by monitoring IL-2 release after incubation with PBMCs. Here, B7-Db showed to 

be superior to B7-scFv. Thus, in combination with B7-Db an enhancement of tumor 

antigen-specific retargeting and activation of T cells could be achieved for scDb and 

scDb-HSA. In summary, combining recombinant bispecific antibodies with improved 

pharmacokinetic properties and tumor directed costimulatory fusion proteins might be a 

promising approach for efficient retargeting and activation of cytotoxic T lymphocytes in 

cancer immunotherapy.

Kathrin Hofer, Holger Kroenig, Heinke Conrad, Barbara Kast, Christian Peschel, Helga 

Bernhard 

Generation of Th1 lymphocyte clones against an 

immunodominant epitope of NY-ESO-1 

Since CD4+ T cells play a critical role in generating and maintaining antigen-specific 

cellular and humoral immune responses our current study focuses on the generation of 

T helper cells from healthy donors and multiple myeloma patients. Our approach is the 

isolation of high avidity tumor antigen specific class - II- restricted T cell receptors 

(TCRs) for an optimized T cell transfer strategy. 

The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer 

antigens eliciting strong humoral and cellular immune responses in tumor patients and 

is a promising candidate antigen for successful adoptive T cell transfer. 

We achieved to generate NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA- 

DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) 

pulsed with the NY-ESO-1 87-111 peptide known to be presented with HLA-DR1+ and 

HLA-DR4+. The specifity of CD4+ T helper cell clones was determined by proliferation 

assays and IFN gamma ELISPOT through screening with NY-ESO-187-111 peptide. By 

limiting dilution of the NY-ESO-1-specific T cell populations we succeeded to isolate T 

cell clones which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO- 

1 protein. 

The ability to isolate NY-ESO-1- specific T helper1 cells facilitates the development of T 

cell transfer regiments which are based on class-II-restricted TCR-transduced T cells for 

a treatment of patients with multiple myeloma.

Johannes Stephani, Ronald Naumann, Hermann Wagner, Tim Sparwasser 

Generation of TLR- „humanized“ Mice with Bacterial Artificial 

Chromosome-Technology 

”Humanized“ transgenic mice expressing human pattern recognition receptors 

specifically on DCs are urgently needed to develop small animal models for testing new 

vaccination strategies, since significant immunologic differences limit the interpretation 

of data obtained from mouse experiments. In comparison to mice, human Toll-like 

receptor 9 (TLR9) has a different ligand specificity and is expressed on different immune 

cells. In mice, TLR9 recognizes unmethylated CpG DNA and is expressed on 

conventional dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs) and B cells. In 

humans, TLR9 is only detectable on pDCs and B cells and mediates immune cell 

activation by slightly different CpG sequences. We attempted to generate a mouse 

model mimicking human TLR9 expression by transgenesis of a Bacterial Artificial 

Chromosome (BAC) containing human TLR9 together with all its cis- and transregulatory 

regions. The purified and linearized BAC has been injected into pronuclei of 

fertilized C57BL/6 mouse eggs, and human BAC-transgenic mice were generated. Two 

BAC transgenic founder mice were obtained which are currently backcrossed to a 

murine TLR9 deficient background. First preliminary immunization data with “human” 

CpG sequences may suggest that the human promoter is functional in mice. Currently 

we are analyzing the expression level and specificity of human TLR9 in mice. This 

mouse model may provide us with new valuable tools to directly examine the role of 

human TLR9 on plasmacytoid DCs and B cells in vaccination studies and may allow us to 

test the therapeutical potential of CpG oligonucleotides in various diseases such as 

infection, allergy and tumor models.

Sandra Ehser, Jing-Jing Chuang, Lucian Jiga, Christian Kleist, Flavius Sandra-Petrescu, 

Gerhard Opelz, Peter Terness 

Generation of tolerogenic dendritic cells by treatment with 

Mitomycin C 

Dendritic cells (DCs) are the most potent antigen-presenting cells and play a central 

role in initiation of immunity or tolerance. Their controlling abilities offer possibilities for 

modulation of immune responses in an antigen-specific manner in organ transplantation 

or autoimmune diseases. To this end, either naturally suppressive DC subpopulations or 

ex vivo manipulated cells can be used. A series of pharmacological agents have been 

shown to alter the properties of DCs. We showed that after a short in vitro treatment 

with the akylating drug mitomycin c (MMC) DCs acquire tolerogenic properties. They 

irreversibly suppress allogeneic T cell responses in vitro and the treatment of recipients 

with MMC-incubated donor DCs strongly prolongs heart allograft survival in rats. In 

order to elucidate the mechanism of suppression we analyzed the supernatants of MMC- 

DCs. They did not mediate any suppressive effect. Therefore, the lacking T cell response 

could not be explained by secreted molecules. For this reason next we looked at the 

gene expression profile of MMC-treated cells. Over 47,000 transcripts and variants were 

analyzed by Affymetrix array, revealing 100 constantly modified gene expressions. 

Intriguingly, we found that one gene cluster was involved in apoptosis and another one 

– as shown in previous studies - in mediation of tolerance. It has been held that 

necrotic cells stimulate, whereas apoptotic cells sometimes inhibit the immune 

response. Our studies indicate that MMC induces apoptosis and upregulates tolerogenic 

molecules, thus, converting DCs into inhibitory cells. MMC-DCs might offer a therapeutic 

tool for antigen-specific suppression of unwanted immune reactions in clinical settings.

Ann-Kristin Mueller, Martina Deckert, Kirsten Heiss, Kristin Goetz, Kai Matuschewski, 

Dirk Schlüter 

Genetically Attenuated Plasmodium berghei Liver Stages 

Persist and Elicit Sterile Protection Primarily via CD8 T Cells 

Live-attenuated Plasmodium liver stages remain the only experimental model that 

confers complete sterile protection against malaria. Irradiation-attenuated Plasmodium 

parasites mediate protection primarily by CD8 T cells. In contrast, it is unknown how 

genetically attenuated liver-stage parasites provide protection. Here, we show that 

immunisation with uis3(-) sporozoites does not cause breakthrough infection in T and B 

cell-deficient rag1-/-and IFN γ-/- mice. However, protection was abolished in these 

animals, suggesting a crucial role for adaptive immune responses and interferon γ. 

Although uis3(-) immunisation induced Plasmodium-specific antibodies, B cell-deficient 

mice immunised with uis3(-) sporozoites were completely protected against wild-type 

sporozoite challenge infection. T-cell depletion experiments before parasite challenge 

showed that protection is primarily mediated by CD8 T cells. In good agreement, 

adoptive transfer of total spleen cells and enriched CD8 T cells from immunised animals 

conferred sterile protection against malaria transmission to recipient mice, whereas 

adoptive transfer of CD4 T cells was less protective. Importantly, primaquine treatment 

completely abolished the uis3(-)-mediated protection, indicating that persistence of uis3 

(-)-attenuated liver stages is crucial for their protective action. 

These findings establish the basic immune mechanisms underlying protection induced 

by genetically attenuated Plasmodium(-) parasites and substantiate their use as 

vaccines against malaria.

Marc Beyer, Sabine Classen, Daniela Eggle, Alexey Popov, Svenja Debey-Pascher, 

Elmar Endl, Percy A. Knolle, Jim Riley, Joachim L. Schultze 

Genomic screening reveals new proteins specifically 

expressed by human regulatory CD4+ CD25high FOXP3+ 

CD127low T cells 

Natural regulatory T cells (Treg cells) in humans can be defined by the expression of 

FOXP3, low expression of CD127 and intermediate to high expression of CD25. While it 

has been clearly established that FOXP3 is an essential factor for differentiation and 

function of these cells, it is also apparent that additional yet unknown genes and their 

respective proteins must exist contributing to full regulatory function of these cells. To 

determine novel proteins involved in activation or function of human Treg cells we 

initiated a systems biology approach using genome-wide transcriptional profiles. A total 

of 192 individual experiments interrogating conventional and Treg cells from healthy 

donors and cancer patients in different states of activation were performed. The first 

surprising result was the identification of a rather small core transcriptome of Treg cells 

comprising of only 43 genes. As expected, genes previously associated with Treg cells 

such as FOXP3 or CD127 were within the specific Treg cell core transcriptome. 

Interestingly, the Treg cell specific genes within the core transcriptome are not enriched 

for cell membrane-associated proteins suggesting that Treg cell lineage is a function of 

fixed transcriptional regulation rather than modulation by extracellular signals. So far, 

only one newly identified transcript was predicted to encode a putative gene containing 

a transmembrane-domain. We first determined the full length transcript of this novel 

gene. Apparently no splice variants exist in humans. By expression cloning we 

established that the transcript is indeed encoding a 18 kDa protein. To study the cellular 

localization of the newly identified protein, we generated a GFP-tagged fusion construct, 

which was inserted into a lentiviral vector. Infection of Jurkat cells with this construct 

revealed that the protein is translated in Jurkat cells and does not exert toxic effects on 

the cells. Interestingly, initial studies to determine its cellular localization hint at an 

intracellular expression of the protein product. Taken together, genome-wide 

transcriptional analysis using the most recent array technology and novel bioinformatics 

approaches have revealed genes specifically expressed within Treg cells. Currently 

ongoing and future studies are focused on deciphering the biology of these novel 

proteins.

Charles Andrew Stewart, Thierry Walzer, Scott Hamilton Robbins, Bernard Malissen, Eric 

Vivier, Immo Prinz 

Germline and rearranged Tcrd transcription distinguish bona 

fide NK cells and NK-like γδ T cells 

NK cells and γδ T cells are distinct subsets of lymphocytes that contextually share 

multiple phenotypic and functional characteristics. However, the acquisition and the 

extent of these similarities remain poorly understood. Here, using Tcrd-H2BEGFP 

reporter mice, we show that germline transcription of Tcrd occurs in all maturing NK 

cells. We also describe a population of mouse NK-like cells that are indistinguishable 

from “bona fide” NK cells using standard protocols. Requirements for V(D)J 

recombination and a functional thymus, along with very low level expression of surface 

TCRγδ but high intracellular CD3 define these cells as γδ T cells. “NK-like γδ T cells” are 

CD127+, have a memory-activated phenotype, express multiple NK cell receptors and 

readily produce interferon-γ in response to IL-12/IL-18 stimulation. The close 

phenotypic resemblance between NK cells and NK-like γδ T cells is a source of 

experimental ambiguity in studies bridging NK and T cell biology, such as those on 

thymic NK cell development. Instead, it ascribes chronic TCRγδ engagement as a means 

of acquiring NK-like function.

Benjamin Wilde, Xin Cai, Sebastian Dolff, Andreas Kribben, Jan Dürig, Christof Specker, 

Thomas Philipp, Oliver Witzke 

GITR and CD134 expression on T-lymphoctyes is associated 

with disease activity in Wegener's Granulomatosis 

Aim/Background: In 2005 it has been reported by Marinaki et al. that the CD25 

expression is increased on CD4+ T-cells in patients suffering from ANCA-associated 

vasculitis. In this study CD4+CD25+ T-cell populations in patients with Wegener’s 

granulomatosis are further characterised. 

Methods: 12 Patients meeting the ACR’s criteria defining WG and 6 healthy controls 

were included in this study. The disease activity and extension were measured by 

common scores such as Birmingham Vasculitis Activity Score (BVAS) and Disease 

Extent Index (DEI). Lymphocytes were analysed by FACS for the expression levels of 

CD134, GITR, CD122, CD103, CD152 and CD80. 

Results: The percentage of patients’ CD25+ cells within the CD4+ T-cell population was 

increased in accordance to Marinaki et al. (27 ±3% vs. 13 ±1% p

Adjobimey Tomabu, Arndts Kathrin, Satoguina Judith, Hörauf Achim 

GITR-GITRL interactions regulate the IgG induction by 


Regulatory T cells play a crucial role in maintaining control of effector lymphocytes in 

different immunological contexts. In chronic onchocerciasis for example, IL-10 

producing antigen specific regulatory T cells (Tr) could be cloned in high frequency, in 

particular from patients with high worm load and little pathology. Many mechanisms 

have been proposed to explain regulatory T cells immunosuppressive activities 

implicating IL-10, TGF-β , CTLA-4, GITR. It has recently been shown that IL-10 

produced by Tr cells induces B cells to secrete IgG4, a non-inflammatory antibody, in a 

cell-contact-dependent manner. The present study was aimed at understanding the 

mechanisms whereby Tr cells preferentially induce IgG4 by B cells. For this purpose, we 

generated FOXP3+GITR+IL10+ Tr clones from human PBMC, using vitamin D3, 

dexamethason and tetanus toxoid as antigen model. These Tr cells were co-cultured 

with autologous purified B Cells to induce IgG4. Using blocking antibodies, we found 

that neutralizing anti-GITR Abs selectively inhibited IgG4 production and increased IgG2 

production. Antibodies against GITRL, IL-10, CTLA-4 and against TGF-β also blocked 

IgG4 production, while anti-ICOS Abs had no effect on IgG4 production. Furthermore, 

the production of IL-10 by the co-cultured cells decreased in the presence of anti-GITR 

antibodies also, the inhibition of IgG4 induction by anti-GITR antibodies was reversed 

by the addition of excess recombinant IL-10 but not of recombinant TGF-β. These 

results indicate that GITR-GITRL interactions modulate IgG4 induction partially through 

induction of other factors chiefly IL10.

Matthias Krusch, Katrin Miriam Baltz, Tina Baessler, Helmut Rainer Salih 

Glucocorticoid-Induced TNF Related Protein (GITR) ligand is 

spontaneously released by tumor cells and diminishes antitumor 

reactivity of NK cells 

Members of the TNF/TNF receptor (TNFR) family mediate multiple cellular functions 

including proliferation, differentiation and cell death. Many TNF family members are 

released as soluble forms, which affects cell-cell interactions by reduction of ligand 

densities and distally modulates effector cells bearing the respective receptor. Here we 

report that human tumor cells spontaneously release a soluble form of Glucocorticoid- 

Induced TNF Related Protein (GITR) ligand (sGITRL), which inhibited cytotoxicity and 

IFN-γ production of GITR-expressing NK cells in a concentration dependent manner. NK 

cell functions were restored by neutralization of sGITRL by addition of a GITR-Ig fusion 

protein in coculture assays. While tumor-derived GITRL did not induce apoptosis, it 

diminished nuclear localized c-Rel and RelB in GITR-expressing NK cells indicating that 

sGITRL negatively modulates NK cell NF-κB activity. To determine whether release of 

GITRL was in fact relevant as an immune escape mechanism of human tumors in vivo 

we analyzed sGITRL levels in sera of cancer patients. While sera of the healthy 

volunteers contained no detectable sGITRL, sera of patients with rectum-, stomach-, 

lung-cancer and germ line tumors contained substantially elevated sGITRL levels. The 

strong correlation of tumor incidence and elevated sGITRL levels clearly suggests that 

sGITRL is released at significant amounts from tumor cells in vivo. Patient sera 

containing sGITRL reduced NK cell reactivity, which could again be restored by addition 

of a GITR-Ig fusion protein. Our data indicate that sGITRL released by tumor cells 

diminishes NK cell-mediated immunosurveillance, and GITRL-neutralization may be 

employed in therapeutic strategies like adoptive NK cell transfer.

Denise Tischner, Nora Müler, Jens van den Brandt, Andreas Weishaupt, Holger 

Reichardt 

Glucocorticoids exert distinct effects on Experimental 

Autoimmune Encephalomyelitis 

Multiple sclerosis (MS) and its animal model Experimental Autoimmune 

Encephalomyelitis (EAE) are chronic inflammatory diseases of the central nervous 

system (CNS) of presumed autoimmune origin. Acute relapses of MS are most 

commonly treated with high doses of glucocorticoids (GCs). However, severe adverse 

effects and incomplete recovery accompany such therapies. Therefore a better 

understanding of the mechanisms of GC action in MS is urgently needed. To distinguish 

effects of GCs on pathogenic and conventional T cells we induced an AT-EAE by 

adoptive transfer of eGFP+ encephalitogenic T lymphocytes into Lewis rats followed by 

treatment with 20 mg/kg dexamethason three days after disease induction. This led to 

a rapid amelioration of the disease and a reduced infiltration of the spinal cord. We 

found that dexamethasone similarly induced apoptosis in pathogenic as well as 

conventional T cells, restored the integrity of the blood-brain barrier and downregulated 

ICAM-1 and IP-10 expression in the spinal cord. Accumulation of 

encephalitogenic T cells in the spleen supported the notion that impaired T cell 

migration to the CNS may contribute to therapeutic efficacy. While GCs did not alter the 

expression of integrins and the chemokine receptor CXCR-3, we identified cytoskeletal 

rearrangements accompanied by the loss of the migratory phenotype. We believe that 

this may partially underlie the reduced lymphocyte infiltration of the CNS. We hope that 

a better understanding of GC action in the treatment of MS helps to improve the 

available therapies.

Sabine Stegmaier, Christof Wagner, Gertrud Maria Hänsch 

Granzyme B expression in mature polymorphonuclear 

neutrophils (PMN) and in their precursor cells 

Polymorphonuclear neutrophils (PMN) are important effector cells of the innate immune 

response and are mainly appreciated for their phagocytic and bactericidal capacity. 

PMN, however, are also effector cells of the antibody-mediated cellular cytotoxicity 

(ADCC). Recently, we described that PMN also contained granzyme B and perforin, the 

major protagonists of cellular cytotoxicity of T cells or natural killer (NK) cells. In 

extension of our previous studies we now attempted to localise granzyme B in the PMN. 

Granules were fractionated using the established protocols. The majority of granzyme B 

was found in association with primary granules, a minor expression was seen in 

secondary granules, and the membrane fraction. According to the “targeting by timing” 

theory the presence in primary granules of granzyme B suggested that it is synthesised 

in a very early developmental state of the cell. Indeed, by cytofluorometry, we detected 

granzyme B in the myloid cell lines HL-60 and U937, and in bone-marrow derived CD34 

+ cells as well. Following in vitro differentiation of the CD34+ cells to PMN, granzyme B 

expression was preserved. In summary, our data provide evidence that PMN contain 

granzyme B, which on one hand enhances their cytotoxic potential, and on the other 

provides an additional means for degradation of extracellular matrices.

Praxedis Martin, Julian Pardo, Reinhard Wallich, Klaus Ebnet, Sandra Iden, Aynur 

Ekiciler, Arno Muellbacher, Michael Huber, Markus M. Simon 

Granzyme B is expressed in mouse mast cells in vivo and in 

vitro and causes delayed cell death independent of perforin 

The proteases granzyme (gzm) A and gzmB have been mainly linked to natural killer 

(NK) cells and cytotoxic T lymphocytes (CTL) and their lytic machinery so far. Together 

with perforin (perf) both gzms are released through exocytosis of NK/CTL secretory 

granule contents into a synapse formed between the killer cell and its target cell. After 

their access to the target cell cytosol they initiate alternative, but strictly perfdependent 

proteolytic pathways leading to apoptosis. However, in addition to these 

intracellular activities, secreted gzmA and gzmB may also function extracellularly by 

cleaving components of extracellular matrices and basal laminae, including fibronectin 

and vitronectin. These processes may result in cell detachment of susceptible targets 

and indirectly in their delayed death, termed anoikis. Recent evidence suggests that 

cells other than NK cells and CTL also express gzms, indicating their involvement in 

additional biological activities. 

Here we present evidence that skin-, but not lung-associated primary mouse mast cells 

as well as in vitro differentiated bone marrow derived mast cells (BMMC) express gzmB, 

but not gzmA or perf. GzmB is associated with cytoplasmic granules of mouse mast cells 

and secreted by BMMC upon Fcε-receptor mediated activation. BMMC from wild type but 

not gzmB-deficient mice cause cell death in susceptible adherent target cells indicating 

that the perf-independent cytotoxic potential of BMMC is executed by gzmB. 

Furthermore, purified recombinant gzmB induces a disorganization of endothelial cellcell 

contacts. The data suggest that activated mast cells contribute via secreted gzmB to 

cell death, increased vascular permeability, leukocyte extravasation and subsequent 

inflammatory processes in affected tissues.

Gerhard Wingender, Jonathan Braun, James Borneman, Mitchell Kronenberg 

Gut derived antigens trigger the final steps of Vα14 iNKT cell 


Invariant NKT (iNKT) cells carry a canonical Vα14 to Jα18 TCR rearrangement in mice, 

recognize glycolipids presented by CD1d and have been implicated in diverse immune 

reactions. Their constitutive activated/memory phenotype and their rapid initiation of 

effector functions after activation are indicative of previous antigen specific stimulation. 

However the nature of this stimulation is so far unresolved. Recently natural antigens 

for the majority of iNKT cells could be defined in the proteobacterium Sphingomonas 

spp. Here we show that Sphingomonas bacteria are commensals in the intestine of 

mice, and that a surplus of bacteria increases the expression levels of activation 

markers by iNKT cells. Furthermore we demonstrate that iNKT cells from germ free 

animals display a less mature phenotype and are hypo-responsive to activation with the 

model antigen α-galactosylceramide. Per-os reconstitution of the germ free animals with 

Sphingomonas bacteria, but not with E.coli, a bacterium lacking specific iNKT cell 

antigens, could recover full maturity of iNKT cells, phenotypically and functionally. 

These data indicate that bacterial iNKT cell antigens can be taken up from the intestine 

and are required for the final differentiation of iNKT cells.

Dagmar Quandt, Hubert Ludwiczak, Barbara Seliger 

Heterogeneous B7-H molecule expression and regulation in 

RCC and melanoma 

The successful fight of the immune system against cancer involves two distinct signals: 

the first is mediated through the interaction of tumor-associated antigens (TAA) 

presented by MHC class I molecules and the specific T cell receptor (TCR), whereas the 

second signal involves the binding of costimulatory partners on both cells. Costimulatory 

molecules are known to efficiently shape the effector immune response towards 

silencing or activation of immune cells. Tumors evade the elimination by T lymphocytes 

due to diverse escape mechanisms including the differential expression of activating or 

inhibitory costimulatory molecules. The newly identified members of the B7-H family 

(B7-H1-4) represent important players in this process. We analyzed a large series of 

melanoma and RCC cell lines for the mRNA and protein expression of the B7-H 

molecules. The results show a heterogeneous constitutive expression pattern for B7-H1, 

2 and 4 at the transcript and/or protein level. In addition, we identified for the first time 

an epigenetic silencing of the B7-H1 expression due to methylation of the promoter in 

melanoma cell lines, which could be reversed by the demethylating agent 

deoxyazacytidine. 

B7-H3 was homogenously expressed on all melanoma and RCC cell lines tested. 

Furthermore, 88% of primary RCC lesions exhibit B7-H3 expression, which significantly 

differed between the lesions. Strong B7-H3 expression correlated with a higher CD8 and 

inverse CD4 T cell infiltration when compared to lesions weakly expressing or lacking B7- 

H3 molecules. Moreover, a strong B7-H3 expression was significantly associated with a 

low pT value in the RCC patients. Immune effector assays to dissect the immunological 

role of the B7-H molecules on tumor cells are under investigation.

Christian Stemberger, Katharina Huster, Martina Koffler, Florian Anderl, Matthias 

Schiemann, Hermann Wagner, Dirk Busch 

Heterogeneous subset generation from a single naïve CD8+ T 

cell upon in vivo priming 

CD8+ T cells are crucial for protection against intracellular pathogens and some tumors. 

Upon first encounter with their cognate antigen, naïve T cells get activated (‘primed’), 

clonally expand, and can develop into very distinct subsets. These comprise short-living 

effector T cells that confer immediate protection by different types of effector functions, 

and memory T cell subsets such as effector- and central memory T cells. Because these 

subsets contribute differently to protective immunity and therefore have different 

implications for the development of vaccination strategies, it is of outstanding interest 

to understand how subset diversity is generated. However, so far it has only been 

possible to tackle questions regarding the origin of diversification via analysis of indirect 

parameters, such as TCR repertoires, and these experiments were limited to global 

analyses of clonotypic T cells. 

To improve analysis of T cell subset generation on a cellular level in vivo, we developed 

a novel adoptive transfer system that allows for the first time to trace the fate of a 

single antigen-specific naïve T cell in a normal (wild-type) recipient mouse. Upon 

immunization, clonal expansion and differentiation of single cell-derived daughter cells 

was monitored in a natural in vivo setting. Phenotypical and functional analyses of 

“single cell”-expanded populations demonstrated that a wide range of diversity can 

develop out of a single naïve precursor cell, including different types of effector cells 

and long-living memory T cell subsets. Most interestingly, we uncovered that subset 

diversification derived from a single cell strictly reflects the differentiation pattern found 

for the endogenous repertoire (in the same individual mouse). This finding indicate that 

the origin of subset diversification lies primarily in the intrinsic plasticity within clonal 

precursors, or in differences in the range of signals provided to their progeny at stages 

during expansion going far beyond the first cell division.

Wibke Bayer, Simone Schimmer, Dennis Hoffmann, Ulf Dittmer, Oliver Wildner 

Heterologous Prime-Boost Vaccination with Ad5 and Fiber 

Chimeric Adenoviral Vectors Enhances Immune Protection 

against Friend Virus 

In a vaccination study against the Friend virus we evaluated an Ad5-based vector and a 

fiber chimera expressing the fiber protein of Ad35 (Ad5F35). The Friend virus is an 

immunosuppressive retroviral complex of spleen focus forming virus and Friend murine 

leukemia virus (F-MuLV) that causes lethal erythroleukemia and splenomegaly in 

susceptible adult mice. The FV is regarded as a useful model to elucidate basic 

requirements for immune protection against retroviral infections. 

We vaccinated C57BL/6 and the highly susceptible CB6F1 hybrid mice with Ad5 or 

Ad5F35 vectors expressing F-MuLV env and gag proteins or with a prime-boost 

combination of the two. After challenge infection, viral load in the spleens of C57BL/6 

mice was reduced ~250-fold and was below the detection threshold in more than 50% 

of the mice. In CB6F1 mice, on day 10 p.i. all vaccinated animals showed significantly 

reduced viremia compared to unvaccinated control mice and the onset of disease was 

significantly delayed. The prime-boost combination of the two Ad vectors resulted in 

improved immune protection with significantly lower viremia compared to vaccination 

with the individual vectors.

Maren Mönkemeyer, Hans Heiken, Rachel Thomas, Reinhold E. Schmidt, Torsten Witte 

Higher risk of CMV reactivation in HIV-1 infected patients 

homozygous for MICA5.1 

Background: 

Infection with human cytomegalovirus (CMV) induces surface expression of MHC class I 

chain-related A (MICA), a ligand for the activating receptor NKG2D. This leads to 

improved recognition and elimination of infected cells by NK cells as well as CD8+ T 

cells. The MICA allele MICA5.1 codes for a truncated, dysfunctional protein. 

The aim of this study was to investigate the contribution of genetic ability to express 

functional MICA protein on the susceptibility to severe CMV reactivation in 

immunocompromised individuals. HCV and GBV-C coinfected HIV-1 infected patients 

were analysed as controls. 

Methods: 

The frequency of the MICA5.1 allele was assessed in 230 Caucasian HIV-1 infected 

patients as well as in 29 healthy controls. MICA5.1 allele was analysed by PCR. The 

association of MICA5.1 homozygosity and risk of CMV reactivation was calculated by 

Pearson Chi-Square. 

Results: 

Comparison of patients with and without a history of CMV disease manifestation 

revealed an enhanced susceptibility to reactivation of CMV for HIV-1 infected patients, 

homozygous for MICA5.1. The percentage of homozygous MICA5.1 individuals was 

similar in HIV-1 infected patients and healthy controls. In contrast, no evidence was 

found for a correlation between a homozygous MICA5.1 genotype and an increased risk 

of infection with GBV-C or HCV. 

Conclusions: 

This study is the first to evaluate in vivo the impact of a MICA polymorphism on risk of 

viral infections. A significant correlation between homozygous MICA5.1 genotype and 

susceptibility to CMV, but not to GBV-C or HCV coinfection, in immunocompromised 

individuals was demonstrated. The elimination of infected cells via NKG2D-expressing 

NK and T cells appears to be a more important immune reaction in CMV than HIV-1, 

GBV-C or HCV infection. 

Supported by BMBF KN Rheuma C2.12

Konrad Alexander Bode, Klaus Heeg, Alexander H. Dalpke 

Histone deacetylase inhibitor butyric acid of bacterial origin 

as mediator of tolerance in the intestinal mucosa 

Posttranslational modifications of histones by acetylation are a major mechanism to 

modify chromatin structure and gene expression in eukaryotic DNA. In a recent work we 

could show that histone HDAC inhibitors like trichostatin A or suberoylanilide 

hydroxamic acid (SAHA) in non-apoptotic concentrations strongly inhibit the TLRinduced 

cytokine expression of dendritic cells. In the present work we investigated 

whether the HDAC inhibitor butyric acid, a product of the metabolism of some anaerobe 

bacteria, is involved in the induction of tolerance towards the physiological bacterial 

flora in the mucosa of the intestine. 

We could show in bone marrow derived dendritic cells that butyric acid inhibits the 

expression of several TLR induced cytokines like IL-12p40, IFN&beta and TNF&alpha on 

protein (ELISA and FACS-analysis) and mRNA (real-time RT-PCR) level. Whereas TLR 

stimulation resulted in transient histone H4 acetylation at selected regions of the IL- 

12p40 promoter as shown by chromatin immunoprecipitation, butyric acid induced a 

hyperacetylation of the whole IL-12p40 locus for a prolonged period. HDAC inhibitors 

had no effects on upstream NF&kappaB activation and nuclear translocation as well as 

on MAP kinase signaling (immuno-blot and gel-shift) but the binding of selected 

transcription factors on the IL-12p40 promoter region were impaired in the presence of 

butyric acid. 

The results give evidence that butyric acid, a product of the metabolism of some 

anaerobe bacteria, affects intestinal immune cells by inhibiting HDACs.

Mareike Schmudde, André Braun, Ulrike Klier, Daniela Pende, Jürgen Sonnemann, 

Lorenzo Moretta, James F. Beck, Barbara M. Bröker 

Histone deacetylase inhibitors sensitize tumour cells for 

cytotoxic effects of natural killer cells 

Introduction: Histone deacetylase inhibitors (HDIs) are currently emerging as potent 

anti-tumour agents. By influencing the transcription of up to 22 % of genes, they induce 

cell cycle arrest, differentiation and/or apoptosis in tumour cells. Beside their direct antitumour 

activity, HDIs enhance the cytotoxic effects of ionizing radiation, chemotherapy 

and recombinant tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). This 

raises the question, whether cytotoxic effector functions of natural killer (NK) or T cells 

are partially responsible for the anti-tumour effects of HDIs. 

Materials and Methods: HDI (SAHA, NaB)-treated tumour cell lines (prostate cancer 

PC3, medulloblastoma DAOY) were exposed to IL2-activated human PBMCs. Cell death 

was quantified by flow cytometric counting of surviving tumour cells and by 51 Cr-release 

assays. The involvement of activating NK receptors in tumour cell recognition was 

determined using masking antibodies in the 51Cr-release assays. Surface expression of 

NK receptor ligands on tumour cells was measured by flow cytometry. 

Results: Both HDIs and PBMCs induced tumour cell death in a dose-dependent manner. 

In combination, their effects were synergistic. Killing of tumour cells depended on the 

activating NK receptors NKG2D, DNAM-1 and the natural cytotoxicity receptors (NCRs). 

HDI-treatment did not result in the engagement of alternative activating NK receptors. 

However, it increased the redundancy of the involved receptors. Furthermore, HDIs 

increased the expression of the NKG2D ligands MICA and ULBP2. 

Conclusions: PBMCs and HDIs synergize in the induction of tumour cell death in vitro. 

This indicates that cytotoxic effector functions of the immune system may contribute to 

the therapeutic activity of HDIs in tumour patients. On a molecular level, this might be 

performed by NK cells, which are more efficiently activated due to increased expression 

of activating NK receptor ligands.

Vanessa Witte, Andreas Baur 

HIV-1 Nef enhances viral gene expression by linking 

transcriptional derepression and activation events 

HIV-associated disease is characterized by a successive dysregulation of the immune 

system due to massive CD4 + T cell depletion. Viral infection of these predominantly 

resting cells drives their activation. This facilitates viral replication but also causes cell 

death. Partly responsible is the Nef protein of HIV-1, which is required for the 

maintenance of high viral loads. Whether this is due to a direct effect of Nef on viral 

transcription remains a matter of debate. 

Luciferase reporter assays revealed a positive effect of Nef on HIV promoter activity, 

which was dependent on cotransfection of Tat and on the presence of the NFkB 

enhancer. Importantly, Nef expression did not initiate nuclear translocation of RelA/p65 

as shown by cellular fractionation experiments and immunofluorescence techniques. 

Instead, PMA- or CD3/CD28-mediated nuclear translocation of RelA/p65 was 

significantly prolonged in Nef-expressing cells. Surpisingly, chromatin 

immunoprecipitation (CHIP) analysis demonstrated that nuclear RelA/p65 only bound to 

the HIV promoter in Nef-expressing cells. Concomitantly, we found that Nef expression 

alone results in the specific Crm1-dependent removal of proteins from the nucleus, such 

as the Polycomb group (PcG) protein Eed, the NFkB kinase IKKalpha or NFkB2/p100, 

and that these proteins interact with histone deacetylases. 

In summary, these results may suggest, that in the absence of Nef, the HIV promoter is 

occupied by inhibitory histone deacetylase-containing protein complexes, which hinder 

HIV gene expression. Nef expression then leads to the removal of these inhibitory 

complexes thus allowing the binding of positive regulatory transcription factors such as 

NFkB. It is intriguing to speculate that by linking activation with derepression Nef finetunes 

HIV transcription thus facilitating gene expression in the presence of suboptimal 

activation stimuli.

Karsten Gülow, Marcin Kaminski, Peter H. Krammer 

HIV-Tat induced generation of Reactive Oxygen Species 

sensitizes T cells towards Activation-Induced Cell Death 

The elimination of activated T cells during the termination phase of an immune response 

is called Activation Induced Cell Death (AICD). AICD is induced via stimulation of the 

death receptor CD95 (APO-1, Fas) by its ligand CD95L. In AIDS patients, AICD is 

strongly enhanced. Here, we show that Reactive Oxygen Species (ROS) generated upon 

T cell activation function as crucial second messengers in activation-induced CD95L 

expression. The oxidative signal combined with simultaneous increase in intracellular 

Ca2+ constitutes for minimal requirement to induce CD95L expression. Either signal 

alone is insufficient. Further, we show that HIV-Tat induces ROS generation and 

depletion of reduced glutathione (GSH). The increase in ROS induced by HIV-Tat 

enhances T cell receptor (TCR) signalling. Thus, cells preincubated with Tat and 

stimulated via the TCR reveal a strong increase in the ROS signal leading to a significant 

increase in CD95L expression and AICD. Since CD4 stimulation can induce Ca2+ influx 

into the cytosol, ROS generation due to Tat treatment is sufficient to induce AICD. 

Therefore, our data provide a possible explanation for CD4+ T lymphocyte depletion 

during progression of AIDS.

Claudia Sievers, Kasia Nasilowska, Kerstin Wolk, Robert Sabat, Hans-Dieter Volk, 

Christian Meisel 

HO-1 inhibits constitutive and IFNg-induced HLA-DR 

expression on myeloid antigen-presenting cells via inhibition 

of IFNg receptor signalling and down-regulation of CIITA 

expression 

Strong and prolonged systemic immunodepression after surgery and major trauma, 

characterised by monocyte deactivation and T cell dysfunction, may predispose critically 

ill patients to infectious complications. The mechanisms that induce and sustain this 

condition are still incompletely understood. Previous studies showed increased 

expression of heme oxygenase-1 (HO-1) in peripheral blood leukocytes in a subset of 

surgical patients who developed post-operative long-lasting immunodepression and 

infectious complications. HO-1 is a stress-inducible heat shock protein with potent antiinflammatory 

activities. We hypothesized that increased expression of HO-1 may play a 

role in the induction of long-lasting immunodepression. Here, we investigated the 

effects of HO-1 on APC and T cells function in vitro by using the specific HO-1 inducer 

cobalt protoporphyrin IX (CoPP). CoPP increased HO-1 expression in human monocytes 

and monocyte-derived dendritic cells, but not in lymphocytes. HO-1 induction resulted 

in diminished constitutive surface MHCII expression on myeloid APC, while MHCI 

expression was unaffected. HO-1 overexpression in monocytes resulted in decreased 

transcription of CIITA and other accessory genes essential for MHCII protein stability 

and peptide loading, including CD74, HLA-DM and Cathepsin S. Decreased STAT-1 

phosphorylation in response to IFNg in CoPP-treated monocytes suggests that HO-1 

impairs cytokine-induced up-regulation of HLA-DR expression also by interfering with 

cytokine-receptor signalling. Enhanced expression of HO-1 in APC resulted in diminished 

antigen-dependent T-cell cytokine production and proliferation. Taken together these 

data indicate that increased HO-1 activity may partake in the induction of 

immunodepression in critically ill patients.

Nadja Brachwitz, Maria Laura Zenclussen, Andre Sollwedel, Ritschel Stefanie, Hans- 

Dieter Volk, Ana Claudia Zenclussen 

HO-1 up-regulation increases the number of uNK at the fetalmaternal 

interface 

We previously suggested a protective function for HO-1 in pregnancy after observing 

diminished levels of HO-1 in human and murine miscarriage. Accordingly, up-regulation 

of HO-1 significantly diminished the abortion rate in mice. At the beginning of 

pregnancy uterine natural killer cells (uNK) start to invade the uterus. It is known that 

they are necessary for a successful pregnancy outcome. Due to the importance of both 

HO-1 and uNK at the fetal-maternal interface, we aimed to investigate whether HO-1 

acts by regulating the number of uNK invading the uterine tissue. 

The expression of perforin (a product of mature NK) and HO-1 was determined by qRT- 

PCR and by immunohistochemistry in uterus, placenta and decidua of normal pregnant 

(CBA/J x BALB/c) and abortion-prone mice (CBA/J x DBA/2J) at different time points of 

pregnancy. Additionally, uNK were quantified at the feto-maternal interface after 

visualization with DBA-lectin staining. Furthermore we performed in vivo studies by upregulating 

HO-1 by Co-PP application on gestation day (gd) 4 or by HO-1-adenoviral 

gene transfer on gd 5 to quantify the number of uNK cells. PBS, Zn-PP or GFP-Ad 

treated animals served as controls. 

An augmentation on HO-1 mRNA and protein levels as well as an increased number of 

uNK beginning on gd 8 could be observed at the feto-maternal interface in both mating 

combinations. Compared to the normal pregnant mice the uNK cell number and the 

perforin mRNA expression were reduced in abortion-prone mice. Up-regulation of HO-1 

prevented fetal rejection, which was accompanied by an enhanced uNK cell number. 

Our data confirm the hypothesis that HO-1 and uNK are relevant for the survival of the 

fetus within the maternal uterus. Furthermore, our results suggest that HO-1 has an 

influence on the migration of uNK at the feto-maternal interface and may be one of the 

mechanisms underlying its protective effect.

Jan C. Dudda, Nikole Perdue, Mary Beauchamp, Daniel J. Campbell 

Homing receptors CD62L and CD103 mark different subsets of 

development and function of Regulatory T cells for 

suppression of autoimmunity 

FoxP3+ regulatory T cells (Treg) control inflammation throughout life. Treg leave the 

thymus with “central” homing receptors (HR) such as CD62L, targeting them to 

lymphoid tissues. The induction of “effector/memory” Treg with “peripheral” HR takes 

place in the lymph nodes (LN), which includes downregulation of CD62L but expression 

of CD103, an integrin involved in epithelial localization. We here tested the impact of 

these HR on the homeostasis and function of Treg after transfer into the autoimmune 

scurfy mouse, which lacks all FoxP3-dependent Treg. CD62L expression and LN 

localization was essential for Treg survival in the neonate and a transfer of wildtype but 

not CD62L-/- Treg suppressed multi-organ autoimmunity. Furthermore, BM-chimeras of 

scurfy and CD62L-/- mice, in which all Treg emerged from the CD62L-/- donor, 

suggested that CD62L was not crucial for Treg survival in the adult, but important for 

the full control of autoimmune scurfy cells. Surprisingly, when we chose a more 

aggressive model transferring scurfy effector cells and CD62L-/- Treg into RAG-/- mice, 

CD62L-/- Treg could expand and control autoimmunity to some extend, after they had 

differentiated into CD103+ effector Treg. Interestingly, the transfer of CD103+ or 

negative Treg alone or in competition with each other into neonatal scurfy mice 

revealed, that CD103 is dynamically regulated and marks a subset of Treg, which is 

effectively suppressing autoimmunity. Thus, LN localization is important for activation of 

naïve-like Treg, whereas effector/memory Treg can home to LN and suppress 

autoimmunity in a largely CD62L independent manner. Additionally, our observation 

that autoimmune suppressors preferentially reside in the CD103+ subset may have 

implications for therapy of autoimmune diseases.

Otilia Postea, Christian Weber, Andreas Ludwig 

Homocysteine-induced adhesive and scavenger activity of 

endothelial cells involves upregulation of the transmembrane 

chemokine CXCL16 by a PPAR-gamma dependent mechanism 

Hyperhomocysteinemia induces endothelial dysfunction and promotes atherosclerotic 

vascular disease. Infiltrates of activated macrophages and T cells are observed in 

human and experimental atherosclerotic lesions. Enrollment of these cells to lesions is 

guided by endothelial-leukocyte adhesion molecules and chemoattractants. The CXCL16 

chemokine is playing a double role, acting as adhesion molecule by interacting with its 

receptor, CXCR6 and as scavenger for oxidized LDL (oxLDL). As the adhesion of 

leukocytes to activated endothelial cells (EC) and uptake of modified lipids are crucial 

stages for the development of atherosclerosis, we investigated the effect of 

homocysteine (Hcy) on the expression and function of CXCL16 chemokine on cultured 

EC (EA.hy 926 cells). Incubation of EC with different pathophysiological relevant 

concentrations of L-Hcy (50 to 200•M) dose-dependently increased T cell adhesion to 

EC as demonstrated in a static adhesion assay. In contrast, D-Hcy and L-cysteine have 

no significant effect. Furthermore, stimulation with L-Hcy increased binding of DiI-oxLDL 

to EC as detected by flow cytometry. L-Hcy-stimulated EC also show a significant 

increase in CXCL16 mRNA and surface expression. Pretreatment of EC with an anti- 

CXCL16 monoclonal antibody reduces T cell adhesion to Hcy-incubated EC and uptake of 

DiI-oxLDL, suggesting that Hcy may influence leukocyte-endothelial cell interaction and 

lipid uptake via the modulation of CXCL16 expression. Antioxidants (Tiron 2,5mM) and 

Pioglitazone (100 •M) significantly reduce Hcy´s stimulatory effect indicating that 

induction of CXCL16 by Hcy involves the disruption of the PPAR-gamma defense 

mechanism against oxidative stress. These data suggest that upregulation of CXCL16 in 

response to homocystemia promotes increased adhesion of CXCR6 expressing 

circulating T cells and scavenging of modified lipids, both events critically implicated in 

the formation and progression of atherosclerotic lesions.

Matthias von Herrath, Christophe Filippi 

How viral infections prevent type 1 diabetes by augmenting 

Treg function 

The most popular hypothesis circulating within the scientific community is that viral 

infections are good candidates for enhancing or eliciting autoimmune disorders such as 

type 1 diabetes (T1D), because they induce strong inflammation, can enhance antigen 

presentation and directly lyse target cells. However, mounting recent evidence shows 

that the opposite scenario, which is prevention or amelioration of T1D by viral infections 

might be at least as common or even a more common outcome. Two recent publications 

in J. Virol. clearly demonstrated that a multitude of Coxsackie B virus and rotavirus 

strains prevented T1D in the NOD mouse model for spontaneous diabetes, and our own 

previous work (Christen et al JCI 2004) showed that LCMV virus commonly prevents 

T1D in two animal models when encountered during the pre-diabetic phase. Thus, it is 

time to rethink this issue. 

A new discovery made by us strongly supports the concept that viral infections 

themselves might be potent enhancers of regulatory T cell (Treg) function: We observed 

that viral (LCMV) infection of NOD mice enhances CD25+FoxP3+ Treg function and 

prevents T1D and that such virally-enhanced Tregs can transfer protection upon 

adoptive transfer into naïve pre-diabetic NOD recipients. In addition, we found that Treg 

lines induced by lymphocytic choriomeningitis virus (LCMV) in vivo, or grown specific for 

the virus or viral epitopes in vitro, were capable to delay autoimmune diabetes in the 

RIP-LCMV model, but failed to significantly affect the anti-viral immune response. 

Thus, encounters with pathogens might be crucial for ‘tuning’ the immune system and 

maintain a sufficient degree of systemic immune regulation, in this way making the 

occurrence of autoimmunity less likely.

Stefan Welte, Kathrin Pietschmann, Lothar Marischen, Susann Beetz, Ina Martens, 

Daniela Wesch, Dieter Kabelitz 

Human γδ T lymphocytes express pattern recognition receptors 

Pattern recognition receptors such as Toll-like (TLR) and NOD-like receptors recognize 

conserved pathogen-associated molecular patterns which distinguish foreign/ dangerous 

organisms from the host cells. With respect to human blood derived γδ T lymphocytes, 

we found reproducible expression of TLR1, 2, 3, 5, 6, 7 and NOD2 mRNA. To analyze 

the functional relevance of the expression of these proteins in more detail, we 

stimulated freshly isolated, highly purified γδ T cells with TLR specific surrogate ligands, 

e.g. bacterial lipopeptides Pam2CSK4 and Pam3CSK4 for TLR2, poly(I:C) for TLR3, 

flagellin for TLR5, and with the NOD2 ligand MDP. In general, all used TLR ligands exert 

no or only limited effects by itself but increased the T cell receptor (TCR) stimulated IFNγ 

production of γδ T lymphocytes. Furthermore, incubation with TLR ligands enhanced 

secretion of TNF-α, RANTES and MIP-1α. MDP caused a small increase in IFN-γ 

production of TCR stimulated cells, also in combination with Pam2CSK4 and Pam3CSK4. Furthermore, we investigated freshly isolated Vδ1 and Vδ2 γδ T lymphocytes, the two 

major subpopulations in the human blood, separately. Both cell populations express a 

similar level of the pattern recognition receptors. In functional tests, Vδ1 γδ T cells 

produce only little IFN-γ upon stimulation, whereas Vδ2 γδ T cells are strong IFN-γ 

producers. The differential stimulation by the different TLR and NOD ligands is currently 

under investigation. 

This study was supported by DFG SPP 1110 “Innate Immunity” (Ka 502/8-3).

Susann Beetz, Tim Meyer, Ina Martens, Thomas Stempfl, Daniela Wesch, Dieter 

Kabelitz 

Human γδ T lymphocytes can initiate an anti-viral immune 

response to double-stranded RNA 

Human blood derived γ&delta T lymphocytes express Toll-like receptor (TLR) 3. After 

stimulation via the T cell receptor (TCR), highly purified γ&delta T lymphocytes can be 

costimulated by the surrogate TLR3-ligand poly(I:C). We performed cDNA microarray 

experiments to investigate differences in gene expression between γ&delta T cells 

stimulated via TCR in comparison to cells stimulated via TCR and with poly(I:C). By 

applying a 1.5fold cutoff, poly(I:C) treatment increased the transcription of 380 genes 

including CD38, CD86, CD274, IL-15Rα, Trail, Fas-Ligand, MHC class I-related molecule 

MICB, granzyme B, perforin and TLR3 itself. The most striking differences concerned 

genes involved in launching an intracellular antiviral response such as myxovirus 

resistance (Mx) proteins, OligoA synthetase, and adenosine deaminase acting on RNA 

(ADAR). Other upregulated genes belong to the immunity/inflammatory cluster and 

include genes for interferon-α, -β, and -γ, TNF-α, CXCL10, and IL-15. Furthermore, the 

production of interferon regulatory factor (IRF) 7 as well as TLR7 mRNAs was triggered. 

The data suggest that human γ&delta T lymphocytes are able to sense viral infection 

and then produce type I interferons and exhibit an enhanced cytotoxic activity. The 

microarray results of some selected genes were confirmed by real-time PCR and flow 

cytometry. In future functional assays the influence of poly(I:C) on the behaviour of 

γ&delta T cells such as cytotoxicity will be investigated.

Thi Thu Hoai Nguyen, Silva Holtfreter, Thi Thu Hong Le, Harald Kusch, Michael 

Hecker, Susanne Engelmann, Alex van Belkum, Uwe Völker, Heiman Wertheim, Barbara 

M. Bröker 

Human antibody response to experimental colonization with 

Staphylococcus aureus 

Thi Thu Hoai Nguyen1, Silva Holtfreter1, Thi Thu Hong Le2, Anne-Kathrin Ziebandt3, 

Harald Kusch3, Quoc Phong Truong2, Leif Steil2, Michael Hecker3, Susanne 

Engelmann3, Alex van Belkum4, Uwe Völker2, Heiman Wertheim4,5, Barbara M. 

Bröker1 

1) Department of Immunology, University of Greifswald, Germany 

2) Department of Functional Genomics, University of Greifswald, Germany 

3) Institute of Microbiology and Molecular Biology, University of Greifswald, Germany 

4) Department of Medical Microbiology and Infectious Diseases, University Medical 

Center, Rotterdam, The Netherlands 

5) Oxford University Clinical Research Unit National Institute of Infectious and Tropical 

Diseases Bach Mai Hospital, Hanoi, Vietnam 

Staphylococcus aureus (S. aureus) is the most common cause of nosocomial infections 

and it can cause a wide range of human diseases ranging from superficial abscesses and 

wound infections to systemic infections, such as osteomyelitis, infective endocarditis, 

and sepsis. At the same time, the microorganism is also a frequent colonizer among the 

normal human flora. In S. aureus carriers we found a strong neutralizing antibody 

response against superantigens secreted by their colonizing strain. It is not known how 

the adaptive immune response against S. aureus proteins is triggered. We colonized 16 

healthy volunteers with the S. aureus strain 8325-4, which was selected because of its 

low virulence, and obtained serum samples before and four weeks after colonization. To 

investigate whether colonization induces changes in the antibody profiles against S. 

aureus, we separated the secreted staphylococcal proteins by two-dimensional gel 

electrophoresis, transferred them onto a PVDF membrane and afterwards performed 

immunoblots with the human sera. The results show a large inter-individual variability 

in the antibody profiles against S. aureus 8325-4. Even before experimental 

colonization, healthy individuals harboured high titre antibodies directed against a broad 

range of extracellular S. aureus proteins. These antibodies are likely due to previous 

encounters with S. aureus. Only rarely we observed additional antibody signals or 

increased signal intensities after experimental colonization with S. aureus. Therefore, 

short term colonization per se does not appear to trigger strong antibody responses to 

S. aureus. We conclude that the high antibody titres seen in most healthy individuals, in 

particular in S. aureus carriers, require either long lasting contact with S. aureus or, 

most likely, minor infections as they commonly occur with this microorganism, 

especially with strains of higher invasive potential.

Anja Mayer, Holger Bartz, Fabian Fey, Alexander Dalpke 

Human bronchial epithelial cells modify function and 

phenotype of dendritic cells in inflammatory settings 

Bronchial epithelial cells (BEC) represent the first line of defence against invading 

microbial pathogens. Epithelial cells not only function as tight barrier but also actively 

contribute to immune recognition of invading pathogens. We can show that BECs 

functionally express various pattern recognition receptors. Moreover, gene array 

analyses show that BECs are able to induce a pathogen-adopted defence program. 

Despite activation of a pro-inflammatory program we noticed that also immunemodulatory 

and genes were induced which might influence professional immune cells. 

Dendritic cells (DCs) are present at the base of bronchial epithelium and serve as major 

antigen-presenting cells in the lung. To investigate the interplay of BECs with dendritic 

cells we performed co-incubation experiments. We observed that dendritic cells secreted 

less IL-12 and TNF-α when co-incubated with BEAS-2B monolayers. DCs also lost their 

ability to release IL-12 when they were cultured with supernatants from BEAS-2B or 

stimulated in a transwell-system. Furthermore, DC phenotype was modified in the 

presence of BEAS-2B supernatant with slight reduction of CD86 and enhanced 

phagocytotic activity. Finally, DC mediated mixed leukocyte reaction (MLR) was 

significantly reduced in the presence of BECs. Current work aims at the identification of 

BEC-secreted immuno-modulatory factors. 

The results indicate that the functional properties of DCs are markedly affected by 

surrounding bronchial epithel cells. Thus, immune homeostasis in the lung is influenced 

by both, epithelial as well as dendritic cells. Similar results within the intestine indicate 

that the microenvironment in general shapes the functional competence of DCs and 

influences microbial recognition processes.

Caroline Maas, Shenchu Jin, Oliver Germandi, Gerd Otto, Peter Galle, Dennis Strand, 

Susanne Strand 

Human Chorionic Gonadotropin protects against T cellmediated 

liver injury in mice by downregulating Bim and 

Puma 

Clinical symptoms of Th1 mediated autoimmune diseases regress in many patients 

during pregnancy. Human chorionic gonadotropin (HCG) plays a major role in early 

human development through a series of well recognized pregnancy-promoting actions 

that are exerted in various reproductive and gestational tissues. Recent research 

indicates that HCG can exert significant physiological actions via its cell surface receptor 

(LH/CG-receptor) in different nongonadal tissues. In our investigations we tested the 

effect of HCG in a mouse model of autoimmune hepatitis. HCG decreased the 

histological manifestations of the Concanavalin A induced cell-mediated 

immunoinflammatory hepatitis, decreased the number of apoptotic hepatocytes in the 

liver and profoundly lowered the increased transaminase levels in the serum. To explore 

the mechanisms underlying the inhibition of apoptosis after HCG treatment, we used 

primary mouse human hepatocytes. We show that HCG can decrease the nuclear 

localization of the transcription factor FOXO3a and concomitantly lower levels of the 

proapoptotic FOXO3a downstream targets, Bim and Puma. 

HCG might be a promising strategy for the clinical treatment of human autoimmune 

disease.

Clarissa Mindnich, Sonja Bonness, Kristine Kohl, Sylvia Schnautz, Dagmar von Bubnoff, 

Dagmar Wilsmann-Theis, Susanne Koch, Thomas Bieber 

HUMAN IN VITRO GENERATED DENDRITIC CELLS EXPRESS 

THE INDUCIBLE NITRIC OXIDE SYNTHASE (iNOS) 

iNOS is a P-450-type cytoplasmic protein that catalyzes the conversion of L-arginine to 

L-citrulline and nitric oxide (NO). NO has been shown to play a role in inflammatory and 

autoimmune tissue injury because of its cytotoxic and immunoregulatory properties. 

The inducible form of NOS is able to produce large amounts of NO and can be expressed 

in a number of mammalian cells after challenge with proinflammatory cytokines. 

Dendritic cells (DC) present antigenic peptides to T cells and thus initiate an adaptive 

immune response. We investigated, whether human in vitro derived DC express iNOS. 

Staining of cryo-fixed human monocyte-derived DC (MoDC) with an iNOS-specific 

monoclonal antibody visualized with phosphatase-coupled secondary reagents revealed 

discrete intracellular localizations of the enzyme. iNOS protein was found in the 

cytoplasm, in a perinuclear region and close to the plasma membrane. Interestingly, the 

magnitude of iNOS protein expression and its localization was influenced by MoDC 

stimulation with Staphylococcus aureus. Also, RT-PCR analysis of MoDC mRNA showed 

enhanced expression of the enzyme after Staphylococcus aureus treatment. As a means 

of measuring functional iNOS activity, we investigated the occurrence of NO in the 

supernatants from unstimulated and Staphylococcus aureus-stimulated MoDC. NO was 

detected in supernatants from Staphylococcus aureus-stimulated MoDC cultures from 

two out of 17 patients with psoriasis, but not in supernatants derived from MoDC from 

healthy controls. We conclude that iNOS mRNA and protein can be expressed by human 

in vitro generated MoDC. However, its functional activity requires additional so far 

unknown factor(s) active under certain pathological conditions.

Annette Paschen, Mostafa Jarahian, Antje Sucker, Sandra Striegel, Iris Moll, Dirk 

Schadendorf, Frank Momburg 

Human Natural Killer (NK) Cells Effectively Kill Autologous 

Melanoma Cells In Vitro but Limited NK Cell Infiltration into 

Tumor Metastasis might Interfere with an Effective Anti- 

Tumor Immunity In Vivo 

Malignant melanoma in its metastatic disease stage is known to down-regulate or even 

completely lose the surface presentation of human leukocyte antigen (HLA) class I 

molecules. Cells displaying an altered HLA class I expression should be eliminated by 

cytotoxic NK cells, as long as ligands of activating NK cell receptors are presented on 

the tumor cell surface. Therefore, we asked about the NK cell recognition of melanoma 

cells with different HLA class I phenotypes. Melanoma cell lines were established from 

HLA class I-positive and -negative metastatic tumor tissues of different patients. In 

general, HLA class I loss on tumor cells was due to mutations affecting the β2microglobulin 

gene. Irrespective of their HLA class I phenotype, tumor cells expressed 

ligands of the activating NK cell receptors NKG2D, DNAM-1 and NCR. Interestingly, in 

vitro activated, polyclonal NK cells isolated from the peripheral blood of a melanoma 

patient were capable of effectively killing autologous HLA class I-positive and -negative 

tumor cells, suggesting that activated NK cells might be exploited for melanoma 

immunotherapy. However, when metastatic tumor tissues were analysed for immune 

cell infiltration, CD56 + NK cells were hardly detectable in most cases, suggesting that 

an inefficient tumor infiltration might be a barrier for an effective NK-based 

immunotherapy of melanoma.

Anja A. Kuehl, Jürgen Westermann, Nina N. Pawlowski, Katja Grollich, Martin Zeitz, 

Jörg C. Hoffmann 

Human peripheral γδ T Cells posses regulatory Potential 

Background and Aim: In animal models of inflammatory bowel disease γδ T cells play 

a protective role by prolonging the survival and diminishing epithelial damage. For 

therapeutical use in inflammatory bowel disease, the regulatory properties of human γδ 

T cells have to be elucidated. Therefore, proliferation, suppression, and cytokine 

secretion of γδ T cells were determined in vitro. 

Methods and Material: Human peripheral γδ T cells were isolated from whole blood of 

healthy donors by MACS technology. The proliferative response was measured by 3 [H]- 

Thymidine incorporation and the cytokine profile of culture supernatants by ELISA as 

well as intracellularly by flow cytometry. Additionally, the suppressive capacity was 

determined by flow cytometry in coculture experiments. 

Results: Human γδ T cells showed in vitro anergic behaviour and suppressed the 

growth of CD4-positive T cells even at low cell ratios. Additionally, they secreted both 

anti- and proinflammatory cytokines. The regulatory properties of human γδ T cells were 

superior to other regulatory T cells (CD4CD25-positive) regarding their suppressive 

behavior and cytokine profile. 

Conclusions: Regulatory γδ T cells could be of therapeutical use in treatment of 

inflammatory bowel disease as they are anergic and act suppressive as well as secret 

protective cytokines. For application of human γδ T cells in therapy their expansion 

under maintenance of their regulatory properties should be elucidated.

Anja Saalbach, Jacqueline Lessig, Jan C Simon, Jürgen Arnhold, Ulf Anderegg 

Human Thy-1 induces secretion of matrix metalloproteinase-9 

and CXCL8 from neutrophils 

Neutrophils are the first cells at sites of inflammation. On their way from blood to the 

site of infection neutrophils have to adhere to endothelial cells (EC), to transverse the 

basement membrane and subsequently to travel through the interstitial matrix. 

Recently, we have shown that Thy-1 is an adhesion molecule on activated dermal EC 

and fibroblasts mediating the binding of neutrophils via Mac-1. Thus, human Thy-1 is an 

alternate EC receptor for the leukocyte integrin Mac-1 that contributes to leukocyte 

recruitment to sites of inflammation thus providing a new pathway for adhesion and 

transmigration of neutrophils. 

Here, we studied whether Thy-1 mediated adhesion of neutrophils mediates only the 

physical contact or further influences neutrophil functions. Since MMP-9 plays an 

important role for the migration of neutrophils through the basement membrane we 

analyzed secreted MMP-9 after interaction of neutrophils with Thy-1. Indeed, binding of 

neutrophils to recombinant Thy-1 stimulated secretion and activation of MMP-9 from 

neutrophils resulting in an enhanced migration through a collagen-IV barrier. 

Accordingly, blocking Thy-1 on activated dermal EC or fibroblasts decreased the MMP-9 

secretion from neutrophils in co-cultures. 

Next, we investigate whether the interaction of neutrophils with Thy-1 regulates the 

secretion of CXCL8 and thus might support the attraction of additional neutrophils at 

sites of inflammation. Binding of neutrophils to Thy-1 induced the release of CXCL8. 

Blocking of Thy-1 on activated dermal EC or fibroblasts in co-cultures with neutrophils 

decreased the CXCL8 secretion confirming the role of Thy-1 in regulation of CXCL8 

release. 

In summary, Thy-1 mediates not only the adhesion of neutrophils to activated dermal 

EC and fibroblasts but also regulates neutrophil function. These results support the 

general concept that the function of ‘adhesion molecules’ may not only be to provide 

mechanical support but might also be to regulate functions such as motility or release of 

chemotactic factors.

Sabrina Laing, Mareike Pilz, Michel Seman, Friedrich Koch-Nolte, Friedrich Haag 

Human TNF&alpha is a substrate for modification by ADPribosyltransferase-1 

(ART1) 

Mono-ADP-ribosyltransferases (ARTs) are GPI-anchored ectoenzymes that covalently 

modify cell surface or soluble target proteins by transferring ADP-ribose from NAD+ to 

arginine residues. In the mouse, ART2, expressed on resting T lymphocytes, plays an 

immunoregulatory role by ADP-ribosylating the P2X7 purinoreceptor, thereby initiating 

rapid apoptosis in T-cells. ART2 is shed from the cell surface in an enzymatically active 

form upon T-cell activation. However, ART2 is a pseudogene in man, and the question 

as to whether ADP-ribosylation plays an immunoregulatory role in the human immune 

system is open. We asked whether in humans the immunoregulatory function of ART2 

might be carried out by ART1. We identified ART1 transcripts in human peripheral blood 

leukocytes, as well as in heart and skeletal muscle, by RT-PCR analysis. We 

hypothesized that ART1, like ART2, might be released from cells and be present in the 

circulation in a soluble form. We thus asked whether soluble ART1 could modify small 

messenger proteins such as cytokines. Indeed, soluble ART1, released from the surface 

of transfected cells by PI-PLC, modified recombinant human TNF&alpha in vitro. 

Furthermore, co-transfection of HEK293 cells with ART1 and human TNF&alpha resulted 

in modification of TNF&alpha at at least 2 distinct sites, i.e. one within the domain shed 

from the cell surface by the action of the metalloproteinase TNF&alpha Converting 

Enzyme (TACE), and one on the stalk that remains connected with the cell membrane 

after cleavage by TACE. Experiments to investigate the functional consequences of ADPribosylation 

of TNF&alpha as well as to identify the ADP-ribosylation sites are currently 

in progress.

Petra Richl, Martin Albers, Henner Morbach, Stephanie Brändlein, H. Peter Vollmers, 

Hermann Girschick 

Humoral immunity against malignant gastric carcinoma cells: 

molecular characterization and age-related frequency of SC-1 

antibody positive B cells 

Objective: The human monoclonal IgM antibody SC-1 was isolated from splenic B cells 

of an adult patient with adenocarcinoma of the stomach. It detects an isoform of the 

DAF/CD55 receptor, expressed specific on gastric adenocarcinoma. Given that this 

antibody is considered to belong to the "natural" or "innate" immunity and is found in 

healthy individuals as well, molecular analysis (use of immunoglobulin genes) and 

analysis of age-related frequency of SC-1 producing B cells is of particular relevance for 

further understanding of natural anti-tumor immunity. 

Methods: By means of an anti-SC-1 antibody we were able to isolate and characterize 

(CD19, CD5, CD27, IgD) B cells from the peripheral blood by multi-color FACS staining. 

In order to uncover an age-related presence of SC-1 positive B cells, we analyzed 

PBMCs from cord blood, children, adults (30, 50 and 70 years old) and compared the 

findings with those from carcinoma patients. Molecular characterization of the B cell 

receptor was carried out by sequence analysis of lambda light chain genes on single cell 

level. 

Results: Across all ages the relative number of SC-1+ B cells was constant at 5% 

whereas the number doubled in carcinoma patients (10%). Combined analysis of 

different epitopes revealed age-related frequencies. Strikingly we revealed a statistically 

significant decrease of SC-1+IgD+ B cells in case of carcinoma and a concomitant 

change of the phenotype to SC-1+IgG+ B cells. 

In both healthy individuals and carcinoma patients SC-1+CD5+ B cells were isolated 

which predominantly carry the lambda light chain 3r (85%). Further analysis revealed a 

selection of short CDRIII regions (27 bp) and a selection of JL 2/3 in SC-1+CD5+ B cells 

of tumor patients. 

Conclusion: The comparative molecular characterization and phenotyping of B cells 

from healthy individuals of different age and carcinoma patients gives information about 

the natural immune-surveillance against tumors, the age-related presence and 

variability. Furthermore, this analysis provides an insight into the clonal expansion and 

differentiation of tumor-specific B cells.

Gordon Grochowy, Michelle Hermiston, Arthur Weiss, Michael Huber 

Hyperactivation of mast cells from CD45 E613R („wedge“) 

mice 

The transmembrane protein tyrosine phosphatase CD45 is involved in the activation of 

the Src kinase Lyn and thus in the antigen-triggered FcεR1-mediated activation of mast 

cells (MCs). Hence, CD45-deficient bone marrow-derived MCs (BMMCs) exhibit 

increased inhibitory Lyn phosphorylation and drastically reduced effector functions 

(degranulation and cytokine secretion). Due to a mutation at the tip of the so-called 

wedge-region, CD45 E613R is unable to dimerize and prone to hyper-activity. 

Correlating, CD45 E613R BMMCs show stronger effector functions after antigentriggering 

than wild-type BMMCs, however, inhibitory phosphorylation of Lyn is 

comparable to CD45-deficient BMMCs. This unexpected phenotype most likely is due to 

attenuated interaction between CD45 E613R and Lyn and a hyper-activation of the Fynregulated 

phosphatidylinositol-3-kinase pathway. Interestingly, depending on the 

addressed receptor systems CD45-deficient and CD45 E613R BMMCs are able to show 

uniform phenotypes as well. Proliferation of both cell types in response to IL-3 and/or 

SF is enhanced compared to wild-type BMMCs. In conclusion, we demonstrate that 

CD45 is required to fine-tune MC responses mediated by different ligand-receptor 

systems. This is reinforced by studying a CD45 inhibitor, which, depending on the 

concentration used, can augment or attenuate MC effector functions.

Ursula Ellinghaus, Rudolf Rupec, Oliver Pabst, Ralf Ignatius, Reinhold Förster, Bernd 

Dörken, Franziska Jundt 

IκBα is crucial for marginal zone B cell development 

Marginal zone B (MZB) cells constitute a distinct B cell lineage located in the splenic 

marginal zone that is specialized in the rapid response to blood-borne pathogens. 

Development of MZB cells depends on the strength of B cell receptor signaling, Notch2 

signaling and subunits of nuclear factor kappa B (NF-κB). The function of Notch2 is 

impressively illustrated by a complete loss of MZB cells in mice with a B-cell-specific 

Notch2-deletion. In contrast, the function of NF-κB remains to be fully elucidated. 

Analysis in mice lacking the NF-κB subunit p50 demonstrated an essential role of p50 

for MZB cell generation, whereas other subunits appear to contribute to a lower extent. 

We investigated the role of IκBα, the main inhibitor of NF-κB, for MZB cell development 

and function. To this aim we generated a B-cell-specific deletion of IκBα in mice 

resulting in a constitutive activation of NF-κB. This caused a 70 % reduction of MZB 

cells as well as putative MZB cell precursors. In contrast, follicular B and B1 cells 

remained unaltered. Interestingly, we observed decreased Notch2 signaling in IκBαdeficient 

splenic B cells, indicated by suppressed transcription of Notch2 and its target 

genes Hes1, deltex1 and Cd21. In contrast, the expression levels of the B-cell-specific 

transcription factors Pax5 and Ebf1 remained unaltered. In order to evaluate the 

functional relevance of a reduced MZB cell population we injected IκBα mice with 

Staphylococcus aureus as a model for infection with blood-borne pathogens. 

Interestingly, IκBα-deficient mice showed a decreased survival compared to wildtype 

mice. This suggests that a reduction of MZB cells in mice with the B-cell-specific 

deletion of IκBα results in an impaired clearence of blood-borne bacterial infection.

R. Riedl, J. Sommer, K. Prinz, A. Egyed, C. Schellack, A. von Gabain, E. Nagy, K. 

Lingnau 

IC31TM: a novel adjuvant that potently activates Type I 

immune responses 

The novel adjuvant IC31TM consists of a combination of a negatively-charged synthetic 

oligodeoxynucleotide and a positively-charged peptide. IC31TM is characterized by a 

broad mechanism of action, as well as an excellent safety profile. Detailed analyses 

showed that the immunostimulatory effect of IC31TM is mediated via the TLR9/MyD88dependent 

signaling pathway of the innate immune system. Additionally, both depot 

formation at the injection site as well as sustained activation of antigen presenting cells, 

followed by antigen uptake and processing, is likely responsible for the induction of the 

observed IC31TM-mediated antigen-specific immune responses. It could be shown in 

mice that IC31TM induces strong T cell responses activating both CD4+ helper T cells as 

well as CD8+ cytotoxic T cells. In addition, strong humoral immune responses with 

increased antibody titers are generated by IC31TM. Because of its activation of innate 

immune cell types, such as NK cells and DC, as well as its stimulation of Type I T cell 

responses, IC31TM may show broad applicability to the treatment of infectious diseases 

and tumors.

Alexandra Doerr, Carsten Watzl, Michael Kirschfink 

iC3b binding to Raji cells modulates Rituximab- induced 

antibody-dependent cellular cytotoxicity (ADCC) 

Malignant cells are protected against autologous complement by various mechanisms 

including the overexpression of membrane-associated complement regulators, and 

soluble complement inhibitors, secreted into the tumour microenvironment. This 

complement resistance represents a major barrier for successful anti-tumour 

immunotherapy. Conflicting results exist on the possible impact of complement on NK 

cell-mediated cytotoxicity. 

In the present study we investigated the ability of complement to increase antibodymediated 

NK cell cytotoxicity, induced by Rituximab, a chimeric anti-tumour antibody, 

which is directed against CD20 expressed on B-cells and successfully applied in 

immunotherapy of Non-Hodgkin-B-cell lymphomas. 

Raji cells were preincubated with C5 depleted human serum as source of complement 

before exposure to freshly isolated NK cells. Complement- and NK cell- mediated 

cytotoxicity was measured by 51 Cr release assay and binding of complement proteins to 

tumour cell surface by cytofluorometry. 

As Raji cells activate the alternative pathway of the complement cascade, a significant 

amount of iC3b was deposited on the tumour cell surface and complement-mediated 

tumour cell lysis (CDC) occured, even in the absence of Rituximab. Rituximab dosedependently 

induced ADCC, as well as CDC and the deposition of iC3b in the presence 

of normal human serum. Tumour-directed NK cell cytotoxicity increased after 

preexposure to serum complement even in the absence of antibody. If Raji cells were 

incubated with low concentrations of Rituximab in the presence of C5-depleted serum 

leading to binding of iC3b on targeted Raji cells, ADCC of the tumour cells was 

augmented. However, if Raji cells were exposed to higher concentrations of Rituximab 

in the presence of C5 depleted serum, higher amounts of iC3b exerted an inhibitory 

effect on NK cell lysis. 

This demonstrates that the amount of deposited iC3b on the target cell determines if 

complement has a positive or negative effect on NK cell-mediated ADCC. 

-->

Yvonne Burmeister, Timo Lischke, Anja C. Dahler, Hans-Werner Mages, Kong-Peng 

Lam, Anthony J. Coyle, Richard A. Kroczek, Andreas Hutloff 

ICOS controls the pool size of effector-memory and 


Inducible co stimulator (ICOS) is an important regulator of T cell effector function. ICOSdeficient 

patients as well as knock-out mice show severe defects in T cell-dependent B 

cell responses. Several in vitro and in vivo studies attributed this phenomenon to 

impaired upregulation of cell surface communication molecules and cytokine synthesis 

by ICOS deficient T cells. However, we now could show in a murine adoptive transfer 

system with antigen specific T cells that signaling via ICOS does not significantly affect 

early T cell activation. Instead, ICOS substantially contributes to the expansion and 

survival of effector T cells upon local challenge with antigen and adjuvant. Importantly, 

the observed biological function of ICOS also extends to FoxP3+ regulatory T cells, as 

can be observed after systemic antigen delivery without adjuvant. In line with these 

findings, absence of ICOS under homeostatic conditions of non-immunized mice leads to 

reduced numbers of both effector-memory and FoxP3+ regulatory T cells. Based on 

these results, we propose a biological role for ICOS as a costimulatory, agonistic 

molecule for a variety of effector T cells with differing and partly opposing functional 

roles. This concept may reconcile a number of past in vivo studies with seemingly 

contradictory results on ICOS function.

Eva Nina Huter, Sabine Stoll, Julia Horn, Juergen Knop, Bodo Grimbacher, Helmut 

Jonuleit 

ICOS plays an essential role in the development of anergic 

and suppressive CD4+ T cells 

The type and maturation state of dendritic cells (DC) determines the differentiation of 

resting CD4+ T cells. While activated, mature myeloid DC support efficient clonal 

expansion of these lymphocytes and their differentiation into IFN-γ-producing effector 

cells, immature DC instead induce anergic, IL-10-producing T cells with suppressive 

properties. Since Inducible Costimulator (ICOS) is involved in the induction of IL-10 in T 

cells and expression of ICOS Ligand (ICOS-L) on mature plasmacytoid DC contributes to 

the induction of suppressive T cells in an inflammatory environment, we analyzed the 

functional role of ICOS in the differentiation of human CD4+ T cells upon their 

interaction with myeloid DC. We report that the induction of T cell anergy by immature 

DC is completely blocked after siRNA-mediated knockdown of ICOS expression. 

Furthermore, anergy induction was also abolished in CD4+ T cells from ICOS-deficient 

patients. In contrast to CD4+ T cells from healthy volunteers, ICOS-deficient CD4+ T 

cells showed strong proliferation in the presence of allogeneic immature DC and 

differentiated into effector T cells with increased IL-4 and reduced but still significant IL- 

10 production. Additionally, after priming and restimulation with allogeneic immature 

DC ICOS-deficient CD4+ T cells were not suppressive in contrast to T cells from healthy 

controls. Taken together, these results indicate a crucial role for ICOS in the induction 

of peripheral tolerance by immature DC.

Johann Röhrl, Thomas Hehlgans 

Identification and Biological Characterization of Mouse Beta 

Defensin 14 – an Ortholog of Human Beta Defensin 3 

Beta defensins are small antimicrobial polypeptides mainly expressed by epithelial cells 

which play an important role in the antimicrobial innate immune response. 

In addition to the direct microbicidal effects of these polypeptides it became evident 

that certain members of the beta-defensin family have the capacity to promote local 

innate inflammatory and systemic adaptive immune responses by interacting with the 

CC-chemokine receptor CCR6. 

We have identified mouse beta defensin 14 (mBD14 or DEFB114) as an ortholog of 

human beta defensin 3 (hBD3 or DEFB103). Based on primary structural analysis 

mBD14 demonstrates 68% homology to its human ortholog, containing three conserved 

cystein linkages, characteric for the beta defensin family. MBD14 is expressed in a wide 

variety of tissues including spleen, colon and tissues of the upper and lower respiratory 

tract. Interestingly, we detected mBD14 expression also in immature CD11c positive 

bone marrow derived dendritic cells. The expression of mBD14 can be induced by TLR 

agonists such as LPS and poly I:C and by pro-inflammatory stimuli e.g. TNF. 

Furthermore, expression of mBD14 seems to be regulated by activation of the 

intracellular pattern recognition receptor NOD2/CARD15 as revealed by reporter gene 

assays. A recombinant mBD14-fusionprotein demonstrated antimicrobial activity against 

E.coli (ATCC 25922) and S. aureus (ATCC 6538). 

In contrast to another recently described member of the mouse beta defensin family 

(mBD4) and its human ortholog human beta defensin 2 (hBD2), mBD14 does not seem 

to interact with the CC-chemokine receptor CCR6.

Laura Rivino, Federica Sallusto, Antonio Lanzavecchia, Jens Geginat 

Identification and characterization of human "contextdependent 

Tr1/memory" cells. 

Two types of regulatory T cells have been described: “natural” CD25+Foxp3+ Tregs and 

“adaptive” Tr1 cells with unknown phenotype. CCR6 expression on in vitro primed 

human T cells required TGF-beta and the absence of polarising cytokines. Circulating 

CCR6+CD25- T cells were Foxp3-, but produced IL-10 with autologous DC that 

suppressed auto-reactive T cell proliferation. Interestingly, T cells reacting with the selfantigen 

MelanA were exclusively CCR6+ in healthy individuals, but were predominantly 

CCR6- in Viltiligo autoimmune patients. CCR6+ T cells responded also to various recall 

antigens and we isolated T cell clones that suppressed responses to autologous DC via 

IL-10, but that produced IL-2 and proliferated with tetanus toxoid. We propose that 

CCR6 is expressed on context-dependent “Tr1/memory” cells that act as conventional 

memory cells in recall responses, but behave Tr1-like upon recognition of cross-reactive 

antigens under steady-state conditions.

Andrea Baetz, Christoph Koelsche, Alexander Dalpke 

Identification of a nuclear localization signal (NLS) in SOCS1 

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of 

janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling 

pathways. Moreover, SOCS proteins act as part of an ECS-type ubiquitin ligase complex 

which extends the functional range of this protein family to a so far not completely 

understood level. We here analyzed temporal-spatial mechanisms of SOCS action by 

generating GFP-SOCS fusion proteins. To our surprise we observed that SOCS-1 but not 

further SOCS family members localized predominantly to the nucleus when expressed in 

HEK293 cells. Similar results were observed in further cell lines and with different tags. 

Sequence analysis revealed a putative bipartite nuclear localization signal (NLS) located 

between the SH2 and the SOCS-box domain. Deletion of this region, introduction of a 

series of R/A point mutations or substitution of this sequence with the respective region 

of SOCS-3 resulted in loss of nuclear and increasing cytoplasmic localization. Fusion of 

the SOCS-1-NLS to cytokine-inducible SH2 region containing protein (CIS) resulted in 

nuclear localization of this otherwise cytoplasmic protein. Although the generated 

mutants of SOCS-1 showed loss of nuclear localization they were still partly effective in 

suppressing IFN-α mediated STAT1 tyrosine phosphorylation and reporter gene activity. 

The results identify a NLS in SOCS-1; however the functional significance differs from 

the well known role in JAK/STAT inhibition.

Theresa Tretter, Ram Kumar Venigalla, Volker Eckstein, Hanns Martin Lorenz 

Identification of human B cells with immunoregulatory 

properties 

Introduction: B cells are well known as mediators of humoral immunity and as antigen 

presenting cells in generation of T cell mediated responses. However observations from 

certain mouse models for autoimmunity point also to a role for B cells in maintaining 

tolerance. So far little is known yet about their immunoregulatory properties, 

specifically in humans and the mechanisms behind. 

Methods: Highly purified CD19+B and CD4+T cells were separated from human PBMC 

by MACS. B cells were prestimulated with SAC, aIgM/IgG or aCD40, and set up in 

cocultures with autologous CD4+T cells under addition of aCD3 +IL-2 or aCD28. CD4+ 

T cell proliferation was determined after 3-6d by 3H Thymidine incorporation and PKH- 

26; apoptosis by AnnexinV. 

Results: under optimal stimulatory conditions T cell proliferation was inhibited by more 

than 50% in presence of SAC-activated B cells and to a lesser extent by aIgM/IgG 

stimulated B cells. There was a strong correlation with activation status of the B cell 

since enrichment of the large SAC activated CD25+ B cell population by FACSsorting 

further enhanced suppression to more than 70%, while the small SAC activated CD25- 

B cell population had no significant effect. Separation of B and T cells by cell culture 

inserts abolished inhibition, suggesting a requirement for direct cell-contact. In addition, 

optimal stimulatory conditions were necessary for inhibitory effects since suppression 

was not observed in absence of CD28 or exogenous IL-2. In addition to growth 

inhibition, T cell specific cytokine production (IFN-gamma, IL-10) was downregulated, 

and a significant proportion of T cells went into apoptosis. 

Conclusions: B cells are able to inhibit T cell mediated responses depending on their 

mode of activation. Further experiments are dealing with the mechanisms of B cell 

mediated suppression and their pathophysiological impact also in autoimmune diseases.

Tereza Havlova, Anja Tessarz, Vaclav Horejsi, Adelheid Cerwenka 

Identification of Key-Players in TREM1/DAP12 Signaling 

Pathway 

Triggering Receptor Expressed on Myeloid cells (TREM-1) is a recently described 

receptor expressed by monocytes and neutrophils, which plays an important role during 

immune responses to microbial infection. The engagement of TREM-1 leads to the 

production of pro-inflammatory cytokines such as TNF-α and IL-8 and thereby 

contributes to inflammatory conditions. TREM-1 associates with the ITAM-containing 

adapter DNAX Activation Protein of 12 kDa (DAP12). In general, ITAM-mediated signals 

lead to cell activation, although DAP12 was recently implicated in inhibitory signaling in 

mouse macrophages and dendritic cells. 

So far, the downstream signaling pathways following receptor triggering have not been 

well characterized. Recently, we identified NTAL/LAB/LAT2 as an important gate-keeper 

of TREM-1/DAP12-induced signaling in myeloid cells. This transmembrane adaptor 

protein is phosphorylated upon TREM-1 ligation in a myelo-monocytic cell line and 

primary human granulocytes. Using siRNA mediated knock-down to decrease NTAL 

expression levels, we observed enhancement of ERK 1/2 phosphorylation, delay in Ca 2+ 

mobilization and a substantial decrease in TNF-α and IL-8 production after TREM-1 

stimulation. 

We are currently focusing on the identification of proteins associated with NTAL in 

context of the TREM-1/DAP12 signaling pathway. In addition, we are analyzing proteins, 

which become phosphorylated upon TREM-1 triggering, via mass spectrometry to obtain 

new insights about the known and yet unknown key-players in this signaling pathway. 

The knowledge gained by this study could help to define novel therapeutic targets for 

the treatment of inflammatory diseases and sepsis.

Katja Kotsch, Vera Merck, Kristina Kunert, Anja Reutzel-Selke, Andreas Pascher, Hans- 

Dieter Volk, Peter Neuhaus, Johann Pratschke 

Identification of molecular candidate marker in zero kidney 

biopsies are indicative for graft quality 

In renal transplantation, allograft biopsies provide valuable diagnostic information. 

Consequently, several attempts have been made to predict the early graft outcome by 

histological and molecular analyses in zero-hour graft biopsies illustrating that subtle 

inflammation and immune activation detected in the intraoperative period are 

associated with adverse allograft outcome post transplantation. The clinical outcome is 

dependent upon various risk factors including donor brain death or prolonged cold 

ischemia (CI). However, current knowledge about the influence of brain death and CI is 

only restricted to a low number of genes and the molecular and cellular mechanisms are 

thus far unknown. Efficient strategies to prevent prolonged cold ischemia or brain death 

related complications require a better understanding of the molecular processes 

reflecting intragraft inflammation present intraoperatively at the zero-hour. We 

therefore studied mRNA gene expression of zero kidney biopsies derived from 63 

cadaveric donors (CAD) (CI=13,3 ± 3,6) and 27 living donors (LD) (CI=2,8 ± 0,7). 

Intraoperative biopsies were taken 30 min. after vascular reperfusion and were 

immediately snap-frozen. Messenger RNA was extracted and gene expression was 

measured by real-time RT-PCR. Recently it has been demonstrated that the induction of 

the T cell marker CD3 or the intercellular adhesion molecule (ICAM)-1 in zero biopsies 

are predictive of acute rejection (Avihingsanon et al. 2005). By comparing zero biopsies 

from CAD versus LD we observed a significant induction of CD3 (p

Katja Kotsch, Vera Merck, Kristina Kunert, Anja Reutzel-Selke, Andreas Pascher, Hans- 

Dieter Volk, Peter Neuhaus, Johann Pratschke 

Identification of molecular candidate marker in zero kidney 

biopsies are indicative for graft quality 

In renal transplantation, allograft biopsies provide valuable diagnostic information. 

Consequently, several attempts have been made to predict the early graft outcome by 

histological and molecular analyses in zero-hour graft biopsies illustrating that subtle 

inflammation and immune activation detected in the intraoperative period are 

associated with adverse allograft outcome post transplantation. The clinical outcome is 

dependent upon various risk factors including donor brain death or prolonged cold 

ischemia (CI). However, current knowledge about the influence of brain death and CI is 

only restricted to a low number of genes and the molecular and cellular mechanisms are 

thus far unknown. Efficient strategies to prevent prolonged cold ischemia or brain death 

related complications require a better understanding of the molecular processes 

reflecting intragraft inflammation present intraoperatively at the zero-hour. We 

therefore studied mRNA gene expression of zero kidney biopsies derived from 63 

cadaveric donors (CAD) (CI=13,3 ± 3,6) and 27 living donors (LD) (CI=2,8 ± 0,7). 

Intraoperative biopsies were taken 30 min. after vascular reperfusion and were 

immediately snap-frozen. Messenger RNA was extracted and gene expression was 

measured by real-time RT-PCR. Recently it has been demonstrated that the induction of 

the T cell marker CD3 or the intercellular adhesion molecule (ICAM)-1 in zero biopsies 

are predictive of acute rejection (Avihingsanon et al. 2005). By comparing zero biopsies 

from CAD versus LD we observed a significant induction of CD3 (p

Wolfram Osen, Mingxia Song, Sabine Soltek, Barbara Leuchs, Julia Steitz, Xuan Duc 

Ngyuen, Dirk Schadendorf, Annette Paschen 

Identification of novel CD4+ T cell epitopes from human 

tyrosinase related protein 2 (TRP-2) by a combinatorial 

approach based on the immunisation of HLA-transgenic mice 

with recombinant Adenovirus and antigen peptide library 

screening 

Melanoma differentiation antigen TRP-2 is a target of spontaneous cytotoxic CD8+ T cell 

(CTL) responses in tumor patients and can thus be considered as a melanoma 

associated tumor antigen (TA). Since induction and maintenance of tumor-specific CD8 

+ T cell responses depends on the activity of specific CD4+ T helper (Th) cells, we set 

out to identify novel TRP-2-specific Th epitopes that could be included in 

immunotherapy approaches against malignant melanoma. Using recombinant 

Adenovirus expressing human TRP-2 (Ad/TRP-2) as an immunogen followed by 

combinatorial peptide library screening in vitro we were able to identify novel 

DRB1*0301-restricted T cell epitopes in HLA-DRB1*0301 transgenic mice. Notably, the 

same epitope was found to be recognized by Th cells of DRB1*03+ donors on syngeneic 

Ad/TRP-2 infected DC, showing that the epitope identified in the HLA-DRB1*0301 

transgenic mouse system is also processed and presented by human DRB1*03+ 

individuals.

Josip Zovko, Marco Herold, Christa Kraus, Andrea Peters, Ingolf Berberich 

Identification of proteins that influence stability and 

functionality of the anti-apoptotic Bcl-2 family member A1/ 

Bfl-1 

Members of the Bcl-2 family control the integrity of mitochondria and thereby influence 

survival and death of cells. Most Bcl-2 family members can localize to intracellular 

membranes via hydrophobic sequences within their C-terminal portion. 

Murine A1 and its human homologue Bfl-1 are anti-apoptotic members of the Bcl-2 

family. A1 is expressed in small amounts in the bone marrow and immature B cells, but 

in high amounts in mature B cells. Thus the protein seems to be important for B cell 

maturation. 

We analyzed the function of the C-terminus of A1. Unless the C-terminal ends of other 

Bcl-2 proteins the tail of A1 does not function as a membrane anchor. Nevertheless, the 

last amino acids of A1 are important for the protein. In fact, the C-terminus of A1 

serves a dual function by being required for the instability and the anti-apoptotic 

potential of the protein. We show that A1 undergoes proteosomal degradation controlled 

by its C-terminus. Interestingly, binding to the pro-apoptotic Bcl-2 factor BimEL results 

in increased stability of A1. This is due to reduced ubiquitination of A1 after binding of 

BimEL. We conclude that the C-terminus of A1/Bfl-1 serves as a docking site for E3 

ubiquitin ligase(s) that control the stability of A1 by targeting the protein to the 

proteasomal pathway. Currently, we are trying to identify such proteins by affinity 

chromatography and mass spectrometry.

Susanne Berchtold, Edda Fahl, Mathias Hornef, Julia Geisel, Julia-Stefanie Frick, Erwin 

Bohn 

IFIT-2 – a putative novel negative regulator of 

proinflammatory responses 

Interferon-induced tetratricopeptide repeat protein (IFIT-) 2 is induced upon acute 

infection with Yersinia enterocolitica in CD11b-positive cells of the spleen of mice as well 

as in the colon of IL-2 deficient mice which develop inflammatory bowel disease. IFIT-2 

is induced by type II and type I interferons or indirectly by LPS in an IFN dependent 

manner. Recently it was reported that mouse IFIT2 (P54) affects protein synthesis by 

interaction with the translation initiation factor eIF3c. Therefore we speculated that this 

gene could represent a negative regulator of host responses by down regulating protein 

synthesis. To address the role of IFIT2, stably transfected RAW 264.7 macrophages 

were established overexpressing IFIT-2. These cells were viable and showed similar 

proliferation as control cells. IFIT-2 overexpression did not alter LPS triggered p38, ERK, 

JNK and IκB phosphorylation indicating that IFIT-2 does not affect LPS mediated signal 

transduction. Overexpression of IFIT-2 in RAW 264.7 macrophages reduced LPS induced 

protein expression in a selective manner at a posttranscriptional level. Thus, TNF- 

&alpha, IL-6 and MIP-2 secretion but not protein expression of IFIT-1 or early growth 

reponse 1 were affected by IFIT-2. IFIT-2 may thereforee represent a novel negative 

regulator of proinflammatory responses.

Uwe Müller, Werner Stenzel, Gabriele Köhler, Gesine Hansen, Nicole Schütze, Reinhard 

Straubinger, Manfred Blessing, Andrew McKenzie, Frank Brombacher, Gottfried Alber 

IL 13 induces disease promoting type 2 cytokines, 

alternatively activated macrophages and allergic 

inflammation during pulmonary infection of mice with 

Cryptococcus neoformans 

In the murine model of Cryptococcus neoformans (C. neoformans) infection Th1 (IL-12/ 

IFN-&gamma) and Th17 responses (IL-23/IL-17) are associated with protection, 

whereas an IL-4-dependent Th2 response exacerbates disease. In order to investigate 

the role of the Th2 cytokine IL-13 during pulmonary infection with C. neoformans, IL-13transgenic 

(IL-13 T/+ ), IL-13-deficient (IL-13 -/- ), and wild-type (WT) mice were infected 

intranasally. Susceptibility to C. neoformans infection was found when IL-13 was 

induced in WT mice or over-produced in IL-13 T/+ mice. Infected IL-13 T/+ mice had a 

reduced survival time and higher pulmonary fungal load as compared to WT mice. In 

contrast, infected IL-13 -/- mice were resistant and 89% of these mice survived the 

entire period of the experiment. Antigen-specific production of IL-13 by susceptible WT 

and IL-13 T/+ mice was associated with a significant type 2 cytokine shift but only minor 

changes in IFN-&gamma production. Consistent with enhanced type 2 cytokine 

production, high levels of serum IgE and low ratios of serum IgG2a/IgG1 were detected 

in susceptible WT and IL-13 T/+ mice. Interestingly, expression of IL-13 by susceptible 

WT and IL-13 T/+ mice was associated with reduced IL-17 production. IL-13 was found 

to induce formation of alternatively activated macrophages (aaMph) expressing 

arginase-1, macrophage mannose receptor (CD206) and YM1. In addition, IL-13 

production led to lung eosinophilia, goblet cell metaplasia and elevated mucus 

production, and enhanced airway hyperreactivity. This indicates that IL-13 contributes 

to fatal allergic inflammation during C. neoformans infection.

Annette Erhardt, Markus Biburger, Gisa Tiegs 

IL-10 AND REGULATORY T CELLS – THE MAIN MEDIATORS OF 

IMMUNOLOGICAL TOLERANCE AGAINST CONCANAVALIN A 

Injection of the plant lectin Concanavalin A (ConA) induces pronounced T- and NKT-cell 

activation followed by the onset of acute liver injury. ConA hepatitis has often been 

described as a murine model for immune-mediated hepatitis in humans. Recently, we 

have shown that ConA pretreated mice developed tolerance against ConA rechallenge 

within 8 days. Suppression of liver damage upon ConA pretreatment was characterized 

by decreased plasma transaminase levels and an anti-inflammatory cytokine response 

with increased IL10 production. Tolerance was fully reversed in IL10 KO mice 

emphasizing the important role of IL10 during ConA tolerance. 

To confirm the relevance of IL10, neutralizing experiments with an anti-IL10R antibody 

were performed blocking the binding site of IL10. Antibody injection prior to ConA 

pretreatment, imitating IL10 KO mice, or prior to ConA restimulation largely reduced 

the tolerogenic effect, suggesting that IL10 participates in long-term differentiation 

processes but also acts as short-term immunosuppressive mediator in vivo. 

Depletion of regulatory T cells (Tregs) and Kupffer cells in vivo prior to ConA 

rechallenge significantly diminished IL10 production, revealing these cells as main 

sources of IL10. Accordingly, Tregs isolated from ConA tolerized mice exhibited 

significantly enhanced IL10 production after ex vivo restimulation in contrast to control 

Tregs. However, in vitro neutralization of IL10 in co-cultures of responder cells and 

Tregs failed to reverse the immunosuppressive effect of Tregs from control as well as 

from ConA tolerant mice. 

In an immuno-therapeutic approach Tregs isolated from tolerized WT mice conferred 

significant protection from ConA-induced liver damage in contrast to Tregs from 

tolerized IL10 KO mice. 

In summary, we demonstrated that ConA tolerance, characterized by both 

immunosuppression and protection from liver injury, is mediated by Kupffer cells, Tregs, 

and IL10. Hence, ConA tolerance appears to be an appropriate model for evaluation of 

therapeutic intervention strategies in complex immuno-regulatory systems.

Anne Schumacher, Paul Ojiambo Wafula, Ana Teles, Hideo Yagita, Hans-Dieter Volk, 

Ana Zenclussen 

IL-10 but not TGF-ß is essential for the suppressor function of 

Treg cells in murine pregnancy 

Problem 

The physiological state of pregnancy is characterised by the tolerance of the maternal 

immune system to paternal/fetal alloantigens. Regulatory T cells (Treg) were described 

to play an important role in the maintenance of this tolerance state and several 

mechanisms were proposed for their regulatory activity. In this context immune 

suppressive molecules such as IL-10 and TGF-ß seem to be essential for Treg function. 

Therefore we aimed to investigate whether the pregnancy-protective effect of Treg is 

mediated by IL-10 or TGF-ß. 

Methods of the study 

CBA/J females were mated with BALB/c males (normal pregnant). On day 14 of 

pregnancy Treg were isolated and transferred into DBA/2J-mated CBA/J females 

(abortion-prone). These animals were additionally treated with either anti IL-10, anti- 

TGF-ß or IgG (isotype control) antibodies on days 0 and 7 of pregnancy. Normal 

pregnant and untreated abortion-prone females were used as controls. 

Results 

Treg + IgG-treated animals had a similar abortion rate as the normal pregnant group. 

In contrast CBA/J females which received Treg and were additionally treated with anti- 

IL-10 presented an increased abortion rate, which was even higher than in the 

untreated abortion-prone group. The application of anti-TGF-ß did not substantially 

modify the abortion rate as compared to Treg + IgG-treated animals. 

Conclusion 

These results suggest that IL-10, but not TGF-ß is essential for the immune regulatory 

function of Treg during pregnancy. It further underlines the assumption that Treg 

mediate their suppressive activity against alloreactive maternal immune cells through 

the release of immune suppressive molecules in vivo, being IL-10 essential in this 

context.

Hyun-Dong Chang, Jun Dong, Andreas Thiel Thiel, Andreas Radbruch 

IL-10 Expression in Th lymphocytes is conditional 

T helper (Th) lymphocytes have the ability to memorize the expression of cytokines 

which they were instructed to express during the primary activation. Cytokine memory 

can thus contribute to the maintenance of chronic inflammation. Cytokine memory is 

accompanied by the stable upregulation of transcription factors and epigenetic 

imprinting of cytokine genes. Here we analyse the memory of Th lymphocytes for the 

reexpression of interleukin-10 (IL-10), critical in the regulation of immune responses 

and reduction of immunopathology. IL-10 is induced by IL-4 and IL-12 and remains 

conditional on the provision of the inducing cytokines. In addition, IL-10 expressing Th 

cells isolated ex vivo using the cytometric cytokine secretion assay do not memorize IL- 

10 expression upon repeated in vitro restimulations. Whereas repeated stimulation with 

IL-4 leads to the establishment of a stable IL-10 memory which goes along with GATA-3 

mediated epigenetic imprinting of the il10 gene, IL-12 induced IL-10 expression in Th1 

cells requires continued IL-12 signaling. In accordance, we could detect no epigenetic 

modifications in the il10 gene in ex vivo isolated and Th1 derived IL-10 expressing cells. 

The maintained dependency of IL-10 expression of Th1 lymphocytes suggests an 

unexpected anti-inflammatory potential of IL-12: In memory Th1 cells IL-10 expression 

remains dependent on IL-12, while reexpression of IFN-γ is independent of the original 

inducer IL-12. The exclusion of IL-10 from the functional memory of Th1 cells may 

reflect the requirement for conditional regulation of inflammatory immune responses.

Manuel N. D. M. Guerreiro, Anne Marie Asemissen, Gianna Schulz, Il-Kang Na, Jochen 

Hühn, Sandra Bauer, Eckhard Thiel, Ulrich Keilholz, Carmen Scheibenbogen 

IL-2 induces IL-10 producing regulatory CD3+ T cells in vitro 

and in vivo 

Purpose: 

IL-10-producing regulatory T cells have recently gained much interest as important 

players in immune regulation. In this study we have analyzed in melanoma patients the 

influence of IL-2 treatment and of in-vitro IL-2 exposure on IL-10-producing regulatory 

T cells. 

Materials and methods: 

PBMC from 6 melanoma patients who had been treated with IL-2 and from healthy 

subjects were analyzed ex vivo or after 72h incubation with IL-2 (50 U/ml) for IL-10 

producing capacity of T cells by intracellular cytokine staining. Further phenotypic and 

functional characterization was performed by flow cytometry. 

Results: 

Less than 1% of unprimed PB T cells produced IL-10 in response to PMA/Ionomycin 

(n=9 healthy subjects). IL-10-producing T cells could be primed by short-term 

incubation with IL-2. After 3 days median 6.29% (range 0.79 – 23.04) of CD4+ and 

10.64% (range 0.64 – 34.99) of CD8+ T cells produced IL-10 in response to PMA/Iono 

(n=9 healthy subjects). CFSE staining revealed that the capacity to produce IL-10 is 

due to priming by IL-2 and not due to expansion of this subset. In PBMCs cultured with 

IL-2 for longer periods further upregulation of IL-10 producing capacity was observed 

on day 7. We next studied IL-10-producing capacity in PBMC from 6 melanoma patients 

who had been treated with IL-2. In accordance with the in vitro studies less than 0.2% 

of unstimulated CD3+T cells produced IL-10 before, and after IL-2 therapy. Upon PMA/ 

Ionomycin stimulation both CD3+CD4+ and CD8+T cells showed enhanced IL-10 but 

not IFN-g-producing capacity in samples obtained 5 days after IL-2 treatment as 

compared to pretreatment samples in 4 of 6 patients. Phenotypic characterization of IL- 

10-producing T cells showed lack of FOXP3 expression, failure to produce IL-4 and IFNg 

and lack of CD25. A major subset of IL-10+ T cells expressed the mucosal chemokine 

receptors CCR6 and/or CCR9, and approximately 30% the inflammatory chemokine 

receptor CXCR3. Interestingly, CD8+ IL-10+ T cells were not detectable in bone marrow 

suggesting that the IL-10+ T cells have a restricted migratory capacity. PBMC primed in 

the presence of IL-2 and stimulated by anti-CD2/-CD3/-CD28 were able to substantially 

inhibit the proliferation of stimulated PBMCs as assed by CFSE proliferation assay. 

Further we could demonstrate the in vitro generation of IL-10-producing influenzapeptide-specific 

T cells by IL-2 with a mean of 1.35 % influenza-specific CD8+IL-10+ T 

cells (range 0.14 – 3.33 %, n= 3) after 7 days. We also could generate MART-1 and 

Tyrosinase-peptide specific CD3+CD8+ IL-10 producing T cells in 4 of 6 melanoma 

patients (0.03-0.13%). The study of the single nucleotide polymorphism –1082 (G/A) 

located in the proximal promoter of the IL-10 gene from 8 healthy subjects did not 

correlate with the frequency of the IL-10+ T cells, suggesting a predominantely 

adaptive response. The effect of IL-7, IL-15, IL-21, also belonging to the gamma chain 

cytokines family, on IL-10-producing and secreting capacity were compared. 

Interestingly, IL-15 had similar IL-10 priming capability as IL-2, while IL-21 did not 

enhance IL-10 production. 

Conclusion 

IL-2 primes IL-10-producing T cells in vivo and in vitro. This finding has important 

implications for the use of IL-2 as vaccine adjuvant in cancer immunotherapy as well as 

for adoptive T cell therapy. Importantly, the gamma chain cytokine IL-21 does not 

enhance IL-10 production.

Kerstin Wolk, Ellen Witte, Ute Hoffmann, Wolf-Dietrich Döcke, Stefanie Endesfelder, 

Khusru Asadullah, Wolfram Sterry, Hans-Dieter Volk, Bianca Maria Wittig, Robert Sabat 

IL-22 Induces Lipopolysaccharide-Binding Protein in 

Hepatocytes: A Potential Systemic Role of IL-22 in Crohn's 

Disease 

Crohn's disease (CD) is a common, chronic, inflammatory bowel disease characterized 

by intestinal infiltration of activated immune cells and distortion of the intestinal 

architecture. In this study, we demonstrate that IL-22, a cytokine that is mainly 

produced by activated Th1 and Th17 cells, was present in high quantities in the blood of 

CD patients in contrast to IFN-γ and IL-17. In a mouse colitis model, IL-22 mRNA 

expression was elevated predominantly in the inflamed intestine but also in the 

mesenteric lymph nodes. IL-22BP, the soluble receptor for IL-22, demonstrated an 

affinity to IL-22 that was at least 4-fold higher than its membrane-bound receptor, and 

its strong constitutive expression in the intestine and lymph nodes was decreased in the 

inflamed intestine. To investigate the possible role of systemic IL-22 in CD, we then 

administered IL-22 to healthy mice and found an up-regulation of LPS-binding protein 

(LBP) blood levels reaching concentrations known to neutralize LPS. This systemic upregulation 

was associated with increased hepatic but not renal or pulmonary LBP mRNA 

levels. IL-22 also enhanced the secretion of LBP in human primary hepatocytes and 

HepG2 hepatoma cells in vitro. This increase was mainly transcriptionally regulated and 

synergistic with that of other LBP inducers. Finally, elevated LBP levels were detected in 

CD patients and the mouse colitis model. These data suggest that systemic IL-22 may 

contribute to the prevention of systemic inflammation provoked by LPS present in the 

blood of CD patients through its induction of hepatic LBP.

Daniel Hebenstreit, Elisabeth Maier, Jutta Horejs-Hoeck, Min Li-Weber, Albert Duschl 

IL-4 suppresses the Gene Expression of TCF-1 in T cells in a 

STAT6 dependent way 

The Wnt signalling pathway plays an important role in numerous developmental 

processes including T cell development. Yet, data on Wnt signalling in mature T cells are 

scarce. T cells have a key function in most immune responses and are also associated 

with a number of pathologic conditions, such as allergy. T cells involved in the latter are 

mainly of the Th2 lineage. Differentiation towards the Th2 type is induced by TCR 

engagement following IL-4 stimulation. 

The present study focuses on the influence of IL-4 signalling on the Wnt downstream 

effector T Cell Factor 1 (TCF-1). Realtime PCR studies on cDNAs from primary human 

and mouse T show a decrease of TCF-1 mRNA after stimulation with IL-4. In contrast, 

this effect does not occur in STAT6 knock out mice. 

By bioinformatics analyses, two STAT6 binding motifs highly conserved between mouse 

and human have been identified in the TCF-1 locus. As demonstrated in EMSA, one of 

these can bind to STAT6 present in nuclear extracts from IL-4 treated primary human T 

cells. To test its repressive effect, this STAT6 binding site was cloned into a plasmid that 

carries a highly IL-4 inducible promoter construct of eotaxin 3 and the luciferase 

reporter gene. Transfection experiments show that the STAT6 binding site from TCF-1 

reduces luciferase activity significantly, whereas a mutated version does not. 

Briefly, our current findings have identified STAT6 to be responsible for the IL-4 induced 

suppression of TCF-1.

Manuel Otte 

IL-4R – signaling through an alternative signal transduction 

pathway 

Manuel Otte, Mario Zaiss, Susanne Bürgis, Anja Thiel, Georg 

Schett and André Gessner 

Institute of Medical Microbiology, Immunology and Hygiene; 

University of Erlangen-Nuremberg 

Interleukin-4 (IL-4) is a pleiotropic cytokine that has different functions on various cell 

types. The IL-4 receptor (IL-4R) binds IL-4 and transduces the signal through the 

Insuline Receptor Substrate (IRS)- and Signal Transducer and Activator of Transcription 

6 (STAT6)- signaling pathways initiating proliferation and gene expression. We 

investigated IL-4 induced cell proliferation in a system of human TF1 cells stably 

transfected with different variants of murine IL-4R α-chains and observed IL-4 induced 

proliferation independent of the IRS- and STAT6-signaling pathways. Therefore we 

postulated that an alternative IL-4R signaling pathway must exist. Interaction studies 

with IL-4R Δ388, the shortest proliferation mediating IL-4R variant, leads to 

identification of p62, JAB1 and p39. These proteins could be possibly involved in 

alternative signaling pathways. Transgenic mice were generated that merely expressing 

the truncated IL-4R Δ388 in the absence of wild type IL-4R. Experiments with different 

cell types of the transgenic mice revealed that the IL-4R Δ388 is still able to promote 

proliferation and anti-apoptosis of CD4+ cells and inhibits osteoclast differentiation of 

bone marrow monocytes. In contrast differentiation of TH2 cells and IgE class switch of 

B cells were not induced via the truncated IL-4R. Thus, the newly established mouse 

model allows to discriminate IRS/STAT6- dependent and independent IL-4 functions.

Julia-Stefanie Frick, Julia Geisel, Frauke Kahl, Hermann Wagner, Carsten Kirschning, 

Ingo Autenrieth 

IL-6 and maturation govern TLR2 and TLR4 induced TLR 

agonist tolerance and cross-tolerance in dendritic cells 

Stimulation of murine bone-marrow-derived-dendritic-cells (DC) with lipopolysaccharide 

(LPS) or the synthetic lipopeptide N-Palmitoyl-S-[2,3bis(palmitoyloxy)-(2RS)-propyl]- 

[R]-cysteinyl-[S]-seryl-[S]-Lys4 x 3 HCl (P3CSK4) induces expression of TNF-a in a 

TLR4- and respectively TLR2-dependent fashion. Pre-treatment of DC with LPS results in 

hyporesponiveness to a subsequent LPS stimulation, termed LPS-tolerance and to 

subsequent P3CSK4 stimulus, termed cross-tolerance. Respectively treatment of DC 

with P3CSK4 resulted in homo-tolerance towards subsequent P3CSK4 stimulation as 

well as cross-tolerance towards subsequent LPS stimulation. Different mechanism seem 

to account for induction of tolerogenic DC. Pre-stimuation with low concentrations of 

LPS or P3CSK4 induced tolerogenic DC in an IL-6-dependent fashion and was neither 

related to activation and maturation of DC nor to downregulation of TLR2/4 expression. 

In contrast, induction of tolerogenic DC by treatment with high concentrations of LPS or 

P3CSK4 was independent of IL-6. In homo-tolerogenic DC degradation of IkB was 

inhibited, as well as in cross-tolerogenic DC pretreated with high concentration of LPS or 

P3CSK4. In contrast, cross-tolerance in DC pretreated with low concentrations of LPS or 

P3CSK4 was not related to inhibition of IkB degradation. The data indicate that in DC 

TLR4 and TLR2 stimulation results in homo- as well as cross-tolerance and that different 

mechanistic effects account for the reduced responsivness of DC.

Marsilius Mues, Marco Mank, Oliver Griesbeck, Hartmut Wekerle, Florian Kurschus 

Imaging Activation: FRET-based Calcium Biosensors in T- 

Lymphocytes 

Interaction between an antigen-presenting cell (APC) and a T-cell leads to the rapid 

formation of an immunological synapse. Upon recognition of the appropriate antigen, a 

swift rise of intracellular calcium is triggered within the T-cell which can be used to 

monitor the activation status of the lymphocyte. However, in vivo calcium imaging in Tcells 

still remains a major challenge as synthetic calcium indicators do not persist 

intracellularly for a sustained period. To overcome this problem we generated 

transgenic mice expressing a novel fluorescence resonance energy transfer (FRET)based 

calcium sensor under control of the T-cell specific human CD2 promoter. This 

sensor features spectrally optimized derivatives of the fluorescent proteins CFP and YFP 

linked by the calcium-sensitive domain Troponin C (Heim et al. 2007, Nature Methods 

4:127-129). Although it was originally designed for calcium imaging in neurons, the 

sensor is also capable of responding to modest alterations in lymphocyte intracellular 

calcium levels. This transgenic mouse will be used for in vivo imaging studies in 

experimental autoimmune encephalomyelitis (EAE). We plan to track the migration and 

activation of T-cells upon the encounter of APCs, e.g. during or after passing the bloodbrain 

barrier and while infiltrating the CNS. For this purpose various inducible, as well as 

spontaneous mouse models of EAE, are available in our lab thus providing the 

opportunity to investigate T-cell activation under different disease conditions.

Thomas G. Berger, Hendrik Schulze-Koops, Michaela Schäfer, Ester Müller, Manfred B. 

Lutz 

Immature and maturation-resistant human dendritic cells 

generated from bone marrow under GMP conditions induce 

allogeneic T cell anergy in vitro 

Immature dendritic cells (DC) have been shown to act tolerogenic. Therfore they 

represent potential clinical tools for tolerogenic cellular immunotherapy in 

transplantation or autoimmunity. A major drawback for their in vivo application is their 

potential to mature during infections or inflammations which would convert their 

tolerogenicity into immunogenicity. Here we extend our findings in the murine system 

and describe the generation of immature DC from human bone marrow (BM) or CD34+ 

precursor cells by low doses of GM-CSF (LowGM) in the absence of IL-4 under GMP 

conditions which are also resistant to maturation by inflammatory cytokine cocktail, 

CD40 ligation or signals through Toll-like receptors (TLR) as detected by surface 

markers and primary stimulation of an allogeneic T cells. This could not be observed 

with BM-derived DC generated with high doses of GM-CSF plus IL-4 (HighGM/4). As 

tolerance mechanism T cell anergy was induced most prominently after two subsequent 

stimulations by both types of immature DC. Anergy induction was more profound with 

LowGM-DC due to their maturation resistance. Together, the generation of immature, 

maturation-resistant LowGM-DC offers the possibility of tolerance induction for clinical 

use.

Daniel Nickel, Sven Poppert, Tatjana Zelenski, Nicole Kästner, Heiko Bruns, Axel 

Schubert, Axel Spahr, Steffen Stenger 

Immune modulation mediated by Aggregatibacter 

actinomycetemcomitans as a possible mechanism for the 

development of periodontitis 

There are more than 200 different species of bacteria colonizing the human gingiva. 

About 5% - among them Aggregatibacter actinomycetemcomitans (Aa) - are frequently 

found in periodontal lesions. To mimic the site of oral inflammation we initially 

investigated the interaction between Aa and human peripheral blood mononuclear cells 

(PBMC). For determining the efficiency of infection we designed probes specific for Aa 

and performed fluorescence in situ hybridization (FISH). The majority of the bacteria 

were in the proximity of or adjacent to PBMC implying the possibility of immunological 

interaction. This finding prompted us to measure cytokine production (TNF, IFN-γ) and 

bacterial survival using a co-culture system of Aa and PBMC. By ELISA we measured 

1300 pg/ml TNF after 24 h and 540 pg/ml after 72 h. The results for IFN- γ were 290 pg/ 

ml after 24 h and 1600 pg/ml after 72 h. Under these conditions (5% human serum) no 

viable bacteria were found after 24 h of incubation. 

We are currently trying to establish conditions that permit survival of both eukaryotic 

and prokaryotic cells. Ultimately we intend to identify immune deviations in patients 

with severe periodontitis that account for the hyper inflammation characteristic for this 

disease.

Jessica Butz, Cordula Fuchs, Barbara Kessler, Heiner Voigt, Daniel Wienhold, Mathias 

Buettner 

Immune reaction of swine after repeated intra-muscular (i. 

m.) immunization with avian influenza virus H5 

In swine there is little evidence of virus host inaction during the recent avian influenza 

virus (AIV) H5N1 epidemics in Asia and South East Asia. To induce and characterize an 

immune response in swine inactivated H5N1 (mallard/Bavaria/1/06)) was injected two 

times intra-muscular (i. m.) followed by a third i. m. injection of live low pathogenic 

H5N2 AIV subtype. 

In total ten animals were immunized, five without and five with parapoxvirus as an 

adjuvant. 

Only after the second and third application H5-specific antibodies were detected in 

swine sera with slightly higher titers in the sera of the adjuvant immunized animals. In 

vitro re-stimulation of peripheral blood mononuclear cells (PBMC) with various live AIV 

subtypes including the vaccine virus and swine pathogenic subtypes did not result in 

significant antigen mediated interferon gamma secretion as measured by ELISPOT. The 

PBMC from immunized animals also showed no or weak interferon alpha secretion after 

AIV stimulation in contrast to other control viral stimuli. Possible immune response 

counteracting activity of virulent avian influenza A subtypes will be investigated.

Marcin Wlodarski, Zachary Nearman, Alan Lichtin, Hans-Dieter Volk, Jaroslaw 

Maciejewski 

IMMUNODOMINANT CYTOTOXIC T LYMPHOCYTE EXPANSIONS 

IN PATIENTS WITH UNEXPLAINED NEUTROPENIA. 

Some cases of idiopathic neutropenia (IN) may result from a T cell-mediated immune 

attack directed against myeloid progenitors. Unlike in large granular lymphocyte 

leukemia (LGL) that is characterized by highly skewed chronic lymphoproliferation, 

cytotoxic T-cell (CTL) expansions in IN may not be discovered using traditional 

laboratory tools. We hypothesized that a precise T-cell receptor (TCR) repertoire 

analysis may uncover CTL expansions in IN that are pathophysiologicaly analogous to 

those seen in large granular lymphocyte leukemia (LGL) and thus can serve as markers 

for CTL mediated process. 

We previously established algorithms for TCR analysis and in-vivo tracking of CTL 

responses that include TCR variable beta (VB) phenotyping followed by subcloning of 

TCR-VB families and clonotypic sequencing, or multiplex VB-PCR and sequencing of the 

entire VB repertoire. For quantitative clonotype tracking we introduced a novel 

clonotypic Taqman-PCR that allows for a very precise detection of patient-specific TCR 

VB chains. Using this approach we studied patients with neutropenia: 12/20 displayed 

CTL expansions that were less dominant than those detectable in LGL but clearly 

distinguishable from subclinical CTL expansions in healthy controls. As a surrogate of 

cytotoxic activity, we found markedly increased production of interferon-G in most 

patients irrespective of the presence of immunodominant CTL clones. 

These results suggest that while CTL clonalities are detectable only in a proportion of 

patients, cytotoxic pathophysiology may be a general mechanism in idiopathic 

neutropenia and immunodominant expansions indicate an autoimmune process. 

Conversely, highly polarized responses in a subset of neutropenic LGL patients may 

represent the “extreme” end of the clonal continuum.

Doreen Haase, Anne Marie Asemissen, Carmen Scheibenbogen 

Immunogenic epitopes of the PAX2 transcription factor 

recognized by colon carcinoma patients 

PAX2 is a member of the highly conserved transcription factor family of paired box 

genes. It confers its major role in the embryonic development of the kidney and parts of 

the central nervous system. In the adult organism PAX2 expression is downregulated 

except for certain cells that are believed to be stem cells. This is in accordance with 

PAX2 being implicated in essential functions important for stem cells like proliferation, 

differentiation and migration. Interestingly, PAX2 expression is restored in a variety of 

solid tumors, including renal cell, prostate and colon carcinomas, as well as in 

lymphomas. Thus PAX2 would be an interesting target of novel therapeutic strategies 

against cancer. 

To identify PAX2 as a tumor rejection antigen, we searched for potential epitopes of the 

protein that are presented by HLA-A2. For prediction of candidate epitopes the 

SYFPEITHI algorithm (www.syfpeithi.de) was used which gives binding probabilities for 

each possible peptide of a given protein to the chosen HLA molecule. PAX2 nonamer 

and decamer epitopes with a high binding probability to HLA-A2 that were cleaved by 

the constitutive and immunoproteasome according to the PAProC database (www. 

paproc.de) were synthesized. 

Screening of the HLA-A2 restricted PAX2 candidate epitopes showed that 10 out of 20 

(50%) HLA-A2 positive colon carcinoma patients had interferon-gamma or TNF-alpha 

producing PAX2 specific CD8+ T cells. It is noteworthy that the epitopes with a positive 

T-cell response cluster in the unique region of PAX2 that shows no homology to other 

PAX family members. None of the 20 healthy donors presented reactivity towards any of 

the PAX2 peptides. 

Currently, different protocols for expansion of PAX2 specific cytotoxic T-cells (CTL) are 

compared. An efficient expansion will allow further characterization of these tumor 

specific CTLs including their ability to secrete cytokines like interferon-gamma and TNFalpha 

as well as their cytolytic activity towards PAX2 loaded cells and PAX2 expressing 

tumor cell lines. 

Taken together our data show the high immunogenicity of PAX2 in cancer patients 

whereas it is not immunogenic in healthy subjects. As a next step it will be important to 

analyse the therapeutic activity of PAX2-directed CTL in preclinical models in order to 

develop PAX2-based T-cell therapy.

Lukas Frenzel, Zeinab Abdullah, Anja Kriegeskorte, Rebecca Borsutzky, Manoj K. 

Gupta, Olaf Utermöhlen, Dirk H. Busch, Martin Krönke, Jürgen Hescheler, Tomo Saric 

Immunological properties of murine embryonic stem cellderived 

cardiomyocytes 

Embryonic stem (ES) cells are regarded as a very promising source of differentiated 

cells for tissue regeneration. ES cell-derived cardiomyocytes could functionally replace 

irreversibly lost cardiac tissue in various animal models of ischaemic heart disease. 

However, clinical application of this therapeutic approach will be hampered by 

immunological rejection of transplanted cells by histoincompatible recipients. To address 

the question of immunological properties of murine ES cell-derived cardiomyocytes we 

have utilized a transgenic murine ES cell line D3aPIG engineered to express GFP and 

antibiotic resistance specifically in ES cell-derived heart cells. This cell line enabled us to 

highly purify GFP-positive cardiac progenitor cells and to specifically address the 

question of their immunogenic properties. To this end, we have determined their 

immunophenotype by flow cytometry, assessed their response to the inflammatory 

cytokine interferon gamma, assayed their physical interaction with cytotoxic T 

lymphocytes (CTLs) and tested their susceptibility to lysis by activated NK cells and 

cytotoxic T cells. These studies have demonstrated that ES cell-derived cardiomyocytes 

constitutively express very low levels of MHC class I molecules on their cell surface, 

which were strongly upregulated by interferon gamma. Interestingly, the cytotoxicity 

experiments revealed that ES cell-derived cardiac cells were resistant to killing by poly I: 

C activated syngeneic and allogeneic NK cells as well as by allogeneic cytotoxic T cells. 

Even strong upregulation of MHC class I molecules on the surface of cardiac cells by 

interferon gamma did not render them sensitive to lysis by immune effector cells, 

indicating that transplanted ES cell-derived cardiomyocytes might be less susceptible to 

rejection as compared to whole organ transplants. Further studies are planned to 

elucidate the molecular basis of this resistance and to assess the engraftment capacity 

and immunogenicity of ES cell-derived cardiomyocytes in vivo upon allotransplantation.

Felix Heymann, Emma E. Hamilton-Williams, Isis Ludwig-Portugall, Susan Quaggin, 

Jürgen Floege, Hermann-Josef Gröne, Christian Kurts 

Immunopathology of T cell-mediated glomerulonephritis 

The different forms of immune-mediated glomerulonephritis (GN) represent a major 

cause of end stage kidney disease. The role of antibodies and immune-complexes has 

been extensively studied, whereas the ability of T cells to mediate glomerular damage 

has been proposed, but awaits experimental verification. We have generated transgenic 

mice expressing a fusion protein consisting of chicken ovalbumin and hen egg lysozyme 

as a model autoantigen in glomerular podocytes under the control of the nephrin 

promoter (NOH mice). Injection of transgenic OVA-specific T cells into NOH mice 

allowed to determine the type of renal immunopathology mediated by T cells specific for 

a glomerular autoantigen. OVA-specific CD8+ effector T cells (OT-I cells) were activated 

by cross-presentation and proliferated in the renal lymph nodes of NOH mice. Injection 

of activated OT-I cells increased cross-presentation in this node, presumably by 

cytotoxic release of antigen from the kidney. Renal immunopathology was only 

observed when with OT-I cells were co-injected with activated OVA-specific CD4+ 

helper cells (OT-II cells). Surprisingly, the typical form of podocyte damage, namely 

foot process fusion, was not detected. Instead, immunopathology was dominated by 

periglomerular mononuclear infiltration, reminiscent of transplant glomerulitis or of 

rapid progressive forms of glomerulonephritis. Adjacent to this infiltrate, the parietal 

glomerular epithelial cells showed signs of injury. Repetitive injection of OT cells 

resulted in proteinuria, reduced creatinin-clearance and structural kidney damage 

accompagnied by massive periglomerular infiltartes. These infiltrates contained many 

dendritic cells expressing CD11c, CD11b, F4/80, and CD8, which showed signs of 

activation. Ablation of these DC resolved the inflammatory infiltrate, demonstrating a 

functional role of kidney DC in mediating kidney damage. In conclusion, we 

demonstrate that T cells can recruit kidney DCs to induce periglomerular infiltrates 

resulting in glomerular immunopathology.

Nadine Nippe, Katja Gutsche, Mechthild Jung, Gerald Grütz 

Immunoregulation of IL-10 induced Autotaxin 

Cytokines are regulators of host response to inflammation. Some cytokines have to be 

associated with the pathogenesis of diseases, whereas others serve to reduce 

inflammatory responses. The mediator of inflammation HMGB1 and IL-1ß are secreted 

by monocytes and macrophages through a non-classical pathway. 

High-mobility group protein 1(HMGB1), a non-histone nuclear protein, functions as a 

late mediator of inflammation. Passive immunization with anti-HMGB1 antibodies 

protects against endotoxin lethality in mice. Active secretion of HMGB1 by monocytes 

and macrophages occurs in response to LPS. We show that its release can also be 

triggered by LPC. 

Secretion of IL-1ß needs two distinct stimuli. An initial inflammatory stimulus, LPS, 

induces synthesis of pro-IL-1ß, processing and release of matured IL-1ß is inefficient. A 

secondary stimulus such as ATP can trigger rapid processing and massive release of IL- 

1ß. 

IL-10 is one of the major immunosuppressive cytokine. It strongly down-regulates the 

expression of proinflammatory cytokines. In order to identify genes which could mediate 

the IL-10 inhibition we analyzed the expression profile of IL-10 regulated genes in 

human monocytes by comparison of expression level of 12000 genes in IL-10 

stimulated monocytes and unstimulated cells. A possible candidate is Autotaxin. We 

show that Autotaxin is highly up regulated by IL-10 in human monocytes. 

Autotaxin is an ecto-enzyme with pyrophosphatase/phosphodiesterase-activity. But 

primarily functions as a lysophospholipase D, degrading lysophosphatidylcholine (LPC) 

into lysophosphatidic acid (LPA). 

Therefore, we hypothesized that Autotaxin could reduces HMGB1 and IL-1ß release by 

degradation of LPC and ATP. We would like to demonstrate first functional results.

Wolfgang G Bessler, Karola Puce, Carsten Kirschning, Maria Huber 

Immunostimulating effects of the bacterial extract OM-89 

OM-89 (Urovaxom®) is a bacterial extract prepared from 18 uropathogenic Escherichia 

coli strains used for the treatment of recurrent infections of the urinary tract. We 

investigated in a mouse model the immunostimulating effects of the bacterial extract. 

After the oral administration of OM-89, we observed ex vivo the production of TNFalpha 

in supernatants of peritoneal cells. Leukocyte activation could also be shown by a 

chemiluminescence assay in blood and liver cells. After repeated oral administration of 

OM-89, an increased serum IgG and IgA response against the Escherichia coli strains 

used for the preparation of OM-89 was observed. We also could demonstrate defined 

adjuvant properties of the extract using ovalbumin as an antigen. Corresponding to our 

findings in the mouse system, preliminary in vitro assays in the human system showed 

an increase in TNF-alpha IL-6, IL-10, and IL-12 production after the stimulation of 

monocyte derived dendritic cells with OM-89. Activation is likely to be mediated via Toll 

like receptors: we could demonstrate the binding of components of the extract to TLR-4 

and marginally to TLTR-2.

Kerstin Annika Sauer, Joachim Heinrich Maxeiner, Petra Scholtes, Roman Karwot, 

Hans-Anton Lehr, Mark Birkenbach, Richard Steven Blumberg, Susetta Finotto 

Immunosurveillance of lung melanoma metastasis in EBI-3 

(-/-) mice by NK-DCs- induced CD8+ T cells 

Antigen presentation plays an important role in lung metastasis, although the 

immunological mechanisms are not completely understood. Epstein-Barr virus-induced 

gene 3 codes for a soluble type 1 receptor homologous to the p40 subunit of IL-12 that 

is expressed by antigen presenting cells following activation. Adoptive intravenous 

injection of the B16-F10 cell line resulted in a significant reduction of lung tumor 

metastasis in EBI-3 (-/-) recipient mice compared to wild type. Consistently, we found 

that EBI-3 (-/-) mice had decreased number of VCAM-1+ endothelial cells. The 

immunological finding accompanying this therapeutic effect was the orchestrate priming 

and activation of T cells by a newly described Dendritic Cell (DC) subset called Natural 

Killer Dendritic Cells (NK-DC). NK-DCs from EBI-3 (-/-) mice released increased 

amounts of IFN-gamma thereby inducing augmented CD8+ T cell responses in the lung. 

This in turn resulted in a TNF-alpha-TRAIL mediated programmed cell death of local 

metastatic tumor cells in the lung of EBI-3 deficient mice. Finally, adoptive transfer of 

EBI-3 (-/-) NK-DC primed CD8+ T cells into tumor bearing wild-type mice ameliorated 

the development of lung metastasis in recipient mice. Taken together, these data 

demonstrate that EBI-3 is a crucial regulator of anti-tumor CD8+ T cell responses in the 

lung by controlling NK-DC activity. 

This work is funded by the Graduiertenkolleg 1043 and Immuno-intervention Cluster of 

Excellence (ICE, Mainz, Germany).

Henoch Hong, Nupur Bhatnagar, Maren Mönkemeyer, Hans Heiken, Reinhold E. 

Schmidt, Dirk Meyer-Olson 

Impact of HIV-1 Vpr on type I and type II interferon 

secretion by plasmacytoid dendritic cells and natural killer 

cells 

Objectives: Type I and type II interferons (IFNs) can induce an antiviral state in target 

cells, which impairs virus replication. In humans, plasmacytoid dendritic cells (pDCs) 

are the most potent type I IFN producers whereas type II IFNs are released by 

activated T cells and natural killer (NK) cells. The molecular and cellular mechanism of 

HIV-induced impairment of IFN secretion is not yet fully understood. Here we addressed 

the question whether HIV-1 Vpr is able to disturb the interplay between NK cells and 

pDCs with regard to their IFN production. 

Methods: We sorted NK cells and pDCs from peripheral blood of healthy donors. Highly 

purified NK cells and pDCs were cultured in the presence or absence of synthetic HIV-1 

Vpr. In order to be able to evaluate the effects of Vpr, pDC/NK cocultures were also 

stimulated with CpG. NK phenotype was assessed by FACS analysis and functions by 

standard 51Cr release assay and IFN-γ ELISA. We analyzed direct effects of Vpr on IFNα 

secretion by pDCs. 

Results: As reported previously, CpG stimulated pDCs were able to activate NK cells as 

determined by higher CD69 expression, increased cytolytic activity and moderate IFN-γ 

release. Vpr did not impair pDC mediated upregulation of CD69 in NK cells and did not 

interfere with increased NK killing activity. However, we found that Vpr substantially 

decreased pDC induced IFN-γ secretion. Furthermore, Vpr significantly hampered IFN-α 

production by CpG activated pDCs. 

Conclusion: The data suggest that Vpr mediates a dysregulation of early IFN-γ 

expression by NK cells and IFN-α production by pDCs, which could be of considerable 

importance in the pathogenesis of HIV infection and deserves further studies.

Nadine Kämper, Claudia Wegscheid, Jörg Keßler, Norbert Koch 

Impact of HLA encoded BAT3 splice variants on MHC class I 

and class II expression 

Division of Immunobiology, Institute of Genetics, University of Bonn 

The BAT3 gene locates in the class III region of the HLA complex on chromosome 6. 

The gene is composed of 25 exons. By alternative splicing of the pre RNA numerous 

variants of BAT3 polypeptides can be generated. It was suggested that BAT3 proteins 

are involved in apoptosis, possibly by regulating proteasomal degradation. BAT3 is a 

proline-rich protein, which contains a zinc finger-like domain, a nuclear localization 

signal and a BAG domain. Aim of our study is to determine expression and subcellular 

distribution of BAT3 splice variants in human cells and their impact on the level of MHC 

class I and class II surface expression. While some BAT3 splice variants show nuclear 

expression other variants were detected in the cytoplasm. Since BAT3 exhibits a short 

half life, it is possible to determine its impact on regulation and induction of MHC genes. 

By transfection of cells with siRNA or with miRNA, the expression of BAT3 was strongly 

reduced. Inspection of MHC and of accessory molecules revealed that in transfected 

cells expression of the MHC class II associated invariant chain (Ii) was depleted. This 

result suggests that the gene regulation of Ii depends on BAT3 expression.

Diana Fleissner, Jan Buer, Astrid Westendorf 

Impact of intestinal dendritic cells for the induction of 

tolerance or pathology 

Several studies have suggested that chronic inflammatory bowel disease may be a 

consequence of antigen specific recognition by appropriate T cells which expand and 

induce immunopathology. To analyse the impact of intestinal dendritic cells (DCs) for 

the presentation of intestinal self-antigens and the induction of immunopathology or 

tolerance we used VILLIN-HA transgenic mice that show specific expression of 

hemagglutinin (HA) from influenza virus A exclusively in enterocytes of the intestinal 

epithelium. Adoptive transfer of naïve HA-specific CD8+ T cells into VILLIN-HA recipient 

mice leads to strong expansion of antigen-specific CD8+ T cells and the development of 

severe intestinal inflammation. In contrast, adoptive transfer of HA-specific CD4+ T 

cells into VILLIN-HA transgenic mice results in proliferation of HA-specific T cells but 

fails to induce intestinal inflammation. Interestingly, cotransfer of HA-specific CD4+ and 

CD8+ T cells further aggravates the pathology. Therefore, auto-reactive CD8+ T cells 

may come first before pathogenic CD4+ T cells ultimately drive disease. Based on these 

findings we analyzed the impact of intestinal DCs for the presentation of intestinal selfantigen. 

Comparison of DCs from the mesenteric lymphnode (MLN) versus lamina 

propria (LP) reveals a slight upregulation of MHC class II, CD80 and CD86 on DCs 

isolated from the LP. Furthermore, DCs isolated from the MLN of VILLIN-HA transgenic 

induce proliferation of HA-specific CD8+ and CD4+ T cells without adding of external HA 

peptide in vitro. These results demonstrate that DCs sample intestinal epithelial 

antigens in the lamina propria and migrate to the MLN where they present the antigen. 

In future experiments we want to characterize the phenotype of DC-stimulated CD8+ 

and CD4+ T cells in more detail to dissect the pathologic and regulatory function of 

these cells. In conclusion, the gut-associated mucosal immune system develops certain 

mechanisms which lead either to immunity or tolerance.

Johannes Lutz, Werner Müller, Chander Raman, Hans-Martin Jäck 

Impaired B Cell Development in the Presence of a Non-Coding 

IgM mRNA 

To establish allelic exclusion (AE) at the Ig heavy chain (IgH) locus VDJ recombination 

of the second locus must be terminated once a productive VDJ rearrangement has been 

made. Many studies have demonstrated that the suppression of further VDJ 

rearrangements is mediated by the newly synthesized IgM heavy chain (•HC) through 

pre-B cell receptor (pre-BCR) signals. However, it is still unclear how rearrangement of 

the second IgH allele is prevented during the time required for the synthesis of the •HC 

and the initiation of pre-BCR signals. Here we present evidence that a stable •HC 

transcript, which reflects the presence of a productive rearrangement, can already exert 

a suppressive effect on VDJ recombination in the absence of a •HC signal. B cell 

development was impaired at the pro-B to pre-B cell transition with an increased 

frequency of •HC-negative pro-B cells in transgenic mice expressing a non-coding •HC 

transcript. This transcript contains a premature translational stop codon at position +3 

and resembles rather a stable productive than an extremely unstable non-productive 

•HC mRNA. Providing a productive transgenic •HC restored B cell development, which 

indicated that the non-coding •HC transcript interfered with no other processes than 

VDJ recombination at the IgH locus. These observations suggest a new role for •HC 

transcripts by indicating the presence of a productive VDJ rearrangement and 

temporarily suppressing VDJ recombination of the second IgH allele until the initiation of 

signals provided by a •HC. 

The work was supported in part by the project grant SFB466 and the research grant JA 

968/2 from the Deutsche Forschungsgemeinschaft (DFG) to H.-M. J.

Verena Moos, Kristina Allers, Thomas Schneider 

Impaired innate functions of monocytes and macrophages in 

Whipple`s disease 

Whipple`s disease is a rare chronic multisystemic infectious disease caused by the 

actinomycete Tropheryma whipplei. Symptoms are heterogenous, but arthropathy, 

weight loss, and diarrhea are present in most cases. Host factors like a reduced capacity 

of T. whipplei-specific Th1 reactivity are suspected to be responsible for the 

pathogenesis of Whipple`s disease. Monocytes and macrophages as important cells of 

the innate immune response have been shown to be involved in the pathogenesis of 

Whipple`s disease, but innate defense mechanism induced by T. whipplei were not 

ivestigated so far. Therefore, we studied phagocytosis and the induction of oxidative 

burst induced by T. whipplei in comparison to related and unrelated bacteria in fresh 

blood of patients with Whipple´s disease. Analysis included serum concentration of 

cyto- and chemokines indicative for macrophage activation, and the phenotypical 

characterization of peripheral and duodenal macrophages. 

T. whipplei is phagocytosed and induces a robust oxidative burst in monocytes of 

healthy controls. However, Whipple`s disease patients showed reduced phagocytosis 

and oxidative burst upon exposure to T. whipplei, whereas their capacity to react to 

other related and not related bacteria was not reduced compared to controls. 

Macrophages of Whipple´s disease patients reveal an alternatively activated phenotype 

in vivo and in situ indicated by the expression of CD163, and the cytokine pattern in the 

sera of Whipple´s disease patients. Hence, we conclude that ineffective innate defense 

mechanism may be responsible for an impaired clearance of T. whipplei and might 

initiate insufficient T. whipplei-specific T cell responses in Whipple`s disease patients.

Kittan Nicolai A., Bergua Antonio, Haupt Sabrina, Donhauser Norbert, Schuster Philipp, 

Korn Klaus, Harrer Thomas, Schmidt Barbara 

Impaired plasmacytoid dendritic cell (PDC) innate immune 

responses in patients with herpesvirus-associated severe 

acute retinal necrosis (ARN) 

Background: Plasmacytoid dendritic cells (PDC), the main producers of type I 

interferons (IFN) in the blood, are important for the recognition and control of viral and 

bacterial infections. Since several viruses are known to induce IFN-alpha production, 

severe courses of herpes virus infections in non-immunocompromised patients may be 

related to numerical or functional PDC deficits. 

Methods: To evaluate this hypothesis, peripheral blood mononuclear cells (PBMC) and 

PDC were repeatedly isolated from eight patients with a history of or an acute episode 

of acute retinal necrosis (ARN), caused by herpes simplex or varicella zoster virus, and 

18 age-matched healthy controls. 

Results: The patients experienced meningitis/encephalitis and frequent infections in 

childhood (n=2), recurrent herpes virus infections at unusual localizations (n=2), ocular 

surgery (n=1), infections and stress around ARN (n=6). The median percentage of 

isolated PDC was significantly lower in patients compared to controls, confirmed by 

FACS analysis using peripheral blood, and extremely low during acute disease. PDC 

counts dropped in five controls suffering from respiratory infections or diarrhea. IFNalpha 

production was significantly lower in patients than controls; anergy to these 

stimuli was observed on four occasions. Real-time analyses for interferon regulatory 

factor (IRF)-7 mRNA and evaluation of PDC surface markers revealed immune 

stimulation in patients compared to controls. 

Conclusion: These data support a model, in which numerical and functional deficits in 

PDC innate immune responses contribute to an impaired control of latent herpes virus 

infections and subsequent development of ARN. The role of CD8+ and natural killer cells 

will be discussed.

Daniela Wesch, Philine Wrobel, Hamed Shojaei, Hans-Heinrich Oberg, Monika Kunz, 

Dieter Kabelitz 

Implications for the design of γδ T cell-based cancer 

immunotherapy 

There is a substantial interest in γδ T cell-based cancer immunotherapy due to their 

potent MHC-nonrestricted cytotoxicity towards various tumor cells. We analyzed the 

susceptibility of a broad range of epithelial tumor cells to V9γVδ2 γδ T cell cytotoxicity. 

The involvement of T cell receptor (TCR) and NKG2D-dependent recognition in tumor 

cell recognition was characterized by antibody blockade. Our experiments revealed 

three patterns of inhibition, i.e. preferential inhibition by anti-TCR antibody or anti- 

NKG2D antibody, or the additive blockade by both antibodies. The extent of inhibition 

did not correlate directly with the level of NKG2D ligand expression on epithelial tumor 

cells. Further, our results indicate for the first time that the NKGD2 pathway is involved 

in the lysis of different melanomas, squameous cell carcinomas of the head and neck 

(SCCHN), lung carcinomas, and pancreatic adenocarcinomas. Lysis of poorly killed 

tumor cells was enhanced by the pre-treatment of γδ T cells with TCR-stimulating 

pyrophosphomonoesters or aminobisphosphonates. The enhancing effect by these 

stimuli was independent of the degree of HLA (mis)match between target and effector. 

Moreover, we currently investigate whether Toll-like receptor agonists, which are 

suggested as adjuvant therapy in clinical trials for different types of cancer including 

adenocarcinomas, have an influence on lysis of tumor cells by γδ T cells. 

Our results indicate that tumors recognized independently of the TCR might be less 

suited for γδ T cell immunotherapy with pyrophosphomonoesters or 

aminobisphosphonates. 

This study was supported by Werner and Klara Kreitz-Stiftung.

Inga Gebuhr, Kathrin Gube, Katrin Vogt, Christian Meisel, Sandra Naundorf, Hans- 

Dieter Volk, Birgit Sawitzki 

Importance of cell surface N-glycosylation for activation of T 

cell subpopulations 

Introduction: Recently, we demonstrated that infiltrating CD4+ T cells of tolerance 

developing kidney grafts recipients express high levels of alpha-1,2-Mannosidase, an 

important enzyme for the N-glycosylation of proteins. The impact of N-glycosylation of 

surface proteins such as costimulatory molecules on T cells for their activation is still 

poorly understood. 

Aim: Here we investigated the role of alpha-1,2-Mannosidase and cell surface Nglycosylation 

for the activation of different T cell subpopulations. 

Methods: Human CD4+, CD4+CD45RA naïve, and CD4+CD45RO memory T cells were 

purified using MACS separation. Cells were cultivated for 2 days in the presence or 

absence of the alpha-1,2-Mannosidase specific inhibitor Kifunensine. Preincubated T 

cells were stimulated with allogenic PBMCs. PHA binding was used to determine 

Mannosidase activity. CD69 expression, cytokine production and Mannosidase 

transcription was analysed. 

Results: The inhibition of Mannosidase activity in CD4+ T cells prior to stimulation 

resulted in a dramatic reduction of cell surface N-glycosylation levels. This reduction 

was accompanied by 2 to 3 fold increase in CD69 frequency, an earlier IL-2 production 

and transcription. This early increase in IL-2 production was due to faster activation of 

ERK. Interestingly, memory T cells displayed 5 fold higher transcription levels and 2 fold 

increased PHA binding capacities when compared to naïve T cells. During allostimulation 

mRNA transcription in memory T cells did not change whereas in naïve T 

cells transcription was transiently down-regulated followed by a 4 fold upregulation. 

Furthermore, naïve and memory T cells also differ in their susceptibility to Nglycosylation 

mediated regulation of T cell activation. Only in naïve T cell inhibition of Nglycosylation 

resulted in increased cytokine production. 

Conclusions: Our data show that N-glycosylation of cell surface proteins negatively 

regulates T cell activation. Furthermore the magnitude and impact of N-glycosylation is 

highly regulated among T cell subpopulations.

Lydia-Mareen Köper, Andrea Schulz, Hans-Jürgen Ahr, Hans-Werner Vohr 

In vitro Differentiation of Skin Sensitizers by Cell Signaling 

Pathways 

Introduction: Animal testing causes ethical problems and due to the EU-guideline 

(76/768/EEC, Feb. 2003), which prohibits the use of animals for the assessment of 

toxicological data for cosmetic ingredients as of 2009, the development of in vitro 

assays has become even more important. In this study, we investigated whether 

analyses of cell signaling pathways can provide a methodology for the detection of 

sensitizing compounds in vitro. 

Methods: Murine and human skin explants as well as reconstituted skin models 

(epidermal model EST-1000 and full-thickness model AST-2000) were exposed to 

sensitizing (Oxazolone and DNFB) or irritant compounds (SDS and TritonX-100). 

Phosphorylation of MAP-kinases (p38, ERK1/2 and JNK1/2), STAT1 and PLCγ were 

determined by cytometric bead array (CBA). 

Results: In skin explants, all three MAP-kinases were exclusively activated after 

exposure to sensitizing compounds. For the reconstituted skin models phosphorylations 

of p38 and JNK1/2 were obtained after stimulation with allergens, whereas treatments 

with irritant compounds lead to ERK1/2 activation. 

Conclusion: MAP-kinase activation provides a promising in vitro tool for the 

discrimination between sensitizers and irritants. The reconstituted skin models AST- 

2000 and especially the EST-1000 showed high induction levels of phospho-p38 specific 

for exposure to sensitizing compounds, comparable to those found in skin explants, i.e. 

complex immune competent tissues.

Sabine Ring, Karsten Mahnke, Alexander Enk 

In vivo activation of injected Tregs precedes the suppression 

of the elicitation phase of Contact hypersensitivity reactions 

independent from spleen and lymph nodes 

We previously demonstrated that adoptively transferred naïve CD4+CD25+ T cells 

(Treg) suppress the elicitation phase of allergic contact dermatitis (CHS), by inhibiting 

the endothelium-interaction and extravasation of effector CD8+ T cells. 

Since activation of Treg is necessary to generate this suppressive activity, we 

determined the activation status of isolated Tregs in the course of injection. Directly 

after isolation, the naïve CD4+CD25+ T cells displayed a non-activated phenotype as 

assessed by low expression of CD69 and CD44, and high expression of CD62L. 2h after 

injection and re-isolation of the dye-labelled Treg we detected significantly increased 

expression of the activation markers CD69, Foxp3 and CD44, whereas CD62L decreased 

in Treg in vivo after hapten application. This indicates that Treg become activated in 

vivo after injection and explains the comparable suppressive function of naïve and 

activated Treg in vivo. To further determine the importance of homing to lymphoid 

organs for Treg action, we splenektomised mice or injected CD62L- Treg. By this way 

we excluded the homing of Treg to spleen or lymph nodes, respectively. In these 

experiments the Treg were still able to suppress the elicitation phase of CHS, indicating 

that peripheral lymphoid organs are not essential for the activation and consequently 

for the suppressive capacity of injected naïve Treg in vivo. 

These data show that naïve Treg upregulate characteristic activation markers in vivo 

after hapten application which results in the suppression of the elicitation phase of CHS, 

independently of peripheral lymphoid organs. Therefore we can speculate that cellular 

interaction(s) in the blood or at the site of inflammation and/or soluble factors are 

involved in mediating suppressive function(s) of Treg in a CHS model.

Sonja Schallenberg, Sabine Ring, Tanja Bedke, Sabrina Schmitt, Kurt Schönfeld, 

Karsten Mahnke, Elisabeth Suri-Payer, Alexander H. Enk 

In vivo depletion of CD4+CD25+Foxp3+ regulatory T cells 

does not affect the growth of established B16 tumors 

Naturally occurring regulatory T cells (Treg) have been described to impede immune 

responses against several tumors, thus depletion of Treg before tumor challenge results 

in diminished tumor growth in several tumor models. 

In order to set up a therapeutical regimen and to improve anti-melanoma therapy we 

used a murine B16 melanoma model. We treated tumor-bearing mice with a 

combination of Treg depletion and stimulation of effector T cells (Teff) via immunization 

with tumor-antigen-loaded dendritic cells (DC). As expected injection of anti-CD25 mAb 

(PC61) before B16 tumor inoculation led to decreased tumor growth. However, 

depletion of CD25+ cells 5-10 days after tumor challenge resulted in enhanced tumor 

growth suggesting that PC61 mAb additionally depletes proliferating CD25+ Teff. To 

test this assumption, we injected CFSE-labeled OVA-specific TCR-transgenic CD4+ cells 

into mice, immunized them with OVA-loaded DC’s and at the same time injected PC61 

mAb. Five days later, draining lymph nodes were isolated and analyzed by flow 

cytometry. To our surprise, recovery, proliferation and IL-2 production of Teff was not 

diminished but rather enhanced compared to the control group without PC61. Next, we 

investigated the efficacy of the Treg depletion during tumor development in different 

organs. We found that Treg (CD25+Foxp3+ cells) in lymphoid organs were depleted, 

whereas they were still detectable inside the tumors. 

These results suggest that the injection of PC61 mAb did not influence Teff activity. 

Further, the tumor creates its own immunosuppressive environment where Treg cannot 

be depleted, either because the mAb does not penetrate well enough into the tumor, or 

because the tumor constantly generates new Treg.

Alla Skapenko, Joachim R. Kalden, Peter E. Lipsky, Hendrik Schulze-Koops 

In vivo function of IL-4-induced Tregs 

We have previously shown that injection of human peripheral blood mononuclear cells 

(PBMC) into the peritoneal cavity of NOD/SCID mice results in the development of a 

Th1-mediated immune response of human cells against murine tissue accompanied by 

an increase in the frequency of CD25+CD4+ T cells. Here, we used this model to 

analyze the role of interleukin (IL)-4 in the generation of human CD25+ regulatory T 

cells in an in vivo situation. NOD/SCID mice were injected intraperitoneally with human 

PBMC and treated with human IL-4 or a neutralizing antibody to human IL-4. IL-4treatment 

down-modulated systemic inflammation as assessed by a decrease of serum 

levels of the human inflammatory cytokines, TNF and IFN-gamma whereas 

neutralization of endogenous IL-4 resulted in an exaggeration of inflammation. Analysis 

of the frequencies of CD25+CD4+ T cells within the human cells recovered from the 

peritoneal cavity of the mice revealed that treatment with IL-4 augmented and IL-4 

neutralization diminished the increase of CD25+CD4+ T cells associated with the 

immune response. Examination of the immunosuppressive ability of recovered human T 

cells in vitro revealed an inhibititory capacity of CD25+, but not of CD25- T cells, for the 

proliferative response of autologous PBMC. Importantly, when injected into animals with 

an ongoing Th1-mediated immune reaction driven by syngeneic human PBMC, purified 

CD25+ T cells were able to reduce serum levels of human IFN-gamma and TNF, 

whereas injection of CD25- T cells resulted in increased levels of both cytokines. Thus, 

CD25+ Tregs that developed in vivo in the presence of IL-4 were functionally competent 

Tregs. This conclusion is supported by the fact that the frequency of CD25+CD4+ T 

cells recovered from the peritoneal cavity at the peak of the xenogeneic reaction in mice 

injected with human PBMC inversely correlated with serum concentrations of human 

IFN-gamma and TNF. Together, the data indicate that CD25+CD4+ T cells which 

accumulate during the Th1-mediated immune response of human cells in NOD/SCID 

mice possess an immunosuppressive phenotype in vivo. Moreover, the data further 

imply that IL-4 controls the generation of competent CD25+ Tregs during an immune 

response in vivo.

Marcus Gereke, Karsten Mahnke, Elmar Jäckel, Jan Buer, Dunja Bruder 

In vivo induction of tolerance via DEC-205 mediated antigen 

delivery – therapeutic safety in the context of infection 

Targeting of antigens to immature dendritic cells has been shown to result in antigenspecific 

T cell tolerance in vivo. In the INS-HA/TCR-HA transgenic mouse model for type 

I diabetes we tested the potential of the dendritic cell-specific monoclonal antibody DEC- 

205 conjugated to the HA antigen (DEC-HA) to prevent disease onset. Whereas 

untreated INS-HA/TCR-HA mice all develop insulitis and about 40 percent of these mice 

become diabetic, repeated injection of newborn mice with DEC-HA protects almost all 

mice from disease development. Histological examination of the pancreata revealed 

significant reduction of peri-islet infiltrations in DEC-HA treated mice and the islet 

structure remained intact. Moreover, HA-specific CD4+ T cells from anti-DEC-HA treated 

INS-HA/TCR-HA mice exhibited increased expression of Foxp3, CTLA-4 and the 

immunosuppressive cytokines IL-10 and TGF-β. These findings argue, that targeting of 

the HA antigen to immature DCs in vivo leads to the expansion of antigen-specific Foxp3 

+ regulatory T cells that suppress autoimmune tissue destruction. 

With respect to a possible application of such approach in human patients it must be 

excluded that DEC-205 mediated antigen delivery in the context of infection, i.e. under 

conditions that result in maturation of DC, would result in T cell activation and 

precipitation of autoimmunity. To address this issue we experimentally infected mice 

before, during or after treatment with antigen coupled to DEC-205 antibodies and 

assess the kinetics of diabetes induction. We demonstrated that treatment with DEC205- 

HA before, during and after infection with bacterial and viral pathogens did not 

precipitate in autoimmunity in diabetes prone mice. In addition we could demonstrate 

an unperturbed adaptive immune response against pathogen with preservation of the 

general immunocompetence. Together, we provide evidence that DEC-205 mediated 

antigen delivery in the context of infection does not result in uncontrolled T cell 

activation and autoimmune disease.

Andreas Wieland, Markus Denzel, Jörg Reimann, Reinhold Schirmbeck 

In vivo produced complexes of antigen with stress proteins 

are potent immunogens 

To facilitate priming of T cells, stress (or heat shock) proteins of the Hsp70/90 families 

have been incorporated into vaccine formulations (by loading antigenic peptides to Hsp, 

or constructing Hsp/antigen fusion proteins). We expressed a chimeric protein 

containing a Hsp-capturing, J-homologous domain and an antigen-encoding sequence to 

produce in situ Hsp/antigen complexes. DNA vaccines expressing antigens with this 

stress protein-capturing domain display enhanced immunogenicity for T and B cells. 

Complexes of antigen associated with constitutively expressed Hsp73 or stress induced 

Hsp70 accumulate to high steady state levels in transfected eukaryotic cells that 

produce these chimeric proteins. We designed a purification method to isolate from 

transfectants native Hsp/antigen complexes that efficiently elicit antigen-specific 

immune responses in mice. Similar to peptide-bound Hsps these complexes facilitate 

priming of CD8 T cells. The delivery of chaperone-associated antigen is thus an 

attractive strategy to elicit multispecific responses of different compartments of the 

immune system.

Undine Meusch, Manuela Rossol, Holm Häntzschel, Christoph Baerwald, Sunna 

Hauschildt, Ulf Wagner 

Increased Infliximab-induced monocyte apoptosis via reverse 

signalling of membrane TNF in patients with rheumatoid 

arthritis 

Treatment of inflammatory autoimmune diseases like rheumatoid arthritis (RA) with 

anti-TNF antibodies is a common way to neutralize soluble TNF alpha and reduce 

inflammation and bone destruction. In the last years another possible mechanism 

explaining the action of anti-TNF antibodies gets into focus. Anti-TNF antibodies are able 

to bind to membrane TNF which leads to signal transduction by a mechanism called 

reverse signalling. In the study presented here, we analyzed apoptosis due to reverse 

signalling in human monocytes of healthy donors and patients with RA. In vitro 

incubation of human monocytes with the anti-TNF antibody Infliximab led to a 

significant increase in apoptosis after 16 h. Interestingly, only one third of the healthy 

donors showed this response whereas 80% of the RA patients underwent apoptosis in 

this in vitro system. Considering only donors who responded with apoptosis, monocytes 

of RA patients underwent apoptosis with a higher rate than monocytes of healthy 

donors. In addition, apoptosis of monocytes of RA patients strongly correlated with the 

expression of membrane TNF. Infliximab-induced apoptosis was independent of caspase 

3, 8 and 9, therefore other pathways were evaluated. Reverse signalling of membrane 

TNF induced phosphorylation of the MAP kinase Erk after 16 h. Accordingly, inhibition of 

Erk activation led to a decreased Infliximab- induced apoptosis. Inhibition of casein 

kinase 1, the enzyme responsible for TNF phosphorylation, resulted in a diminished 

activation of Erk and a decreased apoptosis rate. 

In summary, reverse signalling of membrane TNF induces increased apoptosis in human 

monocytes of RA patients in comparison to healthy donors. Infliximab-induced apoptosis 

of RA monocytes requires cell surface expression of membrane TNF and an involvement 

of the MAP kinase Erk and casein kinase 1.

Joachim Heinrich Maxeiner, Kerstin Annika Sauer, Roman Karwot, Petra Scholtes, 

Rainer Wiewrodt, Hans-Anton Lehr, Susetta Finotto 

Increased lung tumor in NFATc2 (-/-) mice mediated by 

defective CD8+ T cells and increased CD4+CD25+ lung T cells 

Lung cancer, including bronchoalveolar adenocarcinoma is the major cause of death in 

the USA and Europe. The transcription factors nuclear factor of activated T cells (NFAT)s 

are critical in regulating early gene transcription in response to T cell receptor mediated 

signals in lymphocytes. Previous studies on mice lacking NFATc2, a NFAT family 

member, have demonstrated that these mice display hyperproliferation of T and B 

lymphocytes, however little is known about its role in CD8+ T cells. 

In the present study, we show that mice lacking nuclear factor of activated T cells-2 

(NFATc2) develop increased lung metastasis in a bronchoalveolar adenocarcinoma 

murine model twenty-one days after intravenous injection of the L1C2 adenocarcinoma 

cell line. Moreover, significant elevated TGF-beta protein in the airways was 

accompanied by increased number of CD4+CD25+ T cells in the lungs of L1C2 treated 

NFATc2 (-/-) mice. Furthermore, although dendritic cells isolated from the lungs of 

NFATc2 (-/-) mice were of more mature phenotype, they could not induce CD8+ T 

effector (Teff) cell responses as demonstrated by unchanged levels of CD8+CD25+ Teff 

cells and increased development of IL-15 dependent CD8+ T cells (CD8+CD122+) in 

the lung of NFATc2(-/-) mice bearing tumor. In addition, histologically, lung metastasis 

of NFATc2 (-/-) mice were characterized by decreased necrosis in tumor cells as 

compared to those developed in the lung of the wild type littermates. These results 

imply that immunotherapy based on adoptive transfer of dendritic cells might be 

unsuccessful in absence of NFATc2 expression in CD8+ T cells leading to lack of an 

effective CTL response into this tumor model. 

This work is supported by a DFG (FI-817) and SFB 548 grant.

Matthias Kresse, Ingo Uthe, Heike Weighardt, Irmgard Förster 

Inducible ablation of CCL17 positive DC in vivo 

CCL17 (TARC) is a CC chemokine which is expressed by a subpopulation of CD11bpositive 

dendritic cells (DC), in particular following maturation of these cells. CCL17 

expressing DC can be detected in thymus, lymph nodes, Peyer’s Patches, lung, and 

inflamed skin but not in spleen. CCL17 binds to CCR4 which is highly expressed on TH2 

cells but also on other activated T cells. To investigate the role of CCL17 in vivo we 

generated mice expressing a DTR-EGFP fusion protein under the control of the 

endogenous CCL17 promotor using a knockin approach (CCL17/DTR mice). These mice 

developed normally and expression of the DTR-EGFP fusion protein was detectable on 

DC following LPS stimulation. We could demonstrate that application of diphtheria toxin 

(DT) leads to the ablation of CCL17 expressing bone marrow derived DC in vitro. In 

addition, CCL17 expressing DC are efficiently deleted in vivo after application of DT to 

CCL17/DTR mice. To analyse the role of CCL17 expressing DC in TH1 versus TH2 driven 

immune responses we backcrossed CCL17/DTR mice to a C57Bl/6 or Balb/c 

background, respectively. Surprisingly, we observed a significantly reduced size of 

mesenteric lymph nodes and Peyer’s Patches in Balb/c CCL17/DTR mice compared to 

C57Bl/6 CCL17/DTR mice and littermate controls, presumably resulting from enhanced 

expression of the DTR-EGFP fusion protein in dendritic cells of Balb/c mice. The 

functional effects of CCL17 positive DC ablation in innate and adaptive immune 

responses are currently under investigation.

Katharina A. Remer, Calin Apetrei, Tobias Schwarz, Heidrun Moll 

Induction of a local Th1 response after plasmacytoid dendritic 

cell-based vaccination protects mice against infection with 

Leishmania major 

Protective immunity against the protozoan parasite Leishmania is attributed to the 

development of a Th1-type T-cell response, while a Th2-type response results in 

progressive disease cumulating in lethal visceral leishmaniasis. Since distinct DC 

subsets have been proposed to direct the predominant development of either Th1- or 

Th2 T-cell responses, we analyzed the capability of plasmacytoid DC (pDC) to induce 

protection and elicit a Th1 response against Leishmania major in susceptible mice. 

Pulsing with L. major lysate induced the activation and maturation of murine pDC that 

had been isolated from the spleen, as indicated by up-regulation of co-stimulatory 

molecules. Vaccination of susceptible mice with L. major lysate-loaded pDC induced 

highly effective immunity against a challenge infection with L. major parasites. 

Surprisingly, the protection was not accompanied by a polarized systemic Th1 cytokine 

profile, although protected mice had a lower ratio of Leishmania-specific IgG1 to IgG2a 

than mock-treated controls. In contrast, upon restimulation with L. major lysate, cells 

from lymph nodes draining the infected lesions showed a higher IFN-γ to IL-10 ratio 

than the mock-treated controls, indicating the compartmentalized induction of a local 

Th1-type response. The protective immune response induced by vaccination with 

antigen-loaded pDC is mediated by T cells, since adoptive transfer of T cells from 

protected mice confers complete protection against a challenge infection with L. major 

promastigotes to naïve susceptible mice. Again, a local Th1-biased response in the 

presence of a systemic mixed Th1/Th2 response was sufficient to protect susceptible 

animals against an otherwise lethal infection with L. major. These findings demonstrate 

that vaccination with antigen-loaded pDC is able to induce a protective immune 

response against a parasite disease that is mediated by T cells, but unexpectedly 

compartmentalized. Our study underlines that experimental leishmaniasis is a suitable 

model to elucidate the mechanisms underlying DC-based vaccination against infections.

Christine Warmbold, Arthur Ulmer, Thomas Roeder 

Induction of inflammatory signal transduction pathways in a 

Drosophila-derived cell line 

The innate immune system of Drosophila has been introduced as a system for analysing 

basic aspects of inflammation. This includes the discovery of two different signal 

transduction pathways, the Toll-pathway and the IMD-pathway. The Toll-pathway is 

said to response principally to infections with Gram-positive bacteria whereas the IMDpathway 

answers mainly to infection with Gram-negative bacteria. Well described 

ligands for these pathways are for example peptidoglycans and LPS. In vertebrates 

bacterial lipopeptides are well known ligands for the Tol-like receptor 2. Now we have 

investigated that these bacterial compounds are also recognized by the Drosophila 

immune system. 

For our analysis we use the cell line SL2 which is a macrophage-like, epithelial cell line 

isolated from emryos of the D.m. strain Oregon R. 

In this studies the lipopeptides Pam2C-SK4 and Pam3C-SK4 are discovered as new 

ligands for the two pathways (Toll and IMD). Our knowledge is based on luciferase 

reporter assays. Here, a Firefly-luciferase is under the control of promotors for 

antimicrobial peptides that are activated by one of these pathways, namely drosomycin 

by the Toll-pathway and diptericin by the IMD-pathway. The data are normalised by the 

constitutively expressed Renilla-luciferase. 

By using real-time PCR and RNAi-experiments we will confirm our current data and look 

for crosstalks between these two pathways. 

(supported by SFB TR 22, A7)

Julia Strebovsky, Claus Kaiser, Alexander Dalpke, Klaus Heeg, Holger Bartz 

Induction of regulatory T-cells by Toll-like receptor-ligand 

derived deviant dendritic cells 

Dendritic cells (DCs) are professional antigen-presenting cells (APCs) which are the 

most potent stimulators of T-cell (TC) responses. In vitro human CD14+ monocytes can 

be differentiated into DCs by stimulation with granulocyte- macrophage- colony- 

stimulating factor (GM-CSF) and Interleukin-4 (IL-4). Further maturation is achieved by 

subsequent TLR-stimulation (which mimics microbial encounter) or CD40 ligation. 

However, these DC precursor cells also express TLRs (Toll-like receptors), thus they are 

sensitive to TLR stimulation not only during the maturation but also during the 

differentiation period. 

Here we analyzed the effects of TLR stimulation during the process of GM-CSF- 

mediated in vitro generation of DCs from precursor cells. Precursor cells of DCs which 

encounter microbial TLR-dependent stimuli in an early developmental stage 

differentiated into deviant DCs. Those aberrantly generated DCs showed reduced 

expression of CD1a, modified co-stimulatory molecule and reduced cytokine expression. 

They induced less T-cell proliferation in an allogenic MLR. However, deviant DCs had an 

increased expression of PD-ligands suggesting a role in the induction of Tregs. Thus we 

focused on the characterization of TC-responses induced by deviant DCs and 

determined the induction of Tregs which are proven to hold a regulatory capacity. 

Moreover, this regulatory function is shown to be dependent on the presence of IL-2 

which in turn leads to the phosphorylation of STAT5 and the expression of FoxP3.In 

summary we show that early TLR stimulation results in deviant DCs which are shown to 

be potent inducers of Tregs. These findings suggest a model for down-regulation of 

adaptive immune responses by deviant DCs/Tregs in late-stage infection which will be 

presented.

Ria Baumgrass, Vladimir Pavlovic, Britta Lamottke, Maria Lexberg, Joachim Grün, Uwe 

Niesner, Andreas Radbruch 

Induction of Treg cells by manipulation of TCR signalling 

The molecular mechanisms regulating induction of Treg cells are unknown. Recent data 

show that peripheral Treg cells can be induced by concomitant stimulation of 

transforming growth factor beta (TGF-beta) and T cell receptors (TcR). We found the 

TGF-beta interacting factor (TGIF) to be a candidate modulator of this process. TGIF is 

expressed by TcR stimulation and acts as a transcriptional repressor of TGF-beta 

signalling. Impaired TcR signalling by low-dosed CsA leads to a low expression of TGIF 

and therefore to Treg cell induction even at very low concentrations of TGF-beta. The 

induced Treg cells are immunosuppressive both, in vitro and in vivo. TGIF knock-down 

by TGIF-siRNA induced Treg cells from naïve Th cells. Altogether our data provide 

evidence that TGIF acts as a regulator of TGF-beta dependent Treg cell induction. 

Therefore it is a potential novel target molecule for therapeutic manipulation toward 

Treg cell induction in autoimmune diseases.

Stephan Fricke, Nadja Hilger, Manuela Ackermann, Peter Ruschpler, Lutz Uharek, 

Guido Hildebrandt, Jan Matthias Braun, Frank Emmrich 

Induction of xenogenic acute graft versus host disease 

(aGvHD) in mice 

Introduction: Acute graft versus host disease (aGvHD) is one of the major causes of 

unsuccessful haematopoietic stem cell transplantation. aGvHD occurs approximately to 

40%-70% in all cases and is initiated by “supravital” donor T lymphocytes which 

recognize the minor or major histocompatibility antigens of a recipient. Until now, no 

standard therapy for steroid refractory aGvHD is available. The development of new 

therapeutic strategies (anti T cell antibodies or cellular therapeutics) and protocols 

presumes suitable animal models. 

Aims: The induction of graft versus host disease in a murine xenograft model by 

intravenous transfer of human peripheral blood mononuclear cells in human CD4+, 

murine CD4-, human DR+ triple transgenic and wild-type mice should be investigated. 

Methods: Induction and severity of aGvHD in wild-type (C57/Bl/6) and triple transgenic 

human CD4+, murine CD4- and human DR+ mice were investigated. Mice received 

chemotherapy (cyclophosphamide and treosulfan) on day -3 to -1 before intravenous 

transplantation of human PBMCs. All experimental groups were daily examined for 

GvHD symptoms (severe weight loss [>10%], hunched posture, ruffled fur, reduced 

mobility, tachypnoea). For determination of leucocytes by FACS and cytokines by CBA 

blood of all mice were weekly collected by retro orbital bleeding. Finally, histological 

examination of gut and spleen were added. 

Results: By using of the triple transgenic human CD4+, murine CD4-, human DR+ mice 

an acute xenogenic GvHD syndrome could be observed. In contrast, wild-type mice did 

not show a GvHD syndrome (clinical scoring and histology). 

Conclusion: Our results show the induction of xenogenic aGvHD in DR+ transgenic mice 

while MHC-wild-type mice did not develop GvHD. In terms of application of monoclonal 

antibodies as therapeutic potentiality should be considered that they have to be 

produced for specific human epitopes. Therefore, the used humanised triple transgenic 

mouse model is advantageously as interaction of human cell surface molecules could be 

simulated.

Uta Bussmeyer, Arup Sarkar, Kirsten Broszat, Ger van Zandbergen, Christian Bogdan, 

Werner Solbach, Friederike von Loewenich, Tamás Laskay 

Infection with Anaplasma phagocytophilum inhibits IFN- 

&gamma signaling in human neutrophils 

Anaplasma phagocytophilum (Ap) is a tick-borne obligate intracellular bacterium that 

survives and multiplies inside polymorphic neutrophilic granulocytes (PMN). Previous 

findings demonstrated that the bacterium actively subverts antimicrobial effector 

mechanisms of PMN including the oxidative burst after priming with IFN-&gamma. The 

present study aimed to investigate whether an infection with Ap leads to a more general 

impairment of IFN-&gamma signaling in PMN that enables intracellular survival of the 

bacterium. 

The capability of Ap to interfere with IFN-&gamma-mediated activation of PMN was 

assessed by measuring MIG and IP-10 secretion. Neutrophils secreted substantial levels 

of both chemokines when stimulated with IFN-&gamma for 18h. Infection of PMN with 

Ap markedly decreased the secretion of MIG and IP-10 by PMN. To obtain first insights 

into the molecular events leading to the diminished secretion of IFN-&gamma-induced 

chemokines, the phosphorylation of STAT1 was investigated. Western blot analysis 

revealed that IFN-&gamma-induced STAT1 phosphorylation was diminished in Apinfected 

PMN. In further experiments flow cytometry analyses showed a markedly 

decreased expression of the IFN-&gamma receptor alpha chain CD119 on the surface of 

Ap-infected PMN. Moreover, using quantitative RT-PCR a strong upregulation of the 

negative regulator SOCS3 was observed in infected cells. 

Taken together our data show that infection with Anaplasma phagocytophilum results in 

a decreased CD119 surface expression, diminished tyrosine phosphorylation of STAT1 

and augmented SOCS3 gene expression in infected cells and, consequently, results in 

compromised IFN-&gamma-responsiveness of infected PMN. Impaired IFN-&gamma 

signaling in infected cells is likely to contribute to intracellular survival of Ap in PMN.

Vilma Urbonaviciute, Barbara G. Fürnrohr, Silke Meister, Petra Heyder, Martin 

Herrmann, Joachim R. Kalden, Reinhard E. Voll 

Inflammation and immune activation by HMGB1-nucleosome 

complexes – implications for the etiopathogenesis of systemic 

lupus erythematosus 

Pathogenic autoantibodies against dsDNA and nucleosomes (NCs) represent a hallmark 

of systemic lupus erythematosus (SLE). However, the factors breaking the 

immunological tolerance are not fully identified. An important initial step in the 

immunopathogenesis may involve impaired phagocytosis of apoptotic cells with a 

consecutive release of nuclear antigens. The nuclear DNA-binding protein and 

proinflammatory cytokine high mobility group box protein 1 (HMGB1) gets tightly 

attached to chromatin of apoptotic cells and is not released, since apoptotic cells are 

immediately engulfed by phagocytes. We hypothesized, that in case of clearance 

deficiency ”endogenous adjuvant” HMGB1 in complex with NCs can be released and may 

foster an autoimmune response towards NCs, a crucial autoantigen in SLE. 

We found that HMGB1 remains bound to NCs released from apoptotic cells in vitro . Also 

in sera and plasma of some patients with SLE, but not in controls, complexes of HMGB1 

and NCs were detected. Importantly, HMGB1 containing NCs from apoptotic cells 

induced secretion of cytokines including IL-1β, IL-10, and TNFα as well as expression of 

costimulatory molecules on human macrophages and dendritic cells (DC), respectively. 

Neither HMGB1-free NCs from living cells nor from apoptotic HMGB1- or HMGB1/2deficient 

cells induced marked cytokine production or DC activation. Additionally, 

specific inhibition of HMGB1 activity by antagonistic A box domain significantly reduced 

capacity of “apoptotic “ NCs to induce TNFα and IL-10 release by macrophages. 

We conclude that HMGB1 in complex with NCs activate antigen presenting cells thereby 

contributing to breaking of the immunological tolerance against nuclesomes/dsDNA and, 

hence, to the immunopathogenesis of SLE.

Simone Vallbracht, Birthe Jessen, Sonja Mrusek, Anselm Enders, Peter L. Collins, 

Christine D. Krempl, Stephan Ehl 

Influence of a single viral epitope on T cell response and 

disease after infection of mice with respiratory syncytial virus 

Cytotoxic T cells (CTL) are important for virus clearance, but also contribute to 

immunopathology after infection of BALB/c mice with respiratory syncytial virus (RSV). 

The pulmonary immune response to RSV is dominated by a CTL population directed 

against the CTL epitope M2-1 82-90. Infection with a virus carrying a M2-1 N89A 

mutation introduced by reverse genetics failed to activate this immunodominant CTL 

population leading to a significant decrease in the overall antiviral CTL response. There 

was no compensatory increase in responses to the mutated neoepitope, to the 

subdominant epitope F 85-93 or to yet undefined minor epitopes in the N or the P 

protein. However, there was some increase in the response to the subdominant epitope 

M2-1 127-135 which is located in the same protein and presented by the same H-2Kd 

MHC molecule. Infection with the mutant virus reversed the oligoclonality of the T cell 

response elicited by the wild type virus. These changes in the pattern and composition 

of the antiviral CTL response only slightly impaired virus clearance, but significantly 

reduced RSV induced weight loss. These data illustrate how T cell epitope mutations can 

influence the virus-host relationship and determine disease after an acute respiratory 

virus infection.

Torsten Lowin, Rainer H. Straub, Olga Wiesner, Ulf Müller-Ladner, Jörg Schedel 

Influence of cortisol on the expression of integrins and 

intracellular signaling molecules in synovial fibroblasts in 

rheumatoid arthritis 

Purpose: In rheumatoid arthritis (RA) synovial fibroblasts (SF) are localized at sites of 

invasion of the hyperplastic synovial tissue into cartilage and bone, and are able to 

degrade the extracellular matrix (ECM). Integrins mediate adhesion of SF to ECM 

components and influence outside-in and inside-out signaling pathways. As cortisol 

levels are relatively low in chronic inflammation, we examined the influence of different 

cortisol concentrations on the expression of integrins in SF. Furthermore, we 

investigated if cortisol alters intracellular signaling pathways which could, in turn, 

influence the invasive behaviour of RA-SF. 

Methods: Synovial tissue samples from patients with RA and osteoarthritis (OA) 

obtained during arthroplastic surgery were examined using immunofluorescence (IF) 

with anti-alpha5, -alphaν, -alpha5beta1 and anti-beta1 integrin antibodies. To 

characterise the expression profile quantitatively, fluorescence intensity was analysed in 

pixels/cell at the site of invasion and the cartilage-remote region. Isolated RA- and OA- 

SF were incubated with decreasing cortisol concentrations ranging from 10-5 M to 10-9 

M. Integrin expression was evaluated 24 h after stimulation by FACS analysis and 

quantitative PCR (qPCR). To evaluate the specificity of cortisol on integrins, 

cycloheximide (10 µg/ml) and the glucocorticoid receptor antagonist mifepristone (10-5 

M) were used. The activation of intracellular signaling molecules was determined by a 

"cellular activation of signaling ELISA"(CASE). 

Results: IF showed enhanced expression of alpha5, -alphaν, -beta1 integrins at the site 

of invasion in RA synovial tissue compared to OA synovial tissue. The increase of 

integrin expression in RA was greater than in OA varying from 24 % up to 61 % 

Treatment of isolated RA- and OA-SF with cortisol resulted in a concentrationdependent 

upregulation of target integrins with the highest effect seen at 10-5 and 10-6 

M as demonstrated by FACS analysis and qPCR. The effect was abrogated when either 

10 µg/ml cycloheximide or the glucocorticoid receptor antagonist mifeprestone (10-5 M) 

were present. CASE showed an activation by phosphorylation of the intracellular 

signaling kinases ERK, Src, p38 and PI3K upon integrin crosslinking. The presence of 10- 

6M cortisol resulted in a 50 % reduction of kinase phosphorylation. 

Conclusions: In RA, integrins were expressed at the site of invasion to an enhanced 

extent when compared to OA samples. The expression pattern suggests a functional 

role of the investigated integrins in cartilage and bone invasion in RA. Cortisol upregulated 

alpha5 and beta1 integrin expression in a dose-dependent manner. This fact 

and the decreased phosphorylation of intracellular signaling molecules after cortisol 

treatment might lead to a less invasive phenotype of RA-SF.

Veronika Lukacs-Kornek, Verena Semmling, Christian Kurts 

Influence of Cross-Presentation via CCR7 

Cross-presentation of extracellular antigens plays an important role in inducing 

cytotoxic immune responses against bacteria and viruses, as well as in maintaining CD8 

T cell tolerance against self antigens. It requires cellular contact between several rare 

immune cells, such as specific CD8 T cells, specific CD4 T cells and cross-presenting 

dendritic cells. Such encounters are usually regulated by chemokines and their 

receptors. These mediators can guide immunocytes towards one destination, where 

their encounters become more likely or can affect cellular function important in cross 

priming. In the present study, we describe a role of a chemokine, CCL19, which signals 

through CCR7, in cross-presentation. 

Using CCR7-deficient mice we demonstrated that CCR7 decreased activation of CD8 T 

cells by cross priming of cell-associated and of soluble antigens in vivo. To identify the 

cells affected by CCR7 ligands during cross-presentation, we established an in vitro 

cross-presentation assay. We could show that addition of CCL19 to the culture resulted 

in increased cross-presentation, as evidenced by higher expression of early activation 

markers such as CD69 and CD25 and elevated amounts of IL-2 produced by specific 

CD8 T cells. CCR7 did not affect the antigen uptake capacity of dendritic cells (DC) in 

vitro. Furthermore, selective CCR7-deficiency in DC did not prevent the stimulatory 

effect of CCL19 on CD8 T cell activation. However, this effect was completely abolished 

when CCR7-/- CD8 T cells were used. Therefore the increased activation of CD8 T cells 

during cross-presentation was exclusively mediated by a direct effect of CCL19 on the 

CD8 T cells. Future efforts are aimed identifying the intracellular mechanisms employed 

by CCL19 to stimulate CD8 T cells.

Birgit Weinberger, Ilka Weiskirchner, Beatrix Grubeck-Loebenstein 

Influence of latent infection with Cytomegalovirus on Epstein- 

Barr viral parameters in healthy elderly individuals: possible 

interaction of different herpesviruses in immunosenescence 

Latent infection with CMV leads to acceleration and enhancement of age-related 

immunological changes. The influence of other herpesviruses like Epstein-Barr virus 

(EBV) on immunosenescence seems to be less pronounced. Presumably, there are also 

interactions between different herpesviruses and the corresponding immune responses 

in the elderly. 

Antibody titers and viral load in whole blood were analyzed for CMV and EBV in 233 

healthy elderly donors (>60 years) and EBV-parameters were compared in CMV-positive 

and CMV-negative individuals. 

We could show that EBV-DNA load in whole blood is significantly higher in CMV-positive 

elderly individuals and that slightly elevated DNA-levels are seen almost exclusively in 

CMV-positive persons. A positive correlation of titers of EBV-specific antibodies with EBV- 

DNA load was only observed in CMV-positive elderly donors. No such correlation was 

observed in the CMV-negative cohort. 

We conclude that EBV-infection is less controlled by the cellular immune response in 

elderly individuals with latent CMV-infection.

Sandra Martina Dittrich, Elfriede Noessner, Hans Demmelmair, Gernot Desoye, Dolores 

Schendel, Berthold Koletzko, Susanne Krauss-Etschmann 

Influence of n3/n6 polyunsaturated fatty acids on placental 

immune responses 

Introduction 

N3/n6-long chain polyunsaturated fatty acids (LC-PUFA) modulate immune responses. 

In addition, docosahexaenoic acid (n3, DHA) and arachidonic acid (n6, AA) are 

important for the neural development and selectively transported to the fetus. However, 

it is unknown how n3/n6-LC-PUFA affect placental immune responses. 

Material and methods 

Basal mRNA expression levels of IL-6, IL-8, IL-12, TGF-β, MCP-1 and RANTES were 

quantified in choriocarcinoma lines (ACH-3P, BeWo, JAR, JEG) and eight non-placental 

carcinoma lines by real time RT-PCR (iCycler, Fa. Biorad). Selected cell lines were 

incubated with 1•M, 10•M and 100•M DHA, eicosapentaenoic acid (EPA) and AA using 

monounsaturated oleic acid (OA) and saturated palmitic acid (PA) as controls. Cytokine 

expression patterns and the cell proliferation rate (cellscreen, Fa. Innovatis) were 

determined after 1h, 6h, 24h and 48h. 

Results 

Basal mRNA expression levels of IL-6, IL-8, TGF-β, MCP-1 and RANTES were 

comparable in JAR, ACH-3P, RCC 26 (renal cell carcinoma) and MCF-7 (mamma 

carcinoma). IL-12 mRNA was detectable only in BeWo, Du 145 (prostate carcinoma) 

and HEK 293 (human, adenovirally immortalised embryonic kidney cell line). PA 

increased the proliferation rate of JAR and MCF-7. EPA increased the proliferation rate of 

JAR. PA and OA slightly increased IL-6 and RANTES mRNA expression levels, while EPA 

decreased IL-6 mRNA expression levels in JAR. There was no effect of any fatty acid on 

RCC 26. 

Conclusion 

Choriocarcinoma lines had heterogeneous cytokine expression patterns. Even very 

related choricarcinoma lines showed clear differences. EPA, PA and OA modulated 

proliferation rates and cytokine expression levels. However, the changes were small and 

need further confirmation.

Stephan Sudowe, Karina Gisch, Nadine Gehrke, Matthias Bros, Christina Priesmeyer, 

Angelika B. Reske-Kunz 

Inhibition of allergic sensitization and suppression of Th2mediated 

airway inflammation by application of formalinfixed 

Staphylococcus aureus-particles 

Background: Bacterial infections are supposed to act counterregulatory to the 

development of allergen-specific Th2 immune responses. We analyzed whether 

administration of extracellular Staphylococcus aureus inhibited sensitization against 

allergens in a mouse model of type I allergy. Methods: BALB/c mice were immunized 

with alum-adsorbed ovalbumin (OVA) together with formalin-fixed Staphylococcus 

particles. OVA-specific antibody production and cytokine synthesis by spleen cells was 

analyzed. Airway reactivity and cellular infiltration into the airways was assessed after 

intranasal challenge of mice with OVA. In addition, the capacity of Staphylococcus 

particles to modulate cytokine production by bone marrow-derived dendritic cells was 

analyzed in vitro. Results: Simultaneous application of OVA and Staphylococcus 

particles very efficiently inhibited production of specific IgE and IgG1 as well as 

secretion of IL-4 and IL-5 by splenocytes, while enhancing IgG2a formation and 

production of IFN-γ, indicating a shift from a Th2 response towards a Th1-biased 

response. This effect was not dependent on the expression of protein A by 

Staphylococcus. Treatment of mice with Staphylococcus particles during the 

sensitization phase attenuated Th2-dependent inflammatory reactions in the lung 

(airway hyperreactivity, eosinophilia) after local challenge with OVA. Culture of bone 

marrow-derived dendritic cells with Staphylococcus particles induced IL-12p35 and p40 

mRNA expression as well as secretion of IL-12p70, and increased production of IL-10 

mRNA and protein. However, an enhanced frequency or activity of regulatory T cells 

after administration of Staphylococcus particles was not apparent. Conclusions: 

Application of formalin-fixed Staphylococcus particles induced Th1-biased immune 

responses and prevented allergic sensitization as well as symptoms of allergic airway 

inflammation.

Tobias Frankenberg, Susanne Kirschnek, Hans Häcker, Uwe Koedel, Georg Häcker 

Inhibition of apoptosis conserves neutrophil effector function 

and can contribute to inflammation in vivo 

Neutrophil granulocytes have essential functions during immune responses against 

pyogenic bacteria. Following pathogen clearance, neutrophils have to be removed from 

sites of infection, a process that may involve apoptosis. We have previously found that 

in a murine model of Streptococcus pneumonia-induced meningitis, overexpression of 

the anti-apoptotic protein Bcl-2 in the haematopoietic system aggravates inflammationinduced 

tissue damage. The continued presence of neutrophils in these mice despite 

clearance of bacteria suggested that the block of apoptosis caused sustained pleocytosis 

and contributed to the severity of the inflammation. To test this hypothesis, we 

compared in vitro apoptosis and effector function of wt and Bcl-2 overexpressing 

neutrophils derived from isolated bone-marrow progenitor cells retrovirally transduced 

with regulable Hoxb8. In its active state, Hoxb8 promotes progenitor expansion while 

upon inactivation permits cellular differentiation. This culture system thus provides the 

possibility to generate almost unlimited numbers of ‘near-primary’ neutrophils. 

Differentiated Hoxb8 neutrophils displayed the typical nuclear morphology, expressed 

Gr-1, and were phagocytosis-proficient. No obvious differences in terms of 

differentiation and effector functions were observed between wt and Bcl-2 neutrophils. 

Upon prolonged culture the vast majority of differentiated wt neutrophils underwent 

apoptosis. The overexpression of Bcl-2 efficiently protected neutrophils from cell death 

and, importantly, these ‘undead’ cells retained at least some of their effector functions. 

These data show that inhibition of apoptosis can prolong neutrophil lifespan and effector 

function, which very likely contributes to the more severe pathology in Streptococcus 

pneumonia-induced murine meningitis. This model therefore suggests that granulocyte 

apoptosis is an important event for the termination of the innate immune response.

Martin Raftery, Günther Schönrich 

Inhibition of CD1 Antigen Presentation by Human 

Cytomegalovirus 

The betaherpesvirus human cytomegalovirus (HCMV) encodes many molecules that 

block classical and nonclassical MHC molecules. No interaction has been reported, 

however, between HCMV and the only family of nonclassical MHC class I molecules, the 

CD1 family. The CD1 family is particularly interesting as they are present almost 

exclusively on professional antigen presenting cells such as dendritic cells (DC), which 

are in turn a major target for HCMV infection and latency. Furthermore a relationship 

between CD1 antigen presentation and antiviral immunity has recently been 

established. We have determined that HCMV encodes multiple blocking strategies 

targeting group 1 CD1 molecules. HCMV-encoded cmvIL-10 strongly downregulates CD1 

transcription, as does human and ebvIL-10. HCMV also blocks CD1 antigen presentation 

post-transcriptionally. This function is not encoded by a known HCMV MHC blocking 

molecule, and requires the cytoplasmic domain of the CD1 molecule. The HCMV block is 

particularly effective against CD1b, whereas in comparison HSV1-induced block is weak. 

This preferential block implies a role for CD1b in antiviral immunity.

Dorit Fabricius, Sue O’Dorisio, Sue Blackwell, Bernd Jahrsdörfer, Klaus-Michael Debatin 

Inhibition of IFN-α Secretion and Modulation of 

Immunophenotype and Stimulatory Capacity of Human 

Plasmacytoid Dendritic Cell by Vasoactive Intestinal Peptide 

Plasmacytoid dendritic cells (PDC) represent a central link between innate and adaptive 

immunity. The special feature of PDC compared to other DC subsets is that they not 

only are capable of antigen presentation but also unite the capacities of high IFN-α and 

Granzyme B production. The increased frequency in various tumor tissues and in sites 

of active autoimmune processes reflects an essential role of PDC both for tumor and 

auto-immunity. While the role of granzyme B secretion by PDC is still unclear, excessive 

IFN-α production seems to be a key factor in driving pathogenesis in certain 

autoimmune diseases, mainly systemic lupus erythematodes. A better understanding of 

PDC regulation is therefore crucial for more effective immunotherapeutic approaches to 

cancer and autoimmunity. We show here that the neuropeptide vasoactive intestinal 

peptide (VIP) significantly down-regulates IFN-α secretion by PDC and modulates PDC 

immunophenotype. Furthermore, VIP inhibits the potential of PDC to induce proliferation 

of allogeneic CD4+ T cells and VIP-treated PDC decrease the ratio of IFN-γ to IL-4 

secreted by CD4+ T cells. We also demonstrate that PDC actively secrete the cytotoxic 

molecule granzyme B, and that this process can be regulated by VIP and cytokines such 

as interleukin 21 (IL-21). We conclude that modulation of PDC by VIP may be one 

possible escape mechanism of malignancies that secrete this neuropeptide. In contrast, 

VIP may turn out to be an exciting new candidate for immunotherapeutic intervention in 

autoimmune diseases, since it suppresses the pathogenetically critical IFN-α secretion 

and the T cell-stimulatory capacity of PDC.

Rainer Glauben, Arvind Batra, Thorsten Stroh, Elena Sonnenberg, Inka Fedke, Hans 

Anton Lehr, Paolo Mascagni, Martin Zeitz, Britta Siegmund 

Inhibition of NF-κB by histone deacetylase inhibitors in 

models of inflammation-related tumorigenesis 

Histone deacetylase inhibitors (HDACi) have been described initially for their antiproliferative 

effect in cell lines as well as in models of experimental tumor growth. In 

addition, recent studies from our group provide evidence for an anti-inflammatory 

potency of HDACi by using various models of experimental colitis. To characterize the 

combined effect of the anti-inflammatory and anti-proliferative potency of HDACi in 

vivo, the AOM/DSS-model as well as the IL-10KO-model of inflammation-mediated 

colon carcinogenesis were applied. The HDACi SAHA and ITF2357 were administered 

throughout the entire experimental period via oral gavage and the progression of 

tumorigenesis was monitored by lower endoscopy. In both models, HDACi treatment 

resulted in a delay of tumor development as evaluated by endoscopy and histology. In 

addition, the absolute number of adenomas was significantly reduced in the HDACitreated 

groups when compared to vehicle-treated animals as determined by histology. 

Remarkably, the strongest suppression of tumorigenesis was observed in animals 

treated with ITF2357. Recently it was descibed that the transcription factor NF-κB acts 

as a key factor within the process of inflammation-linked carcinogenesis. Furthermore, 

several studies indicate that inhibition of HDAC affects the NF-κB pathway. To further 

explore the mechanistic effects of HDACi treatment in the employed models of 

tumorigenesis, nucleus extraction experiments from colon sections of the ITF2357 

treated as well as untreated AOM/DSS-exposed mice were performed. These 

experiments indicate, that the HDACi treatment inhibits the translocation of p65, a 

subunit of NF-κB, into the nucleus. Consequently, the inhibition of tumor development 

by HDACi is paralleled by a profoundly reduced activation of NF-κB at the site of 

inflammation. Since inflammatory bowel disease (IBD) in humans is associated with an 

increased risk of colorectal cancer, a pharmacological strategy exerting antiinflammatory 

as well as anti-proliferative properties would be highly intriguing for the 

long-term therapy in IBD.

Dirk Reinhold, Alexander Goihl, Bianca Guth, Uwe Lendeckel, Ute Bank, Michael Täger, 

Siegfried Ansorge, Jürgen Faust, Klaus Neubert, Stefan Brocke 

Inhibitors of dipeptidyl peptidase IV (DP IV, CD26)-like and 

of aminopeptidase N (APN, CD13) enzymatic activity suppress 

human T cell activation and IL-17 production in vitro and in 

vivo 

Peptidases like dipeptidyl peptidase IV (DP IV, CD26) and aminopeptidase N (APN, 

CD13) play a regulatory role in T cell activation and represent potential targets for the 

treatment of inflammatory disorders. Compelling evidence has recently demonstrated 

that IL-17-producing CD4 cells (Th17 cells) are a major contributor to the pathogenesis 

of autoimmune inflammation. Since we and others have previously shown that synthetic 

inhibitors of DP IV-like enzymatic activity and of APN suppress disease in models of 

autoimmunity, we tested the hypothesis that these inhibitors target the production of IL- 

17. Here, we demonstrate that the inhibitors of DP IV-like activity, Lys[Z(NO2 )]thiazolidide 

and Lys[Z(NO2 )]-pyrrolidide, as well as the APN inhibitor actinonin inhibit IL- 

17 production and IL-17 mRNA expression in human mitogen-stimulated T cells. 

Moreover, combining DP IV and APN inhibitors increases the suppressive effect on T cell 

proliferation and IL-17 production in vitro in comparison to a single peptidase inhibitor. 

In order to investigate the mechanism by which peptidase inhibitors limit autoimmune 

disease in vivo, we tested the effect of DP IV and APN inhibitors on IL-17 production in 

experimental autoimmune encephalomyelitis (EAE), an animal model of multiple 

sclerosis. Our results show that IL-17 plasma levels are decreased in EAE mice treated 

with combined peptidase inhibitors. 

Collectively, our data suggest that combined inhibition of DP IV-like and APN enzymatic 

activity in pathogenic T cells represents a novel and efficient therapeutic approach 

targeting IL-17 production in autoimmunity.

Simone Klöter, Max v. Holleben, Bernhard Reis, Klaus Pfeffer, Sandra Beer 

Interaction analysis of the adaptor protein SLy2 

Adaptive immunity is crucial for protective host defense and the development of 

immunological disorders. An increasing number of molecules with SH2 or SH3 domains 

involved in the T or B cell receptor signal transduction pathways have been discovered 

in the last years. 

SLy2 (SH3 domain protein expressed in lymphocytes 2) was recently identified as an 

SH3- and SAM-domain containing protein. In humans, the SLy2 gene is located on 

chromosome 21, in mice on chromosome 16. SLy2 is expressed in hematopoetic tissues 

as well as in brain, lung and pancreas. Additionally, SLy2 is up-regulated in activated 

human B cells treated with IL-4, CD40L and anti-IgM. Therefore, SLy2 might be a 

component of signalling cascades that lead to B cell activation and differentiation. 

To elucidate the function of SLy2 we screened for possible interaction partners of this 

adaptor protein. Considering the fact that SH3- and SAM-domains can mediate 

homodimerization of proteins we performed coimmunoprecipitations with differentially 

tagged SLy2 constructs. We were able to show the formation of SLy2 homodimers in 

293T cells via the SH3 domain. 

In order to identify further interaction partners of SLy2 a Yeast Two Hybrid Screen with 

a mouse T cell lymphoma library was carried out. This resulted in two putative 

interaction partners of SLy2: Sin3-associated polypeptide p30 (SAP30) and ribosomal 

protein L12 (rpL12). SAP30 is a component of the histone deacetylase complex 

including HDAC1, HDAC2, mSin3 as well as other proteins and consequently is capable 

of repressing the transcription of different genes. rpL12 is associated with the assembly 

of the 40S and 60S subunit of eukaryotic ribosomes. The interactions of SLy2 with both 

proteins were confirmed by coimmunoprecipitations with lysates from transiently 

transfected 293T cells. This interaction of SLy2 with SAP30 and rpL12 insinuates 

transcriptional and/or translational regulatory functions of SLy2.

Pia Herzberger, Corinna Siegel, Christine Skerka, Volker Fingerle, Ulrike Schulte- 

Spechtel, Bettina Wilske, Volker Brade, Reinhard Wallich, Peter F. Zipfel, Peter Kraiczy 

Interaction of immune regulators factor H and FHL-1 with the 

Lyme disease spirochete Borrelia spielmanii sp. nov. is 

mediated by a plasmid-encoded complement regulatoracquiring 

surface protein-1 

Borrelia spielmanii sp. nov., one of the etiological agents of Lyme disease found in 

Europe escapes complement-mediated killing by recruitment of immune regulators 

factor H and FHL-1 from human serum. Serum-resistant and intermediate serumresistant 

isolates express up to three distinct complement regulator-acquiring surface 

proteins (CRASPs) that bind to factor H and/or FHL-1. Here we report identification and 

functional characterization of BsCRASP-1, the dominant factor H and FHL-1 binding 

protein of B. spielmanii. BsCPASP-1 is a 27.7 kDa outer surface lipoprotein, which after 

processing is predicted to be 24.9 kDa. The gene encoding BsCRASP-1 is a single 

genetic locus that maps to a linear plasmid of approximately 55 kb. Ligand affinity blot 

techniques revealed that both, native and recombinant BsCRASP-1 from different 

isolates, bind to FHL-1 and weaker to factor H. Deletion mutants of BsCRASP-1 were 

generated and a high affinity binding site for factor H and FHL-1 was mapped to a 10 

amino acid residue domain at the C-terminus of BsCRASP-1. Similarly, the predominant 

binding site of factor H and FHL-1 was localized to short consensus repeats 5 to 7. 

Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b 

inactivation when bound to full-length BsCRASP-1 but not to a deletion mutant lacking 

more than 10 amino acid residues at its C-terminus. In conclusion, BsCRASP-1 binds 

the host immune regulators factor H and FHL-1 with high affinity and is the key 

molecule of the spirochetes’ complement resistance. Thus, BsCRASP-1 most likely 

contributes to persistence of B. spielmanii and to pathogenesis of Lyme disease. This 

work was funded by the Deutsche Forschungsgemeinschaft DFG, Project Kr3383/1-1 

and Wa533/7-1.

Nikola Baschuk, Olaf Utermöhlen, Roland Gugel, Gabriele Warnecke, Ulrike Karow, 

Daniela Paulsen, Frank Brombacher, Martin Krönke, Wolfgang Deppert 

Interleukin-4 impairs granzyme-mediated cytotoxicity of 

Simian virus 40 large tumor antigen-specific CTL in Balb/c 

mice 

In this report we analysed the impact of interleukin-4 (IL-4) on tumor-associated simian 

virus 40 (SV40) large T-antigen (TAg)-specific CD8 + cytotoxic T cells during rejection of 

syngeneic SV40 transformed mKSA tumor cells in Balb/c mice. 

Strikingly, challenge of naive mice with low doses of mKSA tumor cells revealed a CD8 + 

T cell-dependent prolonged survival time of naive IL-4 -/- mice. 

In mice immunized with SV40 TAg we observed in IL-4 -/- mice, or in wild type mice 

treated with neutralizing anti-IL-4 monoclonal antibody, a strongly enhanced TAgspecific 

cytotoxicity of tumor associated CD8 + T cells. The enhanced cytotoxicity in IL- 

4 -/- mice was accompanied by a significant increase in the fraction of CD8 + tumor 

associated T-cells expressing the cytotoxic effector molecules granzyme A and B and in 

granzyme B-specific enzymatic activity. 

The data suggest that endogenous IL-4 can suppress the generation of CD8 + CTL 

expressing cytotoxic effector molecules especially when the antigen induces only a very 

weak CTL response.

Michael Meyer-Hermann, Marc Thilo Figge 

Interpreting two-photon lymphocyte motility data of the 

germinal centre: Predictions and analysis by mathematical 

models 

The germinal centre reaction is central to adaptive immunity. Recent progress in 

intravital imaging has provided the first movies of cell trafficking in secondary lymphoid 

follicles [1]. The results of these measurements confirm predictions from our 

mathematical models [2]. In particular, it was predicted and now measured that B 

lymphocytes do not actively move between the zones. However, this result is not 

possible to be deduced from multi-photon measurements alone. It is essential that the 

data are interpreted with mathematical methods. 

We show how mathematical modelling of germinal centre reactions is useful to 

interprete multi-photon intravital imaging data in two steps: At first the models that led 

to the prediction of the undirected motility of lymphocytes are presented. It is shown 

that no directed motility is needed in order to get a fully functional germinal centre 

reaction. In a second step it is shown which results would be expected in multi-photon 

imaging when either undirected motility or directed movement between the zones is 

assumed. This clearly shows that the recent imaging results do not exhibit any link to 

active movement between the zones. 

[1] Allen CD, Okada T, Tang HL, Cyster JG, Science 26 (2007) 528; 

Schwickert et al., Nature 446 (2007) 83; 

Hauser et al., Immunity 2007 in press. 

[2] Meyer-Hermann M, J. Theor. Biol. 216 (2002) 273; 

Meyer-Hermann M, Maini PK 174 (2005) 2489; 

Meyer-Hermann M, Figge MT 2007 in preparation.

Astrid M. Westendorf, Wiebke Hansen, Jan Buer 

Intestinal antigen display promotes the peripheral induction 

of antigen specific CD8 + Foxp3 + T cells 

Little data exist regarding mechanisms of mucosal T cell reactivity or tolerance against 

the variety of antigens in the intestine. In contrast to antigen specific CD4 + regulatory T 

cells, the generation and function of immunomodulatory antigen-specific CD8 + T cells is 

less well defined. To dissect the immunologic mechanisms of CD8 + T cell function in the 

mucosa, reactivity to a self-antigen expressed in intestinal epithelium of mice bearing a 

MHC class-I-restricted T-cell-receptor specific for this antigen was studied. Here, we 

demonstrate that intestinal self-antigen expression leads to peripheral induction of 

antigen-specific CD8 + Foxp3 + T cells rather than the induction of cytotoxic CD8 + 

effector T cells and intestinal pathology. This induction is restricted to the mesenteric 

lymphnode and the lamina propria. Antigen-experienced CD8 + T cells in this transgenic 

mouse model are characterized by significantly upregulation of CD103, CD83, GPR83 

and granzyme A/B expression, molecules also expressed on regulatory CD4 + T cell 

subsets. Despite the fact that naïve and antigen-experienced CD8 + T cell exhibit the 

same proliferative capacity in vitro, CD8 + T cells from this transgenic mouse model 

produce much less IFN-gamma and no TNF-alpha after in vitro stimulation in 

comparison to naïve CD8 + T cells. In addtion, whereas naïve CD8 + T cells further 

aggravate CD4 + T cells proliferation in an inhibition assay CD8 + T cells isolated from 

this transgenic mouse model slightly reduce antigen-specific CD4 + T cell proliferation in 

vitro. In summary, we demonstrate that self-antigen expression of intestinal epithelial 

cells is sufficient to induce CD8 + regulatory T cells which maintain intestinal 

homeostasis by down-modulating effector functions of T cells.

Ilka Knippertz, Andrea Hesse, Eckhart Kaempgen, Gerold Schuler, Alexander 

Steinkasserer, Dirk. M. Nettelbeck 

Intracellular expression of CD40L after adenoviral 

transduction in combination with IFN-gamma treatment 

generates human dendritic cells that both secrete IL-12 and 

have migratory functions 

In Dendritic cell (DC)- based vaccination trials often monocyte-derived DCs (moDCs) 

matured with a cocktail composed of IL-1ß, TNF-alpha, IL-6 and prostaglandine E2 

(PGE2) have been used. It has been shown that PGE2 is indispensable for DC-migration 

but it is also known to inhibit IL-12p70 expression. Moreover other labs reported that 

these two essential functions of DCs are frequently not linked. Here we describe a new 

and interesting method to overcome this problem and to generate mature moDC 

capable of both migrating and producing biologically active IL-12p70. This could be 

achieved by a combined treatment with recombinant human interferon gamma (rh-IFNgamma) 

and adenoviral gene transfer of the trimeric CD40L. Immature moDCs were 

infected with an adenovirus serotype 5 coding for the trimeric human CD40L 

(Ad5hCD40L) in an autologous monocyte-conditioned medium supplemented with GM- 

CSF, IL-4, IL-1ß, TNF-alpha, IL-6 and PGE2. Afterwards (1.5 hours later) rh-IFN-gamma 

was added to the infected cells. 24 hours later cells were checked for their migratory 

capacity, their IL-12 production as well as their CD40L expression and maturation 

status. Interestingly, moDCs infected with Ad5hCD40L and subsequently treated with rh- 

IFN-gamma fully matured, showed a high IL-12p70 secretion ( approx. 700 pg/ml ) as 

well as high migratory capacity. Even when the migrated cells were cultured for an 

additional time period of 24 hours they were still able to secrete notable amounts of IL- 

12p70 ( approx. 160 pg/ml). In summary, using this new maturation protocol, we could 

generate mature DCs which are able to both (i) migrate and (ii) secrete high levels of IL- 

12p70 providing a potentially new therapeutic tool for DC-based vaccination trials.

Silke Overbeck, Peter Uciechowski, M. Leigh Ackland, Dianne Ford, Lothar Rink 

Intracellular Zinc Homeostasis in Leukocyte Subsets is 

Regulated by Different Expression of Zinc Exporters ZnT-1 to 

ZnT-9 

Zinc is an essential trace element, which is a fundamental component of more than 300 

metalloenzymes and plays an important role in the immune system. Because zinc 

deficiency results in decreased immune functions, regulation of intracellular zinc 

homeostasis by zinc binding proteins and zinc transporters is crucial. In the present 

study, we quantified in a first global view the expression of all characterized human zinc 

exporters (hZnT-1-9) in different leukocyte subsets in response to zinc supplementation 

and depletion and analysed their influence on alterations in the intracellular zinc 

concentration. We found that hZnT-1 is the most regulated zinc exporter. Furthermore, 

we discovered that hZnT-4 is localised in the plasma membrane similar to hZnT-1. hZnT- 

4 is most highly expressed in T cells, up-regulated after treatment with PHA and is 

responsible for the measured decrease of intracellular zinc content after high zinc 

exposure. In addition, we found that hZnT-5, hZnT-6 and hZnT-7 in B cells as well as 

hZnT-6 and hZnT-7 in monocytes are up-regulated in response to cellular zinc 

depletion. Those zinc exporters are all localised in the Golgi network and this type of 

regulation explains the observed zinc increase in both cell types after up-regulation of 

their expression during zinc deficiency and, subsequently, high zinc exposure. 

Furthermore, we detected, for the first time, the expression of hZnT-8 in peripheral 

blood lymphocytes, which varied strongly between individuals. While hZnT-2 was not 

detectable, hZnT-3 and hZnT-9 were expressed at low levels. These data provide insight 

into the regulation of intracellular zinc homeostasis in cells of the immune system and 

may explain the variable effects of zinc deficiency on different leukocyte subsets.

Benedikt Fritzsching, Jürgen Haas, Fatima König, Eva Pauly, Johannes Pöschl, Peter 

Krammer, Wolfgang Brück, Elisabeth Suri-Payer, Brigitte Wildemann 

Intracerebral Human Regulatory T Cells: Analysis of CD4 

+CD25+FOXP3+ T Cells in Brain Lesions and Cerebrospinal 

Fluid of Multiple Sclerosis Patients 

Impaired suppressive capacity of CD4+CD25+FOXP3+ regulatory T cells (Treg) from 

peripheral blood of Multiple Sclerosis (MS) patients has been reported my multiple 

laboratories. However, it is unclear if Treg dysfunction in MS patients is limited to a 

reduced control of peripheral T cell activation since most studies analyzed only 

peripheral blood samples. Increasing evidence supports an anti-inflammatory role of 

Treg at the site of organ destruction during autoimmune diseases. To test if human Treg 

migrate into the CNS, we analyzed human tissue from active MS lesions by FOXP3 

immunohistochemistry in human brain biopsies. Similarly, we detected Treg from 

cerebrospinal fluid (CSF) of treatment-naïve MS patients by flow cytometry. Treg were 

detected in more than 50% of MS brain biopsies and in all CSF-samples from MS 

patients. However absolute numbers for FOXP3+ and CD4+ cells were rather low in MS 

lesions and Treg were not detectable in 30% of MS biopsies with CD4+ cell infiltrates. 

Given the high sensitivity of previously activated Treg (CD45ROhiCD95hi) towards 

CD95L-mediated apoptosis (*), we tested if CSF-derived lymphocytes contain a higher 

proportion of CD45ROhiCD95hi Treg than peripheral blood derived Treg from MS 

patients. Indeed, pairwise analysis of Treg in CSF and peripheral blood from the same 

MS patients clearly demonstrated an enhanced proportion of apoptosis-sensitive Treg in 

the CSF. We suggest, that intracerebral elimination of Treg by CD95L-mediated 

apoptosis could critically affect Treg numbers in the MS lesion. 

(*) Fritzsching et al., Cutting Edge, J. Immunology. 2005 Jul 1; 175(1):32-6

Sebastian Kreiter, Mustafa Diken, Abderraouf Selmi, Abdo Konur, Michael Koslowski, 

Christoph Huber, Özlem Türeci, Ugur Sahin 

Intranodal RNA immunisation – a potent method for the 

induction of immunity by selective transfection of DC 

We propose the usage of naked IVT-RNA given as intranodal (i.n.) injection as a new 

vaccination method. We could show by FACS analysis after i.n. injection of eGFP RNA 

into inguinal lymph nodes (LN) of BALB/c mice that DCs were transfected. Histological 

analysis of RNA uptake (Cy5-RNA) in LN showed a positive signal for CD11b+ DCs in 

the paracortical zone with sparing of B-cell follicles. By FACS analysis of similar treated 

LN we could show that a RNA signal was predominatly measurable in CD11c/CD11b+ 

myeloid DCs. Using in vitro and in vivo test systems we proved that human and mouse 

DCs have a high capacity for uptake of RNA in comparison to other cell populations. To 

assess the translational efficacy we used the firefly luciferase system and in vivo 

luminescense. After i.n., i.d. or s.c. RNA injection we measured time kinetics. The 

results showed superior translational efficacy (factor 10-20) for i.n. application in 

comparison with other application modalities 

Using Influenza-HA specific CD4+ and CD8+ T-cells for adoptive transfer experiments 

we tested the ability of i.n. RNA vaccination to expand T-cells. Strong i.n. expansion 

followed by systemic distribution of T-cells was detected. They showed full effector 

functions as shown by IFNγ secretion and cytotoxicity. Priming in naïve mice was tested 

after i.n. immunisations with SIINFEKL RNA resulting in up to 29 % antigen-specific CD8 

+ T-cells. Furthermore we showed T-cell priming against Influenza-HA, hCMV pp65 and 

gp70. We could show superior T-cell expansion with i.n. RNA in comparison to i.d. or s. 

c. immunisation. In tumor protection assay using an A20 Influenza HA transfectant cell 

line all mice were long time protected after 4 i.n. RNA immunisations. 60% of BALB/c 

mice survived in therapeutic assay after 5 i.n. RNA immuniations. 

In summary we have shown that the usage of naked IVT-RNA for intranodal 

immunisation is feasible, preserves lymph node structure, confers selective transfection 

of DCs, leads to expansion of antigen-specific T-cells and allows the induction of antitumor 

immunity.

Stephanie Konrad, Linda Engling, Reinhold E. Schmidt, J. Engelbert Gessner 

INVERSE REGULATION OF THE MURINE FcγRIIB AND FcγRIII 

GENE EXPRESSION BY C5a IS MEDIATED THROUGH DISTINCT 

DNA-RESPONSE ELEMENTS 

The crucial role of Fcγ receptors (FcR) in antibody-mediated autoimmune diseases is 

reflected in gene deletion studies in mice. Like other immune regulatory receptor pairs, 

the FcR system is constituted by a balanced action of activating and inhibitory receptors 

that bind immune-complexed IgG with same affinity. Analyses of animal models have 

shown that the inhibitory FcγRIIB can suppress antibody-mediated autoimmunity, 

whereas activating FcR - FcγRIII promote disease development. Although in vivo 

analysis of alveolar and peritoneal macrophages in mice shown an indispensable role of 

the complement component C5a in the regulation of FcR and the sensing of FcRdependent 

effector cell responses but the molecular mechanism is still unclear. To 

understand the C5a-mediated regulatory mechanisms of FcR expression, we cloned 

both the murine FcγRIII and FcγRIIB genes and demonstrated that inverse gene 

regulation of FcγRIII and FcγRIIB is mediated by functionally distinct cis-active DNA 

promoter elements in their 5´-end regions, which show a similar sequence motive. 

Deletion analysis defined a contribution of the 48-bp(–808/–760) region of the FcγRIII 

promoter and the promoter-associated intronic 42-bp(+442/+484) region of FcγRIIB in 

confering C5a responsiveness of FcγRIII induction versus FcγRIIB suppression. The 

importance of Gi, PI3K and Akt signaling molecules in the C5aR activation pathway that 

differentially control FcR reporter gene activities was further established by 

pharmacological inhibition studies in transfected MH-S and RAW 264.7 macrophage cell 

lines. This study provides insight into the transcriptional regulation of FcR, 

demonstrating the importance of functional different DNA elements in the murine 

FcγRIIB and FcγRIII genes. 

Research supported by SFB587 to JE.G.

Luise Weigand, Xiaoling Liang, Florian Anderl, Judith van der Griendt, Ingrid Schuster, 

Andreas Moosmann, Bernhard Helga, Elfriede Nößner, Dirk Busch, Angela Krackhardt 

Investigation of allorestricted peptide-specific T cell 

responses against Her2/neu – implications for adoptive T cell 

therapy in solid cancer 

Complete remissions have been observed after adoptive T cell transfer in different solid 

cancers including metastatic breast cancer. As allorestricted peptide-specific T cells 

might be a source of specific high-avidity T cell responses against tumor cells, we aimed 

to generate allorestricted T cells with specificity for the model antigen Her2/neu. 

We used T2 cells pulsed with different peptide concentrations of the Her2/neu-derived 

peptide 369 for stimulation of HLA-A2-negative T cells. Peptide-specific T cells were 

isolated by tetramers and streptamers and cloned by limited dilution. Proliferating T cell 

clones were analyzed for their function and T cell receptor repertoire. 

Repeated T cell stimulation with low antigen dose and tetramer-sorting resulted in 

highly peptide-specific T cells. However, tumor reactivity was low. In contrast, one-time 

stimulation resulted in T cells with higher tumor reactivity but partially alloreactivity 

independent of peptide concentration. Interestingly, all CTL clones sorted by tetramers 

showed an intermediate avidity for peptide-pulsed target cells, independent of the 

antigen concentration they have been stimulated with. Sorting with streptamers 

resulted in T cells with high peptide-specificity and these T cells partially showed tumorreactivity. 

Specific T cell clones revealed an almost exclusive usage of the V beta 6.3 

family although different recombined CDR3 sequences have been identified. Finally, 

cocultivation of highly active allorestricted T cells with tumor cells resulted in increased 

IL-8 secretion which has been reported to play an important role in tumorigenesis and 

metastasis. 

In conclusion, allorestricted T cells with specificity for Her2/neu (peptide 369) may 

represent potent tools for targeting Her2/neu-overexpressing tumor cells. However, 

selective tumor-reactivity and potential alloreactivity of allorestricted peptide-specific T 

cell receptors needs to be further investigated by transfer studies. Moreover, the 

potential role of IL-8 in tumor escape in this setting needs to be explored.

Stefanie Margraf, Carsten Watzl 

Investigation of membrane microdomains surrounding 

Natural Killer cell receptors 

Natural killer (NK) cells play a central role in linking the adaptive and the acquired 

immune system. They represent the first lymphoid subpopulation in the defence against 

tumors and viral infection. The activity of NK cells is regulated by the interplay of 

activating and inhibitory surface receptors. The engagement of these receptors as well 

as the localization within different membrane areas is crucial for signalling. We have 

recently shown that the activating receptors 2B4 (CD244) and NKG2D are localized in 

so called ‘lipid rafts’ upon ligand binding or stimulation by mAbs. The localization in 

these specialized membrane domains is crucial for the function of these receptors. Lipid 

rafts are typically analyzed via cold non-ionic detergent lysis and sucrose density 

gradient centrifugation. However, this method has been criticized to result in artefacts. 

A limitation of this method is the isolation and examination of all lipid raft membrane 

fractions and their associated proteins within a cell. Here we use a new approach to 

isolate membrane fragments surrounding a specific surface receptor without destroying 

its natural composition. NK cell receptors are stimulated by antibodies coupled to 

magnetic beads. NK cells are then physically disrupted in a nitrogen-cavitation bomb 

and membrane fractions surrounding the stimulated receptor are immunoisolated by 

separating the magnetic beads. The protein composition of the purified membrane 

clusters is then analyzed via 2D-Gelelectrophoresis. Examination of the natural 

environment of activating and inhibitory NK cell receptors will give us a new insight into 

the structure and composition of lipid microdomains and will help us to understand their 

role in the regulation of NK cell activity.

Vivienne Engelschalt, J. Engelbert Gessner, Richard A. Kroczek 

Involvement of complement and Fc-receptors in the depletion 

of T cells in vivo 

Antibody-mediated depletion of specific cell populations is a promising tool in the 

therapy of cancer and autoimmune diseases. Although depletion of cell populations has 

been successfully performed in a number of systems in vivo, only very few studies have 

dealt with the involved mechanisms. We have chosen to analyze the depletion of CD4 + 

T cells in the mouse as an initial model system. The application of a single dose of 100 

µg of the anti-CD4 mAb YTS191.1 resulted in a 70-80% depletion of CD4 + T cells in the 

spleen and peripheral lymph nodes (popliteal, mandibular, mesenteric, axillary, Peyer’s 

patches) within 72 h, whereas no depletion was observed in the thymus. Significant 

depletion could be observed after 12 h, the maximal effect was achieved around 72 h; 

the kinetics of depletion were comparable in all lymphatic organs. Complement C3deficient 

mice gave the same results as wild-type animals, ruling out a major 

involvement of this factor. In contrast, a dramatic reduction of CD4 + T cell depletion 

was observed in animals lacking the γ-chain common to Fc-receptors, indicating a 

critical involvement of Fc-receptors in antibody-mediated cell depletion. In animals 

lacking only FcγRII, no change in the depletion pattern was observed. Mice lacking 

FcγRI, FcγRIII, or a combination thereof, showed a more discrete reduction in depletion, 

suggesting that the absence of individual Fc-receptors can be compensated by the 

remaining Fc-receptors. Our present investigations are aimed at determining the role of 

NK cells in CD4 + T cell depletion. Further, we will attempt to define the Fc-receptorbearing 

cell type(s) involved in the depletion process.

Alexander Fassold, Werner Falk, Rainer H. Straub 

Is neuropilin-2 a possible target for the treatment of arthritis? 

Background: Sympathetic neurotransmitters in high concentrations exert antiinflammatory 

effects via beta2-adrenergic receptors. Their loss in inflamed tissue 

probably aggravates inflammation. Semaphorin 3C and placental growth factor, which 

bind to neuropilin-2 (NPN-2), are probably involved in repulsion of sympathetic nerve 

fibers (SNF). 

Objectives: To study density of SNF using NPN-2 as the antigenic target. To neutralize 

ligands to NPN-2 with a self-manufactured NPN-2 Fc fusion construct and to investigate 

the effects of inhibition in vitro and in vivo. 

Methods: Immunohistochemistry was used to investigate presence of NPN-2 positive 

nerve fibers in synovial tissue of 10 patients with rheumatoid arthritis (RA) and 10 with 

osteoarthritis (OA). In order to be able to study the effect of the Fc fusion construct, 

sympathetic ganglia of mice were cultured and outgrowth speed analysed. The NPN-2 

Fc-fusion construct was tested in vivo in collagen type II-induced arthritis in DBA/1 

mice. 

Results: In patients with RA compared to OA, density of NPN-2– positive nerve fibers 

was decreased (p

Selina Christen, Edith Hintermann, Monika Bayer, Urs Christen 

JAM-C and its influence on the pathogenesis of type 1 diabetes 

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing 

beta-cells in the pancreas. Recruitment of inflammatory cells, such as T-cells, B-cells 

and dendritic cells is prerequisite to beta-cell-injury. Such a process includes several 

molecular interactions between circulating and endothelial cells. The junctional adhesion 

molecule (JAM) family proteins JAM-B and JAM–C, are expressed on endothelial cells of 

high endothelial vessels and appear to be involved in leukocyte rolling, firm adhesion 

and transmigration. It was recently demonstrated that after blocking of JAM-C ceruleininduced 

pancreatitis was efficiently attenuated in mice. Early intervention with a 

monoclonal antibody directed against JAM-C reduced cytokine production, leukocyte 

influx, and hence tissue damage. 

In order to investigate the influence of JAM-C on trafficking and transmigration of 

antigen-specific, autoaggressive T-cells we used the RIP-LCMV mice as a model system. 

These mice express the nucleoprotein (NP) of lymphocytic choriomeningitis virus 

(LCMV) as a target autoantigen specifically in the beta-cells of the islets of Langerhans 

and turn diabetic after LCMV infection. In such diabetic RIP-LCMV mice the expression 

of JAM-C is detectable around the vessels within the pancreas. Interestingly, 

immunohistological evaluation of pancreas sections revealed that JAM-C was expressed 

directly in between cellular infiltrations and beta-cells. 

Our data suggest that JAM-C might be involved in the final steps of trafficking and 

transmigration of antigen-specific autoaggressive T-cells to the islets of Langerhans and 

might therefore play an important role in the pathogenesis of T1D.

Nils Schoof, Frederike von Bonin, Lorenz Trümper, Dieter Kube 

Janus kinases are targets of tyrphostin AG17 and HSP90inhibitor 

17-AAG in classical Hodgkin Lymphoma 

Classical Hodgkin Lymphoma (cHL) is a malignancy originated of germinal center (GC) B 

cells. Defective immunoglobulin rearrangement should have destined these GC B cells 

for apoptosis. Multiple deregulated signaling pathways are associated with survival and 

proliferation of cHL cells. Chemotherapeutic regimens for cHL are associated with 

stagnant rates of secondary malignancies requiring the development of new therapeutic 

strategies. 

Recently, we and others have shown that permanently activated Signal Transducer and 

Activator of Transcription (STAT) molecules are essential for cHL cell proliferation, and 

inhibitors of the tyrphostin-class are capable of inhibiting STAT tyrosine 

phosphorylation. Here we focused on the Janus kinases (Jaks), the major components 

involved in signal transduction from cytokine receptors to the STAT transcription 

factors. In cHL cells we observed high levels of permanently tyrosine phosphorylated 

Jak1, Jak2, Jak3 and Tyk2. Tyrphostin AG17 reduced tyrosine phosphorylation of Jaks1- 

3 in cHL cell lines in vitro and decreased tumour formation of L428 cHL cells in 

chorioallantoic membrane assay in vivo. Since Jaks are thought to be stabilised by heat 

shock protein 90 (HSP90) and cHL cell proliferation is inhibited by HSP90-inhibitor 17- 

AAG, the effects of 17-AAG on Jak-STAT signaling in cHL cells were investigated. 17- 

AAG led to a complete inhibition of STAT1, -3, -5 and -6 activation. Moreover, 17-AAG 

treatment was accompanied by significant reduction of Jak protein expression. To 

further test the role of HSP90 in stabilisation of Jaks co-immunoprecipitations as well as 

RNAi against HSP90 were performed. 

Our results suggest that recently described effects of 17-AAG on cHL cell proliferation 

are due to inhibition of Jak-STAT signaling. Therapeutics comprising inhibition of Jaks 

either by dephosphorylation or downregulation, with tyrphostin AG17 and 17-AAG 

respectively, may be a promising strategy in cHL and other cancer entities associated 

with permanent STAT activation.

Eva Schlecker, Isabel Hartmann, Michael Ackmann, Elisabeth Weiss 

KIR2DS2 and interaction with DAP12 

The KIR locus is characterized by fluctuating gene content, which mainly depends on 

the presence or absence of activating KIRs, and allelic variation. Whereas the function 

and ligands of the inhibitory KIRs have been extensively studied, similar facts are 

lacking for the activating KIRs. Several studies have demonstrated that activating KIRs 

might be involved in the progression of autoimmune disease and also play a role in 

transplantation situations. For most of the activating receptors no ligands have been 

identified yet. We tried to establish a cell based read-out system to identify ligands for 

activating KIRs by coexpression of KIR2DS2 and DAP12 in Jurkat and HEK 293T cells in 

the presence of luciferase reporter vectors regulated by NFAT or a minimal IFNgamma 

promoter. 

In HEK293T cells DAP12 was higly expressed at the cell surface even in the absence of 

KIR2DS2. Thus, DAP12 cell surface transport does not need transmembrane partners or 

DAP12 associates with a yet unknown polypeptide in these cells. KIR2DS2 was only 

weakly detected at the cell surface in both transfectant cell types. It is possible that the 

GL183 antibody does not recognize KIR2DS2 efficiently. Cross-linking of KIR2DS2, 

DAP12 or KIR2DS2/DAP12 did not result in the activation of the luciferase reporter 

vectors. Moreover, no tyrosine phosphorylation of DAP12 was detected, whereas using 

antibody stimulation of cells expressing hybrid CD3zeta/CD94 and NKG2A molecules 

resulted in tyrosine phosphorylation of CD3zeta. We conclude that on the one hand the 

low binding of the GL183 antibody to KIR2DS2 might be responsible for the lack of 

activation, as several studies have shown that interactions between low-avidity ligands 

could induce an inhibitory signal by recruiting SHP1 to the signalling ITAM complex and 

that DAP12 can also inhibit cellular activation. On the other hand, the association of 

KIR2DS2 with DAP12 might also trigger a repressive state.

Gleb Turchinovich, Jan Kranich, Sonja Schmid, Jürgen Bachl, Jörg Kirberg 

Kruppel-like factor 3 (KLF3) affects marginal zone B cell 


We have demonstrated that the hypermutating Abelson-leukemia virus induced pre-B 

cell line 18-81 harbors a single provirus within the KLF3 locus, leading to constitutive 

KLF3 expression. To study functions of KLF3 in B cells we generated transgenic mice 

over-expressing KLF3 (CD19:KLF3). 

Surprisingly, marginal zone (MZ) B cells were increased 3-10 fold in CD19:KLF3 mice. 

To determine whether the increase is caused cell autonomously, competitive fetal liver 

chimaeras were generated. Here, KLF3 over-expression did not affect the maturation of 

FO B cells as these were derived from normal or CD19:KLF3 cells to the same extent as 

they contributed to immature B cells, respectively. In contrast, the MZ B cell 

compartment became almost exclusively dominated by CD19:KLF3 transgenic cells. To 

ascertain that this altered lineage commitment occurs independent to receptor 

specificity, CD19:KLF3 mice were crossed to B1-8H/3-83κ knock-in mice. Normally, B 

cells expressing the B1-8H/3-83κ combination are mostly in the FO, while cells 

expressing another light chain are enriched in the MZ. Strikingly, KLF3 over-expression 

drives B1-8H/3-83κ expressing cells to mature extensively into the MZ lineage. Thus, 

KLF3 activity alters MZ commitment independent of BCR receptor specificity. However, 

no evidence of increased Ca++ mobilization, tyrosine-phosphorylation, or receptor 

induced proliferation was detectable in KLF3 over-expressing B cells. 

The KLF3 homologous factor KLF2/LKLF regulates S1P-R expression in T cells affecting 

cell migration. We hypothesized that KLF3 over-expression in B cells similarly leads to 

constitutive S1P-R expression and consequently enhanced B cell migration into/or 

retention within the MZ. Normally, LPS stimulation leads to S1P-R down-regulation and 

MZ B cell migration into the follicle. In contrast to our expectation, the migration of MZ 

B cells into the follicle following LPS stimulation was unaffected in CD19:KLF3 transgenic 

mice indicating that S1P-R expression is still under physiological control. 

Thus, further experiments will focus on alternative pathway(s) known to modulate the 

maturation of MZ and FO B cells.

Dietmar Zehn, Michael J. Bevan 

Lack of peripheral control of low avidity self reactive T cells 

Autoimmune diseases develop despite the presence of numerous safeguards to 

eliminate or control T cells directed against self-antigens. This raises the question how 

disease-causing T cells escape elimination or inactivation. We have studied the 

repertoire of T cells directed against a pancreatic antigen that is also ectopically 

expressed in the thymus. T cells responding strongly to this antigen are efficiently 

eliminated. In contrast, central and peripheral tolerance routinely fail to eliminate cells 

that are weakly reactive with this organ specific self-antigen. These low avidity T cell 

cause autoimmunity upon activation via molecular mimicry. While in the periphery, 

these low avidity T cell continuously encounter tissue derived cognate self-antigen 

crosspresented by lymph node dendritic cells. We have analyzed how this chronic 

exposure impacts the low avidity T cells. Our data indicate that the low avidity cells can 

be detected throughout life and retain their disease causing potential as the mice age. 

Moreover, the ability to recruit low avidity T cells into autoimmune responses does not 

rely on the supply of recent thymic emigrants nor does the presence or absence of the 

cognate self-antigen in the periphery alter the reactivity of these low avidity T cells. In 

the model we study, low avidity self-reactive T cells are long lived populations that are 

not changed by peripheral antigen recognition. Thus, we conclude that cells like these 

are a normal part of the T-cell repertoire in healthy individuals. The constitutive 

presence of such cells requires mechanisms that prevent their activation. Unexpectedly, 

the selective elimination of Foxp3+ T cells did not enhance autoimmunity, but rather 

interfered with the ability of low avidity T cells to induce disease.

Gordon Wilke, Gretel Wittenburg, Claudia Berek 

Laser Capture Microdissection and Mircroarray: a 

Characterisation of Follicular Dendritic Cells 

Follicular dendritic cells (FDC) play a crucial role in B cell homeostasis and 

differentiation, nonetheless little is known about this cell type. In the primary follicle B 

cells are embedded into a network of FDC. When germinal centres are induced, FDC 

build the micro-environment where the affinity maturation of the immune response 

takes place. Their tight interaction with B cells and also their fragility make the study of 

FDC a challenging undertaking. 

To further understand the role of FDC within the follicle and their close interaction with 

B cells a new approach was taken. Tissue sections were prepared, stained with FDCspecific 

antibodies and FDC networks of primary follicle directly isolated by Laser 

Capture Microdissection. In addition, spleen suspensions were prepared and follicular B 

cells sorted by FACS. From both cell preparations RNA was extracted and amplified. 

Gene expression was analysed by hybridisation to Affymetrix microarrays. 

Gene expression in FDC networks and in B cells was compared and differentially 

expressed genes in the FDC network identified. The expression of genes characteristic 

for FDC such as CXCL13, VCAM-1 and BP3 demonstrates that this procedure is useful 

for the identification of novel FDC genes. A set of interesting genes was selected and 

their specific expression in FDC verified by immunohistology and in situ hybridisation. 

Murine knock-out models for novel FDC genes were used to demonstrate their effect on 

the development of primary follicles. 

In addition, the question of the origin of FDC was addressed by comparing the 

expression pattern of FDC and stromal cells of splenic follicle anlagen. Finding a high 

degree of accordance in gene expression supports the hypothesis that FDC develop 

from sessile stromal cells.

Katja Farhat, Sabine Riekenberg, Jennifer Debarry, Roland Lang, Jörg Mages, Günther 

Jung, Karl-Heinz Wiesmüller, Artur J. Ulmer 

LIGAND BINDING AND SIGNAL TRANSDUCTION INDUCED BY 

LIPOPEPTIDES 

Toll-like receptors (TLRs) are primary triggers of the innate immune system by 

recognizing various microorganisms through conserved pathogen-associated molecular 

patterns (PAMPs). Among all TLRs, TLR2 is the receptor for a functional recognition of 

bacterial lipopeptides (LPs) and is upregulated during various disorders like chronic 

obstructive pulmonary disease (COPD) and sepsis. This receptor is unique in its ability 

to form heteromers with either TLR1 or TLR6 to mediate intracellular signaling. 

According to the fatty acid pattern as well as the assembling of the polypeptide tail LPs 

can signal through TLR2 in a TLR1- or TLR6-dependent manner. As shown recently 

there are also di- and triacylated LPs which stimulate TLR2 independently of TLR1 and 

TLR6. 

In this study we investigate the signal transduction pathways activated by the different 

TLR2 dimers in murine bone marrow derived macrophages, using the synthetic LPs 

PamOct2C-(VPGVG)4VPGKG, FSL 1 and Pam2C-SK4 to specifically target TLR2/TLR1, 

TLR2/TLR6 and TLR2, respectively. Immunoblotting of MAP-kinases, usage of dominant 

negative forms of adaptor molecules as well as microarray analyzes indicate that all 

dimers use the same signaling cascade to activate transcription factors leading to an 

identical pattern of gene activation. In conclusion, LPs from mycoplasma, Gram-positive 

and Gram-negative bacteria, although they may use different TLR2 dimers, induce the 

same signal transduction pathway during infection.

Christian Draing, Christoph Rockel, Susanne Deininger, Stefanie Sigel, Oliver Dehus, 

Tamara Rupp, Artur Ulmer, Thomas Hartung, Corinna Hermann, Sonja von Aulock 

Lipoteichoic acid from a lipoprotein diacylglycerol transferase 

deletion mutant is a potent immunobiologically active 

compound 

Lipoteichoic acid (LTA) is a potent immunostimulatory surface component of Grampositive 

bacteria as shown by preparation of the full native structure by optimized 

isolation procedures in the absence of contaminations, e.g. lipopolysaccharide, and by 

chemical synthesis of a full structure based on the LTA of Staphylococcus aureus. 

This was recently challenged with an LTA preparation from a lipoprotein diacylglycerol 

transferase deletion (Δlgt) mutant, reported to lack palmitate-labeled lipoproteins and 

immunostimulation. In the present study, however, LTA from the mutant and LTA from 

the respective wild type SA 113 strain induced a comparable release of TNFα in 

incubations with human whole blood or peripheral blood mononuclear cells. In contrast, 

the LTA from the mutant strain was unable to induce cytokine release in the murine 

macrophage cell lines J774.1, MH-S or primary murine macrophage populations from 

the bone marrow or peritoneum. Likewise, the human monocytoid cell line THP-1 was 

unresponsive to the LTA of the mutant strain. The unresponsiveness of the human cell 

line was caused by an inhibitory effect of the FCS used as medium supplement; that of 

the murine cells by differences in the composition of fatty acids compared to LTA from 

the wild type strain and not by the lack of lipoproteins. Thus, LTA and not lipoproteins 

represent the main stimulus of Gram-positive bacteria in human leukocytes.

Anna Schurich, Silke Hegenbarth, Jan Böttcher, Sven Burgdorf, Andreas Dolf, Elmar 

Endl, Christian Kurts, Percy A. Knolle 

Liver sinusoidal endothelial cells are more efficient in crosspresentation 

than CD8alpha splenic dendritic cells expressing 

endocytic receptors devoted to cross-presentation 

Liver sinusoidal endothelial cells (LSEC) constitute a unique antigen presenting cell 

population. LSEC efficiently take up and cross-present antigen to naïve CD8 T-cells 

inducing T-cell tolerance. In order to unambiguously investigate and compare crosspresentation 

in LSEC to conventional APC such as DCs we developed a new isolation 

method that involves functional identification of LSEC in vivo via their extraordinary 

scavenger activity and fluorescence activated cell sorting. This method yielded highly 

pure cell populations (≥ 99.9 %) for functional analysis ex vivo. We compared the 

efficiency of isolated LSEC and splenic DCs for cross-presentation of ovalbumin to CD8 

T- cells ex vivo one hour after intravenous antigen injection. LSEC were more efficient 

in cross-presentation on a per cell basis than CD8α positive splenic DCs. However, 

antigen was not retained in LSEC in vivo, because 90% of antigen was lost after 24h 

and cross-presentation decreased correspondingly. In contrast less efficient crosspresentation 

by DCs was maintained over 24h. It was recently shown that crosspresentation 

of soluble exogenous ovalbumin in DCs is determined by the receptor used 

for endocytosis: the mannose receptor (MR) routing ovalbumin into cross-presentation 

and scavenger receptors into MHC II restricted presentation. We found that MR cell 

surface expression was higher in LSEC than in DCs. MR -/- LSEC, in contrast to DC, do 

not rely exclusively on the MR, but were still capable of cross-presenting ovalbumin 

indicating expression of another receptor involved in cross-presentation. Further 

experiments will characterise this receptor. 

Collectively our data show that LSEC are more efficient than CD8&alpha positive DC in 

cross-presentation of soluble exogenous ovalbumin, utilising endocytic receptors 

devoted to cross-presentation.

Matthias Hardtke-Wolenski, Nadja Saal, Konstantinos Iordanidis, Mark S Anderson, 

Michael P Manns, Elmar Jäckel 

Liver specific immune responses related to AIRE mutations 

The AIRE gene (autoimmune regulator) has been identified as an important mediator of 

central tolerance against ectopically expressed peripheral antigens. Mutatations of AIRE 

are responsible for autoimmune polyendocrinopathy syndrome type I (APSI) in human 

characterized by a multiorgan autoimmune disease. Autoimmune hepatitis (AIH) is part 

of the clinical spectrum in humans and mice. 19% of patients with AIRE mutations 

develop an autoimmune hepatitis which is characterized by autoantibodies against 

cytochrome P450 2A6 and 1A2. 

We investigated AIH in mouse models with various AIRE mutations on different genetic 

backgrounds. Initially we characterized the immunohistochemical staining pattern of 

sera from AIRE -/- mice on rat liver, kidney and stomach sections and on HepG2 cells. In 

parallel, sera analysis has been done on western-blots loaded with whole liver lysate. 

We could demonstrate that the the strength and broadness of the humoral immune 

response correlates well with disease activity of the AIH in AIRE deficient mice. We 

could show that AIH development is dependent on the genetic background used. 

Furthermore within the Balb/c background AIH development is dependent on the 

underlying AIRE mutation. Humoral immune responses in AIRE deficient mice are not 

directed against the antigens described for human AIH. We therefore set out to 

characterize the target antigens of the adaptive immune response in mice with AIH to 

study the defects in their immune tolerance. We therefore developed a column retention 

assay of for non-denatured liver antigens followed by a proteomics analysis with mass 

spectroscopy. With this approach we identified several potential new autoantigens of the 

humoral immune response.

Stephan Borte, Uwe Gerd Liebert, Michael Borte, Ulrich Sack 

Long-term cell-mediated immunity following vaccination with 

live, attenuated measles-mumps-rubella-vaccine in children 

with juvenile idiopathic arthritis under treatment with lowdose 

Methotrexate and/or tumour necrosis factor α receptor 

antagonist 

Juvenile idiopathic arthritis (JIA) represents a heterogeneous group of disorders 

characterized by chronic inflammatory arthritis and turns out to be the commonest 

rheumatic disease seen in childhood worldwide. The multidisciplinary management of 

JIA is founded on anti-inflammatory and immunomodulating drugs. However, infection 

is one of the leading causes of morbidity and mortality in JIA. The risks of serious 

adverse events following vaccination and the immunogenicity of vaccines at all have 

been a matter of controversy and there is some reluctance to vaccination that need to 

be enlightened. We intended to display the course of humoral and cell-mediated 

immunity to measles-mumps-rubella-vaccination (MMR) in healthy children and 

adolescents and to evaluate potential influences of low-dose Methotrexate therapy and 

Etanercept treatment in JIA on success of immunisation and maintenance of long-term 

immunity. Therefore, production of IFNγ by T memory cells upon in vitro stimulation 

with measles, mumps and rubella antigens and seroprevalence of virus-specific IgG 

antibodies were investigated in PBMC and plasma from 16 healthy children and 16 

children with JIA, being treated with low-dose Methotrexate or in combination with 

Etanercept. Our results indicate that MMR-vaccination induced immunity in childhood is 

characterized by steady decline of humoral immunity and development of long-term cellmediated 

immunity. Low-dose MTX therapy following completed MMR-vaccination was 

proved not to hamper Th1-like cell-mediated immunity in vitro. Furthermore, neither 

low-dose MTX nor Etanercept treatment during the course of vaccination showed to 

markedly interfere with intended outcome of immunisation and generation of long-term 

cell-mediated immunity to MMR. No cases of serious adverse events following MMRvaccination 

were observed. In conclusion, these results argue for MMR-vaccination in 

children with JIA using live, attenuated vaccines to ensure protection from infection and 

to prevent morbidity and mortality related to this diseases.

Christian Pötschke, Mandy Busse, Annegret Dummer, Tobias Traeger, Wolfram 

Keßler, Erika Friebe, Marlene Mikulcak, Claus-Dieter Heidecke, Stefan Maier, Barbara 

Bröker 

Long-term effects of polymicrobial sepsis on the adaptive 

immune system 

Generalized polymicrobial infection leading to sepsis remains a common and life 

threatening condition. Following initial hyperinflammation, many patients experience a 

hypoinflammatory phase, for which there is no therapy. Furthermore long-time 

survivors have an increased mortality rate. Using colon ascendens stent peritonitis with 

intervention (CASP-I), a clinically relevant mouse model of diffuse peritonitis, we have 

investigated the long-term consequences of sepsis. 

After CASP-I inflammatory cytokines like TNFα, IL-6, IL-10 and MCP-1 were increased in 

the serum of C57BL/6 mice on the first day of sepsis, while IL-12 and IFNγ became 

measurable on day seven. In the thymic cortex a dramatic increase of apoptosis 

occurred, leading to the elimination of thymocytes by day three. On day 14 thymic 

architecture and thymocyte counts were reconstituted. In the spleen Foxp3-positive as 

well as Foxp3-negative T cells rapidly upregulaed CTLA-4. Later, we observed germinal 

centers and, in the red pulp, structures sharing features with germinal centres. This 

may be related to the strong increase of total IgM and IgG titers in the serum seven 

and 14 days after CASP-I, respectively. 

When a primary immune response to the model antigen TNP-KLH was induced following 

CASP-I, the ex vivo splenocyte proliferation in response to KLH decreased over time 

following sepsis. In contrast, the TNP-specific IgM and IgG concentrations increased. 

Surprisingly, a similar increase of TNP-reactive antibody titers was also observed after 

CASP-I in mice that had not been immunized. 

Our data indicate that generalized bacterial infection leads to the rapid activation of the 

adaptive immune system. The balance between regulatory and effector T cell 

subpopulations may decide about the outcome of sepsis. In pilot studies, the primary T 

cell response to a novel antigen appeared to be suppressed following sepsis. The 

presumed lack of T cell help for B cells in sepsis appeared to be compensated by a 

polyclonal B cell response. How these B cells are activated will be the focus of our future 

studies.

Zulema Cabail, Holger Hoff, Heike Hirseland, Steven Nadler, Gerd R. Burmester, Monika 

C. Brunner-Weinzierl 

Longevity of CD28null T Lymphocytes is abrogated by CTLA- 

4Ig treatment 

CTLA4Ig engages CD80 and CD86 on antigen-presenting cells, which are both ligands 

for CD28 and CD152 on T cells. CTLA4Ig-treatment has demonstrated efficacy in 

treating rheumatoid arthritis and psoriasis. The traditional view of the mechanism of the 

CTLA-4Ig treatment is inhibition of CD28-costimulation preventing activation of 

inflammatory T lymphocytes. 

In autoimmune diseases such as rheumatoid arthritis, CD28null T cells accumulate in 

the joint which show high IFN• production and longevity. In this study, we used 

CTLA4Ig (abatacept•) to investigate the CD80/CD86 costimulatory requirements of 

heterogeneous CD28null T cells. First we could show that despite the loss of CD28 

expression these cells are able to express intracellular CD152. Using a sensitive staining 

method, we demonstrate that they also express CD152 at their cell surface. In vitro 

activation of CD28null CD4 or CD8 T cells in the presence of CTLA4Ig leads to enhanced 

frequencies of AnnexinV+ T Lymphocytes: 10% for CD8 and 25% for CD4 T 

lymphocytes. IDO activation by CTLA-4Ig engagement of CD80 and CD86 was excluded. 

Their Fas -expression at the cell surface was upregulated independently of CTLA-4Ig 

treatment. Apoptosis induction was confirmed demonstrating enhanced Caspase 

activation in CD28null cells stimulated under CTLA-4Ig treatment. These results suggest 

that the efficacy of the CTLA4Ig treatment might be, at least in part, due to absent 

CD152 signalling in inflammatory lymphocytes inducing their elimination by apoptosis.

Anja Siepert, Birgit Sawitzki, H.M. Reichardt, Jochen van den Brandt, Markus Tiedge, 

Manfred Lehmann, Hans-Dieter Volk, Petra Reinke 

Low dose CNI treatment can control effector function of 

depletion resistant 

allo-specific memory T cells (financial support by Else-Kröner- 

Fresenius-Stiftung P14/06//A01/06) 

Donor-specific tolerance to transplanted tissues remains an elusive goal in clinical 

transplantation. Lymphocyte depletion is a commonly used approach in clinical renal 

transplantation as part of standard induction immunosuppressive therapy but also 

tolerance induction protocols. But preliminary studies in humans have shown that 

monotherapies with depleting antibodies can result in severe acute rejection episodes. A 

high pool of donor reactive memory T-cells in transplant recipients is considered to be 

the main reason for these rejection episodes. 

Here we developed an experimental transplant model with an increased allo-specific 

memory T cell pool and tested their influence on graft survival and responsiveness to 

different therapeutic approaches. 

For tolerance induction kidney grafted rats (DA to LEW) were treated with combination 

of the depleting mAbs Ox8 (anti-rat CD8; 4 x 1 mg/kg b.w.) and Ox38 (anti-rat CD4; 4 

x 10 mg/kg b.w.). Allo-specific memory-like T-cells (15 x 106 cells) were transferred 7 

days prior to transplantation. To determine the effect of CNI on memory-like T-cells rats 

were treated additionally with short time or permanent low dose CyA (3 mg/kg b.w. d 0- 

9 or d 0-150). Serum creatinine and survival of allografted rats were used as read-out 

parameters. Graft biopsies from days 5 and 150 pTx were evaluated by 

immunohistology. 

Long-term function of kidney grafts was maintained by perioperative T-cell depletion 

using mAbs Ox8 and Ox38 (MST >150 d). Additional application of allo-specific memory 

T-cells resulted in acute rejection in 5 out of 6 rats (MST >12,7 ± 11,8). Despite 

inducing strong T-lymphopenia, surviving transferred allo-specific memory T cells can 

affect the graft. Short time CyA-application led to prolongation of graft survival time 

with a declining transplant function, 4 out of 6 rats died (MST >51,3 ± 24,8). In 

contrast, permanent CyA therapy induced long-term acceptance of kidney grafts (MST 

> 80 d). 

Depletion of T-lymphocyte combined with low dose permanent CyA is sufficient to 

control effector function of depletion resistant allo-specific memory T cells.

Markus Kleinewietfeld, Mireille Starke, Thomas Blankenstein, Kirsten Falk, Olaf 

Rötzschke 

Low dose cyclophosphamide tumor rejection is independent 

of CD4+ CD25+ regulatory T cells (Treg) 

Cyclophosphamide (Cy) is a chemotherapeutic widely used in cancer treatment. It is an 

alkylating agent that in high dosage (>400 mg/kg) effects the proliferating tumor cells. 

More recently it was rediscovered as an immunomodulatory drug. At a concentration of 

200-400 mg/kg it seems to target preferentially the cycling Tregs, which leads to 

decreased cell numbers, alters the ratio between effector and regulatory T cells and 

inhibits the suppressive capability of Tregs. Moreover, in several experimental tumor 

models striking rejections of tumour cells were observed at concentrations as low as 15 

mg/kg. 

In this study we show that in low dose rejection of tumors by Cy is not driven by 

neutralizing regulatory T cells. In BALB/c mice injected with J558L tumour cells we 

observed tumor rejection of established tumors by a single low dose Cy treatment (15 

mg/kg). This effect was independent of CD25+ T cells, since a depletion with αCD25 

prior the Cy administration did not affect the kinetics of tumor rejection. CD25 depletion 

alone without Cy treatment also induced a delayed rejection. Although, in principle, the 

neutralization of Treg cells can trigger tumor rejection also in this model system, the 

rapid rejection observed after low-dose Cy administration is apparently not a result of 

selective inhibition of Tregs. 

We conclude that low dose cyclophosphamide treatment can trigger rejection of certain 

tumors but depletion or inactivation of CD4+CD25+ T cells is not the driving force of 

tumor rejection. Other mechanisms have to be considered and in particular the 

induction of innate immune responses by low dose cyclophosphamide treatment has to 

be further investigated.

Juliane Ladhoff, Michael Bader, Sabine Brösel, Elke Effenberger, Isabela Schmitt- 

Knosalla, Hans-Dieter Volk, Martina Seifert 

Low immunogenicity of rat embryonic stem cell derivatives 

Introduction: Embryonic stem cells are suggested to be immune-privileged, but they 

carry the risk of tumor development. With proceeding differentiation they lose their 

tumor forming capacity, but they become immunogenic expressing a normal set of 

Major Histocompatibility Complex (MHC) molecules. This immunogenicity might trigger 

rejection processes after application in regenerative therapies. 

Methods: Two cell lines of rat embryonic stem cell like cells (RESC) were driven towards 

the endothelial lineage and analyzed for their phenotype. MHC levels in response to 

interferon-γ (IFNγ) were determined flow cytometrically compared to primary rat aortic 

endothelial cells (EC). Allo-responses were analyzed in vitro by Calcein-based 

cytotoxicity assays, determination of allo-antibody/complement mediated lysis and 

CFSE-based proliferation assays with allogeneic CD4+ T cells. Immune reactions in vivo 

were measured by allo-antibody production. 

Results: RESC derivatives expressed only low levels of MHC class I and several 

endothelial markers, but they do not express MHC class II. In response to IFNγ they 

induced MHC class I, however MHC class II expression remained absent, due to a 

lacking induction of the MHC class II transactivator. RESC derivatives displayed a 

diminished sensitivity to allo-specific T cell attack, were resistant to allo-antibody/ 

complement mediated lysis as opposed to high lysis rates of aortic EC. Furthermore, in 

vitro RESC derivatives exhibited a notably lower capacity to stimulate allogeneic CD4+ T 

cells. Allo-antibody production in vivo sustained these data. 

Conclusions: RESC derivatives elicit less allo-specific immune responses than their 

differentiated adult counterparts. Furthermore, they are resistant against humoral 

mechanisms of allorecognition and are partially protected against cellular immune 

attack. This opens up ways to use stem cell derived endothelium as a powerful tool in 

regenerative cell therapies.

Anette Brass, Shiyuan Hong, Nicole Schwarz, George Dubyak, Michel Seman, Friedrich 

Koch-Nolte, Friedrich Haag 

LPS and interferons induce surface expression and activity of 

ADP-ribosyltransferase ART2.1 on murine bone marrowderived 

macrophages 

Nicotinamide adenosine dinucleotide (NAD), a predominantly intracellular metabolite, is 

released into the extracellular compartment consequent to cell lysis or by regulated 

secretion. Extracellular NAD modulates immune and inflammatory responses by serving 

as a substrate for cell surface ADP-ribosyltransferases (ARTs) that transfer ADP-ribose 

from NAD to arginine residues on target proteins. In the murine immune system, the 

major sources of ART activity are the two ART2 isoforms ART2.1 and ART2.2, which so 

far have only been detected on T cells. On these cells, ADP-ribosylation of the P2X7 

purinoreceptor leads to activation of the receptor and rapid cell death. ART2.1 differs 

from ART2.2 in that it carries an extra disulfide bond, making its activity dependent on 

the presence of thiol reducing agents. We now report that bone marrow-derived 

macrophages (BMDM) from BALB/c mice up-regulate ART2.1, but not ART2.2, in 

response to multiple proinflammatory mediators including agonists for toll-like receptors 

(TLR) and type-1/2 interferons. Stimulation of BMDM with LPS, interferon-&Gamma 

(IFN-&Gamma) or interferon-&beta (IFN-&beta) induced high expression of ART2.1 as a 

GPI-anchored cell surface ecto-enzyme. The catalytic function of the induced cell 

surface ART2.1 was strictly dependent on the presence of extracellular thiol reducing 

cofactors, suggesting that in vivo activity of ART2.1-expressing macrophages may be 

potentiated in hypoxic or ischemic compartments. Consistent with the mutated Art2a 

gene in C57BL/6 mice, LPS- or IFN-stimulated BMDM from this strain lacked expression 

of cell surface ART2 activity in the presence or absence of extracellular thiol reductants. 

In transfection experiments, P2X7 was activated by ART2.1 in the presence of NAD and 

reducing agents. Collectively, these findings implicate NAD-dependent ADP-ribosylation 

as a potential immunoregulatory mechanism for inflammatory macrophages as well as 

for T cells.

Christian Schiller, John-Christian Eilert, Maximilian Nitschké, Alexander Seidl, Michael 

Schleicher, Dolores J. Schendel, Elisabeth H. Weiss 

LST1: a potential transmembrane adaptor protein that 

modulates cell morphology 

The MHC class IV region encodes numerous genes involved in inflammatory responses 

and harbours the LST1 gene which is strongly expressed in immune cells, especially 

monocytes and dendritic cells. LST1 undergoes extensive alternative splicing resulting in 

12 soluble or transmembrane isoforms. It has been proposed that LST1 plays an 

important role in both regulating the immune response and enabling cell-cell 

communication, but the exact mechanisms involved are yet to be elucidated. 

Overexpression of full lenght LST1 in a variety of cell lines results in the formation of 

numerous long and thin cellular extensions, we found these processes to have the same 

physical characteristics as tunneling nanotubes (TNT). However, in HeLa LST1 

transfectants microinjection experiments revealed that these did not enable transport of 

molecules between cells and were thus not functional. 

Membrane orientation of transmembrane LST1 proteins was determined by analyzing 

cell surface expression of FLAG-tagged LST1 isoforms transiently expressed in HEK 

293T cells. Our results show that LST1 encodes type-I transmembrane proteins with a 

short extracellular and a long cytoplasmic domain. Database analysis revealed several 

evolutionary conserved regions in the cytoplasmic domain including two tyrosine 

phosphorylation sites and one ITIM which displayed phosphorylation in pervanadate 

treated LST1 transfectants. We also found two conserved cysteines in the cytoplasmic 

domain to be required for the formation of LST1 homodimers and homotrimers. These 

results lead us to postulate that LST1 multimers may act as transmembrane adaptor 

proteins by transducing signals received from an associated coreceptor, which we are 

currently in the process of identifying.

Niklas Engels, Gökhan Yigit, Christoph Emmerich, Dirk Czesnik, Detlev Schild, Jürgen 

Wienands 

Lytic replication of Epstein-Barr virus can be induced by 

LMP2A 

The B-lymphotropic Epstein-Barr virus (EBV) encodes two integral membrane proteins, 

LMP1 and LMP2A, which can be expressed in infected B cells during difference stages of 

viral latency. LMP2A contains an immunoreceptor tyrosine-based activation motif 

(ITAM) in its N-terminal cytoplasmic tail that is thought to provide a maintenance signal 

for EBV-infected cells. However, no cellular systems are available to study LMP2A 

function independently of other virally encoded proteins. We have generated a Cre/LoxPbased 

system for inducible expression of LMP2A in the DT40 B cell line, which allows 

real time monitoring of LMP2A-induced signaling events. We can show that mere 

expression of LMP2A results in ligand-independent activation of protein tyrosine kinases 

and phospholipase-γ2 (PLC-γ2). Activated PLC-γ2 produces the second messengers IP3 

and DAG, which causes oscillatory cytosolic Ca2+ waves and activation of the Erk-MAP 

kinase pathway, respectively. The latter pathway turned out to be a mandatory positive 

feedback mechanism for LMP2A protein production itself. Eventually, LMP2A-activated 

signaling cascades culminate in expression of BZLF1, the key regulator of lytic EBVreplication. 

Hence, the physiologic function of LMP2A is not to support survival of 

latently infected cells but to act as positive regulator of lytic EBV replication.

Heiko Johnen, Tamara Kuffner, Andrew Cook, Emma Braine, Ben Wu, Roland Stocker, 

Samuel Norbert Breit 

Macrophage Inhibitory Cytokine 1(MIC-1) reduces disease 

severity in mouse models of arthritis and atherosclerosis 

Macrophage Inhibitory Cytokine 1 (MIC-1) is a member of the TGF-beta superfamily and 

is mainly expressed in the CNS, epithelial cells and macrophages. MIC-1 is upregulated 

in response to cellular stress, p53 activation, malignancy, injury and inflammation. 

Elevated serum MIC-1 levels were found in rheumatoid arthritis (RA) patients, and have 

been associated with an increased risk of developing cardiovascular diseases. 

Additionally, MIC1 has been detected in macrophages within human atherosclerotic 

lesions and in rheumatoid synovial tissue. These results suggest a role for MIC-1 in 

inflammatory or autoimmune diseases like atherosclerosis and RA. 

Methods: 

We created MIC-1 transgenic mice using the c-fms (macrophage specific) promoter. The 

effect of MIC-1 overexpression was analyzed by comparing disease progression in the 

ApoE KO mouse models of atherosclerosis, collagen induced arthritis (CIA), and the KRN 

model of serum transfer arthritis. 

Results: 

ApoE KO mice also overexpressing MIC-1 had smaller lesions in the aortic sinus and the 

thoracic aorta compared to ApoE KO mice, but unchanged serum levels of cholesterol, 

CRP, selected inflammatory mediators, and other serum markers of liver and renal 

function. In serum transfer arthritis and CIA, MIC-1 overexpression reduces disease 

severity. In CIA no difference in collagen specific T-cell proliferation or IgG production 

could be detected. 

Whilst the underlying mechanism is as yet not fully understood, the protection from 

disease in these 3 different animal models supports that MIC-1 acts as an antiinflammatory 

cytokine in vivo.

Katharina Kronenberg, Beate G. Exner, Christine Sattler, James A. Hutchinson, 

Gudrun E. Koehl, Stefan Farkas, Hans J. Schlitt, Fred Fändrich, Edward K. Geissler 

Macrophages Driven to a Novel State of Activation have Anti- 

Inflammatory Properties in Mice 

Recurrent episodes of inflammation underlie numerous pathologies, notably the 

inflammatory bowel diseases (IBD). Here, we describe a population of macrophagederived 

cells which mitigate autoimmune colitis in mice, leading to histological 

resolution of the affected intestinal mucosa. Cells were generated from murine 

mononuclear cells in the presence of M-CSF and subsequently stimulated with IFN-g to 

produce what we refer to as “IFN-g-stimulated monocyte-derived cells” (IFNg-MdCs). 

Cells were characterized by FACS and immunomodulation was examined by assays for 

cell death and T cell regulation. Therapeutic effects were assessed in mouse models of 

chronic colitis. IFNg-MdCs express markers including CD11b/c, CD14, CD86, CD123, 

and PD-L1; IFNg-MdCs only arise when macrophages are cultivated in the presence of 

CD4+ T cells, M-CSF and IFN-g. In vitro, IFNg-MdCs profoundly delete lymphocytes (by 

>60%) derived from mice with colitis by a cell-contact, cell activation, and caspasedependent 

mechanism. Intriguingly, lymphocytes surviving in IFNg-MdC cocultures are 

highly enriched for CD4+CD25+Foxp3+ cells, which show up-regulation of IL-10, and 

actively suppress T cell proliferation. CD11b+ cells within the IFNg-MdC population are 

responsible for lymphocyte depletion and CD4+CD25+Foxp3+ cell induction. 

Additionally, using knock-out mice, we show that signaling via IFN-gR and CD40 on 

IFNg-MdCs are necessary for the generation of T regulatory cells. Regarding potential 

therapeutic activity for IBD, IFNg-MdCs reduced established inflammation in two mouse 

models of chronic colitis. We conclude that IFNg-MdCs represent macrophages in a 

novel state of activation, possessing multiple T cell-suppressive effects with therapeutic 

potential to mitigate autoimmune inflammation.

Günes Esendagli, Kirsten Bruderek, Torsten Goldmann, Andreas Busche, Detlev 

Branscheid, Ekkehard Vollmer, Sven Brandau 

Malignant and non-malignant lung tissue areas are 

differentially populated by natural killer cells and regulatory T 

cells in non-small cell lung cancer 

Even though the lung represents a special immune compartment with the capacity of a 

high inflammatory response, ineffective anti-tumor immunity is common in lungassociated 

malignancies. We asked whether a differential composition of the immune 

cell infiltrate in malignant (MLTAs) and non-malignant lung tissue areas (N-MLTAs) 

exists and might potentially contribute to this effect. We performed a comparative 

analysis of immune cells residing in MLTAs and N-MLTAs of non-small cell lung cancer 

(NSCLC) patients. To this end, we used immunophenotyping and functional analyses on 

directly isolated immune cells and tissue arrays on archived paraffin-embedded 

specimens. A strong T cell infiltration was prominent in both tissue compartments 

whereas CD4+CD25+CD127- T regulatory cells were present in MLTAs only. 

Nonetheless, concurrent functional ex vivo T cell analyses revealed no 

significant difference between T cells of MLTA and N-MLTA, suggesting that 

tumor-infiltrating T cells were not functionally impaired. Interestingly, T cell infiltration 

was less pronounced in specimens with a high neutrophilic infiltrate. NK cell infiltration 

was strikingly heterogenous between MLTA and N-MLTA. While NK cells were almost 

absent in the malignant tissue regions, non-malignant counterparts were selectively 

populated by NK cells and those NK cells showed strong cytotoxic activity ex vivo. We 

report that malignant and non-malignant tissue areas in NSCLC are selectively 

infiltrated by certain immune cell types with NK cells being displaced from the tumor 

tissue. These phenomena have important implications for tumor immunology of NSCLC 

and should be considered for the development of future immunologic intervention 

therapies.

Christina Janko, Udo S. Gaipl, Sandra Franz, Nina Ebel, Eberhard Schlücker, Roland 

Meyer-Pittroff, Martin Herrmann, Benjamin Frey 

Mammalian cells under pressure – Cell death pathways and 

immunogenicity of dying cells 

Today, various forms of cancer cause the death of every forth human world wide. The 

classical therapies for solid cancer have limitations in prolonging the live span of the 

patients. Therefore, treatment of cancer requires new therapeutic strategies. Recently 

described therapeutic approaches are based on the vaccination of cancer patients with 

autologous, inactivated tumour cells. The master requirements of cell based, 

therapeutic tumour vaccines are (I) the complete inactivation, (II) the preservation of 

their immunogenicity, and (III) the accordance with statutory provisions. Currently, 

physical treatments (e.g. X-ray, freeze-thaw) and chemotherapeutics are used to 

inactivate tumour cells for vaccination, but all procedures have methodological, 

therapeutical, or legal restrictions. For this reason, we are involved with high 

hydrostatic pressure (p > 50 MPa) for inactivation of tumour cells. 

When tumour cells treated with pressure up to 100 MPa they interrupted proliferation 

but the treatment did only marginally affect cells’ viability. A treatment with pressure 

above 200 MPa, however, resulted in cell death without survivors even in long-term 

culture. We analyzed the matter of cell death and detected, that cells pressurized with 

200 MPa died in consecutive cultures by apoptosis with preservation of the ion 

selectivity of the plasma membrane, phosphatidylserine exposure, and morphological 

alterations typical for apoptosis. In contrast, the cells treated with pressure above 300 

MPa necrotize during treatment. Most importantly, both pressurized apoptotic and 

necrotic cells largely retained their immunogenicity. In contrast to alternative methods 

for the induction of necrosis (heat, detergent, oxidative stress), the nuclei of HHP 

treated cells were efficiently degraded by a Ca2+-dependent DNase within a few hours 

in culture. Furthermore, the viscosity of the cytoplasm increased dramatically. 

Therefore, soluble constituents of the cells are retained within the jellified cytoplasm. 

These features favour HHP as a powerful technique for the inactivation of tumour cells 

employed in whole cell-based vaccination trials.

Christoph Lauer, Michael Basler, Susan D. Demo, Marcus Groettrup 

Manipulation of MHC class I antigen presentation by a LMP7specific 

inhibitor 

The proteasome is responsible for the generation of most epitopes presented on MHC 

class I molecules. Treatment of cells with interferon-? leads to the replacement of the 

constitutive catalytic subunits ?1, ?2 and ?5 by the inducible subunits LMP2 (?1i), MECL- 

1 (?2i) and LMP7 (?5i). The incorporation of these subunits seems to be required for the 

production of several MHC class I restricted T cell epitopes. In this study, we 

investigated the effect of a novel LMP7-specific inhibitor on antigen presentation. 

Fluorogenic assays with substrates specific for the chymotrypsin-like activity 

demonstrated that the inhibitor acts specifically on LMP7 in a concentration range of 

100-300nM. Furthermore, experiments with immunoproteasome deficent mice revealed 

that the generation of the male HY-derived Uty246-254 epitope is not only LMP7dependent, 

as previously reported, but also dependent on LMP2. Treatment of male 

splenocytes with the LMP7-specific inhibitor reduced the Uty246-254 presentation to 

background levels. Flow cytometric analysis of inhibitor treated splenocytes showed a 

reduction of MHC-I expression on wild type, but not on LMP7-deficient cells. Moreover, 

inhibition of LMP7 in cells infected with lymphocytic choriomenengitis virus (LCMV), led 

to decreased presentation of the LCMV-derived GP33-41 epitope. Taken together, we 

were able to show for the first time, that a specific inhibition of the ifn-? inducible 

immunoproteasome subunit LMP7 leads to a reduced presentation of several MHC class 

I restricted epitopes.

Lisa Bruns, Oliver Frey, Christiane Landgraf, Rudolf Volkmer, Thomas Kamradt 

Mapping of T cell epitopes in Glucose-6-Phosphate-Isomerase 

induced arthritis 

Immunization of certain non-transgenic mouse strains with recombinant human Glucose- 

6-Phosphate-Isomerase (huG6PI) leads to the development of polysymmetric arthritis, 

an animal model for Rheumatoid arthritis. We found that DBA/1 mice as well as SJL 

mice are susceptible for G6PI-induced arthritis. We have already demonstrated that CD4 

+ T cells are crucial both in the induction and effector phase of the disease. Therefore 

we used pools of overlapping huG6PI peptides to determine T cell epitopes that are 

relevant for G6PI-induced arthritis. We figured out two potential T cell epitopes: 

huG6PI85-99 and huG6PI469-483. Interestingly, we discovered the same epitopes both 

in DBA/1 and SJL mice. After immunization of DBA/1 mice with huG6PI85-99 and 

huG6PI469-483, the mice developed a mild arthritis with lower incidence. The time 

course of disease of peptide immunized mice differed from mice immunized with 

complete protein. Additionally we found huG6PI-specific antibodies not only in huG6PI 

immunized mice but also in peptide immunized mice. Thus, we conclude that huG6PI85- 

99 and huG6PI469-483 are important T cell epitopes in G6PI-induced arthritis.

Peter Kramer, Frank Siebenhaar, Marcus Maurer, Sven Hendrix 

Mast cell deficient mice display increased brain inflammation, 

neurodegeneration and astrogliosis 

Mast cells (MCs) play a key role in the development and severity of multiple sclerosis 

and its animal model, experimental autoimmune encephalomyelitis. However, the role 

of mast cells in the wound healing response after traumatic brain injury is unknown. 

Here, we compared mast cell-deficient W/Wv-mice and wildtype controls after 

mechanical brain injury. We used the well-studied entorhinal cortex lesion (ECL) model 

to analyze the immunoreactivity patterns of glial fibrillary acidic protein (GFAP, 

astrocyte activation), neurofilament 200 (neurons), isolectin B4 (IB-4, microglia), Ki67 

(proliferation) and FluoroJade B (neurodegeneration) after injury. GFAP 

immunoreactivity and astrocyte proliferation around the lesion was two times higher in 

mast cell-deficient mice compared to controls, while neurofilament 200 expression at 

the lesion site was substantially reduced. Mast cell-deficient mice showed a significantly 

higher number of IB-4-positive cells including a high number of proliferating cells in the 

lesion area compared to controls. Finally, at day 4 after lesion there is a significantly 

higher number of FluoroJade B positive neurons in mast cell-deficient mice. These data 

suggest that MCs protect from brain inflammation, neurodegeneration and astrogliosis.

Yves Montier, Axel Lorentz, Sigrid Krämer, Stephan C Bischoff 

Mast cell mediators stimulate fibroblasts to produce IL-6 that 

vice versa supports mast cell survival 

Background: Human intestinal mast cells (MC) are key effector cells in allergic reactions 

but also involved in host defense and tissue remodeling processes such as wound 

healing, angiogenesis, and fibrogenesis. We have shown previously that human 

intestinal fibroblasts (FB) suppress apoptosis in human intestinal MC independent of the 

mast cell growth factor stem cell factor (SCF) as well as IL-3, IL-4 and NGF. Here, we 

show that IL-6 is the missing factor. Methods: Human intestinal MC and FB were 

isolated from surgical tissue specimens using a four-step enzymatic dispersion method. 

Following overnight culture, MC were separated from adherent FB and purified using 

MACS-technique. Results We found that intestinal FB are capable of producing IL-6 

provided that they were stimulated directly by MC in co-culture or by MC mediators 

such as TNF-α, IL-1β, tryptase or histamine. In order to investigate the role of IL-6 for 

the survival of intestinal MC, MC were incubated with different concentrations of IL-6. 2 

ng IL-6/ml or higher concentrations of IL-6 supported MC survival until 12-16 d of 

culture. But, in contrast to treatment with SCF, all MC incubated with IL-6 died after 20- 

24 d of culture. Interestingly, culture of MC with supernatants of FB stimulated with TNF- 

• or IL-1• gave the same result: MC survived 12-16 d of culture, but they died after 20- 

24 d. MC survival in response to treatment with FB supernatant could be blocked using 

an anti-IL-6 Ab. Conclusion: Our findings suggest that MC mediators (TNF-α, IL-1β, 

histamine and tryptase) stimulate FB to produce IL-6 that vice versa supports MC 

survival.

Julia Scholten, Alexander Gerbaulet, Giuseppe Testa, Thomas Krieg, Karin Hartmann, 

Axel Roers 

Mast cell-specific Cre/loxP-mediated mutagenesis in vivo 

Mast cells are important effector cells in type I allergy, but were recently also shown to 

play key roles in host defence against pathogens. In addition, mast cells were 

implicated in tissue remodeling, wound healing and transplant tolerance. Investigation 

of this important cell type, however, was severely hampered by the scarcity of mast 

cells in the various tissues and the lack of protocols for ex vivo culture of mast cells. 

Until today, in vivo analysis of mast cell-specific functions of individual genes relied on 

the reconstitution of genetically mast cell-deficient mice with mast cells differentiated in 

vitro from bone marrow of mice deficient for the gene of interest. This system yielded 

important information in the past, but suffers severe limitations. Herein, we use the Cre/ 

loxP recombination system for conditional mast cell-specific mutagenesis in vivo. In a 

bacterial artificial chromosome (BAC) containing the entire mouse mast cell protease 5 

(Mcpt5) locus, the coding region of exon 1 was replaced by a Cre cassette. The BAC 

insert was purified and micro-injected into pronuclei of fertilized C57BL/6 oocytes to 

obtain transgenic mice. In order to demonstrate mast cell-specific Cre-mediated 

recombination, six Mcpt5-Cre founder lines were crossed to a Rosa26-EYFP Cre-excision 

reporter line and cell suspensions of various tissues were analysed by flow cytometry. 

Two of the transgenic lines showed highly efficient Cre-mediated induction of the 

fluorescent reporter protein in mast cells, but not in other cell types. The new Cretransgenic 

mouse lines will be useful tools in the elucidation of mast cell biology.

ERIETTA STELEKATI, ZANE ORINSKA, ANNALENA BOLLINGER, SILVIA BULFONE-PAUS 

Mast cells modulate CD8+ T cell responses. 

Mast cells are considered to contribute dominantly to the establishment of an innate 

immune response. However, mast cells can also act as important participants of the 

adaptive immune response, by regulating the phenotype and function of the adaptive 

immunity players (B cells, dendritic cells and T cells). T cells represent a cutting edge 

for the induction of adaptive immunity and CD8+ T cells have been reported to be 

recruited in vivo by mast cells. Therefore, this study was focused on the crosstalk 

between mast cells and CD8+ T cells. The potential of mast cells to induce antigenspecific 

CD8+ immune responses was investigated. We could demonstrate that bone 

marrow derived mouse mast cells (BMMCs) labelled with OVA-derived OT-I peptide were 

able to induce activation, cytokine production and proliferation of transgenic, OT-I 

specific CD8+ T cells. Moreover, blocking of cytokine synthesis in BMMCs by mitomycin 

C treatment and, to a higher extent, fixation of BMMCs by paraformaldeyde treatment, 

decreased their ability to specifically activate CD8+ T cells, thus demonstrating that the 

specific activation of CD8+ by BMMCs is dependent both on membrane-bound peptides 

and on soluble factors produced by BMMCs. Furthermore, Toll like receptor – specific 

pre-priming of BMMCs increased their ability to induce antigen specific CD8+ responses. 

Finally, BMMCs were able to induce antigen specific proliferation of naïve primary CD8+ 

T cells in vivo, as measured by CFSE staining. Taken together, these data demonstrate 

that mast cells can potently induce antigen specific CD8+ responses. This function of 

mast cells can be modulated by Toll like receptor signals. Thus, it is suggested that 

mast cells play a key regulatory role at the cross-roads of innate and adaptive immunity.

Milan Popovic, Ana Teles, Catharina Thuere, Anne Schumacher, Paul Ojiambo Wafula, 

Hans-Dieter Volk, Ana Claudia Zenclussen 

Mast-cell-associated genes Thp1, Mcpt1 and Mcpt5 are upregulated 

after Treg-induced tolerance at the fetal-maternal 

interface: new role for mast cells in pregnancy-induced 

tolerance? 

Objective - CD4+ CD25+ foxp3+ regulatory T cells (Treg) are known to play an 

important role in murine pregnancy outcome by inducing tolerance towards the 

semiallogenic fetus. Mast cells (MC) are known as primary responders in allergic 

reactions. Furthermore, it has been shown that activated MC produce a broad spectrum 

of pro- and anti-inflammatory mediators having very important role in immune 

response to foreign antigens. Recent studies revealed a novel role for MC in the Tregdependent 

allograft tolerance by secreting interleukin-9 (IL)-9. Here, we investigated 

whether MC may be also involved in Treg- mediated tolerance towards the 

semiallogenic fetus. 

Methods – We employed a well characterized murine combination for spontaneous 

abortion (DBA/2J-mated CBA/J females) to identify and quantify MC and their related 

molecules. BALB/c-mated CBA/J females served as controls for normal pregnancy. 

Furthermore, a further group was included, which consisted of animals rescued from 

abortion after transfer of antigen-specific Treg. Localization of MC at the fetal-maternal 

interface was verified by Giemsa staining. The mRNA expression of MC-associated genes 

(tryptophan hydorxylase (Tph)-1 as well as mast cell protease (Mcpt)-1 and Mcpt-5 in 

normal pregnant (NP) mice, abortion-prone (AP) and abortion-prone mice treated with 

Treg (AP+Treg) was analyzed in placental and decidual samples from normal pregnant 

(NP), abortion-prone (AP) and AP mice transferred with Treg by real-time RT-PCR. 

Results and conclusions – Mast cells were localized preferentially in decidua according to 

their granulary morphology, while very few MC could be detected in the placenta. The 

transcript levels of Tph1, Mcpt-1 and Mcpt-5 were comparable during the all pregnancy 

stages (days: 8, 10, 12 and 14) between NP and AP mice. However,treatment with Treg 

on day 14 markedly up-regulated their levels in both, decidua and placenta to the levels 

observed in NP mice. Our results indicate that as already observed in transplantationassociated 

tolerance, MC might contribute to the Treg-induced tolerance at the fetalmaternal 

interface.

Kristina Wiege, Syed Raza Ali, Stephanie Konrad, Roland Piekorz, Bernd Nürnberg, 

Reinhold E Schmidt, J Engelbert Gessner 

MECHANISM OF CELL AND ISOTYPE SPECIFIC Gαi DEPENDENT 

SIGNALING IN IMMUNE EFFECTOR CELLS 

G-protein- coupled receptor (GPCR) signal transduction contributes to the course of 

inflammation processes. In the context of immune complex (IC) mediated lung 

inflammation the GPCR, the complement 5a receptor (C5aR) is significantly involved in 

lung pathology. Previous experimental data verified pertussis toxin sensitive FcRγ 

regulation by C5aR suggesting for Gαi -dependent signal transduction mechanism. From 

the three known isoforms of inhibitory Gαi proteins, Gαi2 and Gαi3 but not Gαi1 are 

expressed on alveolar leukocytes, including macrophages (AM) and neutrophils with the 

exception of a splice variant of Gαi2 (sGαi2 ) which appearance is restricted to AM. Mainly 

Gαi2 appears to be involved in the regulation IC mediated inflammation. Genetic 

deletion of Gαi2 resulted in reduced lung inflammation whereas it was unaffected in 

mice lacking Gαi3 . On AM from Gαi2 deficient mice C5aR expression was reduced; this 

was not seen for neutrophils, suggesting cell and isotype specific functions of (s)Gαi2 / 

Gαi3 . To verify the role of the different isotypes we are using the RNA-Interference 

technique. We could demonstrate that the knock down of Gαi2 mRNA leads to the 

reduced expression of C5aR on peritoneal macrophage cell line. This effect could be 

rescued by the overexpression of Gαi2 . Future work comprises the analysis of sGαi2 and 

Gαi3 overexpression in Gαi2 knock down cells to clarify the role of the specific isoforms 

on the trafficking and functioning of the C5aR. 

Part of the work was supported to K.W. by graduate program (GRK705) of the Deutsche 

Forschungsgemeinschaft

Marco Wendel, Elisabeth Suri-Payer, Adelheid Cerwenka 

Mechanisms of NK cell migration in response to tumors 

Natural Killer (NK) cells are among the first immune cells to enter and eradicate 

growing tumors, in particular tumors with deficiency in MHC class I expression. In 

several studies high numbers of tumor infiltrating NK cells correlated with a good 

prognosis for cancer patients. However, only little is known about the factors regulating 

NK cell migration during anti-tumor immune responses. Our study aimed at identifying 

these factors in the model of subcutaneously growing MHC class I deficient lymphoma 

RMA-S. We observed that IFN-γ was absolutely required for NK cells to accumulate 

within the tumor tissue. Much fewer NK cells were infiltrating the tumors in IFN-γR KO 

mice or when IFN-γ was neutralized and exogenous application of IFN-γ further 

increased NK cell infiltration. We also observed that the depletion of regulatory T cells 

led to increased numbers of NK cells in the tumors, which was also dependent on the 

presence of IFN-γ. Since it has been reported that IFN-γ regulates the expression of the 

chemokine IP-10 and its receptor CXCR3, we determined their contribution to NK cell 

migration in our model. Tumor infiltrating NK cells were significantly decreased in 

CXCR3 -/- mice and the migratory capacity of adoptively transferred CXCR3 -/- NK cells in 

wt mice was impaired. Accordingly, intratumoral injection of IP-10 resulted in locally 

increased NK cell numbers and IP-10 transduced RMA-S cells were rejected in wt mice 

in a NK dependent manner. These data identify the expression of the chemokine 

receptor CXCR3 as a major prerequisite for NK cell migration towards the tumor. 

Exploitation of strategies to augment NK cell accumulation in the tumor tissue might 

prove beneficial in developing more powerful anti-tumor therapies.

Barbara C. Rütgen, Wilhelm Gerner, Armin Saalmüller, Sabine E. Hammer 

MHC typing in swine: The SLA-haplotype repertoire of 

Austrian Large White, Landrace, and Pietrain breeding stocks 

MHC (major histocompatibility complex) genes encode cell surface glycoproteins which 

bind and present antigenic peptides to T cells. The genes within this complex are highly 

polymorphic, suggesting that diversity in MHC genes is a good measure of population 

fitness. Resource herds of swine leukocyte antigen (SLA)-characterized pigs are 

valuable large animal models for biomedical research in terms of immune responses, 

disease resistance, and production traits. This study represents the initial 

characterization of founder haplotypes of commercial pigs in Austria which are F2 

descendants of purebred Large White, Landrace, and Pietrain populations. The 

respective founder SLA-haplotypes were detected by a reverse transcription-polymerase 

chain reaction (RT-PCR)-based SLA typing method to clone and DNA sequence the 

putative alleles at four SLA class Ia loci, designated as SLA-1, SLA-2, SLA-3, and SLA-6 

and four SLA class II loci, SLA-DQA1, SLA-DQB1, SLA-DRA1, and SLA-DRB1. The data 

obtained so far indicate that the Large White, Landrace, and Pietrain pigs have at least 

four SLA class Ia founder haplotypes (4a.0, 4b.0, 13.0, 5.0) and three SLA class II 

founder haplotypes (0.4, 0.2, 0.9). Furthermore, the analyzed specimen point toward 

the occurrence of at least three novel SLA class Ia haplotypes. The newly generated 

sequences will be used to design allele-specific primers for establishing a rapid SLA 

typing assay to discriminate each allele using PCR with sequence-specific primers (PCR- 

SSP). To check the PCR screening approach for false negatives, positive control primers 

were also designed to amplify a portion of the alpha-actin gene and multiplexed with 

the allele-specific primers. By applying a combination of SLA typing by cloning and DNA 

sequencing together with PCR-SSP, we will be able to characterize the entire Large 

White, Landrace, and Pietrain breeding stocks and to identify the SLA haplotype 

distribution present in the respective breeds.

J. Albrecht, T. J. Boeld, K. Doser, R. Eder, J. Stahl, R. Andreesen, J. Ermann, M. 

Edinger, P. Hoffmann 

MHC-compatibility between conventional and regulatory T 

cells is required for suppression of allospecific T cell responses 

Natural CD4+CD25+ regulatory T cells (Treg) contribute to tolerance induction after 

transplantation. We, and others, previously showed that the adoptive transfer of donorderived 

Treg cells prevents lethal graft-versus-host disease (GVHD) after allogeneic 

bone marrow transplantation (BMT) in mice. In contrast, host-type Treg cells failed to 

protect when co-transplanted under identical conditions. This raises the question 

whether MHC compatibility with CD25-CD4+ and CD8+ T cells (Tconv) is a general 

prerequisite for the suppression of alloresponses by Treg cells, or whether merely 

elimination of host-type Treg by allo-aggressive Tconv cells occurred. To address this 

issue, mixed lymphocyte cultures were performed in which CFSE-labelled responder T 

cells (Tresp), Treg cells and antigen presenting cells (APC) were systematically varied 

with regard to their MHC haplotype. When C57BL/6 Tresp cells were stimulated with 

irradiated CB6F1 (BALB/c x C57BL/6) APC, 16.9 + 1.5% of the CD4+ cells and 81.5 + 

2.5% of the CD8+ cells had gone through at least one cell cycle after 6 d. In the 

presence of syngeneic C57BL/6 Treg cells, proliferation of CD4+ and CD8+ Tresp cells 

was decreased to 5.2 + 2% and 26.5 + 3.5%, respectively. In contrast, in cultures with 

allogeneic BALB/c Treg cells, proliferation remained at 10.3 + 0.3% and 89.9 + 0.9% 

for CD4+ and CD8+ Tresp cells, respectively. Stimulation with either mixed C57BL/6 

and BALB/c APC or third party APC (DBA/1) led to comparable results. Lack of 

suppression cannot be explained by early elimination of allogeneic Treg cells, since they 

were still detectable on d6 in co-cultures with MHC mismatched Tresp cells. In 

corresponding in vivo studies, CB6F1 recipients were only protected from lethal GVHD 

when both donor T cell populations were MHC-identical, but not when Tconv and Treg 

cells were derived from the two separate parental strains. These data indicate that MHCidentity 

between Tresp and Treg cells is required for maximum suppression of an 

alloresponse and that Treg cells isolated from a third party donor might not be suited 

for the prevention of GVHD after allogeneic BMT.

Romney Haylett, Lothar Rink 

MHC-II Signaling Directs the Activation of NFAT but not NF-κB 

in B Cells 

For over a decade major histocompatibility complex class II (MHC-II) molecules have 

been acknowledged as signaling receptors although their mode of signaling and exact 

signaling pathways have yet been fully clarified. In this study, the MAP kinase pathway 

leading to ERK1/2 activation was explored for all three HLA isotypes (HLA-DR, -DP, - 

DQ) in the B cell lines BJAB and Raji. Not only could ERK1/2 activity be observed after 

signaling through all three isotypes, but the activation of c-Fos, a well-described 

component of the AP-1 transcription factor known to be phosphorylated by ERK1/2, was 

also established. This led to further studies pertaining to transcription factor activation 

where ligation of MHC-II molecules ultimately led to NFAT1 activation but not NF-κB 

activation in B cells. Future investigations should elucidate whether or not the entire AP- 

1 complex interacts with NFAT in B cells after MHC-II ligation. Although NFAT1 

activation has been described in B cells, relatively few work has been conducted in this 

field. With the novel discovery of MHC-II molecules capable of inducing NFAT activation, 

the understanding of MHC-II signaling and NFAT activation in B cells can be 

tremendously greatened.

Karina Stein, Jennifer Debarry, Anna Hanuszkiewicz, Otto Holst, Jörg Mages, Roland 

Lang, Holger Heine 

Microarray analysis of human dendritic cells stimulated with 

four different bacterial strains with focus on allergyprotecting 

mechanisms 

The incidence of allergic diseases is increasing, especially in industrialized regions. A 

growing number of publications indicate that farming environment in early childhood 

reduces the occurrence of allergic reactions later in life. Recently, we showed that the 

cowshed isolates Lactococcus lactis G121 and Acinetobacter lwoffii F78 prevent allergic 

immune responses in a mouse asthma model. However, the molecular mechanisms 

modulating allergic reactions in humans are only poorly understood. Thus, we 

stimulated human dendritic cells (DC) with these bacteria and two other reference 

strains, E. coli F1111 9-41 and B. subtilis DSM618, and prepared microarray analysis 

after 3, 6 and 12 hrs. A total number of 3623 probe sets in L. lactis-, 4254 sets in A. 

lwoffii- and 4625 sets in E. coli-treated DCs was regulated at least three-fold but only 

1939 probe sets in B. subtilis-treated DCs. Scatterplot analysis revealed that the 

induced expression pattern of E. coli and A. lwoffii stimulated DCs is comparable, with 

already a high number of regulated genes after 3 hrs. In contrast, nearly no genes were 

regulated in L. lactis-treated DCs after 3 or 6 hrs and only a moderate number after 

stimulation with B. subtilis. However, after 12 hrs of stimulation, L. lactis-treated DCs 

showed an expression pattern similar to that induced by A. lwoffii and E. coli whereas 

DCs treated with B. subtilis differed clearly. Cluster analysis confirmed a close relation 

between A. lwoffii-, E. coli- and L. lactis-treated DCs after 12 hrs whereas B. subtilistreated 

DCs formed a separate cluster. Overall, the analysis of cowshed bacteriainduced 

gene expression patterns leads to a better understanding of the molecular 

mechanism preventing allergic immune responses (supported by DFG, SFB/TR22, 

project A2).

Kristina Allers, Désirée Kunkel, Verena Moos, Martin Eisenblätter, Christiane Stahl- 

Hennig, Annette Schrod, Ralf Ignatius, Franz-Josef Kaup, Thomas Schneider 

Migration patterns of activated versus non-activated nonhuman 

primate T lymphocytes: preferential homing of 

activated autologous CD8+ T cells in the rectal mucosa 

Background: Adoptive cell transfer (ACT) is a promising approach to induce antitumour 

immune responses in cancer patients. However, crucial for successful therapy is the 

access of the transferred cells to the tumours. Little is known about the migration 

patterns of in vitro activated primate T cells and their persistence in different anatomical 

compartments after ACT. Here, we describe a model, which enables the long-term 

tracking of T cells in the peripheral blood, secondary lymphoid tissues, or 

gastrointestinal mucosa after re-infusion of autologous peripheral lymphocytes into 

rhesus macaques. 

Methods: PBMC from 4 or 3 rhesus macaques were activated with aCD3/aCD28 or not, 

stained with CFSE and autologously re-injected. Blood samples, lymph node (LN) as 

well as mucosal biopsies (duodenum, rectum) were collected at various time points over 

28 days and analysed for the presence of labeled CD4+ and CD4- T cells using flow 

cytometry. 

Results: On d1 post transfer of activated cells the frequency of labeled cells was 0.2, 

3.8 and 4.6% of total CD4+ T cells in blood, duodenum and rectum, respectively. 

Within the CD4- T cells the frequency of labeled cells was 0.2% in blood and 6.7% in 

the duodenum but as high as 27.5% in the rectal mucosa. 7 days post transfer of 

activated cells 0.3, 0.6, 0.5 and 0.4% labeled CD4+ T cells were present in the blood, 

LN, duodenum and rectum, respectively. Within the CD4- T cells the frequency of 

labeled cells was 0.03% in the blood, 0.3% in LN and 2.9% in the duodenum. In the 

rectal mucosa labeled cells constituted 29.6% of the total CD4- T cells indicating 

persistence of activated CD8+ T cells in the rectal mucosa. 

Conclusions: Injection of CFSE-labeled, autologous T cells into rhesus macaques allows 

the tracking of lymphocyte migration to various anatomical compartments and thus 

representing a pre-clinical model for the evaluation of T cell-based immunotherapy. Nonspecificly 

activated CD8+ T cells preferentially migrate to and persist in the rectal 

mucosa whereas activated CD4+ T cells are found in much less numbers at this site but 

also in other compartments.

Stefan Wiehr, Thomas Herrmann 

Milk-derived immune cells may serve as Trojan Horses in 

Toxoplasma gondii transmission 

Beside vertical transmission of T. gondii from acutely infected mothers in utero, 

transmission of T. gondii via the maternal milk has also been reported. To further 

analyze the lactogenic route of infection our study focused on parameters of humoral 

and cellular immune responses in congenitally versus milk infected rats. F344 rats were 

inoculated i.p. with 1x10e6 NTE or Prugneaud beta-gal tachyzoites at day 14 of 

gestation and blood and milk were taken every second day after delivery. Cells were 

isolated from the milk by density gradient centrifugation and analyzed by FACS and 

confocal microscopy. By using a human foreskin fibroblasts bioassay T. gondii positive 

milk samples could be detected until day 22 p.i. 

T. gondii tachyzoites could be observed inside cells whereas the non-cellular milk 

fraction was predominantly T. gondii negative, suggesting that T. gondii infected cells 

might serve as a vehicle for oral transmission to neonates. Strikingly, a strong antibody 

response was found in milk of infected rats. Moreover, the cellular fraction of the milk of 

infected rats showed a highly elevated number of CD45 positive cells consisting of all 

major alpha-beta and gamma-delta T cell and of granulocytes. To investigate whether 

the presence of tachyzoites in the milk correlated with transmission of T. gondii to 

uninfected offspring, we performed criss-cross experiments. Two months after birth, 

one out of 24 offspring fed with T. gondii positive milk showed a sustained increase of 

T. gondii antigen specific IgG2b as well as antigen specific proliferation with secretion of 

IFN-gamma by mesenteric lymph node cells and splenocytes. Six out of 44 offspring by 

which the parasite was transmitted in utero and 6 of 41 born from infected mothers 

exhibited the same type of immune response. 

These results indicate that T. gondii infected cells found in maternal milk may serve as a 

vehicle for parasite transmission and that both diaplacental and lactogenic infection 

induces a TH1 dominated immune response in the offspring of infected mothers. 

herrmann-t@vim.uni-wuerzburg.de

Marcin Kaminski, Peter H. Krammer, Karsten Gülow 

Mitochondria function as oxidative signalling organelles in 

activation-induced apoptosis of T cells. 

Reactive oxygen species (ROS) generated upon T cell activation are crucial for induction 

of CD95L expression and, consequently, for apoptosis of activated T cells (Activation 

Induced T cell Death - AICD). The molecular source and the signalling steps leading to 

activation-induced ROS production are still unclear. 

Our data show that the proximal T cell receptor (TCR) signalling machinery, consisting 

of ZAP70, LAT, SLP76, PLCγ1 and PKCθ, is crucial for ROS production. Upon activation, 

PKCθ translocates to the mitochondria. The depletion of mitochondrial DNA led to 

identification of mitochondria as source of activation-induced ROS. Pharmacological 

inhibition of the mitochondrial respiratory chain complex I (NADH-quinone 

oxidoreductase) or siRNA-mediated knockdown of the complex I assembly factor 

chaperone NDUFAF1 resulted in blocking of ROS production. Upregulation of MnSOD, a 

crucial mitochondrial antioxidative enzyme, converts ROS released by complex I into a 

hydrogen peroxide signal (H2O2). Since this signal is essential for CD95L expression, 

inhibition of complex I assembly by NDUFAF1-specific siRNA prevents AICD. 

Interestingly, application of metformin, an antidiabetic drug and mild, non-toxic 

complex I inhibitor, also led to decrease in activation-induced ROS production, CD95L 

expression and AICD. 

Therefore, PKC θ-dependent ROS release by mitochondrial complex I constitutes a 

crucial signalling event leading to AICD of T cells.

Martina Anzaghe, Zoe Waibler, Holger Ludwig, Shizuo Akira, Siegfried Weiss, Gerd 

Sutter, Ulrich Kalinke 

Modified vaccinia virus Ankara induces Toll-like receptor 

independent type I interferon responses 

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus strain 

undergoing clinical evaluation as a replication-deficient vaccine vector against various 

infections and tumor diseases. To analyze the basis of its high immunogenicity, we 

investigated the mechanism of how DNA encoded MVA induces type I interferon (IFN) 

responses. MVA stimulation of bone marrow derived dendritic cells (DC) showed that 

plasmacytoid DC were main IFN-alpha producers that were triggered independently of 

productive infection, viral replication, or intermediate and late viral gene expression. 

Increased IFN-alpha levels were induced upon treatment with mildly UV irradiated MVA 

suggesting that virus encoded immune-modulator(s) interfered with the host cytokine 

response. Mice devoid of Toll-like receptor (TLR) 9, the receptor for double stranded 

DNA, mounted normal IFN-alpha responses upon MVA treatment. Furthermore, mice 

devoid of adaptors of TLR signaling, MyD88 or TRIF, and mice deficient of protein kinase 

R (PKR) showed IFN-alpha responses that were only slightly reduced when compared to 

wild-type mice. MVA induced IFN-alpha responses were critically dependent on 

autocrine/paracrine triggering of the IFN-alpha/beta receptor (IFNAR) and were 

independent of IFN-beta, thus involving “half” a positive feedback-loop. In conclusion, 

MVA mediated type I IFN secretion was primarily triggered by non-TLR molecules, was 

independent of virus propagation and critically involved IFN-feedback stimulation. These 

data provide the basis to further improve MVA as a vaccine vector.

Christian Jacobi, Jürgen Roemisch, Stefan Meuer, Thomas Giese 

Modulation of gene expression by IVIG in healthy donors and 

multiple sclerosis patients 

Intravenous immunoglobulin G (IVIG) has become an established first- and second-line 

line treatment in a number of immune mediated diseases during recent years. IVIG is 

now proposed to modulate a wide range of molecules (e.g. bacterial/viral antigens, 

toxins, Fc receptors, complement, autoantibodies), to act on different cell types (e.g. B 

cells, T cells, macrophages, dendritic cells, endothelial cells), and to modulate various 

immunological pathways at both the humoral and cellular levels including inflammation, 

antigen presentation, cell growth and apoptosis. Using a variety of specific assays many 

distinct effects of IVIG have been demonstrated in vitro. To provide further insight into 

the mechanisms involved in IVIG-dependent immunmodulation under physiological 

conditions, we have established a human whole blood gene expression assay in vitro. 

There, we analyzed the effect of IVIG on CD2-, CD3-, LPS- and PMA/Ionomycin 

stimulated leukocytes in whole blood of 20 healthy donors, 15 untreated MS patients 

and two MS patients during relapse. IVIG induces among others the expression and 

release of Interferon-gamma, MCP1 and IP10. Interestingly, IVIG reduces the MCP1 and 

IP10 expression and release upon LPS stimulation, whereas Interferon-gamma 

expression is further enhanced. Since both chemokines are physiologically induced by 

Interferon, this effect of IVIG is unanticipated and deserves further investigation for its 

possible role in anti-inflammatory properties of IVIG. No significant differences in the 

IVIG induced expression profile between healthy donors and MS patients could be 

observed.

Anastasia Schneider, Tatiana Binder, Rodica Bernatowicz, Anke Zobywalski, Christine 

Falk, Anton Hartmann, Dolores Schendel, Susanne Krauss-Etschmann 

MODULATION OF HUMAN DENDRITIC CELLS BY SIX 

DIFFERENT PROBIOTIC BACTERIAL STRAINS 

Background 

Lactobacilli (Lb) and bifidobacteria (Bb) are probiotic bacteria and have been suggested 

to protect against allergic diseases. Since the underlying immune mechanisms are 

unclear at present, we tested whether six different probiotic strains polarize the 

maturation of human monocyte-derived dendritic cells (moDC). 

Methods 

UV-inactivated and live Lb rhamnosus GG (LGG, Valio Ltd, Finland), Bf. species 420 

(Bifidobacterium lactis, Danisco Deutschland GmbH), Lb acidophilus LA-5, Lb paracasei 

subsp. paracasei LC-01, Bb animalis BB-12 and Bb longum BB-46 (Chr.Hansen GmbH, 

Denmark) or LPS were co-cultured with moDC at ratios of 1-100 bacteria per one DC for 

24 h. UV-inactivated bacteria were counted visually after DAPI-staining. Viability of live 

bacteria was more than 90% as evaluated with a live/dead-staining kit (Invitrogen 

GmbH). Surface expression of costimulatory molecules on DC was analyzed by flow 

cytometry. Cytokines (IL-1beta, -6, -10, -12, TNF-alpha, IFN-gamma) and chemokines 

(IL-8, MIP-1alpha, MIP-1beta) were quantified in culture supernatants by multiplex 

assay (Bio-Rad Laboratories GmbH). Results are expressed as percentage of LPS 

stimulation. 

Results 

Priming with UV-inactivated BB-12 induced the highest synthesis of IL-6, -8, -10, -12, 

TNF-alpha and MIP-1alpha by moDC, while UV-inactivated LA-5 induced the highest 

synthesis of IL-1beta, IFN-gamma and MIP-1beta. In preliminary experiments, UVinactivated 

or live probiotics induced similar expression of costimulatory molecules on 

moDC. 

Conclusions 

The response of human moDC to UV-inactivated probiotics depends on the bacterial 

strain. UV-inactivated and live probiotics lead to similar expression profiles of 

costimulatory molecules on moDC. The analysis of the cytokine production of DC will 

show, whether this extends to soluble signals.

Irene Wittmman, Diana Aichele, Gerhard Groer, André Gessner, Markus Schnare 

Modulation of innate immune responses by recombinant 

murine bactericidal permeability / increasing protein through 

neutralizaton of LPS and Gram-negative bacteria 

Rationale: Recognition of LPS by TLR4 initiates inflammatory responses inducing potent 

anti-microbial immunity. However, uncontrolled inflammatory responses can be 

detrimental. In order to prevent the development of septic shock during an infection 

with Gram-negative bacteria the immune system has developed regulatory mechanisms 

to neutralize LPS by specialized proteins. Here we report the recombinant expression 

and functional characterization of the mouse homolog of human bactericidal 

permeability / increasing protein (BPI). 

Methods: Mouse BPI was stably expressed and highly purified by affinity 

chromatography. RAW 267.4 cells as well as bone marrow derived dendritic cells were 

stimulated with various TLR-ligands in the presence or absence of recombinant mouse 

BPI (rmBPI). Activation of the cells was determined by ELISA (TNF, IL-12), NO-assay as 

well as flow cytometry (CD86). In addition RAW macrophages expressing a NFκBreporter 

construct were stimulated with UV-treated Gram-negative (E. coli, S. 

typhimurium, N. meningitis, P. aeruginosa) as well as Gram-positive (Staph. aureus, L. 

monocytogenes, group B Streptococcus) bacteria in the presence or absence of rmBPI. 

The cellular activation was determined by TNF-ELISA and NFκB activity. Finally 

replicating E. coli were cultured with RAW macrophages in the presence or absence of 

rmBPI. The bacterial numbers as well as the cell activation was analyzed. 

Results: Purified rmBPI is a 60kD protein that was able to specifically neutralize LPSmediated 

activation of macrophages and to block LPS-dependent maturation of dendritic 

cells. Importantly, rmBPI neutralized the capacity of UV-irradiated and live Gramnegative 

bacteria to activate immune cells but it did not influence the stimulatory 

properties of Gram-positive bacteria. Finally rmBPI did not influence the bacterial 

growth. 

Conclusion: Together these data demonstrate that murine BPI is a potent LPS 

neutralizing protein that may play an important role as a negative modulator of innate 

immune responses during Gram-negative infections.

Stephan Schierer, Andrea Hesse, Ina Mueller, Eckhart Kaempgen, David T. Curiel, 

Gerold Schuler, Alexander Steinkasserer, Dirk M. Nettelbeck 

Modulation of viability and maturation of human monocytederived 

dentritic cells by oncolytic adenoviruses 

In spite of intensive efforts to modify oncolytic adenovirus (ads) for improved specificity 

and efficacy of cancer cell lysis, it is becoming increasingly clear that the killing of noninfected 

cancer cells in parallel to viral cell lysis will be crucial for oncolytic ads to be 

effective in the clinic. An attractive scenario towards this goal is the induction of 

systemic anti-tumor immunity by adenoviral oncolysis. In order to establish the basis 

for the development of adenoviral oncolytic vaccination we examined the effects of 

oncolytic ads on the biology of human dendritic cells (DCs). DCs are the most potent 

antigen presenting cells, key regulators of immune induction and have been intensively 

exploited for various vaccination protocols. The prime objective of this study was to 

investigate how optimized, melanoma-targeted oncolytic ads affect the viability and 

maturation of human monocyte-derived DC and the potency of such DCs to activate T 

cells. 

Both immature DCs (iDCs) and mature DCs (mDCs) were transducible with luciferase 

encoding ads at high titers. Fiber chimeric ads with an ad serotype 3 derived knob 

domain (5/3fiber) showed an improved transduction efficacy compared with an ad with 

serotype 5 (5fiber) fiber. After infection of iDC and mDC with oncolytic ads (5 or 

5/3fiber) at high titers (5000vp/cell), no significant toxicity was observed. Oncolytic ads 

showed no or strongly attenuated DNA replication in human DCs, thus confirming their 

replication specificity. As determined by staining of cell surface maturation markers of 

DCs two to three days post infection with oncolytic ads (5 or 5/3fiber), we observed, 

dependent on the donor, no or a partial DC maturation. With respect to the maturability 

of DCs with cytokines and/or LPS, no negative influence of infection with oncolytic 

adenovirus was documented. However, we did find an increase in the IL-12 secretion of 

LPS resp. LPS/IFNg matured DCs after infection with oncolytic ads. Finally DCs, infected 

with oncolytic ads for 3 days, matured and then loaded with peptide were still able to 

prime and stimulate specifically CD-8 T-cells with similar or superior efficacy compared 

to uninfected DCs, dependent on the donor. 

In conclusion, oncolytic ads do not harm viability and maturability of monocyte derived 

DCs, but also do not induce complete maturation of these cells. Therefore, oncolytic ads 

might represent a promising tool for oncolysis-induced anti-tumor immune activation, 

however, this strategy might require additional maturation signals for DCs, for example 

by expression of immunostimulatory genes inserted into the genome of oncolytic ads.

Philip Kruse, Cornelia Rosner, Lutz Walter 

Molecular characterisation of the killer cell immunoglobulinlike 

receptors (KIR) of the rhesus macaque (Macaca mulatta) 

The killer cell immunoglobulin-like receptors (KIR) belong to the immunoglobulin 

superfamily and are expressed mainly on the surface of natural killer (NK) cells. These 

receptors interact with the major histocompatibility complex (MHC) class I molecules 

and can mediate either an activating or an inhibitory signal to the NK cell. In contrast to 

the activating receptors almost all ligands of the inhibitory receptors are known in 

humans. 

We have started to analyse KIR genes and their function in the rhesus macaque 

(Macaca mulatta), a frequently used primate model for the study of infection diseases, 

organ transplantation and in development of vaccines. A cDNA library of enriched NK 

cells from 30 rhesus macaques has been established. Up to now, more then 15 new KIR 

cDNA clones could be isolated. Phylogenetic analysis with already known rhesus 

macaque KIR sequences confirms rapid evolution of KIR genes in primates. Further 

analyses indicate the occurrence of two different activating KIR gene lineages in rhesus 

macaques, which have developed independently from each other and both differ from 

the lineage of human activating KIR genes. 

The main differences of the rhesus macaque activating KIRs and the KIRs in hominoid 

primates can be found in the transmembran region. Rhesus macaque activating KIRs 

contain an arginin instead of a lysine in humans in the transmembran region. Usually, 

this lysine residue interacts with the DAP12 adaptor molecule, which mediates 

activating signals to the NK cell. Therefore, it is still open which adaptor molecule 

interact with the activating KIRs of the rhesus macaque. Currently we are investigating 

whether these activating KIRs do interact with adaptor molecules other than DAP12.

Cary Mac Millan, Alexander Hann, Peter Bannas, Wolfgang Koestner, Friedrich Buck, 

Friedrich Haag, Friedrich Nolte 

Molecular characterization of ADP-ribosylated T cell 

membrane proteins 

NAD-dependent ADP-ribosylation is a posttranslational protein modification catalyzed by 

ADP-ribosyltransferases (ARTs). ART2.2 is a GPI-anchored ecto-enzyme expressed on 

murine T cells, Tregs, IELs, and NKT cells. Exposure of T cells to the ART substrate, 

NAD, results in ADP-ribosylation of cell surface proteins, affecting important T cell 

effector functions, including Calcium signalling, clustering of the T cell receptor, 

adhesion to target cells, and apoptosis by as yet unknown mechanisms. ART2 is a 

promiscuous enzyme which can ADP-ribosylate different proteins in vitro. On the T cell 

surface, the specificity of ART2 is restricted by its association with lipid rafts, which 

focuses ART2 onto specific target proteins (1). Some of the known targets for ADPribosylation 

on T cells (P2X7, LFA-1, and CD45) are involved in T cell activation and 

associate with lipid rafts in an activation dependent manner. In order to identify further 

functionally interesting ART target-proteins we used the NAD analogue etheno-NAD as 

substrate to etheno-ADP-ribosylate cells surface proteins on lymphoma cells (2). Etheno- 

ADP-ribosylated targets were purified from cell lysates by affinity chromatography on a 

monoclonal antibody specific for etheno-adenosine and were analyzed by mass 

spectrometry. The results revealed a number of interesting proteins, including CD25, 

CD205, and CD229, as candidates for further functional studies. 

(1) Bannas, et al. Blood, 105: 3663-70 (2005) 

(2) Krebs, et al. Anal. Biochem 314:108-15 (2003)

Viet Bui, Andreas Diefenbach 

Molecular cloning and functional characterization of a novel 

stimulatory immunoreceptor expressed by myeloid cells 

Innate immune cells express stimulatory receptors to recognize diseased cells or 

pathogens. We have identified and cloned a novel stimulatory immunoreceptor 

expressed in mice. This novel receptor contains a single Ig-V domain and is a member 

of the Ig-superfamily. A charged residue in the transmembrane domain indicated 

association with a signaling adaptor molecule. Indeed, the receptor could not be 

expressed in cell lines lacking signaling adaptor molecules. Our studies demonstrated a 

requirement of the signaling adapter KARAP/DAP12 for surface expression and 

signaling. We have generated a monoclonal antibody specific for this receptor. 

Preliminary experiments revealed the receptor to be expressed by myeloid cells such as 

dendritic cells, macrophages, and neutrophils while being absent from T, B, NK, and 

NKT cells. Interestingly, we identified another gene within the same locus encoding 

another immune receptor sharing 87% identity in the extracellular domain. These data 

suggest that both receptors may recognize the same cognate ligand. In contrast to the 

stimulatory receptor, this second receptor possesses a consensus ITIM motif in its 

cytoplasmic tail region suggesting it to be an inhibitory receptor. This inhibitory receptor 

shares the same expression pattern as the stimulatory receptor. Whereas stimulation of 

myeloid cells leads to the down-regulation of the stimulatory receptor, its inhibitory 

counterpart seems to be up-regulated. Based on these initial findings, we hypothesize 

that these two receptors are paired stimulatory/inhibitory receptors expressed by 

myeloid cells that likely recognize the same cognate antigen. The inverse nature of their 

expression would suggest that this receptor pair is an important molecular switch 

regulating the function of myeloid cells.

Susan M Schlenner, Lars A Schneider, Thorsten B Feyerabend, Markus Wunderlin, 

Hans-Reimer Rodewald 

Molecular mechanism of mast cell-mediated innate defense 

against endothelin and snake venom sarafotoxin 

Mast cells are protective against snake venom sarafotoxins that belong to the 

endothelin peptide family. The molecular mechanism underlying this recently recognized 

innate defense pathway is unknown but secretory granule proteases have been invoked. 

To specifically disrupt a single protease function without affecting expression of other 

proteases, we have generated a mouse mutant lacking selectively mast cell 

carboxypeptidase A (Mc-cpa) activity. Using this mutant, we have now identified Mc-cpa 

as the essential protective mast cell enzyme. Mass spectrometry of peptide substrates 

after cleavage by normal or mutant mast cells showed that removal of a single amino 

acid, the C-terminal tryptophan, from endothelin and sarafotoxin by Mc-cpa is the 

principle molecular mechanism underlying this very rapid mast cell response. Mast cell 

proteases can also cleave endothelin and sarafotoxin internally but such ‘nicking’ is not 

protective since intra-molecular disulfide bridges maintain peptide function. We 

conclude that mast cells attack endothelin and sarafotoxin exactly at the structure 

required for toxicity, and hence sarafotoxins could not ‘evade’ Mc-cpa’s substrate 

specificity without loss of toxicity.

Jennifer Debarry, Anna Hanuszkiewicz, Otto Holst, Holger Heine 

Molecular mechanisms of human dendritic cell stimulation by 

the Allergy-protective Lactococcus lactis strain G121 

Exposure to farming environment during early childhood strongly influences the 

development of allergic reactions later in life (“hygiene hypothesis”). As the first year of 

life is important for the establishment of the THelper cell balance, environmental 

influences such as increased exposure to certain bacteria might play a pivotal role in 

adjusting this balance by promoting TH1 responses and thereby inhibiting TH2 responses 

which are associated with allergies and asthma. The Gram-positive bacterium 

Lactococcus lactis strain G121 was isolated from a cowshed and could be shown to 

reduce allergic reactions in a mouse allergy model. Furthermore, we were able to show 

a TH1 polarizing effect of L. lactis G121 in human dendritic cells (DCs) which might be 

the underlying mechanism of the allergy-protective properties. Activation of DCs is 

mainly mediated through NOD2 and the TH1 polarization by L. lactis G121 is impaired 

after blocking the access to intracellular receptors via inhibition of phagocytosis. With 

respect to the involved signal transduction pathways we next analyzed the NF-κB 

pathway. In contrast to stimulation with E. coli we were unable to detect any substantial 

translocation of p65 into the nucleus, even when the cells were monitored for up to 3 

hrs. However, translocation of the c-Rel subunit into the nucleus was strongly 

detectable within 120 min of stimulation. C-Rel is essential for the induction of TH1 polarizing cytokines since it controls IL12p35 and IL23p19 gene expression. In addition, 

we detected a sustained activation of DCs for up to 48 hrs with respect to the TH1 polarizing IL12p35 and IL12p40 gene expression. This could be an important 

mechanism for a prolonged triggering of an anti-allergic immune response (supported 

by DFG, SFB/TR22, project A2).

Malte Bachmann, Jens Paulukat, Josef Pfeilschifter, Heiko Mühl 

MOLECULAR MECHANISMS OF IFNγ-INDUCED IL-18 BINDING 

PROTEIN PROMOTER ACTIVATION AS DETECTED IN HUMAN 

DLD-1 COLON CARCINOMA CELLS 

Due to its prominent activity as IFNγ inducing factor, IL-18 has been introduced as a 

pivotal mediator of inflammation. Accordingly, IL-18 bioactivity is strongly associated 

with the pathogenesis of acute and chronic inflammatory diseases, among others, 

sepsis, rheumatoid arthritis, and Crohn`s disease. IL-18 Binding Protein (IL-18BP) is a 

naturally occurring inhibitor that counteracts IL-18 bioactivity. We and others could 

previously demonstrate that IL-18BP is strongly induced by IFNγ, resulting in a negative 

feedback mechanism that has been recognized in cell culture and in vivo. Here we 

sought to investigate in DLD-1 colon carcinoma cells molecular mechanisms that direct 

IL-18BP expression under the influence of IFNγ. The capability of IFNγ to induce IL-18BP 

was confirmed on the promoter level by performing luciferase reporter studies. Those 

experiments revealed that a proximal GAS element (gamma-activated sequence;-24bp 

to -32bp) plays a pivotal role in IL-18BP expression by IFNγ activated DLD-1 cells. IL- 

18BP was dependent on STAT1 activation as shown by siRNA technology. Indeed, EMSA 

and ChIP analysis proved STAT1 binding to the GAS element at the proximal IL-18BP 

promoter position. Full induction of IL-18BP by IFNγ was in part dependent on de novo 

protein synthesis. In fact, induction of IRF-1 by IFNγ and the presence of an IRF-1 

binding site close to the GAS element under investigation suggests a role for IRF-1 in 

IFNγ induced IL-18BP. Altogether, data on DLD-1 colon carcinoma cells presented herein 

indicate that direct action of STAT1 on the proximal GAS element of the IL-18BP 

promoter is key to IFNγ induction of this most relevant immunoregulatory cytokine 

antagonist.

Maria Papatriantafyllou, Thilo Oelert, Günter Hämmerling, Bernd Arnold 

MOLECULAR MECHANISMS OF PERIPHERAL CD8 T CELL 

TOLERANCE 

Clarifying the mechanisms of immunological tolerance is essential for the development 

of effective therapies against cancer and autoimmunity. Central tolerance is achieved by 

clonal deletion or the intrathymic induction of regulatory T cells. However, not all 

autoreactive T cells are eliminated in the thymus and self-tolerance is further assured 

by rendering autoreactive T cells that escape negative selection, tolerant in the 

periphery. In our model a regulatory CD8 + CD25 - T cell population mediates peripheral 

tolerance against self antigen. In neonates CD8 T cells pass through the endothelium 

and have access to tissues (e.g. liver, skin). Those that have specific T cell receptor 

(TCR) for autoantigens are rendered tolerant and suppress immune response against 

self during the adult phase. In detail, transgenic mice express anti-Kb TCR (Des-TCR) in 

CD8 T cells and the Kb molecule under the control of the 2,4Ker-IV promoter in the skin 

but not in the thymus (DesTCRxKerKb mice). As a result, the CD8 T cells recognize the 

antigen in the periphery during the neonatal phase, become tolerant and suppress the 

immune response against Kb self antigen. This model is being used to investigate the 

gene expression pattern of tolerant CD8 T cells and clarify the molecular mechanisms of 

peripheral CD8 T cell tolerance. Either naïve, activated or tolerant CD8 T cells are sorted 

from the spleen of DesTCR mice or Des-TCRxKerKb mice respectively and their gene 

expression profile is determined with the oligonucleotide microarrays from Affymetrix. 

According to the microarray data genes that are involved in the suppression of the 

immune response are upregulated in tolerant CD8 T cells, such as components of the 

TGF-β1 and Notch pathways. The most prominent candidates are under investigation 

and their contribution to CD8 T cell tolerance will be discussed.

Tobias Schwerd, Johannes C. Hellmuth, Andreas Schmidt, Hendrik Poeck, Michael 

Wenzel, Stefan Endres, Simon Rothenfusser 

Molecular mechanisms of virus recognition by Rig-I-like 

helicases 

Viral infections are a constant threat to the integrity of human beings. Survival of the 

host organism depends on the rapid induction of innate immune responses including the 

production of interferon type I. The newly described family of cytoplasmic Rig-I-like 

helicases sense viral infection via the recognition of viral RNA, and trigger a potent 

antiviral defense. This family comprises Retinoic acid-inducible gene I (RIG-I) and 

melanoma differentiation associated gene 5 (MDA-5) two non-redundant signalling 

receptors and the regulatory protein Lgp-2.Rig-I was recently shown to bind and detect 

RNA of negative stranded RNA viruses, via a virus-specific 5’-triphosphate modification 

absent in normal cytoplasmic RNAs. This provided a structural basis for the distinction 

of self- and non-self RNA. We are interested in the molecular details leading from ligandbinding 

by these helicases to the activation of anti-viral signalling processes. 

Classically, helicases are known to unwind double-stranded (ds)RNA in an ATPdependent 

fashion. Using an in vitro assay we demonstrate, that the ATPase activity of 

RIG-I is triggered in the presence of dsRNA. Interestingly, RIG-I-dependent ATP 

hydrolysis can also be activated by in vitro transcribed but not synthetic single-stranded 

(ss)RNA. In both settings activation of RIG-I’s ATPase activity is independent of 5’-end 

modifications. To investigate the correlation between helicase activity and signalling we 

tested a similar set of ligands in IFN-beta luciferase reporter assays. Our experiments 

indicated that helicase activity triggered by dsRNA is required but not sufficient for IFN 

promoter activation. In addition, pull-down assays show that an intact ATP-binding 

motif is not needed for ligand binding. 

In conclusion, we propose that double-strandedness is an important molecular feature 

for recognition and downstream signaling of viral RNA by Rig-I. This may facilitate the 

development of new therapeutic molecules in tumor immunology and viral infection.

Petra Riedl, Kurt Reifenberg, Joerg Reimann, Reinhold Schirmbeck 

Mono-specific, hepatic CD8 T cell responses primed by 

cationic peptide/oligonucleotide complexes suppress viral 

replication in HBV transgenic mice 

Preclinical mouse models are informative to explore the possibilities for specific immune 

interventions in chronic viral infection. We describe a novel Hepatitis B Virus (HBV)specific 

transgenic (tg) mouse model (1.4HBV-Smut mice). Due to a point mutation in 

the translational start codon of the small HBV surface protein (HBs), no infectious viral 

particles can be assembled. 

We tested if hepatic CD8 T cell responses can be elicited by a peptide-based vaccination 

strategy in which an antigenic, Kb-restricted HBs peptide (S190-197; VWLSVIWM) is 

fused to a cationic HIV-tat derived peptide (RKKRRQRRR). Positively charged fusion 

peptides are quantitatively complexed with negatively charged immunostimulating 

oligonucleotides (ODN). In non-transgenic mice, HBs-encoding plasmid DNA- and 

peptide- based vaccination protocols efficiently primed Kb/S190-197-specific CD8 T cell 

responses. In contrast, only the peptide/oligonucleotide vaccine (but not the pCI/S DNA 

vaccine) elicited Kb/S190-197 specific CD8 T cell responses in 1.4HBV-Smut mice. CD8 

T cells accumulate in the livers of tg mice and efficiently inhibit HBV replication. Thus, 

minimal cationic peptide/ODN vaccines are attractive tools that elucidate cellular and 

molecular events that lead to a monospecific, therapeutic CD8 T cell response in the 

HBV expressing liver.

Sonja Schmucker, Mario Assenmacher, Anne Richter 

Monocytes and myeloid dendritic cells are both able to induce 

primary activation of naïve MART-1-specific CD8 + T cells in 

vitro 

Adoptive T cell therapy for cancer and viral infections in immuno-compromised patients 

is hampered by the lack of reliable protocols for primary activation and expansion of 

naïve tumor or virus-specific T cells. 

In this study we examined the capacity of monocytes and myeloid dendritic cells (MDC) 

to in vitro prime naïve MART-1-specific CD8 + T cells. 

Therefore we magnetically isolated CD14 + monocytes, CD1c + myeloid dendritic cells 

(MDC), and naïve CD8 + CD25- CD45RO- CD45RA + CD62L + T cells from peripheral blood 

mononuclear cells (PBMC) of healthy HLA-A2-positive donors. Naïve T cells were 

stimulated with either autologous MART-126-35 peptide-pulsed CD14 + monocytes or 

CD1c + MDC in the presence of CD28 antibodies, IL-7 and IL-15. For further expansion 

IL-2 was added subsequently from day three on. At day eight to ten the frequency, the 

degree of expansion, and the expression of CD45RA, CD45RO and CD62L of HLA-A2/ 

MART-1-Tetramer + CD8 + T cells was determined. Additionally, production of IFN-? by 

the expanded T cells was analysed after restimulation with MART-1-pulsed monocytes. 

In PBMC of healthy donors naïve HLA-A2/MART-1-Tetramer + CD8 + T cells are 

detectable at frequencies of around 0.07% of the CD8 + T cell population. Eight to ten 

days after in vitro primary activation the frequencies of HLA-A2/MART-1-Tetramer + CD8 

+ T cells increased to 0.3% - 5.7%, irrespective of whether monocytes or MDC were 

used as antigen presenting cells (APC). We found a 90-fold expansion of MART-1specific 

T cells, which showed a CD45RA + CD45RO-/+ CD62L- effector/effector memory 

phenotype. Restimulation of the activated and expanded CD8 + T cells with MART-126-35 peptide- but not irrelevant peptide-pulsed monocytes led to IFN-? production in up to 

70% of the HLA-A2/MART-1-Tetramer + CD8 + T cells, confirming antigen specificity and 

indicating functionality of the cells. 

In conclusion we established an in vitro protocol for priming and expansion of MART-1specific 

CD8 + T cells based on the use of peptide-pulsed monocytes or MDC as APC

Beatrice Jahn-Schmid, Gottfried Fischer, Gabriele Gadermaier, Matthias Egger, 

Fatima Ferreira, Christof Ebner, Barbara Bohle 

Mugwort pollen allergy as a unique model for the 

investigation of allergen-specific CD4+T cells using HLA 

classII/peptide tetramers 

Mugwort pollen allergens represent the main cause of pollinosis in late summer in 

Europe. Ninety-five percent of mugwort–allergic patients are sensitized to the major 

allergen Art v 1. In contrast to other common pollen allergens which contain multiple T 

cell epitopes, Art v 1 contains only one single immunodominant T cell epitope (Art v 

125•36). We characterized the minimal epitope of Art v 125-36 in detail and 

investigated a possible association of Art v 1-reactivity with HLA class II-phenotypes. In 

addition, the potential use of HLA-classII tetramers was evaluated. 

Art v 1-specific T cell lines (TCL) and clones (TCC) were established from 51 patients 

with clinically defined mugwort pollen allergy and IgE specific for Art v 1. In 96% of the 

patients a cellular response to Art v 125-36 was obtained and a core region of 5-10 

amino acids containing 3-5 amino acids essential for T cell reactivity was defined by 

using truncated and single-substitution analog peptides for T cell stimulation. The 

frequency of HLA-DRB1*01 in patients recognizing Art v 125-36 was significantly 

increased as compared to healthy controls (69% vs. 21%; odds ratio: 8,45; p

Andreas Junker, Jana Ivanidze, Joachim Malotka, Ingrid Eiglmeier, Hans Lassmann, 

Hartmut Wekerle, Edgar Meinl, Reinhard Hohlfeld, Klaus Dornmair 

Multiple Sclerosis: T cell receptor transcriptome in distinct 

brain regions 

We investigated the T cell receptor repertoire in distinct lesions and normal appearing 

white matter (NAWM) of multiple sclerosis patients. To this end we analyzed 19 lesions 

(inactive demyelinated, 15; slowly expanding chronic, 3;active lesions, 1) and five 

NAWM regions from postmortem brains of four MS patients by CDR3 spectratyping. For 

each anatomical site 325 semi-nested PCR reactions were performed. About 800 V_- 

NDN-J_ combinations were sequenced. 

We identified several distinct T cell clones at different anatomical sites in all patients. 

Some clones were present in all investigated lesions, and additionally, even in NAWM. A 

single clone was detected in nine different sites in one patient. None of the clones was 

shared among different patients. Analysis of the hypervariable NDN region revealed 

silent nucleotide exchanges. 

Individual CD8+ T cells were isolated from cryosections by laser microdissection and 

characterized by single-cell PCR. We found at least some of the pervasive T cell clones 

belonged to the CD8+ compartment. 

This study shows that pervasive T cell clones exist in distinct regions of MS brain which 

are not restricted to lesions, but are also present in NAWM. All clones are 

"private" (unique) to individual patients. Several silent nucleotide exchanges suggest 

that the corresponding T cell clones were stimulated by particular antigens. Some of the 

highly pervasive clones were CD8+, supporting the pathogenic relevance of this T cell 

subset.

Felix C. Popp, Elke Eggenhofer, Prezemyslaw Slowik, Philipp Renner, Katharina 

Kronenberg, Hans J. Schlitt, Pompiliu Piso, Marc H. Dahlke 

MULTIPOTENT MESENCHYMAL STROMAL CELLS INDUCE LONG- 

TERM ALLOGRAFT ACCEPTANCE MEDIATED THROUGH IDO 

Multipotent mesenchymal stromal (MS) cells isolated from adult bone marrow inhibit the 

T cell response in mixed lymphocyte cultures. Modulating the immune system through 

MS cells may play an important role in organ transplantation since new concepts of cell 

based immunosuppression are urgently needed. 

Here we applied MS cells four days in advance of performing a completely MHC 

mismatched heart transplantation followed by a seven day course of mycophenolate 

mofetil (MMF). The median survival of the transplanted grafts was greater than 100 

days as compared with 15 days when applying MMF alone. 

After applying 1-methyl tryptophan, a potent inhibitor of IDO, animals rejected heart 

graft early. However, real time PCR for IDO mRNA levels produced no significant 

differences. Presumably MS cells induce tolerance during a narrow time window after 

transplantation. This assumption is supported by the fact that the timing of MS cell 

application is crucial for achieving long term allograft acceptance. 

In conclusion, MS cells induced long-term graft acceptance in our heart transplantation 

model. Blocking IDO resulted in rejection, indicating a central role of IDO in MS cell 

mediated graft acceptance. To further elucidate the mechanism behind MS cell mediated 

tolerance, animals need to be analyzed at time points closer to the heart 

transplantation.

Verena Besche, Christina Glowacki, Nadine Wiechmann, Andrea Renzing, Ngoc-Anh 

Dang, Stephan Sudowe, Jürgen Knop, Angelika B. Reske-Kunz, Matthias Bros 

Murine dendritic cells exert tolerogenic function at their 

immature state and upon differentiation in the presence of 

dexamethasone 

Under homeostatic conditions, dendritic cells (DC) are involved in the maintainance of 

peripheral tolerance against self as well as harmless environmental antigens by inducing 

antigen-specific regulatory T cells. Upon inflammation glucocorticoids are generated 

endogenously as a negative feedback regulatory mechanism by cells of the adrenal 

cortex. Due to their potent anti-inflammatory effects, glucocorticoids are frequently 

prescribed in the treatment of severe allergic diseases. Here we show that addition of 

the synthetic glucocorticoid dexamethasone (DEX) to murine bone marrow derived DC 

(BM-DC) cultures resulted in reduced expression of costimulatory molecules and 

accessory molecules involved in DC-T cell interaction as compared with untreated 

immature BM-DC. Moreover, DEX largely prevented upregulation of these molecules 

upon stimulation with lipopolysaccharide (LPS). DEX upregulated expression of 

glucocorticoid-responsive genes shown to exert anti-inflammatory activities like FcgRIIB 

and IL1RA. Functionally, DEX-treatment prevented the acquisition of potent T cell 

stimulatory capacity upon stimulation with LPS. Both immature BM-DC and DEX-treated 

cells exerted tolerogenic function, since prestimulated alloreactive T cells were 

refractory to restimulation and suppressed proliferation of naive T cells upon allogeneic 

stimulation.

Ulrich Salzer, Chiara Bacchelli, Stephanie Jennings, Allesandro Plebani, Helen Chapel, 

Hans D Ochs, Simon Urschel, Bernd H Belohradsky, H Bobby Gaspar, Bodo Grimbacher 

Mutations in TACI/TNFRSF13b in patients with CVID – a 

genetic, immunological and clinical study in a large patient 

cohort 

Background: TACI (TNFRSF13b) is an important regulator of B cell responses and 

differentiation. We recently showed that TACI sequence variants are associated with 

common variable immunodeficiency (CVID) and that some of these variants are present 

in the general population at lower frequencies. 

Results: We studied a cohort of 570 new CVID patients. 54 patients (9.5%) showed at 

least one mutated TACI allele. 12 patients (22%) showed homozygous or compound 

heterozygous (c.h.) mutations in TNFRSF13b, while most of the patients (n=42; 78%) 

were heterozygous. 16 different mutations were identified, of which 13 have not been 

described. TACI-C104R and A181E were the most frequent alleles (n=39; 72%), being 

present also in about 2% of healthy controls, whereas their combined frequencies in 

CVID were about 7%. Importantly, all of the variants seen in controls were 

heterozygous and did not include frameshift or nonsense changes, which were exclusive 

to the CVID population. Homozygous or c.h. mutated receptors did not bind the ligand 

APRIL. Functional analysis of heterozygous TACI mutants implied that these may act 

differently or are being compensated by the redundant system of BAFF/APRIL receptors. 

Analysis of B cells in 31 TACI-deficient patients did not show a distinct B cell phenotype. 

The patients presented with the full clinical spectrum of CVID. Autoimmunity was seen 

in 11 of 39 patients (28%) and clinical signs of lymphoproliferation were present in 23 

of 41 patients (56%). 

Conclusions: TACI is a highly polymorphic gene, shows a complex pattern of sequence 

variants and is mutated in 10% of CVID patients. While B cells with homozygous or c.h. 

mutated TACI receptors showed specific functional defects, the role and significance of 

heterozygous mutations is still unclear. In addition, the occurrence of heterozygous 

TACI-C104R and A181E alleles in normal controls suggests that these variants act more 

as disease modifiers rather than being disease causing in CVID. However, the 

immunological and clinical phenotype of TACI-deficient patients was indistinguishable 

from that of other CVID patients. 

Supported by DFG SFB620/C2; NIH/NIAID: USIDnet grant # NO1-A1-30070 (B. G.), 

the Primary Immunodeficiency Association and the Medical Research Council, UK (C.B.).

Max Bastian, Tobias Braun, Heiko Bruns, Martin Röllinghoff, Steffen Stenger 

Mycobacterial Lipopeptides Elicit CD4+ Cytolytic T 

Lymphocytes in Mycobacterium tuberculosis Infected Humans 

An ex vivo model was developed to study frequency, phenotype and effector functions 

of human T lymphocytes recognizing hydrophobic antigens of Mycobacterium 

tuberculosis (M.Tb). To obtain unbiased results we chose to characterize T-lymphocytes 

responding to a chloroform methanol extract (M.Tb-CME) containing the whole 

spectrum of mycobacterial glycolipids and lipopeptides. A significant proportion (290 

IFN-g+ T cells/ 10EE5 PBMCs) of T lymphocytes recognized M.Tb-CME and developed to 

effector memory cells as determined by the expression of CD45RO and the chemokine 

receptors CXCR3 and CCR5. Antigen-specifically expanded lymphocytes were isolated 

and further analyzed for their potential to provide protection against M.Tb. Purified cells 

fulfilled all criteria required for an efficient immune response against tuberculosis: i) 

release of macrophage-activating Th1 cytokines and chemokines required for the spatial 

organization of local immune responses ii) cytolytic activity against antigen-pulsed 

macrophages and iii) recognition of M.Tb-infected macrophages resulting in killing of 

intracellular bacteria. Phenotypically, M.Tb-CME expanded cells were CD4+, challenging 

current concepts that cytotoxic and antimicrobial effector cells are restricted to the CD8 

+ T-cell compartment. The T cell responses to the hydrophobic M.Tb-CME preparation 

were MHC class II restricted. Pretreatment of M.Tb-CME with proteinase or delipidation 

by alkaline hydrolysis abolished the stimulatory capacity. This indicates that lipidated 

peptides of M.Tb induce CD4+ cytolytic and antimicrobial T-lymphocytes that recognize 

their epitope on infected macrophages. Our data support the emerging concept that T 

cells specific for hydrophobic antigens play an important role in the immune defense 

against microbial pathogens and may extend the scope of immunogenic compounds that 

should be included in novel vaccine formulations against tuberculosis.

Peter Reichardt, Bastian Dornbach, Song Rong, Stefan Beissert, Faikah Güler, Karin 

Loser, Matthias Gunzer 

Naive B cells generate regulatory T cells in the presence of a 

mature immunological synapse 

Naïve B cells are ineffective antigen presenting cells and considered unable to activate 

naïve Tcells. However, antigen specific encounter of these cells leads to stable cell pairs 

that remain associated over hours in lymph nodes in vivo. The mechanism of signalling 

and the physiological outcome of such pairs has not been evaluated. We show here that 

antigen specific conjugates between naïve B cells and naïve T cells display a mature 

immunological synapse (mIS) as signalling device in the contact zone which is absent in 

specific T cell-dendritic cells (DC) pairs. Thereby, B cells induce substantial proliferation 

but contrary to DC no loss of L-Selectin in T cells. Surprisingly, while DC-triggered T 

cells develop into normal effector cells, B cell stimulation over 72 hours induces 

regulatory T cells that inhibit de novo priming of fresh T cells in a contact dependent 

manner in vitro. Due to their high levels of L-selectin these cells home to lymph nodes 

where they also potently inhibit T cell priming in vivo. Such, immune responses in an 

hypersensitivity model and rejection of organ grafts was prevented. Thus, presentation 

of specific antigen by naive B cells drives naïve T cells into a regulatory phenotype. This 

might explain old findings on tolerance induction by B cells, identifies the mIS as a 

central functional module of this process and may represent a powerful approach for 

therapeutic intervention.

Nils Kruse, Arnhild Schrage, Katrin Neumann, Katharina Eulenburg, Friderike 

Blumenthal-Barby, Simon Fillatreau, Percy A. Knolle, Martin Zeitz, Alf Hamann, Katja 

Klugewitz 

Naive CD4+ T cells primed by Liver Sinusoidal Endothelial 

Cells (LSEC) do not differentiate into cytokine producing 

effector cells and fail to induce a delayed type 

hypersensitivity reaction (DTH) 

The liver, which is known to have immunomodulatory functions, favours the induction of 

tolerance rather than immunity. For example LSEC, one population of hepatic nonprofessional 

antigen presenting cells (APC) prime CD8 + T cells leading to anergy or 

deletion. But it is controversial if LSEC can prime naive CD4 + T cells. Whereas LSEC 

isolated by counterflow elutriation primed naive CD4 + T cells, others have reported that 

LSEC isolated by depletion of CD45 + cells were not sufficient to induce proliferation of 

naive CD4 + T cells. 

In our study we investigated if naïve CD4 + T cells can be primed by LSEC alone by 

using a novel experimental approach. To exclude the influence of professional APC, we 

immunomagnetically purified LSEC from bone marrow chimeric mice that express MHC 

class II exclusively on non-hematopoietic cells. We analyzed the phenotype and the 

functional properties of the LSEC primed CD4 + T cells. 

We demonstrated that LSEC alone can prime naive CD4 + T cells in vitro, determined by 

proliferation. However the LSEC were weak presenters, as high antigen concentrations 

were required. The CD4 + T cells neither differentiated into IFNγ expressing Th1 cells nor 

produced IL-4 or IL-10. In an in vitro suppression assay this T cell population 

suppressed the proliferation of naive CD4 + T cells. But markers characteristic for 

regulatory T cells (CD25, Foxp3 and αE) were negligible. Concerning their 

proinflammatory capacity LSEC primed CD4 + T cells did not induce a DTH reaction in 

contrast to CD4 + T cells primed by professional APC. Determining the stability of their 

phenotype in vivo we observed that the cells did not express IFNγ after transfer into 

syngeneic mice, even in the presence of the specific antigen. 

Taken together CD4 + T cells can be primed by LSEC and acquire an anergic phenotype. 

This modulation might be involved in establishing and maintaining hepatic tolerance not 

only for CD8 + T cells but also for CD4 + T cells.

Benedikt Fritzsching, Eva Pauly, Nina Oberle, Navina Kuss, Katrin Hartmann, Peter 

Ruf, Johannes Pöschl, Peter Krammer, Elisabeth Suri-Payer 

Naive regulatory T cells: activation and apoptosis 

sensitization of this novel Treg subpopulation during the 

human neonatal period 

CD4+CD25+FOXP3+ regulatory T cells (Treg) are potent immunosuppressive T cells. 

Reduction of peripheral blood Treg numbers or function have been reported to be 

associated with human autoimmune disease. However, the mechanisms responsible for 

Treg -reduction are not clear. We have recently reported high sensitivity of the majority 

of Treg towards CD95L-mediated apoptosis*. However, a Treg subpopulation remains 

consistently apoptosis-resistant. Gene micro array and 6-color flow cytometry analysis 

including FOXP3 revealed that naïve Treg constitute this so far neglected apoptosis 

resistant Treg population. Naïve Treg show a phenotype similar to naïve conventional T 

cells (CD45RA+, CD45RO-, partly CD31+, CCR6-, CD95-). Whereas naïve Treg 

represent only a minority of Treg in adults, almost all cord blood Treg show such a 

naïve phenotype. Here, we analyzed the postnatal activation status of Treg during the 

first 28 days of healthy newborn infants. Whereas most conventional T cells showed a 

naïve phenotype, Treg rapidly acquired an activated phenotype. Similar to adult Treg 

these in vivo activated neonatal Treg showed an enhanced apoptosis sensitivity. Treg 

activation and apoptosis sensitization occurred during the early postnatal expansion 

phase of lymphocytes. This observation might reflect an important tolerance mechanism 

for the control of the proliferating T cell compartment in the first weeks of a newborn´s 

life. 

Benedikt.Fritzsching@med.uni-heidelberg.de 

* Fritzsching et al., Cutting Edge, Journal of Immunology l. 2005 Jul 1; 175(1):32-6

Manoj Kumar, Norman Putzki, Hans Christoph Diener, Hans Grosse-Wilde, Ernst 

Kreuzfelder 

Natalizumab seems to effect mainly B lymphocytes in patients 

with Multiple Sclerosis 

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system which is 

characterized by inflammation, demyelination, gliosis and axonal damage. Natalizumab 

[anti-very late antigen-4 (VLA-4) antibody] is approved as monotherapy for the 

treatments of relapsing remitting MS. It exerts its immunologic effects by targeting the 

VLA-4, alpha4 integrin receptor, the site responsible for the migration of leukocytes 

from the blood into inflamed tissue. 

Twenty-four patients with relapsing remitting course of MS were included in our study. 

To investigate the influence of natalizumab therapy on the counts of leukocytes and 

their subpopulations as well as VLA-4 expression in B lymphocytes in whole blood, we 

used flow cytometry before treatment initiation (baseline) and during follow up to 6 

months. 

Relative (%) and absolute counts (cells/microL) of lymphocytes were significantly (p < 

0.05. Kruskal-Wallis test) increased following treatment (e.g. 6 months: 42 %, 3081 

cells/ microL) with natalizumab compared with baseline values (26%, 1815 cells/ 

microL). Within lymphocytes, there is increase in relative and absolute counts of B cells 

(e.g. 6 months: 22%, 720 cells/ microL) compared with baseline (17%, 307 cells/ 

microL) but T lymphocytes and natural killer cells were not significantly different during 

ongoing treatment. We measured VLA-4 expression in B lymphocytes and found that 

there is significant decrease in percentage as well as mean fluorescence intensity (MFI) 

of VLA-4 positive B cells during therapy (e.g. 6 months; percentage: 86 %, MFI; 739 

channels) compared with baseline (percentage: 39%, MFI: 706 channels). 

Therefore natalizumab may increase counts of lymphocytes. This seems due to 

increased B cell counts. Increase in B lymphocytes counts may be explained by 

suppression of transmigration because percentage of VLA-4 positive B cells and VLA-4 

expression decreased during therapy.

Stephanie Joachim, Martin Wax, Daniela Kraft, Norbert Pfeiffer, Franz Grus 

Neurodegeneration of the optic nerve and changes in 

antibody profiles after immunization with heat shock protein 

27 

In previous studies complex antibody profiles against retinal antigens were found in 

patients with glaucoma. Antibodies against heat shock protein 27 (HSP27) have been 

identified in these studies in sera of glaucoma patients. The aim of this study was to 

analyze, if immunization with HSP27 can cause retinal ganglion cell loss in rats similar 

to the loss found in glaucoma patients. 

Lewis rats were immunized with 100 mg HSP27 (plus Freund’s adjuvant and pertussis 

toxin) and divided into three groups: group 1 was euthanized after 4 weeks (n=6), 

group 2 after 5 weeks (n=6), and group 3 after 6 weeks (n=9). Control animals that 

received no immunization were euthanized after 6 weeks (n=10, group 4). Intraocular 

pressure was measured during the course of the study and blood was collected at 

several time points. Serum was used to detect IgG antibody patterns against retina via 

Western blotting and Protein G bead techniques. The antibody patterns were analyzed 

by multivariate statistical techniques. Eyes were harvested the day the animals were 

euthanized and were fixed and flatmounts were stained with Brn-3a, which detects the 

retinal ganglion cell (RGC) nuclei. We used a computer assisted quantitation of RGC 

density. 

No significant changes in intraocular pressure were observed during the course of the 

study. The animals immunized with HSP27 showed a lower number of RGCs in 

comparison to controls (P

Barbara Daller, Michaela Jungbeck, Klaus Pfeffer, Daniela N. Männel, Thomas Hehlgans 

Neutralization of LIGHT ameliorates DSS-induced intestinal 

inflammation 

Activation of the lymphotoxin-b receptor (LTbR) is essential for the development, 

maturation and maintenance of secondary lymphoid organs. Recent studies identified 

LTbR activation as a critical signaling pathway in the gut and highly relevant to 

intestinal inflammation. In order to investigate the role of the LTbR ligand LIGHT 

(lymphotoxin-like inducible protein that competes with glycoprotein D for binding to 

herpesvirus entry mediator on T cells) in the pathology of colitis, we used LIGHTdeficient 

mice in a model of acute colitis induced by oral administration of dextran 

sulfate sodium (DSS). 

LIGHT-deficient mice developed almost no intestinal inflammation according to 

histological score, weight loss, and colonic myeloperoxidase activity. Therefore, mouse 

anti-mouse LIGHT monoclonal antibodies were generated by immunization of LIGHTdeficient 

mice and standard hybridoma techniques. ELISA and Western blot analysis 

demonstrated specific binding of the monoclonal antibodies to mouse LIGHT protein. 

Furthermore, two of the monoclonal antibodies neutralized mouse LIGHT in vitro and in 

vivo. This was demonstrated by a significantly reduced intestinal inflammation of 

C57BL/6 mice treated with anti-mouse LIGHT antibodies during the induction of acute 

DSS-colitis. 

LIGHT serum levels in patients with Crohn´s disease and ulcerative colitis were found to 

be significantly elevated. Thus, the present study clearly demonstrates a role for LIGHT 

in enhancing intestinal inflammation and also indicates that neutralization of LIGHT 

might be beneficial in treatment of acute inflammatory diseases.

Katrin Birkholz, Niels Schaft, Jan Dörrie, Michael Schwenkert, Christian Kellner, Georg 

Fey, Gerold Schuler 

New strategies for antigen loading of human monocytederived 

DC 

Although responses in dendritic cell (DC)-based vaccination of melanoma and renal cell 

carcinoma patients are encouraging, further improvement of the efficiency of these 

vaccines is needed. A comparison of different methods, such as electroporation of 

defined tumor-antigen RNA or total tumor RNA, phagocytosis of tumor cells, direct 

peptide loading, or the use of antibody-antigen constructs, is necessary to optimize DC 

antigen loading. To examine this, we chose MAGE-A3, a cancer-testis antigen expressed 

in malignant melanoma, multiple myeloma, and many other tumors, as a model 

antigen. As a read-out for antigen-presentation efficiency, CD4 and CD8 T cells, 

transfected with RNA encoding TCR recognizing MHC-presented MAGE-A3 epitopes were 

used. As one possible loading strategy, we cloned antibody-antigen constructs, 

consisting of a single-chain Fragment variable (scFv) directed against DEC205, an 

endocytosis receptor expressed on the surface of DC, genetically linked to different 

parts of MAGE-A3. After receptor-mediated endocytosis, the MAGE-A3 antigen should 

be delivered into the DC, and subsequently presented on MHC class II molecules. The 

expressed and purified fusion proteins were detected in western blot analysis and 

displayed specific binding to mature (m)DC and DEC205-transfected CHO cells. In 

another loading strategy, MAGE-A3-DCLAMP RNA was used. The DCLAMP sequence 

targets the antigen to lysosomes, which leads to MHC class II presentation. Indeed, 

electroporation of mDC with MAGE-A3-DCLAMP RNA resulted in HLA-DP4-restricted 

presentation of the MAGE-A3 peptide KKLLTQHFVQENYLEY. Taken together, we have 

established tools to optimize loading of DC with tumor antigen which might lead to 

better anti-tumor DC vaccines.

Timo Herrmann, Nousheen Zaidi, Hubert Kalbacher 

New strategies for the analysis of processed antigens in 


During antigen processing, protein antigens are taken up by antigen presenting cells 

(APCs) like dendritic cells (DCs) and subequently proteolytically processed by different 

proteases. Antigenic peptides are then displaced on MHC class II molecules and 

presented to T-helper cells to modulate immune response. Understanding how foreign 

antigens are proteolysed to antigenic peptides is therefore key to understanding both 

the normal and abnormal function of the immune system. However the analysis of 

processed protein antigens in antigen presenting cells is still difficult because of the very 

low concentration of internalized species but moreover because of the very high 

complexity of processed self proteins in a given cell. 

For this reason, new strategies have been developed in our lab, based on specifially 

labeled proteins and antibody-based affinity capturing with biomagnetizable beads in 

combination with high resolution mass spectrometry. Recombinant proteins, e.g. myelin 

basic protein (MBP) or myelin oligodendrocyte glycoprotein (MOG) and large peptide 

fragments of known epitopes were labeled with fluorescein labels (FITC) or biotin and 

used for pulse chase experiments in DCs. FACS analysis was used to control the 

internalization. The DCs were then subjected for subcellular fractionation allowing to 

analyze organelles relevant to antigen processing (endosomes/lysosomes) by western 

blot analysis and high sensitive RP-HPLC with fluorescence detection. Finally in a key 

step, capturing of the labelled antigens was achieved by Ab-coated magnetic beads. The 

antibodies were generated in our lab, carefully purified and characterized and then 

covalently coupled to activated beads. The captured-peptides were eluted in a one-stepprocedure 

directly onto a MALDI-target and characterized. 

This approach allows the exclusive analysis of pulsed antigen fragments and even more 

to analyze processed ligands associated to MHC class II molecules. 

It could be used as a readout for functional studies of antigen processing, e.g. knockout 

systems, si-RNA-studies or specific cell-penetrating inhibitors, regarding processing 

relevant proteases.

Hamid Kashkar, Anke Deggerich, Jens-Michael Seeger, Benjamin Yazdanpanah, Katja 

Wiegmann, Dirk Haubert, Carola Pongratz, Martin Krönke 

NF-kappaB independent down-regulation of XIAP by 

bortezomib sensitizes Hodgkin Lymphoma B-cells against 

cytotoxic drugs 

The proteasome inhibitor, bortezomib, has been shown to possess promising anti-tumor 

activity and significant efficacy against a variety of malignancies. Different studies 

demonstrated that bortezomib breaks the chemoresistance in different tumor cells 

basically by altering nuclear factor-kappaB (NF-kappaB) activity. NF-kappaB has been 

shown to be constitutively active in the vast majority of primary Hodgkin-Reed- 

Sternberg (H-RS) cells in lymph node sections and in Hodgkin’s lymphoma (HL) cell 

lines and was suggested to be a central molecular switch in apoptosis resistance in HL. 

Here we report a bimodal effect of bortezomib in HL cells. Whereas high-dose 

bortezomib induced direct cytotoxicity which correlated with decreased NF-kappaB 

activity, low-dose bortezomib sensitized HL cells against a variety of cytotoxic drugs 

without altering NF-kappaB action. Strikingly, bortezomib induced marked XIAP downregulation 

at the post-translational level which was independent of the NF-kappaB 

status. Similarly, RNA interference (RNAi)-mediated XIAP down-regulation generated 

susceptibility to cytostatic agents. The results identify XIAP as an NF-kappaB 

independent target of bortezomib action that controls the chemoresistant phenotype of 

HL cells.

Michael Kiessling, Peter H. Krammer, Karsten Gülow 

NF-kB suppression sensitizes cells towards ROS-induced 

apoptosis 

Various types of cancer show resistance towards apoptosis induction. One reason for 

the resistance is constitutive activation of the nuclear factor-kappa B (NF-kB) pathway. 

Selective inhibition of NF-kB by overexpression of the super-repressor form of IkBα led 

to increased apoptosis in Jurkat T-cells. However, the mechanism of this enhanced cell 

death remained unclear. 

Here, we show that higher levels of reactive-oxygen-species, whose accumulation is 

increased in IkB&alpha overexpressing cells, promote c-jun N-terminal kinase (JNK) 

phosphorylation. Treatment of cells with antioxidants such as N-acetyl cysteine (NAC) 

abrogated PMA-induced ROS formation, JNK activity and thus, apoptosis. Further, 

downregulation of JNK by siRNA resulted in reduced apoptosis. One putative target for 

ROS may be MAPK phosphatases (MKP), whose catalytic cysteine residue is oxidized by 

ROS and therefore inactivated.

Maren Claus, Sabine Wingert, Carsten Watzl 

NK cell activity is regulated by CD48 - 2B4 (CD244) 

interaction in cis and in trans 

Function of human NK cells is regulated by a variety of different cell surface receptors. 

SLAM-related receptors (SRR) are important modulators of NK cell activity. Most SRR 

are activated by homophilic interaction. Thus, SRR do not only function as activating NK 

cell receptors, but also as activating NK cell ligands. 2B4 (CD244) is the only SRR that is 

activated by binding to a distinct ligand: CD48 is a GPI-anchored surface molecule that 

is widely expressed on hematopoietic cells. Recent work demonstrated that, like other 

SRR, 2B4 expressing target cells can also induce NK cell cytotoxicity by interacting with 

NK cell expressed CD48. These findings suggest an additional function of the 2B4-ligand 

CD48 as an activating receptor on NK cells. 

In the present study, we show that 2B4 does not only bind in trans to CD48 on 

neighboring cells, but can also interact in cis with CD48 molecules on the same NK cell, 

thereby modulating NK cell activity. The expression level of CD48 on NK cells is about 

20fold higher than that of 2B4. Using soluble CD48-ILZ fusion proteins, we demonstrate 

that removal of endogenous CD48 from the cell surface by phosphatidylinositol-specific 

phospholipase C (PI-PLC) increases the binding of soluble CD48 to NK cell 2B4. This 

suggests that the interaction between CD48 and 2B4 in cis prevents the 2B4 receptor 

from recognizing CD48 in trans. To test if this also has functional consequences we 

decreased the density of CD48 on the cell surface of NK92 cells by PI-PLC treatment. 

This significantly increased 2B4-mediated cytotoxicity against CD48 expressing target 

cells. Taken together, our findings provide evidence for the modulation of NK cell 

activity by the density of 2B4 and CD48 on both the NK cell and the potential target 

cell. Further, the present data suggest functional consequences of an in cis interaction 

between 2B4 and CD48 for NK cell activity.

Matthias Krusch, Sorin Armeanu, Ulrich Martin Lauer, Alexander Steinle, Michael 

Bitzer, Helmut Rainer Salih 

NK cell lysis of hepatoma cells following specific induction of 

NKG2D ligands by the proteasome inhibitor Bortezomib 

Natural killer (NK) cells as components of the innate immunity substantially contribute 

to anti-tumor immune responses. However, the tumor-associated ligands engaging 

activating NK cell receptors are largely unknown. An exception are the MHC class-I 

chain-related molecules MICA and MICB and the UL16-binding proteins (ULBP) which 

bind to the activating immunoreceptor NKG2D expressed on cytotoxic lymphocytes and 

potently stimulate anti-tumor immune responses. Here we report that treatment of 

human hepatocellular carcinoma (HCC) cells with the proteasome inhibitor Bortezomib 

stimulates recognition of cancer cells by cytotoxic lymphocytes via NKG2D. Bortezomib 

treatment induced transcription of MICA and MICB in the HCC cell lines PLC, HUH-7 and 

Hep3B leading to increased total and cell surface MIC protein expression. The induction 

of MIC molecules increased lysis of HCC cells by NK cells, which was abolished by 

blocking NKG2D-NKG2D ligand interaction using anti-MIC F(ab’)2 fragments. 

Importantly, Bortezomib treatment did not induce MIC protein expression in primary 

human hepatocytes and did also not alter NK cell viability and effector functions. Taken 

together, our data demonstrate that Bortezomib mediates specific priming of malignant 

cells for NK cell effector mechanisms. These results suggest the clinical evaluation of 

Bortezomib in solid tumors such as HCC, especially in combination with immunotherapy 

approaches like adoptive NK cell transfer.

Marion Schneider, Xuefang Ren, Ying Wang 

NK cell targeting of phagocytic dendritic cells 

NK dysfunction appears to be responsible for i)massive hemophagocytosis and ii) 

exceedingly high concentrations of interferon-g in a disease called hemophagocytic 

lymphohistiocytosis (HLH). Genetic NK deficiency is either due to mutations of the 

perforin gene, vesicular trafficking molecules such as Munc13-4 or syntaxin11. In 

addition, transient NK dysfunction may occur in infectious diseases such as EBV, 

leishmaniosis and also in autoimmune diseases. Using in vitro cultures supplemented 

with high dose IL-2 (10 3 IU/ml), NK function can be reconstituted in vitro. In the 

current study, hemophagocytes were enriched from patients with HLH and phenotyped 

by flow cytometry. Phagocytes were then tested as targets in cellular cytotoxicity 

assays using IL-2 activated effectors of these patients. 

Results: HLH derived phagocytes exhibited a dendritic cell phenotype expressing low 

CD80 and CCR7, but high densities of CD40, CD86, CD11c, CD63, CD64, and HLA-DR. 

CD209 was variably positive as well as BDCA4, CD1a, CD178 (FAS-Ligand) as well as 

TRAIL (tumor necrosis factor related apoptosis inducing ligand) receptor DR4. 

Phagocytes often lacked polarization and ultrastructural studies demonstrated even 

distribution of Golgi stacks in the cell. cells were derived from patients with HLH as well 

as from healthy donors. NK cytolysis by IL-2 acitvated killer effectors (derived from 

patients as well as healthy donors) was positive against 51chromium labeled cultured 

phagocytes as well as against K562 target cells and against allogeneic, EBV-transformed 

B cells. Lytic function was in a large portion independent of calcium and granule release. 

In defined effector target combinations, phagocytes were totally resistant to NK 

cytolysis. In the presence of TRAIL blocking antibodies, we found that DR4 positive 

phagocytes were killed by IL-2 activated receptors but DR4 negative phagocytes were 

not killed. We conclude that TRAIL and Trail Receptor DR4 play an important role in NK 

control of hemophagocytic cells overactivated in HLH. This context opens new aspects of 

treatment and cure.

Cédric VONARBOURG, Andreas DIEFENBACH 

NK CELLS EXPRESSING CLASS II MHC MOLECULES: SEPARATE 

LINEAGE OR JUST NK CELLS ? 

NKG2D is a stimulatory immunoreceptor constitutively expressed by all NK cells, a 

subset of NKT cells and the gd T cells of the skin. NKG2D interacts with a group of class 

I MHC-like molecules (NKG2D ligands, NKG2DL) that are upregulated on stressed cells 

whereas normal cells do not express these molecules. The NKG2D receptor/ligand 

system functions as a sentinel system to alert the immune system to endogenous 

cellular stress. Recent data from our lab indicated that vaccination of mice with NKG2DL 

+ tumor cells (but not with the parental NKG2DL- cells) leads to enhanced priming of 

tumor antigen-specific CD8 T cell responses. We wanted to investigate the molecular 

mechanism of how NKG2D-mediated innate immune activation leads to the 

enhancement of adaptive immunity. We hypothesized that NKG2D could regulate the 

function of antigen presenting cells (APC). During these studies, we identified a 

population of NKG2D+ cells co-expressing class II MHC which resides in the lymph 

nodes (ca. 30-40% of all NK cells) whereas this population was rather small in the 

spleen (

Petra Prinz, Ainhoa-Marie Figel, Judith Hosse, Peter J Nelson, Julia S Schleypen, Nicole 

Baur, Dolores J Schendel, Christine S Falk, Elfriede Noessner 

NK cells with distinct phenotypic and functional profile in the 

context of myeloid infiltration and renal cell carcinoma milieu 

Renal cell carcinoma (RCC) harbor high numbers of infiltrating lymphocytes with 

apparent limited efficacy in tumor control. Two groups of clear cell RCC were identified, 

one containing high (>20% of the lymphocyte population, n=14), the other low (

Rachel Thomas, Maren Mönkemeyer, Gamze Kabalak, Reinhold E. Schmidt, Torsten 

Witte 

NKG2D polymorphism associated with reactivation of CMV in 

HIV-1 infected patients 

Background 

NKG2D is an activating C-type lectin-like receptor expressed on NK cells and cytotoxic T 

lymphocytes. Upon ligand binding (ligands: stress-inducible MICA/B, ULBPs) NKG2D 

signals via the associated DAP10 adaptor protein, inducing cytotoxicity in NK cells and 

co-stimulation of the TCR. 

The two alleles of NKG2D differ by one nucleotide, guanine (G) or adenine (A), which 

results in an amino acid residue substitution in the transmembrane region at the 

binding site to DAP10. One of these alleles may be less functionally efficient in costimulating 

the TCR, and is associated with SLE, Sjögren`s syndrome and scleroderma. 

The aim of this study is to investigate any association of these alleles with HIV and 

CMV. 

Methods 

PCR and RFLP analyses were used to determine the allelic distribution of NKG2D in 240 

HIV patients and 239 healthy controls (all Caucasian). Fisher’s exact test was used to 

calculate the significance of the association. 

Results 

The A allele of NKG2D was found to be associated with HIV (with a frequency of 36% in 

HIV patients, 27% in healthy controls), as well as with CMV reactivation in HIV patients 

(48%). Associations with other co-infections (including GBV-C and HCV) have yet to be 

determined. 

Conclusions 

This study is investigating the importance of NKG2D signalling in HIV and CMV 

infections. The NKG2D-mediated control of CMV infection in HIV co-infected patients 

appears to play an important role, as the functionally less efficient allele is correlated 

with CMV reactivation. 

Supported by BMBF KN Rheuma C2.12

Benjamin Stoelcker, Kirsten Krätzel, Günther Eissner, Michael Pfeifer, Christian Schulz 

NKG2D-triggered effector function of bronchial epithelial cell 

activated alloreactive CD8+ T cells 

Allogeneic hematopoietic stem cell transplantation (SCT) has emerged as a curative 

therapeutic option still holding severe side effects including pulmonary toxicity. The 

exact role of GvHD for the lung injury after SCT has still to be determined. Induction of 

GvHD is considered to depend upon interactions between donor T cells and host APCs, 

but it has recently been shown that non-haematopoetic allograft cells can directly 

activate host CD8+ T cells. This prompted us to investigate in vitro the potential of 

bronchial epithelial cells to activate allogeneic CD8+ T cells and to study the induced 

cytotoxic effector functions. 

The bronchial epithelial cell line BEAS-2B was shown to express MHC I, MIC A and B, 

ULBP-2 and -3, ICAM1, CD70, B7-H1,-H2,-H3, but no or only very low levels of CD80 

and CD86. Purified allogeneic CD3+ CD8+ CD4- CD16/56- T cells cocultured with 

irradiated BEAS-2B in the presence of low dose IL-2 produced significant amounts of 

IFNγ, upregulated alloantigen activation markers CD69 and HLA-DR and showed high 

proliferation as compared to IL-2 alone stimulated T cells. Cytotoxicity assays 

demonstrated that specific, Granzyme B-mediated cytolytic activity against BEAS-2B 

was induced in the alloactivated CD8+ T cells. They showed increased expression of 

NKG2D and their cytolytic activity was inhibited using a blocking anti-NKG2D antibody, 

but not by blocking MHC I with antibody W6/32. IL-15, which has been shown to be 

able to contribute to TCR-independent CTL effector function could not be detected in the 

supernatants of the BEAS-2B/CD8+ T cell cocultures. This, together with the low dose 

IL-2 used argues against the induction of LAK cells. Cold target inhibition assays with 

K562 cells revealed that cytotoxicity was not the result of contaminating NK or NKT 

cells. 

Our in vitro data let us speculate that bronchial epithelial cell triggered NKG2Dsignalling 

contributes to a MHC-unrestricted CTL effector function in lung-directed 

alloreaction. This might be of clinical relevance for the course of GvHD after SCT.

Hansjörg Thude, Kathrin Rebstock, Bärbel Lutz, Jörg Blume, Dagmar Barz 

No association of the CD45 C77G gene polymorphism with 

inflammatory bowel disease in German patients 

Background: Inflammatory bowel diseases (IBD) like the Crohn`s disease (CD) and the 

ulcerative colitis (UC) are related with environmental, genetic, and immunological 

factors. Many aspects of the pathophysiology and the interactions are still unknown. In 

the present study we investigated whether C into G transversion at position 77 of exon 

A of the CD45 gene contributes to IBD. It has been described that this transversion 

prevents splicing of exon A of the CD45 gene and leads to a variant expression pattern 

of CD45RA on leucocytes. Material and methods: In a first step we analysed CD45RA/ 

CD45RO expression patterns on lymphocytes from patients with CD (n=178), UC 

(n=122), and healthy control individuals (n=360) by flow cytometric analysis. 

Subsequently all members of the study were genotyped for CD45 C77G polymorphism 

by polymerase chain reaction-allele specific restriction enzyme analysis. Results: Two 

patients with CD, six patients with UC, and eight control individuals displayed the 

variant CD45RA expression pattern. All identified individuals with variant CD45RA 

expression carried the C into G transversion in a heterozygous form. No homozygous 

individual was detected. The corresponding allele frequency of G was 0.55% in patients 

with CD, 2.45% in patients with UC, and 1.11% in control individuals demonstrating no 

significant difference (P=0.375 for CD vs controls and P=0.125 for UC vs controls) in 

the distribution of the 77G allele. Conclusion: These data shows that the C77G 

transversion is unlikely to be involved in IBD susceptibility in a German population.

Sascha Rutz, Marko Janke, Manuela Krueger, Alexander Scheffold 

Notch induces interleukin-10 production by T helper 1 cells 

T helper 1 (Th1) cells direct pro-inflammatory reactions and enable cellular immune 

responses against intracellular pathogens. If not properly controlled, Th1 cells may also 

cause immunopathology. IL-10 is a potent regulator of Th1 responses. Recently, IL-10 

production by Th1 cells has been identified as a critical self-limiting mechanism during 

infection. 

We identify Notch as a potent inducer of IL-10 in developing and established Th1 cells. 

Notch-modified Th1 cells still produce high levels of interferon-γ, but completely lose 

their pro-inflammatory capacity and instead potently suppress Th1-mediated responses. 

IL-10 induction is strictly dependent on IL 12/STAT4, and can be triggered by Delta-like 

Notch-ligands. 

We show that Notch/Dll interactions directly modify the inflammatory potential of Th1 

cells via the induction of IL-10, which may be part of a Th1-intrinsic self-limiting 

mechanism. These data suggest that triggering the Notch pathway facilitates a 

conversion of pro-inflammatory effector cells into regulatory cells; a finding of great 

interest regarding new therapeutic strategies in Th1-mediated chronic inflammatory 

diseases.

Anne Endmann, Dirk Bumann, Susanne zur Lage, Marcin Lyszkiewicz, Siegfried Weiss, 

Holger Lößner 

Novel Salmonella vaccine strains with in vivo amplifiable 

antigen expression plasmids 

Live attenuated Salmonella enterica serovars are explored as mucosal vectors for the 

delivery of heterologous antigens or DNA vaccines. Conventional expression systems 

are based on maintenance of the expression plasmids at multi-copy numbers 

representing a high metabolic burden for the bacteria. We have developed a novel 

Salmonella vaccine strain with amplifiable expression plasmids bearing two replication 

origins. The first replicon derived from the F factor or plasmid pSC101 ensures the 

minimal-copy number state. A second medium-copy replicon derived from plasmid RK2 

is responsible for conditional amplification of the expression plasmids under control of 

Salmonella-specific in vivo inducible promoters. This replicon mediated strong 

amplification of the plasmid copy number up to 50-100 copies. Consequently, enhanced 

expression of plasmid-encoded antigen was observed in vitro and in vivo. Furthermore, 

the expression level of the antigen could be further increased and tightly controlled in 

vivo by regulating its transcription via in vivo inducible promoters at the same time. The 

novel Salmonella vaccine strains were assessed regarding their colonization properties 

after oral administration and their immunogenicity in an adoptive transfer model. 

Intravenous immunization with such novel strains resulted in substantial ovalbuminspecific 

CD4+ T cell responses.

Britta Schneider, Jeannette Gerspach, Sabine Münkel, Harald Wajant, Peter Scheurich, 

Klaus Pfizenmaier 

Novel TRAIL fusion proteins as promising candidates for 

cancer therapy 

Homotrimeric soluble TRAIL is currently been clinically exploited as cancer therapeutic, 

but shows little antitumoral activity in monotherapy. In an attempt to further improve 

TRAIL’s tumour selective activity we generated new TRAIL fusion proteins comprising an 

antibody fragment (scFv) for targeting ErbB2. Further, a variant was designed, where 

TRAIL is expressed as a single polypeptide chain (scTRAIL). Apoptotic activity of 

scTRAIL is equivalent to a homotrimeric TRAIL molecule. We compared tumour 

targeting and apoptosis induction of scTRAIL based and of conventional, homotrimeric 

TRAIL fusion proteins with nontargeted TRAIL. Interestingly, among the tested TRAIL 

molecules, the tumour antigen targeted scTRAIL fusion protein showed highest 

apoptotic activity in vitro. The underlying mechanisms of this difference in apoptotic 

activity will be discussed. Superior apoptosis induction of targeted TRAIL molecules 

represent a promising strategy to improve TRAIL’s anti-tumoral action and minimizes 

potential non-targeted actions on normal tissues.

Verena Susanne Meyer, Dagmar Sigurdardottir, Wolfgang Kastenmüller, Georg 

Gasteiger, Ingo Drexler, Stefan Stevanovic 

Novel vaccinia virus T-cell epitopes identified by mass 

spectrometry of HLA ligands 

Modified vaccinia virus Ankara (MVA) is currently under consideration as a third 

generation vaccine against smallpox as well as tested as recombinant viral vector 

vaccine for immunotherapy of infectious diseases and cancer. CD8 T-cell responses 

against MVA are well documented, but only a few vaccinia virus T-cell epitopes are well 

characterized. 

In this study, we describe a novel approach to identify vaccinia virus-derived MHC class 

I peptide ligands. Peptides isolated from HLA molecules of MVA-infected and noninfected 

B-cells were differentially labeled by chemical modification, separated by HPLC 

and sequenced using ESI mass spectrometry. Using this approach, we identified several 

MVA-specific HLA-A*0201 and HLA-B*0702 ligands. The majority of these peptide 

determinants were derived from early viral proteins. Only one ligand corresponded to a 

late gene product. Importantly, we confirmed the presence and functionality of CD8 Tcells 

recognizing these ligands in PBMCs derived from MVA-vaccinated donors. Two of 

the 6 epitopes were identical to recently published determinants found to be reactive in 

Dryvax vaccinees. The data will help to further characterize and to evaluate the 

immunogenicity of vaccines based on orthopoxviruses.

Kurt Schönfeld, Sabine Ring, Tanja Bedke, Sonja Schallenberg, Karsten Mahnke, 

Alexander H. Enk 

Nucleofection is an effective method to transduce regulatory 

T cells with different homing receptors. 

Regulatory T cells (Tregs) are a well described subset of CD4 T cells. They are 

characterized by the expression of CD4, CD25 and the transcription factor FoxP3, as 

well as by their ability to suppress the proliferation of CD4 T cells. 

We have recently shown that in a murine contact hypersensitivity (CHS) model, injected 

Tregs are able to suppress the contact allergen induced ear swelling reaction through 

inhibition of the CD8 cells. So far it is not clear as to how Tregs suppress CD8 cells and 

whether homing to the skin and or to the skin draining lymph nodes is mandatory for 

their function. Thus, to investigate whether homing of Tregs to different tissue sites 

affects their ability to suppress CHS reactions, we set out to clone the cDNA of the 

murine homing receptors CCR7, CCR9, CCR10, Itgae and PSGL-1 and to transfect 

Tregs. 

Using EL-4 T cell line as well as expanded and freshly isolated Tregs we were able to 

establish reliable transfection of Tregs with the respective plasmids using the Amaxa 

nucleofection method. Using this method we yielded transfection rates of 50%, (EL-4) 

20% (freshly isolated and stimulated Tregs) and 10% (expanded Tregs), respectively. 

In further experiments we plan to inject these cells into mice and to analyse their 

migration pattern as well as their suppressive function in the CHS model. These 

experiments will clarify as to how different adhesion molecules guide Treg homing in 

vivo and whether differential homing affects the suppressive activity of the Tregs.

Sabrina Schmitt, Karsten Mahnke, Kurt Schönfeld, Svetlana Karakhanova, Alexander 

H. Enk 

Number and function of regulatory T cells in GvHD increase by 

Extracorporeal Photopheresis 

Extracorporeal Photopheresis (ECP) is a procedure commonly used to reduce transplant 

rejection. It is also applied in diseases like GvHD, cutaneous T cell lymphoma and other 

medical conditions involving overboarding immune reactions. 

The mechanism by which ECP exerts its immunosuppressive properties remains elusive. 

However, since regulatory T cells (Treg) are a major cellular component that contributes 

to immunosuppressive mechanisms, we set out to investigate whether ECP affects Treg 

function. 

For this end we analysed peripheral blood of patients with GvHD after bone marrow 

transplantation who received ECP-treatment. The patients were ECP treated on 2 

consecutive days and blood samples were taken before and after each session. First, we 

prepared PBMC and compared the respective amounts of various cell types within the 

peripheral blood of the patients before and after treatment via FACS analysis. Here we 

observed an increase of CD4+ CD25+ FoxP3+ Treg directly after each ECP cycle and 

also in the general course of treatment (6 cycles, over 3 months analysed). 

Moreover, to study functional properties of this distinct population we analysed the 

suppressive effect of MACS-bead isolated Treg before and after ECP using conventional 

suppression assays. As controls Treg were isolated from untreated healthy volunteers. 

These assays revealed that Treg before ECP showed a significantly reduced suppression 

of T cell proliferation, whereas after ECP, Treg equalled Treg isolated from healthy 

volunteers in their suppressive capacity. 

In conclusion ECP may exert its immunosuppressive functions by upregulating the 

number and the suppressive capacity of regulatory T cells and may thus be an 

appropriate therapy to prevent over-reactions of the immune system after stem cell 

transplantations.

Martin Kolev, Marieta Ruseva, Steffen Thiel, Frederik Hansen, Jens Jensenius 

On the role of MBL in a murine model of oral infection with 

Salmonella typhimurium 

Salmonella enterica is a gram-negative intracellular microorganism, which grows in the 

mononuclear phagocytic system of the host and causes disease. The complex 

interaction between the both host and the bacterium is still unknown. Multiple humoral 

and cellular effector mechanisms, implicated in host resistance to Salmonella infections 

have been described.• 

Complement plays a major role in host defense against bacteria and fungi. • 

MBL (mannan binding lectin)-a plasma protein belonging to a family of proteins known 

as collectins. It interacts with a wide array of microorganisms, including Salmonella spp. • 

Mice and calf models have been used to study the mechanism of Salmonella infection in 

vivo. • 

The model we used relies on pretreating mice with Streptomycin, which kills the normal 

gut flora and allows for subsequent infection with Salmonella.• 

We infected them with Salmonella enterica serovar Typhimurium SL1344 - a naturally 

occurring strain that is streptomycin resistant.• 

We followed the course of Salmonella infection for 3 days in wild-type (WT) female 

C57BL/6 mice and compared disease severity with female MBL double knock-out (DKO) 

mice (both MBL-A and MBL-C deleted). We observed about 100 fold increased bacterial 

loads of the cecum in MBL DKO compared to wild-type animals. These results were also 

confirmed by H&E staining of ceca cryosections. There was also an about 10-fold 

increase in bacterial loads in fecal pellets of MBL DKO mice, when compared with WT. 

However, no difference in liver loads between WT and DKO was observed. The latter 

suggests that the infection after 3 or 4 days is systemic. The greater severity in DKO 

was also reflected by rapid weight loss. • 

The results suggest an important role of MBL in the immune response to systemic 

Salmonella infection.

Monika Braun, Christine Sers, Ruprecht Kuner, Barbara Simm, Barbara Mosetter, 

Dolores Schendel, Christine Falk 

Oncogenic KRAS-signalling and DNMT activity are involved in 

immune escape of tumor cells 

The transformation process of cancer cells is often accompanied by an accumulation of 

genetic and epigenetic abnormalities that lead to indefinite proliferation and evasion of 

immune recognition by immune effector cells such as CTL and NK cells. The impact of 

these malignant alterations on immune evasion strategies by tumor cells is still poorly 

understood. HCT116 colon carcinoma cell lines were selected with respect to mutated Ki- 

Ras that is associated with oncogenic constitutive MEK/ERK signalling. In addition, 

variants of this cell line with aberrant DNA-methylation due to deletion of DNAmethyltransferase 

DNMT1 and/or 3b were available. 

These cells were treated with a MEK/ERK pathway inhibitor (U0126) and analysed for 

mRNA expression profiles by microarray-analysis and RT-PCR, for promoter-methylation 

patterns by bisulphite sequencing and for surface expression by FACS analysis. The 

functional relevance for immune recognition was tested using HLA class I-restricted CTL 

and allogeneic NK cells. In addition, the effect of IFN-α versus IFN-γ treatment in 

combination with Ras-signalling inhibition and DNMT-deficiency was analysed with 

respect to sensitivity to lysis by CTL and NK cells. 

The analysis revealed that some immune genes are co-regulated via DNMT and MEK/ 

ERK signalling. HLA-A and NKG2D-ligand ULBP2 surface expression was up-regulated by 

inhibition of MEK/ERK signalling and/or deletion of DNMT, with highest expression levels 

in those cells where both systems were blocked. Using an HLA-A2-restricted CTL clone 

as effector cell, these changes in the density of HLA-A could be sensed and led to 

enhanced tumor lysis. In addition, treatment with IFN-γ could further enhance CTL lysis, 

but diminished NK-mediated killing. In contrast, IFN-α treatment could increase CTL 

lysis without decreasing the NK cell response. Taken together, our data indicate that 

both, oncogenic Ras-signalling and DNMT activity contribute to the immune escape 

against CTL recognition. Importantly, this escape could be reversed by IFN-α but not 

IFN-γ with respect to both, CTL and NK recognition.

Varsha Kumar, Jens Stein 

Optical ProjectionTomography as a tool for studying the threedimensional 

structure of Seconday Lymphoid Organs* 

The internal organization of complex organs, such as secondary lymphoid organs (SLO), 

is essential to understand their function. So far, most information regarding SLO 

organization and restructuring during inflammation has been obtained using traditional 

two-dimensional (2D) sectioning of tissue followed by immunofluorescent or 

immunohistologic labeling. However, differences obtained due to the plane of cutting of 

the specimen and the lack of a three-dimensional (3D) context makes interpretation of 

2D sections not always reliable. Optical Projection Tomography (OPT) is a recently 

developed imaging technique ideally suited to examine internal 3D structures of wholemount-labeled 

specimen (0.5 to 15 mm diameter), which elies on the reconstruction of 

high-resoulution virtual sections using computer-based back-projection algorithms. Our 

first whole-mount staining of B cell follicles in LN and spleen for OPT provided us with 

high resolution, qualitative 3D overviews of B cell follicle structure. To the best of our 

knowledge, these images covering entire LNs and extensive spleen sections constitute 

the first 3D overviews over these important microcompartments. This allowed us to 

identify the polarized localization of B cell follicles in LN and previously unnoticed “B cell 

bridges” between subsets of individual follicles, which would have been difficult to 

assess in 2D sections. More importantly, we quantitatively analyzed B cell follicle 

number, volume, and relative distances, with image analysis software (Imaris, Bitplane, 

Switzerland) and showed that number and volume of B cell follicles to the total volume 

of LN remains constant in any given LN. Furthermore, we have also established staining 

techniques to label vascular network which will allow us to examine, by OPT, the 3D 

relationship between various microcompartments before and after inflammation. Thus, 

our studies not only aim to characterize the 3D anatomy of murine SLO, but also to 

form a more objective basis to model changes during inflammatory processes.

Mathias Fousse, Ulrich Mack, Tobias Hodapp, Urban Sester, Gerhard W. Sybrecht, 

Hans Köhler, Martina Sester 

OPTIMIZED DIAGNOSIS OF LATENT TUBERCULOSIS 

INFECTION BY ANALYSIS OF ESAT-6 AND CFP-10 SPECIFIC T- 

CELL REACTIVITY USING A RAPID FLOW-CYTOMETRIC ASSAY 

We have recently shown that a flow-cytometric whole blood assay based on tuberculin 

(PPD) and the M. tuberculosis specific proteins ESAT-6 and CFP-10 may be superior in 

diagnosing a latent tuberculosis infection as compared to the established skin test. This 

study was carried out to establish a clinically relevant threshold of PPD reactivity 

including cytokine profiles and T-cell reactivity towards ESAT-6 and CFP-10. 

Whole blood from 439 patients was stimulated in vitro with PPD, ESAT-6 and CFP-10. 

After 6 h, the number and cytokine pattern (IFNγ, IL-2) of T cells as potential marker for 

disease activity was determined using flow-cytometry. In 310 patients, skin-testing and 

flow-cytometry was compared using ROC analysis. 

Skin test induration and PPD reactive T-cell frequencies showed a significant correlation 

(r=0,60, p

Gudrun Szalay, Martina Sauter, Carmen Ruoff, Reinhard Kandolf, Karin Klingel 

Osteopontin is not involved in antiviral immunity but in 

cardiac fibrosis during chronic enterovirus myocarditis 

Osteopontin (OPN) is a pleiotropic cytokine involved in tissue remodeling, chemotaxis 

and cell-mediated immunity. OPN is expressed by different cell-types like macrophages, 

T-cells, epithelial cells and smooth muscle cells, and expression is strongly up-regulated 

in inflamed tissues. After coxsackievirus B3 (CVB3) infection, OPN was detected as one 

of the highest regulated genes in murine hearts by microarray analysis. Therefore, we 

wanted to elucidate whether OPN plays a role in heart remodeling and/or in cellmediated 

immunity to enterovirus infection. 

We used quantitative RT-PCR to examine cardiac OPN expression in CVB3-infected mice 

at different time points post infection (pi). Mice, susceptible for chronic myocarditis, 

showed a higher OPN expression during acute and chronic myocarditis with a maximum 

at day 8 pi, than mice resistant to chronic myocarditis. OPN gene deficient (OPN-ko) 

mice revealed a course of myocarditis with viral titers, IFNγ and IL-10 secretion patterns 

and no chronic inflammation, which is typical for resistant mice. By in situ hybridization 

and immunohistochemistry we detected macrophages as producers of OPN within 

inflammatory lesions in the heart. Transcript levels for matrixmetalloproteinase-3, 

tissue inhibitor of metalloproteinase-1, urokinase-plasminogen activator and procollagen 

I – all molecules involved in cardiac remodeling – were high in susceptible mice, 

whereas in resistent mice and OPN-ko mice only minimal levels were detected. In 

addition, morphometric analysis demonstrates a more distinct myocardial fibrosis in 

susceptible mice than in resistent mice and OPN-ko mice only had minimal fibrosis. 

OPN is not involved in the cell-mediated immunity against CVB3. However, OPN 

expression induces the expression of proteins responsible for remodeling thereby 

leading to pronounced myocardial fibrosis.

Stefan Ehlers, Sahar Aly, Klaus Wagner, Sven Malm, Tamas Laskay, Jörg Mages, 

Roland Lang, Ian Orme, Franz Bange 

Oxygen Status of Lung Granulomas in M. tuberculosis- 

Infected Mice and Guinea Pigs and Causal Involvement of 

CXCR3-targeted Chemokines in Granuloma Necrosis 

It is often assumed that Mycobacterium tuberculosis (Mtb) -induced granulomatous 

lesions, particularly those undergoing central caseation, are anoxic, and that the 

survival of Mtb in these lesions requires the integrity of its non-oxidative respiratory 

pathways. Using the hypoxia marker pimonidazole we provide immunohistochemical 

evidence that in the most frequently used animal model system of inbred mice, Mtbinduced 

granulomas, even after more than one year of aerogenic infection, are not 

severely hypoxic. Direct measurements of oxygen tension with a flexible microelectrode 

in mouse lungs chronically infected with Mtb revealed a wide range of oxygen partial 

pressures in different parts of the lungs which, however, rarely approached the anoxic 

conditions consistently found in necrotizing tumors. We further show that a Mtb mutant 

defective in nitrate reductase (narG) necessary for survival under anaerobic conditions 

in vitro, can persist in the lungs of chronically infected mice to a similar extent as wild 

type Mtb. In contrast, necrotizing granulomas in guinea pig lungs and lymph nodes 

infected by aerosol with Mtb consistently showed staining patterns indicative of severe 

hypoxia, when examined with pimonidazole immunohistochemistry. Interestingly, 

necrotizing granulomas in mice aerogenically infected with Mycobacterium avium 

TMC724 also had hypoxic zones immediately adjacent to their caseating centers. 

Granuloma vascularization was significantly decreased in central, but not peripheral, 

areas of granulomas of infected wild-type (WT) compared to IFN-gamma knock-out 

(GKO) mice infected with M. avium TMC724. This was associated with a reduced mRNA 

expression of angiostatic chemokines, such as CXCL9-11, in GKO mice. 

Histomorphology in CXCR3-KO mice, however, showed similar caseating granulomas as 

WT mice 16 weeks after TMC724 infection. 

Conclusion: The combined evidence of microbiological, immunohistological, and 

biophysical approaches strongly supports the conclusion that Mtb-induced granulomas 

are not anoxic in mice. This is in contrast to Mtb-induced lesions in guinea pigs which 

appear to be severely hypoxic. Therefore, the in vivo validation of metabolic pathways 

that are induced in Mtb under severely reduced oxygen tension in vitro may not 

unequivocally be performed in the mouse model of tuberculosis and may need to resort 

to other experimental model systems, for example the guinea pig. 

Using the M. avium model of lung immunopathology, we demonstrate that, while IFNgamma 

causes a dysbalance between angiostatic and angiogenic mediators and a 

concomitant reduction in granuloma vascularization, CXCR3-targeted chemokines are 

not sufficient to induce granuloma necrosis. 

This work was supported in part by grants to SE (SFB367-C9, NGFN2/NIE-S05T22), TL 

(SFB367-B10, La1267/1), RL (NGFN2/NIE-S31T29) and to IMO (NIH/AI054697).

Michal Smida, Anita Posevitz-Fejfar, Burkhart Schraven, Jonathan A. Lindquist 

PAG downregulation results in increased proximal signaling 

but reduced functional outcomes in primary T cells 

PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains) is a 

transmembrane adaptor protein that recruits Csk (C-terminal Src kinase) to the plasma 

membrane, where it then phosphorylates inhibitory tyrosines within Src kinases. 

Recently, we have also shown that PAG negatively regulates Ras activation by recruiting 

RasGAP. Thus, PAG functions as a negative regulator of both Src kinases and Ras. Since 

PAG knockout mice possess no apparent phenotype, the importance of PAG as a 

negative regulator has been questioned. Therefore we investigated PAG function in 

primary human T cells and in Jurkat T cells using RNA interference. 

Suppression of PAG expression leads to an enhancement of kinase activity in both Fyn 

and Lck, which is reflected by increased phosphorylation of their activatory tyrosines. As 

a consequence, we detect enhanced basal tyrosine phosphorylation in resting cells, 

which is also more sustained upon TCR stimulation. Additionally, we have investigated 

specific proteins involved in the proximal signaling. We observed an enhanced activation 

of ZAP70 and PLC gamma and a 5-fold increase in Ras activation. However, IL-2 

promotor activity appears to be decreased and also the proliferation of PAG deficient 

cells was reduced, as measured by both CFSE labeling and thymidine incorporation. 

Thus, although the removal of a negative regulator leads to enhanced proximal 

signaling, this does not necessarily lead to an increase in proliferation or IL-2 

production, but rather may potentiate other outcomes of T-cell activation, e.g. cell 

death.

Gurumoorthy Krishnamoorthy, Florian C. Kurschus, Klaus Dornmair, Reinhard 

Mentele, Hans Lassmann, Hartmut Wekerle 

Paradoxical autoimmunity: Molecular self mimicry initiates 

spontaneous autoimmunity in myelin-specific T cell receptor 

transgenic mice deficient of cognate self antigen 

We describe here the identification of neurofilament medium (NF-M), a neuron-specific 

cytoskeletal intermediate filament protein, as a cross-reactive autoantigen for a myelin 

oligodendrocyte glycoprotein (MOG)-specific T cell receptor (TCR). Studying the 

mechanisms of spontaneous autoimmunity in MOG-specific TCR transgenic mice (2D2), 

we bred the TCR transgene into MOG deficient (MOG-/-) mice of appropriate C57BL/6 

background. Surprisingly, MOG-deficient 2D2 TCR transgenic mice (2D2 x MOG-/-) 

developed experimental autoimmune encephalomyelitis (EAE) with frequency and 

symptoms undistinguishable from single transgenic MOG+/+ 2D2 mice. About 10 - 20% 

of the 2D2 and 2D2 x MOG-/- mice developed spontaneous EAE at around 5-8 weeks of 

age. The T cells isolated from the inflamed central nervous system (CNS) predominantly 

contained transgenic T cell populations ruling out any endogenous T cells might be 

responsible for autoimmunity. The clinical and histological features of the EAE were 

similar between these two strains. 

Having excluded failed gene ablation in our MOG-/- strain, we scanned MOG-/- CNS 

tissue for 2D2 T cell activating activity. We identified one fraction which induced a 

strong proliferative response of 2D2 T cells. Mass spectrometry analysis identified 

neurofilament proteins as major components. Sequence alignment of NF-M and MOG 

revealed a motif shared by the MOG 35-55 peptide and a segment of NF-M. We 

confirmed cross-reactivity between MOG and NF-M on the level of recombinant proteins 

and synthetic peptides. 

This is the first report of a pathogenic, myelin specific T cell clone cross-reacting with 

another CNS self-protein which can serve as target of an autoimmune attack.

Hanna Erdmann, Paul Crocker, Bernhard Fleischer, Thomas Jacobs 

Pathogenic Trypanosoma cruzi interacts with the immune 

modulatory molecule mouse Siglec-E (sialic acid-binding Iglike 

lectin-E) 

The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a 

chronic infection, in which a strong suppression of the immune response leads to a lifelong 

persistence of parasites in host cells. The trypanosomal trans-sialidase is described 

to be a major virulence factor. This enzyme allows the parasite to acquire sialic acids 

from its environment by cleavage from host glycoconjugates and direct transfer to GPIanchored 

mucin-like molecules on its own cell surface. In the current investigation we 

provide evidence that the murine molecule Siglec-E directly interacts with sialylated 

ligands on the surface of T. cruzi. Siglecs are sialic acid-binding Ig-like lectins that are 

expressed on most cell types of the immune system. They have been shown to act as 

inhibitory receptors, ascribed to the presence of conserved immunoreceptor tyrosinebased 

inhibition motifs (ITIMs) in their cytoplasmic regions. The murine Siglec-E is 

expressed on phagocytic cells as well as on NK cells. Using Siglec-E transfected CHOcells 

and a Siglec-E-Fc fusion molecule we demonstrate that Siglec-E binds with high 

affinity to both extracellular trypomastigotic and intracellular amastigotic parasites of 

the pathogenic T. cruzi Tulahuen strain. In contrast, we can detect only a weak binding 

of Siglec-E to extracellular trypomastigotic parasites of the apathogenic T. cruzi 

Tehuantepec strain, whereas the binding of Siglec-E to intracellular amastigotic 

parasites of this apathogenic strain is completely absent. 

These results raise the possibility that the pathogenic parasite dampens immune 

responses via binding to the inhibitory receptor Siglec-E, allowing the parasite to persist 

and to establish a chronic infection.

Jan Dirks, Urban Sester, Daniela Presser, Heinrike Wilkens, Hans Köhler, Martina Sester 

PD-1 expression on CMV specific CD4 T cells is associated 

with viremia and reversible functional anergy in patients after 

organ transplantation 

Recently, chronic infections with LCMV, HIV or HCV were shown to be associated with 

anergic T cells characterised by high expression of PD-1. We have previously shown that 

uncontrolled CMV replication after transplantation correlates with a drop in CMV specific 

T cells. This study was extended to analyse whether functional anergy of specific T cells 

in individuals with and without CMV viremia may affect control of CMV replication. 

CMV specific CD4 T cells from 12 controls, 25 hemodialysis patients, 43 renal and 14 

lung-transplant patients were quantified using flow cytometry and analysed for their 

expression of PD-1 and cytokines IFNγ and IL2. Specific proliferation was analysed by 

CFDA-SE dilution. CMV-DNA was quantified using PCR. 

In viremic patients, a significantly higher proportion of CMV specific CD4 T cells were PD- 

1 positive (median 42.3%) as compared to non-viremic patients (7.4%), hemodialysis 

patients (8.8%) or controls (3.1%, p=0.001). In line with functional impairment, PD-1 

positive T cells produced significantly less IFNγ as compared to PD-1 negative cells (MFI 

101 vs. 210, p

Hiroaki Azukizawa, Nobuo Kanazawa, Manfred B. Lutz 

Peripheral de novo induction of CD4+ CD25+ Foxp3+ 

regulatory T cells by steady state migratory dendritic cells 

Transport of microbial antigens from infection sites to the draining lymph nodes by 

dendritic cells (DC) is well established. Accumulating evidence suggests that DC 

migration also occurs under steady state conditions resulting in tolerance induction to 

peripheral self antigens. However, the precise migratory DC phenotype and the CD4+ T 

cell tolerance mechanism induced by such migratory DC remain unclear. We show that 

steady state migration of skin DC depends on CCR7 but not on inflammation or 

microbial stimuli, since skin-derived DC are present in peripheral lymph nodes of germfree 

mice and mice deficient for in CD40L, TNFR1/2 or MyD88. At their surface 

migratory DC express elevated levels of MHC II and costimulatory molecules but they 

do not produce cytokines, thus representing a semi-mature state. Only migratory DC 

within draining lymph nodes contained peripheral self antigens as apoptotic cells. In 

transgenic mice expressing OVA as cell-associated neo-self antigen in the skin (K5mOVA), 

or mice subcutaneously implanted with OVA-secreting osmotic pumps, only 

migratory skin-derived DC but not immature lymph node-resident DC were able to 

present OVA in vitro or in the peripheral lymph nodes to adoptively transferred OVAspecific 

CD4+ T cells (OT-2), as evaluated by the use of K5-mOVA x CCR7-/- mice, 

lacking migratory DC. In both models of antigen transport, adoptively transfered OT-2 x 

RAG-/- T cells, which are devoid of natural Tregs, became activated in the peripheral 

lymph nodes and developed into CD4+ CD25+ Foxp3+ regulatory T cells within 2 

weeks. Together, our data indicate that semi-mature, steady state migratory DC induce 

Treg against soluble and cell-associated self-antigens in peripheral lymph nodes under 

steady state conditions.

Fabien Agenes, Jean-Pierre Dangy, Jörg Kirberg 

Peripheral inter-clonal competitiveness requires T cell 

receptor contact to restricting MHC molecules 

In the adult, peripheral lymphocyte numbers are relatively constant, indicating that 

there are homeostatic control mechanisms. Known requirements for peripheral T cell 

survival, as well as homeostatic proliferation, are the TCR interaction with restricting 

self-MHC molecules and the availability of lymphokines, such as IL-7. It is possible that 

by competition towards these factors T cells reach a constant steady state number; 

however, the mechanisms by which these restrains interrelate are not completely 

understood. 

It has been noted that upon adoptive transfer, homeostatic proliferation of donor T cells 

can occur even in the full complement of host cells, provided the latter express a 

different TCR. In other combinations, however, donor T cells are blocked by the 

preformed presence of unrelated T cells. Thus there might be a “hierarchicalcompetitiveness”. 

We find such hierarchical-competitiveness in the order OT-1 TCR > 

P14-TCR > F5-TCR. As the OT-1 TCR is restricted to Kb, and the others are to Db, it 

was possible to remove the OT-1 TCR ligand exclusively. Using various Kb-mutant mice 

we demonstrate that proliferation of OT-1 T cells in hosts harboring P14-TCR expressing 

cells, and, likewise, the competence of OT-1 T cells to block proliferation of P14-TCR 

expressing cells, requires OT-1 TCR binding to Kb. Detecting no functional crossreactivity 

of the OT-1 TCR, this excludes that direct competition for MHC molecules 

causes hierarchical-competitiveness. Furthermore, parabiosis experiments with Kbmutant 

mice exclude that the hierarchical-competitiveness is caused by competition for 

direct cellular contact to, or modulation of, the restricting MHC-bearing cells. Thus, it is 

the apparent avidity of the TCR to restricting MHC molecules that defines a relative 

competence to compete, secondarily, for a trophic resource available in limited supply 

and not bound to the APC. Such mechanism would allow for a localized competitiveness 

operating beyond a T cells’ specificity.

Kristina Streyl, Mikhail Nosov, Vijay Kumar Ulaganathan, Hartmut Wekerle, Alexander 

Flügel 

Peripheral milieus license autoaggressive effector T cells to 

invade their target organ 

Using a transfer model of experimental autoimmune encephalomyelitis (tEAE) we 

explore the question why the paralytic disease occurs only after an obligatory prodromal 

phase of several days after transfer of the autoaggressive T cells. Traditionally CNS 

invasion during EAE is believed to follow a biphasic course. The first infiltration wave 

occurs hours after T cell transfer. These “pioneer” cells are thought to prime the 

immune privileged CNS tissue and thus, pave the way for a second inflammatory wave, 

which occurs at the onset of clinical disease. Using retrovirally labeled green fluorescent 

protein expressing encephalitogenic T cells (T GFP cells) we now found that the 

numbers of pioneer T cells in the preclinical CNS parenchyma analyzed by 

cytofluorometry and microscopically were negligible low (

Sebastian Bunk, Iuliana Suznea, Jan Rupp, Matthias Maass, Michael Przybylski, Albrecht 

Wendel, Corinna Hermann 

Persisting Chlamydia pneumoniae infections are associated 

with differential immune responses 

The respiratory pathogen Chlamydia pneumoniae can disseminate from the lung via 

infected phagocytes and persist in the host tissue for years. A reliable diagnosis, which 

allows discrimination between past and persistent infection is a prerequisite to study its 

association with chronic inflammatory diseases. Our project aimed to investigate 

whether persistent C. pneumoniae infections are associated with specific antibody 

response patterns. 

Therefore, C. pneumonia proteins were prepared from elementary bodies, separated by 

2D-gel electrophoresis and blotted to nitrocellulose membranes. The membranes were 

incubated with 39 human sera and bound IgG antibodies were quantitatively analysed 

by a LAS-3000 imaging system. Nineteen of the 39 sera revealed evidence for 

persisting C. pneumoniae infections as determined by PCR analysis of monocytes or 

vasculatory samples. Using high resolution MALDI-FTICR-MS, we identified 38 

Chlamydia proteins originating from 33 genes which were frequently recognized among 

the 39 sera. About half of the proteins, including some with high antigenic potential, 

represent Chlamydia antigens not described before. Comparison of antibody response 

pattern between sera from subjects with and without evidence for persisting C. 

pneumoniae, resulted in differential reactivity of the two groups of sera towards twelve 

proteins. While the reactivity of sera from PCR-positive donors was increased for eight 

proteins, reactivity was decreased for four proteins, thereby reflecting altered protein 

expression patterns of in vitro models of C. pneumoniae persistence. Taken together, 

these results provide first evidence for serological differences associated with the status 

of C. pneumoniae infections.

Nirmal Robinson, Tom Li Stephen, Sonja Meemboor, Wiltrud M. Kalka-Moll, Georg Plum 

Phagosomal and endosomal trafficking studies by adenoviral 

Rab-GFP fusion proteins in primary cells 

Antigen presentation by major histocompatibility complex (MHC) class II on antigen 

presenting cells (APCs) is facilitated after a sequence of events that starts with a 

phagocytic process, followed by phagosomal processing of the antigen, phagolysosomal 

degradation of the phagocytosed material and loading of antigenic peptides onto MHC 

molecules. A detailed picture of this process termed phagosome maturation is beginning 

to emerge, involving regulators of membrane trafficking in mammalian cells and 

phagosomal interactions with endosomal organelles and the trans-Golgi network. So far, 

cell biology studies of antigen presentation can be performed in primary cells after 

fixation and permeabilization for staining with specific antibodies or probes for other 

endosomal pathway . Investigation in live cells is solely possible in cell lines after 

transfection with expression vectors for GFP-tagged endosomal marker proteins. In an 

effort to open up ways to elucidate the phagosomal processing events after antigen 

uptake in live primary antigen presenting cells of murine and human origin we have 

developed a set of adenoviral Rab-GFP fusion protein expression vectors that allow 

observation and tracking individual phagosomes. Transfection with the early endosomal 

marker Rab5-GFP, late endosomal marker Rab7-GFP, and transferrin receptor recycling 

marker Rab11-GFP was achieved in 92% to 93% of the mouse dendritic cells, in 89% to 

94% of human macrophages, and in 70% to 82% of mouse macrophages. Rab-GFP 

fusion proteins were readily expressed in these primary cells using these highly efficient 

adenoviral constructs allowing phagosome and endosome trafficking by fluorescently 

labeled fluid phase marker. Our data show that the intracellular endocytic trafficking 

events preceeding antigen presentation in primary APCs can be effectively studied in 

live cells by using our adenoviral expression systems.

Thomas Giese, Claudia Sommerer, Martin Zeier, Stefan Meuer 

Pharmacodynamic controlled tapering of Cyclosporine A – a 

new step towards individualized immunosuppressive therapy 

in transplanted patients 

Background. At present it is unclear which dose of Cyclosporine A (CsA) is optimal with 

respect to immunosuppressive efficacy and drug specific side effects at the level of the 

individual patient. Recently we proposed the quantitative assessment of NFAT regulated 

gene expression in peripheral lymphocytes as a new tool to measure the individual 

degree of immunosuppression by CsA and demonstrated that a significant correlation 

exists between suppression of NFAT-regulated genes and clinical complications such as 

infections and malignancies. The reduction of CsA dosage under close pharmacodynamic 

monitoring may lead to reduced drug specific side effects while maintaining optimal 

graft protection. 

Methods. Eight stable renal transplant recipients were enrolled and longitudinally 

followed for 27 (12-44) months. Median CsA dose was 1.83 mg/kg/day at the time of 

enrolment and was tapered to 1.19 mg/kg/day. NFAT-regulated gene expression was 

measured in peripheral blood lymphocytes stimulated with PMA and ionomycin ex vivo. 

All patients had an ultrasound guided allograft biopsy. 

Results. The stepwise reduction of CsA was inversely correlated to the residual NFATregulated 

gene expression. In seven patients there were no signs of acute rejection in 

the whole study period. One patient had a BANFF Ia rejection. In this patient the mean 

residual NFAT-regulated gene expression had increased up to 47% in the 6 month prior 

to rejection. All patients without rejection had a median NFAT-regulated gene 

expression of 25%. 

Conclusion. The measurement of residual NFAT-related gene expression is a helpful 

and reliable tool in individualizing the immunosuppressive therapy in long-term allograft 

recipients.

Carmen Scheibenbogen, Anne Letsch, Antonia Busse, Sandra Bauer, Alexander 

Schmittel, Wolf-Karsten Hofmann, Lutz Uharek, Eckhard Thiel, Ulrich Keilholz 

Phase II trial of vaccination with WT1 peptide, GM-CSF, and 

KLH in patients with acute myeloid leukemia: immunological, 

molecular, and clinical results 

Purpose: The transcription factor Wilms tumor protein (WT) 1 belongs to a new 

generation of tumor antigens, which are essential for tumor cell proliferation. A phase II 

trial of vaccination with an HLA-A2-restricted peptide was performed in patients (pts) 

with acute myeloid leukemia. 

Methods: Pts received repeated vaccinations with 0.2 mg WT1.126-134 peptide (day 3), 

62.5 mcg GM-CSF (days 1-4) as DC-stimulant and 1 mg KLH (day 3) as T helper 

protein. WT1-specific T cell responses were analysed by tetramer and cytokine flow 

cytometry. WT1 levels were assessed by qRT-PCR. 

Results: 

Of 29 pts enrolled 26 were evaluable for clinical and 25 for immunological outcome. One 

complete remission (514 days) and 13 stable diseases (99 to 339 days) were observed, 

5 with >50% blast reduction and 3 with hematological improvement. The median time 

to treatment failure (TTF) was 143 days. WT1 mRNA-levels as molecular marker of 

leukemia load decreased in 52% (2 to>50-fold). There was a significant association 

between decrease in WT1 mRNA levels and TTF (p=0.026). 

WT1 tetramer+ CD8+ T cells were found in 7 out of 25 pts before vaccination. At week 

10 a >2-fold-increase was observed in 13 of the 18 pts without a preexisting WT1 

specific T cell response in PB, whereas in the 7 pts with preexisting responses these 

could not be boosted. In 19 out of 25 pts T cell responses could be analysed in parallel 

in bone marrow at week 18, showing similar frequencies of WT1 tetramer+ CD8+ T 

cells in bone marrow (0.36%) and in PB (0.26%). WT1 specific cytokine producing T 

cells were found in 13 out of 25 pts at week 10 with 7 pts producing IFNg, 11 TNFa, and 

5 IL-2. In selected pts WT1-specific T cells were characterized as both effector and 

memory T cells and demonstrated cytotoxic and proliferative capacity and specific 

cytokine production in response to autologous leukemic blasts. 

Conclusion: This study proves immunological and clinical efficacy of WT1 peptide 

vaccination in AML. Two ongoing studies investigate WT1 vaccination in WT1 expressing 

carcinomas and as adjuvant therapy in high-risk AML.

Kai Schledzewski, Klein Diana, Martin Falkowski, Gerhard Moldenhauer, Jo van 

Ginderachter, Julia Kzhyshkowska, Lou de Leij, Gernot Geginat, Bernd Arnold, Sergij 

Goerdt 

Phenotype analysis, cytokine induction and gene expression 

profiling identify LYVE-1+ tumour associated macrophages to 

be a specialized subset of M2 macrophages 

Tumor-associated macrophages (TAMs) significantly contribute to regulation of tumor 

growth and metastasis. When analysing murine tumors immunohistochemically 

regarding lymphangiogenesis, we unexpectedly found a significant percentage of TAMs 

to express the lymphatic endothelium-specific hyaluronan receptor LYVE-1. Further 

analysis of these special TAMs in B16F1 melanoma and TS/A mammary carcinoma 

revealed that LYVE-1 expression occurred in a subset of CD11b+, F4/80+ tissue 

macrophages that preferentially co-expressed the sinusoidal endothelial scavenger 

receptor stabilin-1. In vitro, LYVE-1 expression was induced in 25-40% of murine bone 

marrow-derived macrophages upon exposure to B16F1 melanoma-conditioned medium 

and IL-4/dexamethasone. By FACS analysis, 11.5% of bone marrow-derived 

macrophages were LYVE-1+, stabilin-1+ double-positive, while 9.9% were LYVE-1+, 

stabilin-1- and 33.5% were LYVE-1-, stabilin-1+. As both LYVE-1 and stabilin-1 are thus 

shared endothelial-macrophage receptors, we further analyzed the molecular repertoire 

of these special LYVE-1+ macrophages by gene profiling. By comparing LYVE-1+ versus 

LYVE-1- bone marrow-derived macrophage populations using Affymetrix mouse genome 

MOE430 2.0 array chips, we identified a set of 121 genes highly overexpressed in the 

LYVE-1+ macrophage population. In addition to well known markers of alternatively 

activated (M2) macrophages such as arginase, CD163, and fibronectin, novel molecules 

including Mgl2 were identified demonstrating that LYVE-1+ macrophages are a 

specialized subset of M2 macrophages. This specialized M2 profile was confirmed by RT- 

PCR analysis of LYVE-1+ TAMS isolated ex vivo from TS/A tumors. To identify the 

factors necessary to induce LYVE-1+ macrophages in vitro, we tested a broad array of 

Th1- and Th2-associated factors. We could show that a mixture of Th2 cytokines 

administered sequentially mimicked the effects of the B16F1 conditioned medium. In 

order to further test the functions of LYVE-1+ M2 TAMs in vivo, bone marrow chimeras 

with bone marrow-specific cytokine receptor knock out as well as LYVE-1 and stabilin-1 

single and double knock out mice will be analyzed in the LYVE-1+ TAM-rich TS/A tumor 

model.

Tobias Käser, Wilhelm Gerner, Sabine Hammer, Martina Patzl, Armin Saalmüller 

Phenotypic and functional characterisation of porcine CD4 

+ CD25 high regulatory T cells 

Over the last years regulatory T cells (Tregs) were defined as CD4 + CD25 + T 

lymphocytes expressing the transcription factor Foxp3 (Forkhead Box P3) with the 

ability to down regulate various immune responses. In swine the existence of CD4 + CD25 

+ T-lymphocytes was described before but nothing is known about the function of this 

minor cell population to date. Therefore we studied porcine CD4 + CD25 + T cells with 

regard to major attributes of murine and human Tregs: their phenotype concerning the 

expression of several Treg-relevant antigens, including Foxp3, their IL-10 production 

and their suppressive capacity. Our results revealed that porcine CD4 + CD25 + T cells 

with high CD25 expression count for about 2 to 9% of the CD4 + T cell subset. They 

demonstrate a strong Foxp3 expression and a heterogeneous CD45RC-, CD8?- and MHC- 

II-defined phenotype. Additionally they show an enhanced IL-10 production. Cocultivation 

of increasing numbers of CD4 + CD25 high T cells with a constant number of 

CD4 + CD25 - responder-T cells caused a decrease in proliferation of the entire culture. 

This demonstrates the suppressive capacity of the CD4 + CD25 high T cell subset and- 

together with their Foxp3 expression- the existence of porcine Tregs.

Stefan Maßen, Dirk Jäger, Inka Seil, Barbara Mosetter, Christine Falk 

Phenotypic and functional characterization of KIR-expressing 

cytotoxic T cells 

Introduction: Expression of NK receptors of the KIR (Killer-Ig-like-receptor) family is 

mainly observed on NK cells. However, some cytotoxic T cells (CTL) have been shown to 

express KIR following allogeneic or tumorspecific stimulation, they have also been 

detected in peripheral blood of patients with autoimmune diseases like rheumathoid 

arthritis. 

Materials and Methods: We generated cytotoxic T cell lines by stimulating isolated 

CD8 positive T cells with peptides derived from the tumor associated antigens Rab38 

and NY-BR-1 that are mainly expressed by melanoma and breast cancer cells, 

respectively. The phenotypic characterization with respect to KIR and NK receptor 

expression was determined by multicolour flow cytometry. The specificities of the CTL 

lines were examined in chromium-release assays and CD107a-degranulation assays 

targeting Rab38- or NY-BR-1-loaded T2-cells. Cytokine secretion patterns were 

quantified by multiplex-analyses (Luminex). 

Results: Through an extensive phenotypic analysis, the CTL showed a broad expression 

of KIR receptors in addition to the activating receptors NKG2D and 2B4. The 

functionality of these receptors and their activating and inhibitory potential, tested in 

redirected-degranulation experiments, demonstrated that the cytotoxicity of the CTL 

lines was clearly dependent on the TCR complex. Activating as well as inhibitory KIR 

receptors have only a minor regulatory potential, even though the activating NK 

receptor, NKG2D, displayed some co-stimulatory effect. All CTL lines showed de novo 

secretion of GM-CSF, IFN-γ and TNF-α as a result of the TCR-complex-stimulation. In 

addition, the constitutive secretion of MIP-1β was significantly increased upon TCR 

triggering. 

Taken together, these data indicate that even in an autologous peptide-specific 

stimulation, some CTL maintain their KIR expression, although these KIR may be 

functionally dissociated from the TCR signaling cascade. In addition, these KIR-positive 

CTL displayed mixed cytokine/chemokine patterns distinct from classical TH1/TH2 

patterns.

Esther Wilk, Tibor Horvath, Katy Kalippke, Nadine Wilke, Reinhold E. Schmidt, Roland 

Jacobs 

Phenotypical and functional characteristics of CD20 + T cells 

In humans a small percentage (3-4%) of T cells coexpress CD20, which is commonly 

considered a marker of mature B cells. In order to characterize these cells in more 

detail, their function and phenotype were analyzed. The study revealed that CD3 + CD20 

+ cells are present in different compartments including blood, spleen and liver. The cells 

comprise a heterogenous T cell population with 60% coexpressing CD8 and 40% 

expressing CD4. Constitutive production of cytokines (i.e. IFNγ, IL-2, TNFα) and strong 

expression of CD95 indicate a high activation state of the cells and susceptibility to FasLmediated 

apoptosis. Together with their low proliferative capacity in response to various 

stimuli (e.g. PHA and CD3 crosslinking) our findings suggest that CD20 + T cells 

represent a terminally differentiated cell type with immune regulatory capacity. In 

summary, we present new phenotypical and functional characteristics of CD20 + T cells, 

providing a basis to further study the in vivo role of this T cell subset. This might be of 

particular interest in patients undergoing rituximab treatment since this antibody 

targets CD20 + cells and effectively depletes all CD3 + CD20 + T cells along with the 

intended B cells.

Bernhard Reis, Roland Piekorz, Bernd Nürnberg, Klaus Pfeffer, Sandra Beer 

PI3Kγ and δ play combined roles in B cell development 

Class I phosphoinositide 3-kinases are a family of intracellular signalling molecules 

which regulate cell differentiation, survival, proliferation, and migration through central 

downstream targets including Akt. The specific roles of the PI3K catalytic subunits p110γ 

and p110δ in lymphocyte development and function were demonstrated by generating 

gene-deficient mice. It was shown that in the thymus combined activities of PI3Kγ and 

PI3Kδ during positive and negative selection are essential for T cell differentiation and 

survival. Here, we extent the functional analysis of these PI3K isoforms in B cell 

differentiation and function. We show that peripheral B cell numbers are dramatically 

reduced in PI3Kγ/δ double deficient mice due to a developmental block at the pro-B cell 

stage. Moreover, upon antigen receptor triggering or LPS stimulation double deficient B 

cells failed to proliferate properly. Importantly, on a molecular level, the cells showed 

an impaired upregulation of key regulators of cell cycle progression and survival 

including D-type cyclins and bclx. Thus, both PI3Kγ and PI3Kδ display Isotype-specific 

and -redundant roles in B cell development and function.

Kerstin Schilling, Bin Hu, Karine Missy, Klaus-Dieter Fischer 

Pix proteins in B cell Signalling 

alphaPIX is a multi-domain signaling protein with a RhoGEF domain that activates Rac 

and CDC42. AlphaPix associates with proteins involved in cytoskeletal-membrane 

complexes and cell migration. It has been shown that PIX proteins play roles in some 

immune cells, including neutrophils and T cells. We have generated alphaPIX knockout 

mice and analyzed their immune system. alphaPIX protein was specifically expressed in 

immune cells, unlike its homolog betaPIX, which was expressed in immune cells and in 

a wide range of other cells. Although mice lacking alphaPIX had reduced numbers of 

mature lymphocytes and defective immune responses, the number of marginal zone B 

cells was inceased. In addition, basal migration of B cells and other lymphocytes was 

enhanced. Antigen receptor-directed proliferation of alphaPIX - T and B cells was also 

reduced, were as LPS induced proliferation of B cells was normal, suggesting for the 

first time that alphaPIX is important for B cell receptor (BCR) signaling. In addition, 

alphaPIX - B cells showed multiple molecular defects, including increased overall 

tyrosine phosphorylation and activation of ERK kinase, and reduced phosphorylation of 

PAK kinase following antigen receptor stimulation. These results reveal specific roles for 

alphaPIX in the immune system and suggest that alphaPIX is an important signaling 

intermediate downstream of the B cell receptor.

Katrin Moser, Oliver Winter, Nicole Haupt, Martin Szyska, Bimba F. Hoyer, Andreas 

Radbruch, Falk Hiepe, Rudolf A. Manz 

Plasma cell longevity in NZB/W mice 

Through the production of autoantibodies, plasma cells contribute to the pathogenesis 

of Systemic lupus erythematosus (SLE). In non-autoimmune individuals, long-term 

plasma cell survival occurs mainly in the bone marrow where lifetime can reach several 

months to years. The longevity of these cells depends on factors like IL-6 and SDF-1, 

produced by accessory cells. In NZB/W mice, a model for SLE, high numbers of plasma 

cells also survive in the spleen. We here address the question whether this abnormal 

plasma cell survival in spleens of NZB/W mice is due to an increased potential of this 

tissue to support plasma cell longevity and/or due to an enhanced intrinsic survival 

capacity of NZB/W plasma cells. To analyse the intrinsic survival capacities of plasma 

cells from different genetic backgrounds we performed in-vitro cultures with isolated 

plasma cells. Without addition of survival factors nearly all plasma cells died within 3 

days of culture. Addition of IL-6 resulted in an increase in surviving plasma cells which 

was much more pronounced with plasma cells derived from autoimmune NZB/W and 

NZB mice compared to plasma cells from non-autoimmune Bl6 and NZW mice. 

However, plasma cells of BALB/c origin showed the same survival capacity as NZB/W 

derived ones. Possibly indicating that higher sensitivity for IL-6 mediated survival can 

contribute, but is not sufficient to explain the increased plasma cell survival in NZB/W 

spleens. Therefore we also tested the microenvironment of plasma cells in the spleen of 

NZB/W mice in tissue sections for the presence of increased production of plasma cell 

survival factors. SDF-1 signals were found in close proximity to some, but not all 

plasma cells. Increased numbers of a subset of myeloid related cells located in close 

proximity to plasma cells and expressing multiple cytokines known to stimulate plasma 

cell survival were detected in NZB/W spleens. This finding stresses a main impact of the 

splenic microenvironment for the increased survival rate in NZB/W spleens.

Oliver Winter, Katrin Moser, Nicole Haupt, Martin Szyska, Andreas Radbruch, Rudolf A. 

Manz 

PLASMA CELL SURVIVAL AND DEVELOPMENT IN THE BONE 

MARROW 

Plasma cells secrete antibodies which are a key factor for the adaptive immune defence. 

Bone marrow is the major site where long-lived plasma-cells reside. Survival of these 

cells in specific microenvironments (niche) depends on survival factors supplied by 

proximate cells, including APRIL, IL-6, TNF-a. SDF-1+ reticular stromal cells found in 

association with plasma cells are likely to contribute to this niche. 

To further characterize these niches, we immunized B6 and Balb/c mice with the 

antigen ovalbumin, sacrificed them at several time points during the immune response 

and tracked ovalbumin specific plasma cells via confocal microscopy. Stromal cells 

found in close association with plasma cells were considered to contribute potentially to 

niches. These cells were further characterised by flow cytometry. 

Plasma cells enter the bone marrow via the vascular niche. Here, they are found during 

the first days following their immigration into the bone marrow in contact with B78+ 

cells of the endothelial linage, possibly including mature endothelial cells and endothelial 

precursor cells. These plasma cells also make contact to Gr1 high cells, likely 

resembling mature granulocytes emigrating from the bone marrow. 

Later, at about 7 days after entering the bone marrow, plasma cells are found more 

distant from B78+ cells. They now are localized adjacent to VCAM-1+ reticular stromal 

cells where they also specifically bind to a particular type of myeloid related cells that 

produce multiple important plasma cell survival factors, including Il-6, TNF-a and APRIL. 

Our data suggest that following immigration of plasma cells via the vascular niche, longterm 

plasma cell survival later is supported in a second niche. Here, VCAM+ reticular 

stromal cells seem to provide a meeting point for long-lived plasma cells with myeloid 

related cells that support plasma cell survival through the production of multiple 

important plasma cell survival signals.

Julian Pardo, Christin Urban, Arno Müllbacher, Reinhard Wallich, Christoph Borner, 

Markus M Simon 

Pleiotropism of granzyme B-induced cell death; A means of 

the host to counter pathogens evasion strategies of CTLmediated 

recovery 

Granzyme B (gzmB) of cytotoxic lymphocytes is essential for recovery from intracellular 

pathogens, but the molecular basis is still unresolved. Here, we analyzed gzmBmediated 

death pathways under more physiological conditions using ex vivo virusimmune 

CTL that express perf and gzmB, but not gzmA (gzmB+CTL). We show that 

gzmB+CTL kill targets independent of caspases and mitochondrial signaling. In addition 

the data reveal that gzmB+CTL independently induce pro-apoptotic processes either via 

caspases 3/-7, leading to plasma membrane perturbance and ROS production, or via 

Bid/Bak/Bax, resulting in cytochrome c release and that both pathways elicit ??m 

suppression. Our data provide evidence for a pleiotropic pro-apoptotic function of gzmB 

suitable to counteract evasion strategies of pathogens and to control tumors.

Mario Fabri, Alessandra Zingarelli, Eva Flenner, Martina Bessler, Helena Hafke, Wiltrud 

Maria Kalka-Moll 

Polysaccharide modulates CD8+ T cell responses by 

enhancing TCR cross-linking 

Polysaccharides have been classically considered T cell-independent antigens. In 

contrast, zwitterionic capsular polysaccharids (ZPS) from bacteria such as Bacteroides 

fragilis and Streptococcus pneumoniae elicit potent CD4+ T cell responses in a MHC 

class II-dependent manner. ZPSs, which are characterized by having both positively and 

negatively charged substitutans on each repeating unit of a highly repetitive structure, 

induce CD4+ T cell-dependent formation of intraabdominal abscesses. Previous 

investigations on ZPS-induced intraabdominal abscesses have exclusively focused on 

the role of CD4+ T cells. Herein, we sought to elucidate the immune response of CD8+ 

T cells to ZPSs. We characterize the function of CD8+CD28- T cells in a experimental 

model of ZPS-induced abscess formation in vivo and in vitro. Moreover, we provide 

evidence that ZPSs modulate CD8+ T cell activation by a previously unknown 

mechanism implying enhanced cross-linking of TCRs on CD8+ T cells. These data 

provide substantial new insights in the unique functions of ZPSs to induce and regulate 

T cell-dependent immune responses of both CD4+ an CD8+ T cells.

Hans-Willi Mittrücker, Steinhoff Ulrich, Stefan H. E. Kaufmann 

Poor correlation between BCG vaccination-induced T cell 

responses and protection against tuberculosis 

Mycobacterium bovis bacille Calmette-Guérin (BCG) is the most widely used live 

bacterial vaccine. However, limited information is available correlating route and dose of 

vaccination and induction of specific T cell responses with protection against 

tuberculosis. We compared efficacy of oral and systemic vaccination and correlated 

vaccine-induced T-cell responses with protection in experimental tuberculosis of mice. 

Following oral and systemic vaccination, we observed profound differences in 

persistence and dissemination of BCG, and frequencies and location of specific IFN-γsecreting 

CD4+ and CD8+ T cells. Yet, both vaccination routes caused comparable 

levels of protection against aerosol challenge with M. tuberculosis. Protection correlated 

best with rapid accumulation of specific CD8+ T cells in infected tissues of challenged 

mice. In contrast, specific IFN-γ production by CD4+ T cells reflected the load of M. 

tuberculosis rather than the strength of protection. Our data question the measurement 

of IFN-γ secretion by CD4+ T cells and emphasize the need for new biomarkers for 

evaluation of tuberculosis vaccine efficacies.

Michael Probst-Kepper, Andrea Kroeger, Robert Geffers, Christian Erck, Miguel 

Godinho, Vitor Martins dos Santos, Hansjörg Hauser, Jan Buer, Rudi Balling, Siegfried 

Weiss 

POSITIVE FEEDBACK-CONTROL BETWEEN GARP AND FOXP3 

IN REGULATORY T CELLS 

CD4 + CD25 hi regulatory T cells (Treg) have emerged as a unique population of 

suppressor T cells that critically depend on the transcription factor FOXP3 for their 

development and function. Therefore, FOXP3-transduction was suggested to generate 

antigen-specific Treg cells. However, this approach is insufficient for human effector CD4 

+ T cells (Th). Most likely this is due to the inadequate induction of the Treg-specific 

receptor GARP (glycoprotein-A repetitions predominant). We identified GARP as master 

switch receptor for Treg cells, since retroviral over-expression of GARP in antigenspecific 

Th cells was sufficient to induce sustained high levels of FOXP3, regulatory 

function, and an extended Treg/FOXP3-signature. Conversely, down-regulation of GARP 

in Treg cells by short-interfering RNA reduced FOXP3 expression and suppressor 

functions and vice versa, down-regulation of FOXP3 reduced GARP expression and 

suppressor functions. Thus, a positive feedback-loop was established. Two minor 

constituents of this regulatory circuit were identified, the endopeptidase legumain and 

the β-galactoside binding protein LGALS3. Both up-regulated FOXP3 although not 

reaching the levels induced by GARP. Whereas casein-kinase I phosphorylation at 

position-6 of LGALS3 was essential for FOXP3 induction, the kruppel-like factor 2 

(KLF2), involved in the regulation of T cell quiescence, was induced independent of this 

phosphorylation. A preliminary computational model based on a hybrid functional Petri 

net was developed to describe the main regulatory interactions. Thus, GARP represents 

a promising tool for stable conversion of antigen-specific Th towards regulatory T cells, 

the characterization of the underling regulatory network will be pivotal for an 

understanding of the regulatory program.

Nasr Hemdan, Frank Emmrich, Joerg Lehmann, Ulrich Sack 

Possible induction or exacerbation of autoimmune diseases 

by heavy metals through changing cytokine profiles 

The development of autoimmune diseases involves a combination of appropriate genetic 

predisposition and encounter with environmental risk factors such as immunotoxic 

agents. Because of their immunomodulative potency, some heavy metals have been 

implicated in the induction and exacerbation of autoimmune diseases. We aimed at 

testing the association of exposure to heavy metals and the possible roles of different 

cytokines in autoimmune disorders. BALB/c mice were exposed intraperitoneally to 

cadmium (Cd) acetate or mercuric (Hg) chloride, and following different exposure 

regimes, mice were killed and levels of Th1/Th2 and the pro-inflammatory cytokines 

were assessed. Our results show that, in response to heavy metal exposure, a 

significant increase in IL-2, IL-4, IL-5 measured after 5 weeks of exposure to Hg, and 

accompanied by decrease in IFN-γ release indicating possible proliferation of Th2 rather 

than Th1 subsets. However, the increase in Th2 cytokines was not significant in the 3week 

exposure group, and was accompanied by an increase in IFN-γ release. This 

indicates changing of cytokine profiles along different exposure periods, and thereby 

possible distinct roles of different cytokines in different phases of autoimmune diseases. 

The proinflammatory cytokines TNF-α and IL-6 show insignificant increase in 

comparison to control mice. In case of Cd-exposure, 5-week exposure lead to 

stimulation of IFN-γ release which was prominent following long exposure accompanied 

by increase in the Th2 cytokine IL-5. These results also indicate differences in response 

to heavy metals depending on the metal ion itself as well as duration of exposure, and 

highlights the dependence on T cells, or at least specific cytokines, for example IFN-γ, in 

different phases of autoimmune diseases.

Anja Dahten, Dennis Ernst, Dana Hoser, Margitta Worm 

PPARγ-ligation improves development of allergen-induced 

skin inflammation in a murine model of dermatitis 

Background: Recent studies point to the pathophysiological role of the nuclear receptor 

PPARγ in the inflammatory immune response. We have previously shown that a specific 

ligand of PPARγ attenuates the systemic immune response via regulation of humoral 

immunity and inhibition of T cell-derived inflammatory cytokine production. The 

objective of this study was to investigate the impact of PPARγ-ligand treatment on the 

local immune response in a murine dermatitis model. Methods: We established a murine 

model with reproducible, OVA-induced skin inflammation. In this model PPARγ-ligand 

was applied at different time points via intraperitoneal or epicutaneous routes. Skin and 

blood samples were obtained for analysis respectively. Affected skin areas were 

assessed by a standardised clinical skin score (CSS), histological and 

immunohistochemical analysis. OVA-specific IgE, IgG1 and IgG2a levels were measured 

by ELISA on different time points (day 1, 21, 35 and 70). Results: Systemic application 

of PPARγ-ligand reduced the severity of OVA-induced eczematous skin lesions up to 

70%. These observations were confirmed by histological examinations of skin biopsies, 

showing decreased thickness of dermis and epidermis (p < 0.001) accompanied by 

significantly reduced infiltration of CD4 and CD8 positive lymphocytes and mast cells. 

OVA-specific IgE and IgG1 responses were significantly inhibited on days 21/35, but 

IgG2a synthesis was not affected. Conclusion: Our results demonstrate PPARγ-ligand 

treatment inhibits the development of allergen-mediated dermatitis by local and 

systemic mechanisms. These findings are important for the development of novel 

therapeutic strategies involving a PPARγ-ligand based treatment in allergic diseases.

Luisa Klotz, Indra Dani, Linda Diehl, Ari Waisman, Thomas Klockgether, Percy Knolle 

PPARgamma ablation in CD4+ T cells augments T cell 

responses resulting in enhanced T cell infiltration of the CNS 

and increased disease severity during experimental 

autoimmune encephalomyelitis 

The peroxisome proliferator-activated receptor gamma (PPARgamma) belongs to a 

group of ligand-activated transcription factors involved in the regulation of metabolism 

and inflammation. PPARgamma is expressed in cells of the peripheral immune 

compartment like lymphocytes and dendritic cells, but also within the CNS, mainly in 

microglial cells and in neurons. Interestingly, oral administration of PPARgamma 

agonists ameliorates the clinical course and histopathological features in experimental 

autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. However, 

it is still unclear which cell type is primarily involved in PPARgamma-mediated 

suppression of (auto)-immunity. Therefore, we generated mice with a T cell specific 

knock-out of PPARgamma employing a Cre-recombinase mediated ablation of 

PPARgamma which is limited to CD4+ cells. In these mice (CD4-PPARg-ko) and their 

Cre negative wild-type littermates, we investigated both disease course and T cell 

responses during actively induced MOG-EAE. 

CD4-PPARg-ko mice exhibited both an earlier disease onset and a signficantly increased 

disease severity during the induction phase of EAE when compared to wild-type mice. 

Accordingly, T cell infiltration of the CNS was significantly increased in CD4-PPARg-ko 

mice. Moreover, both priming of naïve CD4-PPARg-ko T cells in vitro as well as 

restimulation of CD4-PPARg-ko T cells primed in vivo yielded significantly augmented T 

cell responses characterized by an increase in T cell proliferation as well as production 

of proinflammatory cytokines. 

These data demonstrate that ablation of PPARgamma enhances T cell reactivity both in 

vitro and in vivo, resulting in pronounced T cell responses and consecutively increased 

disease severity in a model of CD4+ T cell mediated autoimmunity. Selective activation 

of PPARgamma in T cells therefore represents a promising future target for control of T 

cell mediated autoimmunity.

Jürgen Haas, Benedikt Fritzsching, Petra Truebswetter, Peter Krammer, Elisabeth Suri- 

Payer, Brigitte Wildemann 

Prevalence of Newly Generated Naïve Regulatory T-Cells is 

Critical for Treg Suppressive Function and Determines Treg 

Dysfunction in Multiple Sclerosis 

Suppressive function of CD4+CD25highFoxP3+ regulatory T-cells (Treg) is impaired in 

patients with relapsing remitting multiple sclerosis (MS). The mechanism underlying the 

Treg functional defect is unknown. Treg mature in the thymus and the majority of cells 

circulating in the periphery rapidly adopts a memory phenotype. Since our own previous 

findings suggest that the thymic output of T-cells is impaired in MS patients we 

hypothesized that an altered Treg generation may contribute to the suppressive 

deficiency associated with MS. We therefore determined the role of Treg which enter the 

circulation as recent thymic emigrants (RTEs) and, unlike their CD45RO+ memory 

counterparts, express CD31 as a typical surface marker. 

We show that numbers of CD31+ co-expressing CD4+CD45RA+CD45RO-Foxp3+Treg 

(RTE-Treg) within peripheral blood decline in dependence of age and are significantly 

reduced in MS patients, whereas the entire Treg population is equally distributed as 

compared to healthy control donors. Depletion of CD31+ cells from total Treg 

neutralizes the difference in inhibitory potencies of patient and donor Treg detectable 

when using total Treg in the co-culture experiments. Furthermore, patient derived Treg 

but not healthy Treg exhibit a contracted TCR V-beta repertoire. 

These observations suggest that a shift in the homeostatic composition of Treg subsets 

related to a reduced thymic-dependent de novo generation of Treg with compensatory 

expansion of less suppressive memory Treg may contribute to the Treg defect 

associated with MS. 

Supported by Hertie-Stiftung (1.01.1/04/003), Deutsche Forschungsgemeinschaft (SFB 

405, 5H, and SFB 571, B7), the Young Investigator Award from the Faculty of Medicine, 

University of Heidelberg to BF, and Serono GmbH Deutschland.

Reinhard Maier, Rita De Giuli, Veronika Nindl, Simone Miller, Volker Thiel, Roland 

Züst, Ari Waisman, Birgit Ledermann, Burkhard Ludewig 

Preventing autoimmune myocarditis through cardiac myosinspecific 

tolerance 

Virus-induced myocarditis might broaden the pathogen-specific immune response 

towards heart antigens such as the abundantly expressed myosin heavy chain alpha 

(myhca). The chronic immune response against cardiac self-antigens may lead to 

dilative cardiomyopathy (DCM), which represents a prevalent cause of human heart 

disease and heart failure. Immunization of BALB/c mice with the myhca-derived peptide 

(amino acids 614-629) elicits myocarditis induced by peptide-specific CD4 T cell 

responses, and therefore uncouples the chronic autoimmune phase from putative viral 

infections. In order to further characterize the mechanisms and effector molecules 

involved in autoimmune myocarditis and to evaluate therapeutic strategies, we have 

generated a novel transgenic mouse model. In this model (Rosa-IM mouse), the 

expression of the myhca peptide is directed to the MHC class II pathway through a 

chimeric invariant chain (Ii-myhca) in which the CLIP peptide sequence is replaced by 

the myhca614-629 peptide. The construct is designed in such a way that the expression 

is only achieved in the presence of Cre-recombinase. The ubiquitous expression of 

myhca in all MHC class II positive cells induced specific immune tolerance and as a 

consequence mice were protected from myocarditis. The Rosa-IM mouse represents 

therefore a versatile tool to dissect the basic mechanisms in autoimmune myocarditis, 

to delineate the decision making process between activation and tolerization of heartspecific 

CD4 T cells and to study the participation of different antigen presenting cells 

like dendritic cells, B cells and macrophages in the disease process.

Patrick Vollmar, Stefan Nessler, Bianca Wolff, Sudhakar Reddy Kalluri, Hans-Peter 

Hartung, Bernhard Hemmer 

Preventive and therapeutic effects of the antidepressant 

venlafaxine on murine experimental autoimmune 

encephalomyelitis 

Antidepressants, which are in use for the treatment of major depression, are known to 

impact on the immune system. In this study, we examined the immunomodulatory 

properties of venlafaxine, a selective serotonin-/norepinephrine reuptake inhibitor, in 

murine experimental autoimmune encephalomyelitis (EAE), a Th1-mediated central 

nervous system demyelinating disease model of multiple sclerosis. EAE was induced in 

SJL mice by adoptive transfer of PLP-specific T-cells. Mice received different doses of 

venlafaxine before transfer and after onset of disease. Sustained oral treatment with 

6mg and 60mg/kg per day significantly ameliorated the clinical course of disease 

compared to vehicle during both preventive and therapeutic intervention. The 

ameliorating effect was more pronounced in the high dose group with respect to the 

suppression of relapses in the chronic phase of disease. Continuous drug delivery for 14 

days was sufficient to delay the onset and peak of disease significantly. However, 

ameliorating effects were more pronounced in the sustained treatment experiments. 

Disease modifying effects are mediated by venlafaxine’s potential to reduce the 

secretion of the Th1 cytokines TNF-alpha and IFN-gamma in encephalitogenic T cell 

clones and lines. It further suppressed significantly the mRNA gene expression of 

proinflammatory cytokines IFN-gamma, TNF-alpha and the chemokine RANTES in the 

spinal cord of EAE mice. These findings demonstrate the strong immunomodulatory 

property of the antidepressant venlafaxine and pioneer further studies to clarify whether 

venlafaxine may exert similar effects in MS.

Ivan Bogeski, Valentin Mirceski, Markus Hoth 

Probing the redox activity of T-lymphocytes deposited at 

electrode surfaces with voltammetric methods 

Reactive oxygen species and redox signaling in general have a very important, 

physiological role in the regulation of the immune response. In addition, increased 

redox signaling is an attribute to many cancer cells thus having a pathological effect. 

The detection and the evaluation of these signaling events are very often accompanied 

with difficulties. Here, we describe a novel electrochemically-based technique for 

monitoring the T-lymphocyte redox state. 

T-lymphocytes were attached on the surface of a working electrode, which was 

previously modified with 2-Palmitoylhydroquinone as a redox mediator. Using cyclic 

voltammetry, we were able to indirectly (via the redox mediator) monitor an electron 

transport from the cells towards the working electrode, which enabled us to precisely 

evaluate the redox state and the redox potential of the cells. This new technique is 

rather simple and sensitive and may be used in the future as a valid diagnostic 

procedure in various branches of bio-medical science.

Lars-Oliver Tykocinski, Anna Sinemus, Esmail Rezavandy, Bruno Kyewski 

Promiscuous gene expression in the thymus - is epigenetic 

regulation the key? 

The scope of central tolerance is to a large extent dictated by expression of tissuerestricted 

antigens (TRA) by medullary thymic epithelial cells (mTECs), a process known 

as promiscuous gene expression (pGE). While increasing insight into the tolerance 

modes linked to pGE has been gained in the last years, the molecular mechanisms 

involved in the regulation of pGE in mTECs remain largely obscure. Promiscuously 

expressed genes tend to co-localize in clusters in the genome and imprinting of the 

insulin-like growth factor 2 gene is selectively lost in mTECs. Thus, apart from the 

involvement of the transcriptional regulator Autoimmune Regulator (Aire), epigenetic 

mechanisms may have an important contribution in the regulation of pGE in mTECs. To 

assess the role of epigenetic modifications in the regulation of pGE, we compared 

histone modifications and the DNA methylation status in a cluster of TRA genes in 

mTECs and the respective tissue cells. Our current results document a close correlation 

between promiscuous gene transcription and a permissive chromatin configuration at 

the single gene level in mTECs but not locus-wide chromatin alterations as observed in 

the tissue cells.

Thorsten Joeris, Petra Krienke, Ulrike Kuckelkorn, S.H.E. Kaufmann, Ulrich Steinhoff 

Proteasome assembly: Competitive integration of constitutive 

and IFNγ inducible catalytic subunits 

Proteasomes are multicatalytic protease complexes responsible for non-lysosomal 

degradation of proteins. In vertebrates 20S proteasomes occur in two major forms: 

constitutive proteasomes (c20S) and the IFNγ inducible immunoproteasomes (i20S). 

The two forms differ in their catalytically active subunits and consequently in their 

overall specificity and activity. Due to the diverse effects of proteasome composition on 

multiple cellular processes, it is of interest to understand the basic mechanisms of c20S 

and i20S formation. So far, formation of i20S is believed to be a result of cooperative 

integration of the IFNγ inducible subunits β1i, β2i and β5i. According to this concept β5i 

is necessary for processing of β1i and β2i precursor subunits during proteasome 

assembly. Here, we analyse the formation of 20S proteasomes during infection of WT 

and β5i deficient (lmp7 -/- ) mice with Listeria monocytogens. In lmp7 -/- mice β5 can 

easily process β1i and β2i resulting in the formation of mixed proteasomes (m20S). The 

observed accumulation of unprocessed β1i and β2i in precursor proteasomes is caused 

by a lack of β5 subunits and not by its inability to pair with β1i and β2i. As β5 is not 

upregulated by infection, it can not compensate the absence of β5i in lmp7 -/- mice. Our 

in vivo data question the current model of cooperative i20S formation. Instead, we 

provide evidence that the mechanism underlying i20S formation is substantially 

regulated by simultaneous overexpression of IFNγ inducible subunits, which enables 

them to outcompete the constitutive subunits at protein level during proteasome 

assembly.

Kirsten Neubert, Silke Meister, Damian Maseda, Kerstin Amann, Reinhard Voll 

Proteasome inhibition ameliorates lupus symptomes in NZB/ 

W mice 

Systemic lupus erythematosus (SLE) is an autoimmune disease which is characterized 

by circulating IgG autoantibodies predominantly directed towards nuclear antigens. The 

proteasome inhibitor bortezomib (Bz) is used for the treatment of multiple myeloma, a 

malignant plasma cell neoplasia. One mechanism of Bz might be the blockade of the key 

transcription factor NF-κB, which is also important for survival of B lymphocytes 

especially mature B cells. Therefore, we investigated the effects of NF-κB inhibiting 

agents such as Bz in a mouse model for SLE. 

To address this question, we treated NZB/W mice with Bz twice weekly over 10 months. 

Bz-treated mice have significantly prolonged survival time and decreased proteinuria 

compared to the control mice. Histologically, there were no or only minor signs of 

glomerulonephritis in Bz-treated mice. The IgG anti-dsDNA and anti-Histone antibodies 

were strongly reduced during the whole treatment. The IgG concentrations in sera were 

significantly decreased during the first months of treatment, respectively. After the first 

month of treatment the IgG serum concentrations increased again and reached the 

levels of control mice. 

Flow cytometric analyses of the splenic lymphocyte compartment from NZB/W mice, 

which were Bz-treated over 8 weeks, revealed a strong reduction of T and B cell 

numbers. Interestingly, Bz had no significant effect on the B cell numbers in the bone 

marrow. 

These data indicate that Bz prolongs the survival and ameliorates the clinical 

parameters of lupus-like disease in NZB/W mice. We suggest that both T and B 

lymphocyte subsets are affected by Bz, potentially due to inhibition of NF-κB activation 

along with induction of terminal endoplasmic reticulum stress leading to apoptotic cell 

death of lymphocytes.

K. Doser, J. Albrecht, T. J. Boeld, R. Eder, J. Stahl, R. Andreesen, P. Hoffmann, M. 

Edinger 

Protection from graft-versus-host disease by donor CD4+CD25 

+ regulatory T cells improves B lymphocyte reconstitution 

after allogeneic bone marrow transplantation 

Allogeneic bone marrow transplantation (BMT) is a well-established therapy for 

hematologic malignancies. However, graft-versus-host disease (GVHD) caused by cotransplanted 

mature donor T cells, remains a significant problem. The major target 

organs of GVHD are skin, liver and intestines, but also lymphoid organs, thereby 

impairing immune reconstitution and increasing the susceptibility to opportunistic 

infections. We, and others, previously demonstrated that the adoptive transfer of donor 

CD4+CD25+ regulatory T (Treg) cells prevents GVHD. We now investigated how their 

suppressive activity influences immune reconstitution after allogeneic BMT by examining 

the regeneration of the B cell compartment. For this purpose, lethally irradiated BALB/c 

recipients were transplanted with either 5 x 106 T cell-depleted bone marrow cells (TCD 

BM) from C57BL/6 donors alone, or TCD BM plus 5 x 105 CD4+CD25- donor T cells 

(Tconv) with or without equal numbers of donor Treg cells. As expected, mice that 

received only TCD BM did not develop GVHD and reconstituted their B cell compartment 

from transplanted BM within 20-40 d, whereas those that received additional Tconv cells 

developed severe GVHD and lacked donor as well as host B lymphocytes in peripheral 

blood (PB) until their early death or sacrifice by d 100. In contrast, most animals that 

received Tconv and Treg cells at a 1:1 ratio were protected from GVHD and showed a 

complete, yet delayed reconstitution of their B cell compartment, with appox. 50% 

reconstitution by d45-60 and 100% by d100. Comparative analyses of BM, PB and 

spleen revealed that GVHD interferes with B cell reconstitution not solely by destroying 

peripheral lymphoid organs, but also by eliminating early B cell precursors in the BM. 

We thus speculate that the dysregulated production of pro-inflammatory cytokines 

during GVHD is toxic for early B cell precursors and/or that the alloresponse destroys 

the BM niche for developing B cells. Importantly, co-transplanted donor Treg cells 

prevent this pathology and therefore improve rather than impair immune reconstitution 

after allogeneic BMT.

Aleksandar Backovic, Nikolaus Wick, Georg Wick 

PROTEIN SIGNATURES ON SILICONE SURFACE AS 

BIOMARKERS FOR IMMUNOLOGIC-FIBROTIC SIDE EFFECTS 

TO IMPLANTS 

An inflammatory response with subsequent fibrotic reactions are the most common side 

effects of medical silicones. However, although there is a growing number of medical 

interventions that require long term active or passive silicone implants, there has been 

surprisingly little effort to understand the molecular mechanisms behind the adverse 

effects they might induce. In the present study we focused our attention onto proteins 

adhering to the surface of medical silicones, as they have been identified as the key 

activators of the host defense mechanisms that precede fibrotic changes in the tissue 

surrounding the implant. Interestingly, abundant mononuclear cells, including dendritic 

cells and macrophages can be found in the fibrotic tissue surrounding silicone implants. 

In the discovery phase, a proteomics approach was used to identify proteins adsorbed 

from the serum of silicone mammary implant (SMI) carriers to the surface of silicone in 

vitro. Out of the 184 proteins which adsorbed to silicone, 14 showed differences in the 

adhesion pattern between the non-symptomatic SMI carriers compared to the group of 

patients developing a fibrotic reaction (peri-SMI fibrotic capsule) to silicone. In the 

subsequent development phase, a simple silicone linked immuno-sorbent assay 

(SILISA) was developed that can simultaneously detect the signature of the 14 

differentially adhered proteins in a high throughput fashion. In a cohort study of 100 

SMI carriers the SILISA successfully discriminated patients with adverse reactions to 

silicone implants. Furthermore, the same test can be used to assess various silicone 

types for the development of fibrotic side effects, and in such way tailor the choice of 

implants to the test results of individual patient. Both, large scale prospective and 

retrospective blinded studies are being conducted to validate these preliminary results, 

and to determine accuracy, precision, linearity, range, and the robustness of the test 

system. 

The project has been supported by the Competence Center Medicine Tirol (KMT) and the 

Lore and Udo Saldow Foundation.

Bettina Tosetti, Eva Glowalla, Martin Krönke, Oleg Krut 

Proteomics Based Identification of Cell Wall-associated 

Proteins as Protective Vaccine Candidates against 

Staphylococcus aureus 

S. aureus is an important human pathogen with an increasing clinical impact due to the 

extensive spread of antibiotic resistant strains. Therefore the development of a 

protective polyvalent vaccine is of great clinical importance. We employed an 

intravenous immunoglobulin (IVIG) preparation as a source of antibodies directed 

against S. aureus surface proteins for the identification of novel vaccine protein 

candidates. IVIG induced a strong opsonophagocytic activity of human neutrophils for 

S. aureus. In order to identify proteins that are targeted by IVIG, subtractive proteome 

analysis (SUPRA) of S. aureus surface proteins was performed. Proteins solely reacting 

with IVIG, but not with IVIG depleted of S. aureus-specific opsonising antibodies 

(dSaIVIG), were predicted to serve as vaccine candidates. Nearly 40 promising vaccine 

candidates were identified by this preselection method using MALDI-TOF analysis. Three 

of these candidates, enolase (Eno), oxoacyl reductase (Oxo) and a hypothetical protein 

(hp2160), were expressed as GST-fusion proteins, purified and used for the enrichment 

of corresponding IgGs from IVIG by affinity chromatography. Affinity purified anti-Eno, 

anti-Oxo and anti-hp2160 antibodies showed significant opsonising activity enabling 

uptake and killing of S. aureus by human neutrophils. Significant antibody responses 

were elicited in mice immunised with recombinant antigens. After challenge with S. 

aureus, reduced staphylococcal spread was detected in immunised mice by an in vivo 

imaging system. The recovery of S. aureus CFUs from organs of immunised mice was 

diminished by 10-100-fold. Furthermore, immunisation with hp2160 led to an 

remarkably improved survival rate compared to control mice. The results of this study 

suggest our approach to be a valuable tool for the identification of novel vaccine 

candidates and therapeutic antibodies.

Dirk Haubert, Nina Gharib, Francisco Rivero, Katja Wiegmann, Marianna Hösel, Martin 

Krönke, Hamid Kashkar 

PtdIns(4,5)P-restricted plasma membrane localization of FAN 

is involved in TNF-induced actin reorganization 

The WD repeat protein FAN is a member of the family of TNF receptor adaptor proteins 

that are coupled to specific signaling cascades. However, the precise functional 

involvement of FAN in specific cellular TNF responses remained unclear. Here we report 

the involvement of FAN in TNF-induced actin reorganization and filopodia formation 

mediated by activation of Cdc42. We show that the PH domain of FAN specifically binds 

to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P) which targets FAN to the 

plasma membrane. Site-specific mutagenesis revealed that the ability of FAN to mediate 

filopodia formation was blunted either by the destruction of the PtdIns(4,5)P binding 

motif, or by the disruption of intramolecular interactions between the PH domain and 

the adjacent BEACH domain. Using pull-down experiments, FAN was shown to interact 

with the actin cytoskeleton machinery in TNF-stimulated cells. The results of this study 

suggest that PH-mediated plasma membrane targeting of FAN is critically involved in 

TNF-induced Cdc42 activation and cytoskeleton reorganization. Furthermore, in vivo 

analysis of FAN-deficient mice revealed impaired TNF-induced dendritic cell migration 

pointing to a role of FAN in TNF-dependent dendritic cell motility.

Carina Conrads, Ramona Siemer, Mario Assenmacher, Claudia Niemand 

Quality assessment of enriched CD4+CD25+ regulatory T cells 

Many publications during the last years have reported naturally occurring CD4+CD25+ 

regulatory T cells to play an important role in autoimmunity, transplantation tolerance 

and tumor immunity. Intracellular FoxP3 staining is an important method for the 

analysis of regulatory T cells. In addition functional capacity of regulatory T cells is often 

assessed by in vitro suppression assays. 

We have developed a ready-to-use, well-defined, and robust method using 

MACSiBeadTM Particles for in vitro suppression assays. MACSiBeadTM Particles loaded 

with CD2, CD3 and CD28 antibodies act as artificial APCs and are simply added in a 1:1 

bead to cell ratio to the cell culture. 

Human CD4+CD25+ regulatory T cells were isolated from peripheral white blood cells 

using the MACS technology (n=9) with a mean purity of 87.8% (range 76.5-93.6%). 

More than 95% of isolated cells were CD4+, of which 78.5% were positive for FoxP3 

(range 63.8-88.5%). 

Isolated CD4+CD25+ regulatory T cells and autologous CD4+CD25- responder T cells 

were polyclonally stimulated with CD2, CD3 and CD28 antibody loaded MACSiBeadTM 

Particles (“Treg Suppression Inspector”) for 4 days and proliferation was analyzed by 

3H-thymidine incorporation for 16 hours. Isolated regulatory CD4+CD25+ T cells 

showed a mean suppression rate of 67.6% (range 38-92%) at a 1:1 ratio of regulatory 

T cells to responder T cells. 

We show that quality assessment of enriched CD4+CD25+ regulatory T cells is possible 

by staining for FoxP3 and using MACSiBeadTM Particles.

Tatiana Binder, Rodica Bernatowicz, Michael Rothballer, Michael Schmid, Susanne 

Krauss-Etschmann, Dolores J. Schendel, Anton Hartmann 

QUANTITATION OF LIVE VERSUS DEAD PROBIOTIC BACTERIA 

Background: The effect of probiotic bacteria on the immune system is subject of 

intensive research. However, live probiotic bacteria might induce different signals 

versus dead probiotic bacteria in bioassays, e.g. using dendritic cells. Therefore, we 

asked how standard culture affects the viability of different probiotic bacteria. 

Methods: Lactobacillus rhamnosus GG (LGG, Valio Ltd, Finland), Lactobacillus reuteri 

ATCC 55730 (BioGaia AB, Sweden), Bifidobacterium sp. 420 (Bifidobacterium lactis, 

Danisco Germany GmbH), Lactobacillus acidophilus LA-2, Lactobacillus acidophilus LA-5, 

Lactobacillus paracasei subsp. paracasei LC-01, Bifidobacterium animalis BB-12 and 

Bifidobacterium longum BB-46 (Chr. Hansen GmbH, Denmark) were cultured in MRSbroth 

(Merck KGaA) under anaerobic conditions for 24 hours. After cultivation the 

bacterial cells were stained with a LIVE/DEAD staining kit (Invitrogen GmbH). The 

percentage of dead bacteria within the total population of bacterial cells was counted 

visually via fluorescence microscopy. 

Results: The viability of LGG, B. lactis, LA-2, LA-5, LC-01, BB-12 and BB-46 ranged 

between 94%-99%. In contrast L. reuteri yielded only around 80% live bacteria after 24 

hours of cultivation. 

Conclusions: Our data show that viability of different probiotic strains can vary 

considerably under standard culture conditions. Therefore, the precise assessment of 

the viability of bacteria is important for functional immune assays. Currently, there are 

no straightforward bioassays at the single cell level available to distinguish among 

active, inactive or stressed live bacterial cells.

Guido Wabnitz, Urban Sester, Henning Kirchgessner, Yvonne Samstag 

Ras/PI3Kinase/cofilin independent activation of human 

CD45RA+ and CD45RO+ T-cells by superagonistic CD28 

stimulation 

T-cell activation requires costimulation of TCR/CD3 plus accessory receptors (e.g. 

CD28). A hallmark of costimulation driven T-cell activation is the dynamic 

reorganization of the actin cytoskeleton important for receptor polarization in the 

immunological synapse. The classical model of T-cell costimulation was challenged by 

detection of superagonistic CD28 antibodies. These antibodies induce T-cell proliferation 

and - as demonstrated here - production of IFN-γ, CD25 and CD69 even in the absence 

of TCR/CD3 triggering. 

Here we analyzed whether superagonistic CD28 stimulation induces costimulatory 

signaling events. Costimulation leads to phosphorylation of the actin bundling protein Lplastin 

and dephosphorylation of the actin reorganizing protein cofilin. Interaction of 

cofilin with actin is crucial for receptor polarization. Dephosphorylation of cofilin requires 

activation of Ras and PI3Kinase. Interestingly, superagonistic CD28 stimulation 

activates human peripheral blood T-cells (PBT) independently of Ras and PI3Kinase. 

Accordingly, it does not lead to cofilin dephosphorylation and receptor polarization. 

Likewise, L-plastin is not phosphorylated. Thus, superagonistic CD28 stimulation does 

not mimic costimulation. Instead, it leads to a Ras/PI3Kinase/cofilin independent state 

of "unpolarized T-cell activation". Finally, we demonstrate that superagonistic activation 

of human PBT requires calcium flux and activation of Src-kinases as well as PKC, Raf-1 

and Erk1/2.

Rebekka Wehner, Bärbel Löbel, Martin Bornhäuser, Ernst Peter Rieber, Marc Schmitz 

Reciprocal activating interaction between native human blood 

dendritic cells and NK cells in antitumor immunity 

Dendritic cells (DCs) are characterized by their unique capacity to induce primary T cell 

responses, providing the opportunity of DC-based cancer vaccination strategies. 

Additional findings reveal that DCs may also play a crucial role for the activation of 

innate antitumor immunity. Recently, we defined 6-sulfo LacNAc (slan) DCs (formerly 

termed M-DC8+ DCs) as a major subpopulation of human blood DCs which are capable 

of activating tumor-reactive cytotoxic T cells and of mediating tumor-directed 

cytotoxicity (Immunity 2002;17:289-301; Blood 2002;100:1502-1504, Immunity 

2006;24:767-777). Here, we investigated the reciprocal interaction between slanDCs 

and NK cells and the underlying mechanisms. 

To determine whether slanDCs promote NK cell activation, DCs were 

immunomagnetically purified from the blood of healthy donors and cultured for 6 h in 

the absence of exogeneous cytokines to allow spontaneous maturation. Subsequently, 

DCs were coincubated with NK cells in the presence of lipopolysaccharide (LPS). We 

found that LPS-stimulated slanDCs strongly stimulate proliferation and IFN-gamma 

secretion of NK cells. Furthermore, slanDCs markedly augmented the expression of the 

activating NK cell receptors NKp46 and NKp44. In addition, NK cell-mediated 

cytotoxicity against K-562 tumor cells and freshly prepared acute myeloid leukemia 

blasts was significantly improved by slanDCs which is dependent on interleukin (IL)-12. 

Further studies revealed that fresh NK cells efficiently enhance the secretion of the 

immunomodulatory cytokine IL-12 by slanDCs via IFN-gamma. In addition, the ability of 

slanDCs to produce the immunosuppressive cytokine IL-10 was markedly reduced by 

NK cells. 

In conclusion, we found that slanDCs strongly promote proliferation, IFN-gamma 

secretion, and tumor-directed cytotoxicity of NK cells. Reciprocally, NK cells efficiently 

modulate the production of IL-12 and IL-10 by slanDCs. These results indicate that the 

bidirectional cross-talk between native human DCs and NK cells may play a pivotal role 

in the regulation of antitumor immunity.

Andra Schromm, Jörg Howe, Artur Ulmer, Karl-Heinz Wiesmüller, Tobias Seyberth, 

Günther Jung, Manfred Rössle, Michel H.J. Koch, Klaus Brandenburg 

Recognition of bacterial lipopeptides by human macrophages 

is critically determined by physico-chemical parameters 

The importance of lipoproteins from the cell wall of Gram-positive and Gram-negative 

bacteria for the initiation of an innate immune defense is being increasingly recognized. 

It is well-established that lipoproteins are potent stimulants of the human innate 

immune system and elicit a variety of proinflammatory immune responses. However, in 

contrast to endotoxins (lipopolysaccharide, LPS), which are the main amphiphilic 

component of the outer membrane of Gram-negative bacteria, the molecular principles 

underling the immune stimulatory activity of lipopeptides are only partially understood. 

Investigations of synthetic lipopeptides corresponding to N-terminal partial structures of 

bacterial lipoproteins defined the chemical prerequisites for their biological activity and 

in particular the number and length of acyl chains and sequence of the peptide part. 

Here we present experimental data on the biophysical mechanisms underlying 

lipopeptide bioactivity. Investigation of selected synthetic diacylated and triacylated 

lipopeptides revealed, that the geometry of these molecules (i.e. the molecular 

conformations and supramolecular aggregate structures) and the preference for 

membrane intercalation provide an explanation for the biological activities of the 

different lipopeptides. This refers in particular to the agonistic or antagonistic activity (i. 

e., their ability to induce cytokines in mononuclear cells or to block this activity, 

respectively). 

The analytical data show that our concept of ‘endotoxic conformation’, originally 

developed for LPS, can be applied also to the investigated lipopeptides, and suggest 

that the molecular mechanisms of cell activation by amphiphilic molecules are governed 

by a general principle.

Ram Kumar Chowdary Venigalla, Theresa Tretter, Stefan Krienke, Regina Max, 

Norbert Blank, Volker Eckstein, Anthony D Ho, Hannes Martin Lorenz 

Reduced CD4+CD25- T Cell sensitivity to the suppressive 

function of CD4+CD25highCD127-/low Regulatory T Cells in 

active SLE Patients 

Introduction: 

SLE is a systemic autoimmune disease with an inflammatory phenotype, accompanied 

by high numbers of activated T cells (CD4+CD25+) in peripheral blood, which can lead 

to contamination of the nTreg population (CD4+CD25++) and discrepancies regarding 

their numbers and function. To overcome this problem, we have chosen low expression 

of CD127 along with CD4+CD25++ as a marker of true nTregs for cell sorting. This 

allowed us to gain highly purified nTregs and to study their role in patients with SLE. 

Methods: 

CD4+CD25++CD127-/low nTregs and CD4+CD25- T responder cells, (Tresp) were 

separated by FACSsorting. Proliferation was quantified by 3H Thymidine incorporation 

and Immunophenotype by FACScan. 

Results: 

We observed a slight but significant increase in the percentage of CD4+CD25highfoxp3 

+ T cells in active SLE patients (2.65 ± 0.4%) (SLEDAI >3, n = 13), and only marginal 

increase in inactive SLE patients (2.0 ± 0.4%) (SLEDAI ≤3, n = 11) compared with 

normal donors (1.75 ± 0.1%, n =19), correlating well with numbers of CD4+CD25+ 

+CD127-/low nTreg. Proliferation of nTregs from patients sorted for CD4+CD25++ vs 

CD4+CD25++CD127-/low significantly decreased from 9704±1775 cpm to 2585±495 

cpm, (ND:2028±548 cpm to 1291±388 cpm), confirming reduced effector cell 

contamination. In suppressor assays Tresp of normal donors were regulated to a 

comparable extent by Tregs from normal donors (81 ± 2%) and active SLE patients (73 

±3%; p = 0.01), while Tresp from active SLE were less suppressed, regardless if Treg 

from healthy donors (54 ± 7%; p = 0.0009) or SLE patients (53 ± 6%, p = 0.0001) 

were present. This Tresp cell resistance showed a direct correlation to disease activity, 

with inactive SLE patients showing the lowest Tresp resistance. 

Conclusions: 

Highly purified nTreg from SLE patients show no defect in suppressor function compared 

to normal donors. However CD4+CD25-Tresp cells from active SLE patients overcome 

suppressive function of normal and SLE-Treg, which might contribute to pathogenesis of 

SLE. 

.

Annegret Plege, Katja Borns, Reinhard Schwinzer 

Reduction of human anti-pig T cell responses by transgenic 

expression of human PD-L1 on pig cells 

Objective: Antibody-mediated blocking of receptor-ligand interactions providing 

positive costimulatory signals (e.g. CD28/B7) is one approach to inhibit T cell mediated 

graft rejection after transplantation. Furthermore, promoting the interactions of cell 

surface molecules which deliver negative signals (e.g. PD-1/PD-L1) might also decrease 

T cell reactivity. To test this concept we asked whether human anti-pig T cell reactivity 

can be modulated by overexpression of the human negative costimulatory molecule PD- 

L1 on porcine cells. 

Methods: The pig B cell line L23 was transfected with the pIRES-AcGFP vector 

containing human PD-L1. Stable transfectants (L23-hPD-L1 cells) were established, 

phenotypically characterized and used for in vitro stimulation of purified human CD4+ T 

cells and as targets for cytotoxic effector T cells. 

Results: Wild-type L23 cells as well as mock transfected controls triggered strong 

proliferative responses in human CD4+ T cells. However, when L23-hPD-L1 

transfectants were used as stimulators, T cell proliferation was significantly reduced (30- 

50%). The decreased stimulatory capacity of PD-L1 transfectants could be reversed by 

treatment of the cells with monoclonal antibodies to PD-L1 or PD-1 suggesting that the 

inhibitory effects are mediated by the interaction of the PD-L1 transgene with PD-1 on T 

cells. L23 cells were highly sensitive to lysis by human cytotoxic effector T cells. In L23hPD-L1 

cells, however, the intensity of cell mediated cytotoxicity was significantly 

reduced. 

Conclusion: These data indicate that in human T cells activated by PD-L1 transfectants 

the balance between positive and negative costimulation is shifted towards negative 

signals thereby diminishing the intensity of the response. Transgenic expression of 

human PD-L1 in pig cells and tissues could be an approach to prevent T cell reactivity 

after pig to human xenotransplantation.

Niko Föger, Andrew Chan 

Redundant versus unique roles of the actin regulatory 

proteins coronin-1 and coronin-2 in the immune system 

The integrity of the actin cytoskeletal network is critical for many biological processes. 

Coronins constitute an evolutionary conserved family of proteins implicated in the 

regulation of actin cytoskeletal dynamics. Focusing on the physiologic function of 

coronin-1, a coronin family protein preferentially expressed in immune cells, we have 

demostrated a requirement for coronin-1 in maintaining the cellular steady-state F-actin 

content, chemokine-mediated functions in T cells and, most surprisingly, maintaining 

normal cellular survival. Coronin-2 is closely related to coronin-1 and the two genes are 

co-expressed in hematopoietic cells. In a genetic approach to address the question of 

functional redundancy in vivo, we have generated coronin-2 deficient mice and crossed 

them with coronin-1 deficient mice to obtain coronin-1_coronin-2 double deficient mice. 

Analysis of the T cell compartment in coronin-1_coronin-2 double deficient mice 

indicates a significantly exacerbated phenotype as compared to the respective single 

knock-outs. Further phenotypical and functional analysis of lymphocytes from these 

different coronin knock-out strains will define the relative roles of coronin-1 and coronin- 

2 in regulating actin cytoskeletal dynamics in lymphocytes and reveal the degree of 

potentially unique versus redundant functions of these proteins in the immune system.

Manuela Ahrendt, Reinhard Pabst, Ulrike Bode 

Regeneration of transplanted lymph node fragments in the 

mesentery to study effects of the gut lymph on lymph node 

cytokines and chemokines 

The mesenteric lymph nodes (mLN) are important for immune responses and 

acquisition of oral tolerance. However, the influence of the gut in forming and regulating 

the microenvironment of mLN and mediating immune responses is only partly known. 

To study this, rat and mouse mLNs were removed and either fragments of mesenteric 

(mLNtx) or peripheral lymph nodes (pLNtx) were transplanted into this region. Two, 4, 

6, 8 and 10 weeks after transplantation the lymph node tissue was excised and different 

cytokines and also chemokines were analysed by real-time PCR. The regeneration of the 

transplants was documented by a re-established flow of lymph from the gut wall to the 

lymph node tissue and by immunohistology of typical lymph node compartments. 

IL-10 and CCL8 mRNA were expressed at a similar level to mLN in normal untreated 

animals, whereas IL-4 regenerated completely after re-construction of the regenerated 

lymph nodes. Interestingly, IL-2 was expressed in the pLNtx comparable to the pLN 

control. In accordance with this expression pattern, CCR9 expression of the mLNtx was 

similar to that of the mLN control, whereas in the pLNtx CCR9 expression was 

comparable to that of the pLN control. 

On the one hand mLNtx and pLNtx expressed cytokines, e.g. IL-4 and CCL8, in a similar 

way after transplantation, suggesting that IL-4 and IL-10 were recruited from the 

draining gut wall. On the other hand IL-2 and CCR9 mRNA were expressed comparable 

to the control lymph nodes, indicating that these molecules were produced by the 

surviving cells. 

The regeneration of transplanted LN fragments in the mesentery is a useful technique to 

define the role of the gut wall for protein levels of different cytokines and chemokines in 

the mLN in inducing immune responses and oral tolerance in the gut immune system.

Michael Andrzejewski, Nicole Schwarz, Christian Weber, Andreas Ludwig 

Regulated shedding of transmembrane chemokines by the 

metalloproteinase ADAM10 facilitates detachment of adherent 

leukocytes 

CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family since 

they occur not only as soluble but also as membrane-bound molecules. Expressed as 

type I transmembrane proteins, the ectodomain of both chemokines can be 

proteolytically cleaved from the cell surface, a process known as shedding. Our previous 

studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the 

largest proportion of constitutive CX3CL1 and CXCL16 shedding but is not involved in 

the phorbolester-induced release of the soluble chemokines (inducible shedding). Here 

we introduce the calcium-ionophore ionomycin as a novel, very rapid and efficient 

inducer of CX3CL1 and CXCL16 shedding. By transfection experiments in Cos-7 cells 

and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective 

metalloproteinase inhibitors we demonstrate that the inducible generation of soluble 

forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal 

cleavage fragments remaining in the cell membrane reveals multiple cleavage sites 

used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. 

Addressing the functional consequences of induced shedding we demonstrate that 

ionomycin-induced CX3CL1 shedding via ADAM10 can lead to the detachment of 

monocytic cells from their cellular substrate, pointing towards a release mechanism 

potentially important for leukocyte diapedesis.

Dirk Brenner, Alexander Golks, Mareike Becker, Christian R. Frey, Rostislav Novak, 

Friedemann Kiefer, Peter H. Krammer, Rüdiger Arnold 

Regulation of Activation-induced Cell Death by T Cell 

Receptor-Proximal Signalling 

Lymphocyte homeostasis is strictly controlled to maintain physiological levels. 

Activation-induced cell death (AICD) is one mechanism to delete superfluous and 

autoreactive lymphocytes by restimulation of their immunoreceptors. So far 

Immunoreceptor-proximal mechanisms leading to AICD are elusive. Here we 

characterize Hematopoietic Progenitor Kinase 1 (HPK1) as a differentially regulated TCRproximal 

signalling protein involved in AICD of primary T cells. 

We show that HPK1 is a functional component of the endogenous I-κB kinase (IKK) 

complex and prove HPK1 to be essential for TCR-mediated IKK and NF-κB activation. 

We demonstrate proteolytic processing of HPK1 into HPK1-C specifically in AICDsensitive 

primary T cells. The cleavage product HPK1-C sequesters the inactive IKK 

complex and suppresses NF-κB upon TCR restimulation. T cells of HPK1-C transgenic 

mice are sensitized towards TCR-mediated AICD. While it is well established that AICD 

of T cells partially depends on the CD95/CD95L system we show T and B lymphocytes 

from HPK1-C transgenic mice undergo AICD independently of CD95/CD95L. We show 

that CD95L-dependent and HPK1/HPK1-C-mediated cell death pathways complement 

each other in AICD of primary human T cells. Our results define HPK1 as a novel 

regulator of AICD in lymphocytes.

Christina Hartwig, Miriam Mazzega, Hanne Constabel, Georg Behrens, J. Engelbert 

Gessner, Thomas Tschernig 

Regulation of allergic inflammation in a murine asthma 

model: Antigen uptake via Fcγ-receptors by DC-subsets, 

impact on tolerance and immunity 

Murine Fcγ-receptors (FcγRI-IV) are expressed on many inflammatory cell types. The 

role of these receptors in the initiation or maintenance of allergic inflammation has not 

yet been well defined. To identify the impact of FcγR on the antigen uptake and antigen 

presentation of dendritic cells (DC), FcγR-deficient (γ-chain [γ -/-], FcγRII -/-) and 

control mice were sensitized and challenged with OVA or PBS. Relevant asthma markers 

such as IgE and IgG titre in serum, the number of eosinophils in BAL, the cytokine 

spectrum in serum and BAL-fluid as well as a possible pulmonary hyperresponsiveness 

were analysed. The numbers of eosinophils were significantly reduced in γ -/- mice after 

OVA treatment, whereas the levels of IgE and IgG were comparable with control mice. 

FcγRII -/- developed a strong inflammatory response, characterized by an eosinophil 

and neutrophil influx and an increased IgE level. 

Furthermore, the impact of FcγR on antigen presentation and T cell proliferation was 

determined in vitro and in vivo. In vitro, T cell proliferation was more strongly 

stimulated by OVA/ α-OVA immune complexes than by soluble OVA in C57Bl/6 wt and 

FcγRII -/- mice. In contrast the γ -/- mice show a greatly reduced T cell proliferation and 

no differences between soluble OVA or OVA/ α-OVA treated cells. To clarify the role of 

specific DC subsets CD8+, CD4+ and CD4/8 – spleen DCs were measured separately. 

To analyse the antigen uptake via immune complexes, T cell proliferation were also 

determined in vivo. OVA exposed C57Bl/6 wt mice and γ -/- mice show similar 

responses to those in vitro. Surprisingly, not only the T cell proliferation in γ -/- mice 

but also the proliferation in FcγRII -/- was reduced. 

These results demonstrate that FcγR are involved in the pathogenesis of experimental 

asthma, most probably by facilitating enhanced uptake of immune complexes by DCs. 

SFB 587, B5, B10 German Research Foundation

Daniela Sánchez, Sigrid Krämer, Yves Montier, Herbert Schmidt, Stephan C Bischoff 

Regulation of human intestinal fibroblasts by Endotoxins and 

Shiga toxins 

Introduction: Previous in vitro studies demonstrated activation of colonic fibroblasts by 

bacterial products indicating a role of fibroblasts in innate immunity. In this study, we 

investigated the expression of different cytokines, collagens and metalloproteinases 

(MMP) by human intestinal fibroblast after stimulation with the bacterial cell wall 

polymers LPS, LTA or Shiga toxin-containing bacterial supernatants respectively. 

Methods: Fibroblasts were isolated from surgical tissue specimens and cultured for 1-2 

weeks until they formed a subconfluent layer. Fibroblasts were challenged with different 

concentrations of LPS and LTA or with supernatants from E. coli bacteria expressing 

Shiga toxin 1 or Shiga toxin 2. Results: Stimulation of intestinal fibroblasts with LPS and 

LTA enhance the expression and release of proinflammatory cytokines (TNF-alpha, IL-8, 

IL-6); however, expression of collagen 3 and 13, as well as MMP3 was not affected. 

Furthermore, supernatants of Shiga toxin-releasing E. coli strains also significantly 

enhanced the mRNA-expression of TNF-alpha, IL-8 and IL-6. Conclusion: Human 

intestinal fibroblasts are capable of expressing different cytokines upon stimulation with 

LPS, LTA and supernatants of Shiga toxin-releasing E. coli strains. This data supports 

the concept that human intestinal fibroblasts have a role in innate immune response 

and act as a pro-inflammatory and immune regulatory but not profibrotic cell in 

bacterial infections.

Norbert Hüser, Annette Fasan, Monika Semmrich, Bernhard Holzmann, Melanie 

Laschinger 

Regulation of LFA-1 activity: Importance for leukocyte 

recruitment and alloantigenic T cell activation in cardiac 

allograft rejection 

The leukocyte-specific integrin LFA-1 is considered to be important for immune 

responses leading to organ transplant rejection. Regulating the affinity of LFA-1 for its 

ligand ICAM-1 is known to be crucial for its function. However, the importance for deactivating 

LFA-1 remains largely unknown. Using a mutant mouse that locks LFA-1 in an 

active state (LFA-1 d/d ) we investigated the function of LFA-1 de-activation and deadhesion 

in a model of heterotopic cardiac transplantation. 

Defect in LFA-1 de-activation ameliorates graft survival in LFA-1 d/d recipient mice. A key 

event in graft rejection is its infiltration by allogen-primed T cells. Constitutively active 

LFA-1 hinders activation and proliferation of allo-reactive T cells. We found expression 

of the chemokines CXCL10/IP-10 and CCL5/RANTES to be markedly reduced in grafts of 

LFA-1 d/d recipients. This defect translates into a low number of CD4 and CD8 T cells 

detected in the transplanted heart. Furthermore, major leukocytes initiating the 

inflammation at early stages of graft rejection are not efficiently recruited into cardiac 

allografts of LFA-1 d/d hosts. The reduced amount of TNFα and IFNγ in the allograft 

seems to account for this defect. We conclude that controlling LFA-1 de-activation is 

required for the generation of immune responses that are vital for allograft rejection.

Jens Derbinski, Sheena Pinto, Stefanie Rösch, Klaus Hexel, Bruno Kyewski 

Regulation of promiscuous gene expression within defined 

chromosomal regions 

Promiscuous expression of tissue-restricted auto-antigens in medullary thymic epithelial 

cells (mTECs) imposes central T cell tolerance and is essential for protection from organspecific 

autoimmune diseases. The molecular regulation of this unusual gene expression 

is not well characterized and its delineation from cell lineage-specific gene expression 

control remains unclear. 

Two alternative models have been postulated (i) emulation of cell-lineage specific gene 

signatures or (ii) loss of gene repression resulting in an apparently stochastic gene 

expression pattern. Each model predicts different gene expression patterns at the single 

cell level. 

In previous experiments using pooled mTECs we observed contiguous promiscuous 

gene transcription in the casein locus. Here, we compared the expression profile of the 

casein gene locus in mTECs and mammary gland epithelial cells by single cell PCR. 

Mammary gland cells showed highly correlated intra- and inter-chromosomal coexpression 

of milk proteins and one of its control elements (i.e. the casein genes, 

lactalbumin, WAP and Elf5). In contrast, at the single cell level we did not observe 

contiguous expression of the casein gene locus in mature CD80hi mTECs and the 

expression of these genes was not correlated and did not reveal discernible patterns. 

The apparent stochastic expression pattern within the casein locus in mTECs clearly 

delineates the molecular mechanism(s) of promiscuous gene expression from cell 

lineage-specific gene control.

Katja Thümmler, Andreas Ramming, Alla Skapenko, Hendrik Schulze-Koops 

Regulation of specific immunity by homotypic T cell/T cell 

interaction 

Specialized T cells with a regulatory phenotype are most important in controlling specific 

immunity to self-antigens and, thus, in maintaining peripheral tolerance. Different T cell 

subsets with a regulatory phenotype have been described that may develop in the 

periphery in response to T cell stimulation. Differentiation of T cells, in turn, is regulated 

by cytokines and a variety of cell surface receptors, for which activated effector T cells 

express the appropriate ligands. Here, we tested the hypothesis, that homotypic T cell 

interactions may permit the induction of T cells with a regulatory phenotype. CD4 T cells 

were isolated from the peripheral blood of healthy donors and activated under Th1 or 

Th2 conditions. The resulting effector cells were fixed with paraformaldehyd and 

cocultured together with syngeneic freshly isolated resting CD4 memory or naive T cells 

but in the absence of specific T cell stimulatory factors such as mAbs to T cell surface 

molecules, antigens or mitogens. After five days of coculture, the phenotype and 

function of the resulting cells were analyzed by assessing their production of cytokines 

by ELISA, flow cytometric analysis of cytoplasmic cytokines and realtime PCR and by 

determining their ability to prevent the proliferation of autologous responder T cells. T 

cells that had been generated in the presence of fixed Th2 effector cells produced IL-4, 

but little IFN-γ. In contrast, Th1 effector cells promoted the development of IL-10 and 

IFN-γ double-producing T cells. Of interest, both T cell populations that were generated 

by homotypic T-T cell interactions strongly inhibited the proliferation of CD25 negative 

responder T cells in a dose dependent manner. Although T-T cell-generated effector 

cells did not express Foxp3 their regulatory capacity was comparable to that of 

conventional CD25 positive Tregs. The results indicate that homotypic T-T cell 

interaction induces the generation of Th1-like or Th2-like effector T cells with a 

regulatory phenotyp, providing a novel potential negative feedback mechanism to 

control sustained T cell driven immunity.

Carl Friedrich Classen 

Regulation of the granzyme B inhibitor proteinase inhibitor 9 

(PI-9) in monocytes: Immunomodulating effects of 

cotrimoxazole 

Proteinase inhibitor 9 (PI-9) - the only known endogeneous natural antagonist of the 

lymphocyte protease granzyme B (GrB) - is an intracellular serpin expressed in 

lymphocytes and monocyte-derived cells. By intracellular flow cytometry, we have 

previously shown that ex-vivo stimulation by lipopolysaccharides leads to upregulation 

of PI-9 within 24 hours in the monocyte, but not the lymphocyte fraction; this can be 

inhibited by the NF-kappaB inhibitor pyrrolidin dithiocarbamate (PTDC). Co-trimoxazole 

- the combination of sulfamethoxazole and trimethoprim - is widely used as antibiotic 

for bacterial and pneumocystis infections. Since other sulfonamide metabolites like 

sulfasalazine have been described to exert immunomodulatory effects, we now asked 

whether co-trimoxazole might influence expression of PI-9 in monocytes and 

lymphocytes. A 24 hours incubation assay was done using heparinized full blood of 

healthy volonteers, and PI-9 expression was analysed by intracellular flow cytometry. 

We found that co-trimoxazole leads to PI-9 upregulation in monocytes at therapeutical 

concentrations (800 µM resp. 140 µM sulfamethoxazol/trimethoprim), similar to the 

upregulation induced by lipopolysaccharides. Combination of co-trimoxazole with 

lipopolysaccharides did not further enhance PI-9 expression. In lymphocytes, no 

alteration of PI-9 expression was induced. Since it has been shown that PI-9 overexpression 

in antigen presenting cells, e.g. dendritic cells, leads to an enhanced 

immune response, our finding may be relevant for specific immune regulation. 

Pharmacological modification of PI-9 expression may represent an interesting tool, both 

for the study of immune regulatory mechanisms or, even, for therapeutical 

interventions in immune diseases.

Jennifer Freyer, Stefan Floess, Alf Hamann, Jochen Huehn 

REGULATION OF THE MURINE TRANSCRIPTION FACTOR 

FOXP3 

The transcription factor Foxp3 is specifically expressed in natural CD4+ regulatory T 

cells and critically involved in their development and suppressive function. Although a 

lot of knowledge has been accumulated regarding the function of Foxp3, relatively little 

is known about the molecular regulation of Foxp3 expression. We have recently 

identified an evolutionarily conserved region within the foxp3 locus upstream of the 

translational start site, containing numerous CpG motifs, which in Tregs were selectively 

demethylated compared to non-regulatory T cells. This conserved region, which we 

named TSDR (Treg specific demethylated region), turned out to be associated with 

acetylated and trimethylated histones specifically in Tregs, indicating an open chromatin 

structure. Luciferase assays showed that the TSDR encompasses transcriptional activity, 

which was affected by in vitro methylation. Currently, we are generating deletion 

mutants of the TSDR to more precisely identify the critical elements involved in the 

molecular control of Foxp3 expression.

Janine Wehrhahn, Robert Kraft, Sunna Hauschildt 

Regulation of TRPM2 channels in human monocytes and 

macrophages 

TRPM2 belongs to the superfamily of transient receptor potential (TRP) proteins and 

functions as a calcium-permeable, nonselective cation channel. TRPM2 is specifically 

activated by intracellular ADP-ribose and can be opened during oxidant stress. 

Here we show for the first time the occurrence of TRPM2 in human monocytes and 

monocyte-derived macrophages on mRNA-, protein- and functional level. We studied 

freshly isolated monocytes and macrophages in comparison with 16 h cultivated 

unstimulated and LPS-stimulated (100ng/ml) cells. 

Besides the total TRPM2-mRNA we could detect the splice variants TRPM2-S and TRPM2- 

ΔC in both monocytes and macrophages. The splice variant TRPM2-ΔN was absent. 

Using semi-quantitative real time PCR we found that in monocytes TRPM2-mRNA was 

significantly down-regulated after 16 h cultivation in the absence but not in the 

presence of LPS. In human macrophages the TRPM2-mRNA-level was not affected by 

different incubation conditions. 

Western blot analyses showed that in human monocytes TRPM2 protein expression 

follows the same pattern as the mRNA-expression. In macrophages however the TRPM2 

protein-level does not exactly mirror the mRNA-level. 

Whole-cell patch clamp recordings using ADP-ribose in the pipette solution consistently 

revealed TRPM2-like cation currents in both monocytes and macrophages. The 

functional activity of TRPM2 correlated with the mRNA and protein data in monocytes 

and the mRNA data in macrophages. 

The expression and functionality of the cation channel TRPM2 in human monocytes and 

macrophages provide a potent mean to regulate intracellular calcium levels and 

biological answers. The inhibition of the TRPM2 channel will help to clarify its exact 

function in human monocytic cells.

Christian Erbel, Roland Klingenberg, Sultan Celik, Benjamin Funke, Hardy 

Schumacher, Nadine Wambsganss, Thomas Dengler 

Regulatorische T Zellen und Signalkaskaden über IL-17 und 

IL-10 als atheroprotektive Komponenten in 

atherosklerotischen Läsionen 

Atherosklerotische Läsionen enthalten typischerweise ein Infiltrat aus Makrophagen und 

T Zellen. Ziel dieser Studie ist der Nachweis regulatorischer T Zellen (Treg) als potentiell 

atheroprotektiver Komponente in atherosklerotischen Läsionen. 

Plaquegewebe aus den Karotiden von 47 Patienten, die sich einer TEA unterzogen, 

wurden gesammelt. Mit Hilfe der quantitativen PCR und der Immunohistochemie wurden 

die Immunzellen in dem Gewebe typisiert. Klinische Daten der Patienten wurden 

ausgewertet. Die Plaques wurden nach der Symptomatik der Patienten in eine 

symptomatische und eine asymptomatische Gruppe eingeteilt. Die Patientendaten 

wurden nach Vorhandensein einer Statin- und/oder Aspirintherapie unterteilt. 

Die Analyse aller Plaques zeigte lymphozytäre Infiltrate, insbesondere Tregs in fast allen 

Proben. Treg waren signifikant häufiger in Plaques von asymptomatischen Patienten im 

Vergleich zu Karotidenproben von symptomatischen Patienten (p=0,006) vorhanden. 

Zudem scheinen Treg unter einer Statintherapie oder ohne Aspirintherapie deutlich 

häufiger vertreten zu sein. Weiterhin war in Treg-positiven Karotisplaques eine erhöhte 

Expression des antiinflammatorischen Zytokines IL10 nachweisbar, welche u.a. von 

Treg produziert wird. Zudem zeigte sich ein signifikanter Unterschied in der Expresison 

von IL-17, vermehrt exprimiert im asymptomatischen Patientenkollektiv (p=0,03) und 

häufiger bei Patienten mit einer Statin- (p=0,02) oder Marcumar/Clopidogreltherapie. 

Die vorliegende Studie weißt Treg als mögliche atheroprotektive Komponente im 

immunzellulären Infiltrat von atherosklerotischen Läsionen nach und gibt somit einen 

Anhalt für eine mögliche Bedeutung von Treg für die Plaquegenese und Symptomatik, 

insbesondere aufgrund der Assoziation mit erhöhten Spiegeln von IL-10 in Treg-haltigen 

Plaques und der vermehrt auftretenden Treg in Patienten mit einer Statintherapie. 

Erstmals wird die Anwesenheit von IL-17 nachgewiesen, vornehmlich in 

asymptomatischen sowie bei Patienten mit einer Statintherapie.

Sabine Riekenberg, Katja Farhat, Jennifer Debarry, Holger Heine, Karl-Heinz 

Wiesmueller, Roland Lang, Artur J. Ulmer 

Regulators of G-protein signaling are modulated by bacterial 

lipopeptides and lipopolysaccharide 

Regulators of G-protein signaling (RGS) accelerate the GTPase activity of G&alpha 

subunits, thus driving G-proteins in their inactive GDP-bound form. This property 

defines them as GTPase activating proteins (GAPs). In our study we analyzed the effect 

of different Toll-like receptor (TLR) agonists on RGS1 and RGS2 expression in murine 

bone marrow-derived macrophages (BMDM) and J774 cells. 

To identify different signal transduction pathways and patterns of gene expression we 

stimulated BMDM cells for 2 and 6 h with different lipopeptides like PamOct2C(VPGVG)4- 

VPGKG, FSL-1 and Pam2C-SK4 to activate signal transduction through TLR2 in a TLR1-, 

TLR6-, or TLR1 & 6 independent manner and for 3 h with LPS to activate TLR4 signaling, 

respectively. Affimetrix micro array analyses showed a strong down-regulation of RGS1 

and RGS2 at 2 h and 6 h of stimulation with the different TLR2 ligands. Furthermore, we 

detected an up regulation of RGS1 as early as 15 min after stimulation. We confirmed 

these data by real-time PCR, flow cytometry and fluorescence microscopy. In addition 

we found a strong up regulation of both, RGS1 and RGS2 mRNA, after stimulation with 

LPS in MyD88-/- mice, indicating that a MyD88-independent pathway of TLR4 signaling 

is responsible for this up regulation. The application of poly I:C, a TLR3 ligand, and the 

usage of Trif-/- mice confirmed this conclusion. 

We suggest that modulating of RGS1 and RGS2 by TLR2 and TLR4 ligands play an 

important role during inflammatory and immunological reactions. 

(Supported by Deutsche Forschungsgemeinschaft UL68/3-2)

Arthur Liesz, Elisabeth Suri-Payer, Claudia Veltkamp, Henrike Dörr, Clemens Sommer, 

Thomas Giese, Roland Veltkamp 

Regulatory T Cells (Treg) are Important Cerebroprotective 

Immunomodulators in Acute Experimental Stroke by an 

Interleukin-10 Dependent Pathway. 

Inflammatory cascades contribute substantially to secondary infarct growth after 

ischemic brain damage. Because CD4+CD25+Foxp3+ Treg are important negative 

immunomodulators in various inflammatory diseases, we studied the role of Treg in 

experimental stroke and the involved immunosuppressive mechanisms. 

Permanent focal cerebral ischemia was induced by middle cerebral artery occlusion 

(MCAO) in C57BL/6 mice and Treg were eliminated in one group of mice by preischemic 

anti-CD25 mAb injection. While no difference was detectable between groups at 24h, 

infarct volumes became significantly larger between 3-7d after MCAO in Treg depleted 

mice. Correspondingly, transfer of only CD4+CD25- T cells into RAG-/- mice resulted in 

larger infarcts than transfer of total CD4+ cells. Treg started to invade the brain 3d 

after MCAO (shown by FACS and immunohistology). The lack of Treg resulted in 

significantly higher microglial activation (Iba1+ cells) 24h after MCAO. Further, levels of 

proinflammatory cytokines (RT-PCR and ELISA) were significantly more elevated in the 

ischemic brain hemisphere and the serum of Treg depleted mice at various time points 

after MCAO compared to control mice. The intraventricular injection of IL-10 prevented 

the secondary infarct growth in Treg depleted mice. Finally, RAG-/- mice receiving 

wildtype CD4+CD25- T cells plus Treg with impaired IL-10 secretion had significantly 

larger infarcts than mice receiving additional wildtype Treg. 

In conclusion, we show that Treg are physiological master anti-inflammatory modulators 

in ischemic stroke which substantially reduce secondary infarct progression. This effect 

is mainly mediated by early IL-10 signalling, inhibition of microglial activation and 

cytokine expression as well as delayed Treg invasion.

Paula Kolar, Holger Hoff, Karin Knieke, Kolja Hegel, Dagmar Quandt, Monika Brunner- 

Weinzierl 

Regulatory T cells require costimulation by CTLA-4 (CD152) 

Unwanted T cell responses in the periphery are suppressed by regulatory CD4+CD25+ T 

cells (Treg). Here we show that Treg cells strongly co-express Foxp3 and intracellular 

CTLA-4, a major inhibitory molecule of T cell responses. Additionally, we could show 

that Treg cells are the only population of T helper cells which express surface CTLA-4 ex 

vivo implying a constant or immediate need for CTLA-4 signaling. This assumption is 

extended using serological blockade of CTLA-4 during in vitro inhibition of inflammatory 

effector T cells by Tregs cells leading to reduced inhibitory capacity. Interestingly, 

blocking of CTLA-4 on Treg cells during in vitro stimulation in the absence of CD25- 

target cells rescues their proliferation, but not their cytokine production. Strikingly, 

serological blockade of CTLA-4 during activation of Treg cells leads to enhanced AICD. 

We could show that CTLA-4 induced signaling interferes with CD95/CD95L mediated 

activation of caspases. Our results suggest that the surface expression of CTLA-4 on 

Treg cells serves Treg cells´ basic needs to preserve the stringency of their anergic 

status and their survival under non/low-inflammatory conditions.

Ioanna Galani, Marco Wendel, Carola Schellack, Elisabeth Suri-Payer, Adelheid 

Cerwenka 

REGULATORY T CELLS SUPPRESS IFN-γ DEPENDENT 

LEUKOCYTE ACCUMULATION AND MACROPHAGE ACTIVATION 

IN LYMPHOMA 

Regulatory T cells (Treg) have been shown to suppress immune responses of tumorspecific 

T cells; yet, little is known about the impact of Treg on innate immune cells in 

tumor models. Since many tumors lose expression of MHC class I, our study aimed at 

defining strategies to strengthen immune responses against a high tumor burden of the 

MHC class I-deficient lymphoma RMA-S. We demonstrate that Treg depletion led to 

tumor rejection and generation of immunological memory. This tumor rejection was 

associated with a substantial, IFN-γ dependent increase in numbers of tumor-infiltrating 

leukocytes, including macrophages. Tumor-infiltrating macrophages from Treg depleted 

mice expressed increased amounts of MHC class II, produced enhanced levels of 

inflammatory cytokines and inhibited tumor cell proliferation. Our study reveals that in 

an in vivo tumor model, numbers and activity of macrophages are controlled by Treg. 

These data identify macrophages as novel targets for Treg mediated immune 

suppression in cancer.

Mario Zaiss, Jochen Zwerina, Karin Polzer, Eva Gückel, Alla Skapenko, Hendrick 

Schulze-Koops, Nikki Horwood, Andrew Cope, Georg Schett 

Regulatory T cells suppress osteoclast formation- a new link 

between the immune system and bone 

Objective. 

To investigate whether regulatory T cells can suppress osteoclast differentiation 

and to define a new potential link between the immune system and the skeleton. 

Methods. 

Regulatory CD4+CD25+foxp3+ T cells were isolated and purified from the spleen and 

co-cultured with CD11b+ osteoclast precursor cells isolated from the bone marrow. 

Osteoclastogenesis and bone erosion was assessed by TRAP staining and pit resorption 

assay, respectively. In addition, trans-well experiments and cytokine blocking 

experiments were performed to define the mechanisms of interaction between 

regulatory T cells and osteoclasts. 

Results. 

CD4+CD25+foxp3+ T cells dose dependently inhibited MCSF- and RANKL dependent 

osteoclast formation in contrast to CD4+CD25- T cells. Also pit formation was inhibited 

by up to 80%. The blockade of osteoclast formation was not based on an alteration of 

RANKL/OPG balance but essentially dependent on direct cell-cell contact via CTLA-4. 

Regulatory T cell-mediated expression of TGF-β, IL-4 and IL-10 contributed to the 

inhibitory effect on osteoclastogenesis, but was not essential. 

Conclusion. 

These data show that CD4+CD25+foxp3+ regulatory T cells suppress osteoclast 

formation. These data provide a new link between the immune system and bone and 

extend our knowledge on the regulation of bone homeostasis by the immune system.

Florian Eberle, Mehtap Sirin, Klaus Heeg, Alexander Dalpke 

Relevance of RNA-mediated immunostimulation for RNA- 

Interference (RNAi) 

Double-stranded RNAs can regulate endogenous messenger RNA stability which is 

referred to as RNA interference (RNAi). This process can be mimicked by small synthetic 

RNA, so called small inhibitory RNA (siRNA). In addition to the newly identified role of 

RNA in gene-silencing, it is known that RNA is also a target for immune recognition 

principles. Even more, siRNA can induce activation of some cell-types of the innate 

immune system thus mediating off-target effects. Immune-recognition of RNA is 

mediated by three members of the Toll-like-receptor family (TLR 3,7 and 8) as well as 

by the cytosolic RNA-binding proteins RIG-I and MDA5. Additional, there are evidences 

that RNA recognition occurs in a cell-type specific manner. 

We aimed to identify RNA motives, which determine the immunostimulatory potential of 

RNA and RNA oligonuclotides, such as siRNAs. We show that functional siRNAs were 

immunostimulatory on several cell-types of the innate immune system. By introducing 

uridine modifications, immunostimulation could be reduced significantly, whereas 

modifications of other nucleotides showed only marginal effects. We further show that 

bacterial RNA preparations were immunostimulatory not only on cells of the immune 

system but also were able to stimulate normal tissue cells. We can also show that 

recognition of bacterial RNA differs significantly from recognition of RNA 

oligonucleotides.

Christian Hofmann, Thomas Harrer, Kathrin Eismann, Silke Bergmann, Matthias Schmitt- 

Haendle, Gerold Schuler, Jan Dörrie, Niels Schaft 

REPROGRAMMING T CELLS WITH A HIV-1-SPECIFICTY BY 

ELECTROPORATION OF TCR-ENCODING RNA 

HIV-1 establishes a persistent infection in humans and destroys the patient’s CD4+ 

immune cells, which ultimately leads to paralysis of the immune system. However, high 

levels and a broad specificity of anti-HIV-1 CD8+ cytotoxic T lymphocytes (CTL), 

especially against conserved epitopes, are considered to be critical for long-term control 

of HIV-1 replication. Unfortunately, most HIV-infected patients are unable to generate 

such a powerful immune response. A possible immunotherapy is the adoptive transfer 

of T cells, which were reprogrammed by introduction of an HIV-specific T cell receptor 

(TCR). Until now, these HIV-specific CTL were generated by retroviral transduction of 

TCR encoding cDNA. However, this strategy harbors the threat of stable genetic 

alteration of autologous cells. Therefore, we studied TCR transfer by RNA 

electroporation into CD8+ T cells. An HIVpol-specific TCR, which recognizes the HLA-A2 

restricted peptide ILKEPVHGV, was used. These reprogrammed T cells produced the proinflammatory 

cytokines IL-2, TNF, and IFNgamma after stimulation with peptide-loaded 

target cells, and efficiently and specifically killed these targets, even after 

cryopreservation. The cytolytic function of the reprogrammed T cells persisted for at 

least 72 h after transfection. Peptide-titration studies revealed that the lytic avidity of 

the TCR-RNA-electroporated CD8+ T cells was in the same range as that of the parental 

CTL clone. Taken together, we show here for the first time that functional transfer of 

virus-specific TCR by RNA electroporation is feasible. This technology represents an 

innovative, secure, and easy method to produce virus-specific T cells, and may 

represent a new tool in the fight against HIV infection.

Esther Wilk, Katy Kalippke, Sabine Buyny, Reinhold E Schmidt, Roland Jacobs 

Response of CD56 dim and CD56 bright NK cells to Interleukin 21 

IL-21 is a cytokine with pleiotropic effects on various cell types including DC, B, T, and 

NK cells. In order to evaluate the effects of IL-21 on human NK cell subpopulations 

functional studies were completed on CD56 dim and CD56 bright NK cells, both bearing IL- 

21 receptors at identical densities. Stimulation with IL-21 strongly induced proliferation 

of CD56 bright NK cells. Cytotoxicity against K562 target cells was preferentially 

augmented in CD56 dim whereas the expression of granzyme K increased mainly in 

CD56 bright NK cells. Intracellular analysis of STAT proteins revealed IL-21 induced 

phosphorylation of STAT1 and STAT3 in CD56 dim NK cells, and to an even higher degree 

in CD56 bright NK cells. In the CD56 bright NK cell population, IL-2 led to a slight 

phosphorylation of STAT1 and 3, which was synergistically increased when cells were 

treated with IL-2 and IL-21 in combination. STAT5 was strongly phosphorylated in 

CD56 bright NK cells by low dose IL-2, while IL-21 did not affect STAT5. Our data 

indicates that the NK cell directed cytokines IL-2 and IL-21 not only differently affect 

functions in NK cell subpopulations but can also act in a synergistic fashion. 

Supported by DFG Priority Program SPP1110, project Ja1058.

Elke Scandella, Evelyn Lattmann, Sanjiv Luther, Simone Miller, Tobias Junt, Burkhard 

Ludewig 

Role for lymphoid tissue inducer-like cells in the restoration 

of immunopathological destruction of the lymphoid stroma 

cell network 

Many viral infections, including human Hepatitis B and C or immunodeficiency viruses 

(HIV) are associated with an immunopathological damage of secondary lymphoid 

organs leading to immunosupression. Similarly, infection of mice with the lymphocytic 

choriomengitis virus (LCMV) leads to destruction of secondary lymphoid organ structure 

which is mediated by virus-specific cytotoxic T cells (CTL). In this study we show that 

the destruction also affects follicular dendritic cells and gp38+ T zone stroma cells which 

are the major producers of the constitutive chemokines CXCL13, CCL19 and CCL21b 

and are therefore essential for the maintenance of lymphoid structures. Furthermore, 

LCMV infected gp38+ stroma cells were lysed by activated virus-specific CTL. The loss 

of stroma cell network is corroborated by a transient reduction of constitutive 

chemokines. A gene-array analysis revealed that genes which are crucial for the 

development of lymphoid organs, e.g. lymphotoxin-b, IL-7Ra or VCAM-1, were upregulated 

concomitantly, thus indicating the immediate initiation of a transcriptional 

'reorganization program'. Moreover, we found accumulation and proliferation of CD3- 

CD4+IL7-Ralpha+ positive cells, which phenotypically resemble lymphoid tissue inducer 

(LTi) cells and are required for lymphoid organ development. In addition, mice lacking 

LTi cells display a delay in the re-expression of CCL19, CCL21 and CXCL13 following 

LCMV infection.

Adan Chari Jirmo, Claus-Henning Nagel, Beate Sodeik, Reinhold E Schmidt, Georg 

Behrens 

Role of direct versus cross-presentation after acquisition of 

HSV-1 by murine DC for generating antiviral immunity 

Background:Phenotypically, murine splenic dendritic cells (DC) can be divided into at 

least three subsets. Differences in surface markers appear to correlate with functional 

differences including antigen acquisition, processing, and presentation. Using HSV-1 as 

an infectious model we aimed to explore the contribution of direct and indirect antigen 

preseantion to generate cytotoxic T lymphocytes (CTL). 

Methods: Spleen DC from C57BL/6 mice were sorted into different subsets their ability 

to present various forms of HSV-1 to HSVgB-specific TCR-transgenic T cells (gBT-1) was 

analysed. Using infected target cells and viral mutagenesis we established conditions to 

assess direct infection vs cross-presentation. In addition, we determined influence of 

TLR ligands and blocking phagocytosis on the ability of cross-presenting CD8+ DC to 

stimulate Ag-specific CD8+ T cells. 

Results: Although all splenic DC subsets express HSV-1 entry mediating receptors, 

produce late HSV-1 antigens, and effectively presented HSV-1 antigen to CTL upon 

direct infection in vitro, only the CD8+ DC subpopulation were capable of presenting 

cellular viral and non-viral antigen in the context of MHC class I (cross-presentation). 

However, this ability by CD8+ DC to cross-present is not due to the fact that they are 

the only subsets that are capable of taking up cellular antigen both in vitro and in vivo. 

Using gB- or gH-deficient HSV-1 (“single round virus”) we were unable to detect any 

contribution of direct presentation to the overall CTL generation in vitro after acquisition 

of virus from infected target cells. In addition, we observed almost complete diminished 

antigen presentation of cellular Ag after inhibition of DC phagocytosis. 

Conclusion: We conclude that cross-presentation is an intrinsic ability of CD8+ DC and 

the dominating antigen presentation pathway for generation of an effective CTL 

response in HSV-1 infection.

Katharina König, Linda Diehl, Carsten Golletz, Thomas Quast, Waldemar Kolanus, 

Percy Knolle, Reinhard Büttner, Lukas C Heukamp 

ROLE OF FHL2 IN THE REGULATION OF DENDRITIC CELL 

MIGRATION 

Regulation of cell migration is a hallmark of dendritic cells function and plays a central 

role in the initiation and regulation of immune responses. Under steady state conditions 

immature dendritic cells continuously acquire antigen in the periphery and migrate to 

draining lymph nodes, where they present exogenous antigens to T cells. 

Furthermore, cell migration is likely to be modulated in important pathological situations 

such as cell invasion and in tumor progression. Cellular motion through complex 

substrates is only partially understood. There is a large body of evidence suggesting 

that integrin-mediated force generation is required for cell locomotion on twodimensional 

(2D) substrates, but translocation of cells within (3D)-tissues can 

apparently resort to other mechanisms, in which selective involvement of the Rho 

GTPases RhoA and Rac1 has been proposed to play a major role. We have discovered 

that migration of murine, bone-marrow derived dendritic cells is differentially regulated 

in 2D and 3D-assay conditions. Furthermore, we found that the RhoA-regulated Fourand-a-half-Lim-Domain 

protein FHL2 plays important and so far unappreciated roles in 

these processes. 

FHL2 deficient bone marrow derived dendritic cells show a significantly increased 

migratory speed, a morphologically mature phenotype and altered expression of α and 

ß-integrins.

Kerstin Juelke, Andreas Thiel, Chiara Romagnani 

Role of inflammatory cytokines in modulating NK cell 

competence and maturation 

The role of NK cells in several protective responses has been extensively elucidated, 

while their contribution in self tolerance maintenance is still unclear. Recently it has 

been shown that self tolerance of NK cells can occur independently of MHC-mediated 

inhibition and hyporesponsiveness plays a role in self-tolerance of NK cells as it has 

been proposed for B and T cells. 

The comparative analysis of resting human CD56dim NK cells sorted according to the 

presence or lack of expression of KIR and NKG2A revealed that, although KIR- NKG2A- 

NK cells are hyporesponsive in response to MHC class I target cells, once stimulated 

with inflammatory cytokines, their cytotoxic hyporesponsiveness is reverted. Moreover, 

we showed that KIR- NKG2A- NK cells are at least as good as KIR+ NKG2A+ in 

proliferating and producing IFN-gamma in response to cytokines and that these 

functions are not related to NK cell competence but rather to NK cell maturation. In 

addition, we demonstrated that KIR- NKG2A- NK cells can acquire expression of KIR and 

NKG2A upon cytokine stimulation. These findings suggest that although NK cell 

hyporesponsiveness represents a mechanism of self tolerance maintenance in steady 

state conditions, during an immune response KIR- NKG2A- NK cells can produce IFNgamma, 

proliferate and acquire the expression of new inhibitory receptors which might 

allow them to regain competence. Thus, we propose a revised concept of NK cell self 

tolerance as a rather dynamic process in which NK cells might contextually change their 

competence properties during inflammation. These findings might have important 

implications in the pathogenesis of autoimmunity.

Romy Laugks, Patricia Schmidbauer, Bernhard Holzmann, Melanie Laschinger 

ROLE OF LFA-1 ACTIVITY REGULATION IN CELL CYCLE 

CONTROL OF PROLIFERATING T LYMPHOCYTES 

The leukocyte specific integrin LFA-1 (αLβ2) is crutial in the interaction between T 

lymphocytes and antigen-presenting cells. It is well established that LFA-1 needs to be 

activated in order to bind ligands. However, the function of LFA-1 de-activation is not 

completely understood. By the deletion of the cytoplasmic GFFKR motif of the αL 

subunit in mouse germ line we previously generated a mouse mutant that locks LFA-1 

in an inactive state (LFA-1d/d). The defect of LFA-1 de-activation leads to an impaired 

de-adhesion from its ligand ICAM-1 on endothelial cells reflected in a reduced migration. 

We found the antigen-specific proliferation of LFA-1d/d T cells to be dramatically 

reduced. The analysis of interaction between LFA-1d/d T cells and antigen-presenting 

dendritic cells exhibits the disability of T cells expressing LFA-1 in a constitutively active 

state to de-adhere from dendritic cells. Furthermore, we observed a different cell cycle 

profile of LFA-1d/d T cells compared to wildtype cells suggesting an influence of LFA-1 

in early cell cycle control. 

These data imply that LFA-1 de-activation is essential for cell cycle control during 

antigen-specific proliferation and clonal expansion of naïve T lymphocytes.

Chiara Cordiglieri, Werner Dammermann, Bo Zhang, Barry Potter, Andreas Guse, 

Alexander Flügel 

Role of NAADP-mediated Ca 2+ signalling in encephalitogenic 

CD4 + T cells 

Nicotinic acid adenine dinucleotide phosphate (NAADP) was recently discovered as a 

novel second messenger involved in the initiation and propagation of Ca 2+ signalling in 

T cells, but its impact on T cell function is still unknown. 

We developed small molecular antagonists based upon nicotinic acid that specifically 

block Ca 2+ mobilization by NAADP. These compounds suppressed antigen-driven 

proliferation and cytokine release in primary human and rat CD4 + T cells. 

To better address the importance of NAADP-mediated Ca 2+ signalling in the functions 

of CD4 + T cells, we used these antagonists in vivo, in a rat model of experimental 

autoimmune encephalomyelitis (EAE), the classical T cell mediated autoimmune model 

for Multiple Sclerosis. 

NAADP antagonists ameliorated the clinical courses of both active and adoptive transfer 

EAE. Molecular analyses and live two photon imaging of retrovirally-tagged fluorescent T 

cells revealed that NAADP antagonists mainly acted in the effector phase of EAE, where 

CNS inflammation is initiated by encephalitogenic autoaggressive T cells. NAADP 

antagonists interfered with the migration of the T cells to their target organ and with 

their efficient activation, thus ameliorating CNS inflammation and paralytic disease. 

These results show that the NAADP/Ca 2+ signaling pathway is crucial for the 

pathogenic potential of autoaggressive T cells and thus qualifies as novel target for the 

treatment of T cell-mediated autoimmune diseases.

Annette I. Garbe, Taras Kreslavsky, Harald von Boehmer 

Role of Notch in survival, proliferation and differentiation of 

alpha beta T cell precursors 

Notch signaling can synergize with both the pre-TCR and the gamma delta TCR to 

determine alpha beta lineage commitment. In case of gamma delta TCR expressing 

precursors the selected cells terminate gamma delta TCR expression and undergo Tcra 

rearrangement. Under physiological conditions gamma delta TCR expressing precursors 

have a competitive disadvantage to pre-TCR expressing cells. In recent studies we could 

show that precursors with a gamma delta TCR compete poorly for Notch ligands 

because they compete better with pre-TCR expressing cells when offered an excess of 

soluble ligands. Overexpression of intracellular Notch abolishes the competitive 

advantage entirely. Our results indicate that Notch signaling is not only essential for 

survival of DN3 precursors of alpha beta lineage cells but is essentially required for 

proliferation of DN3 and DN4 cells. The synergy of TCR and Notch signals becomes 

visible in increased CD5 expression that can be diminished by specifically interfering 

with Notch signaling. Preliminary data indicate that Notch signaling does not only 

contribute to alpha beta lineage choice by helping survival and proliferation of precursor 

cells: The differentiation into CD4+CD8+ alpha beta lineage cells of DN3 precursors 

with increased survival because of Bcl-2 overexpression still requires Notch signaling 

indicating that Notch contributes to survival, proliferation as well as differentiation of 

alpha beta lineage precursors.

Varsha Pattu, Bin Qu, Eva.C Schwarz, Tanja Mayer, Carolin Bick, Reiko Trautmann, 

Markus Hoth, Jens Rettig 

Role of SNARE proteins in vesicle fusion at the Immunological 

Synapse of Cytotoxic T lymphocytes. 

Cytotoxic T lymphocytes(CTLs), kill target cells through secretion of lytic vesicles 

containing cytotoxic components such as perforin and granzymes. Fusion of these 

vesicles occurs at the contact zone between the target cell and CTL, the immunological 

synapse(IS). We are interested in the molecular mechanism behind this fusion event. 

The specific Soluble NSF attachment receptor (SNARE) protein isoforms are well known 

to be required for vesicle fusion in neuronal synapses, but a potential function in CTLs 

remains unkown. We have established an efficient method of focal stimulation of human 

primary CD8 T lymphocytes by anti-CD3/anti-CD28 antibody coated beads. The effector 

status of these cells was confirmed by staining with specific markers such as CD25 and 

CD62L. We analysed the expression patterns of different SNARE proteins by reverse 

transcriptase PCR and found several candidates that are upregulated upon stimulation. 

Immunocytochemistry with isoform specific antibodies was used to further narrow down 

our candidates based on co-localisation with lytic vesicle markers. Functional studies 

like capacitance measurements and cytotoxicty assays were established to gain further 

insight into the field.

Stefano Majocchi, Natalio Garbi, Günter J Hämmerling 

Role of Tapasin-ERp57 heterodimers in the generation of MHC 

class I/peptide complexes 

Antigen presentation by MHC class I molecules is necessary for CD8 T cell activation. 

Optimal peptide loading onto MHC-I molecules occurs mainly in the peptide-loading 

complex in the endoplasmic reticulum. The loading complex is a multimeric structure 

that consists of MHC-I heavy chain, β2-microglobulin, the MHC-I-specialised chaperone 

tapasin (Tpn), the thiol oxidoreductase ERp57, the lectin binding chaperon calreticulin 

and the peptide transporters associated with antigen processing TAP1 and TAP2. 

The generation of peptide-receptive MHC-I molecules and their association into the 

peptide loading complex is relatively well understood. Recently we have shown that 

ERp57 has a critical structural role in the loading complex by increasing the affinity of 

Tpn for MHC-I molecules. This is achieved via a covalent association between Tpn and 

ERp57 mediated by a disulfide bond between C95 and C57, respectively. This 

association is believed to be stable, although ERp57 C60 can mediate the release of 

ERp57 from tapasin under certain conditions in vitro. In contrast, very little is known 

about the release of MHC-I/peptide complexes from the loading complex. In order to 

investigate whether dissociation of Tpn and ERp57 is required for the release of MHC-I/ 

peptide from the loading complex, we have reconstituted ERp57 KO cells with wt or 

mutant forms of ERp57 that stabilise the Tpn-ERp57 covalent association. Results on 

the association of peptide-receptive MHC-I to the loading complex and release of MHC-I/ 

peptide from the loading complex in the ERp57 mutant cell lines will be presented.

Carolin Konermann, Daniel Degrandi, Cornelia Beuter-Gunia, Sandra Beer, Klaus 

Pfeffer 

Role of the IFN-γ inducible 65kDa GTPases in pathogen 

defense 

IFN-γ induces a number of cellular programs functional in innate and adaptive resistance 

to infectious pathogens. Microarray expression studies of IFN-γ stimulated macrophages 

displayed a new family of murine GTPases, the 47kDa and 65kDa guanylate-binding 

proteins (GBPs), whose expression was highly upregulated. The members of the GBP 

family are structurally characterized by the canonical G(X)4GKS and D(X)2G motifs. 

Some GTPases carry a CAAX site for isoprenoid modification which regulates 

intracellular localization. Several studies revealed a pathogen specific protective 

function of members of the 47kDa family in mice. However, the role of 65kDa mGBPs 

during infection remains ill-defined. 

Via qRT-PCR and Western Blot analyses we confirmed the microarray data and analyzed 

the expression levels of the 65kDa GBPs mGBP1 to 5 in macrophage cultures stimulated 

with several cytokines and TLR ligands. Especially upon IFN-γ stimulation the mGBPs 

were strongly upregulated. Induction of these GTPases was STAT1 dependent, shown by 

the absence of mGBP expression in stimulated STAT1-/- fibroblasts. Furthermore, the 

GBPs were strongly induced in vivo in several organs of mice infected with L. 

monocytogenes or T. gondii. The degree of GBP induction after infection was different 

for each GTPase, indicating a non-redundant, pathogen specific role of these proteins. 

Fusion-proteins as well as intracellular staining of endogenous GTPases displayed 

localization in intracellular vesicle-like structures. After stimulation with IFN-γ and 

infection of macrophages or fibroblasts with T. gondii mGBP1, 2 and 3 migrated to the 

parasitophorous vacuole (PV) whereas mGBP5 did not recruit to the PV. The ability to 

accumulate at the PV was dependent on the isoprenylation but not GTP-binding or 

hydrolysation as shown with mutated fusion-proteins. 

These results demonstrate that the mGBPs belong to a novel class of GTPases 

specifically induced by the immunomodulatory and pro-inflammatory cytokine IFN-γ and 

therefore could be effectors of immunity in the defense against intracellular pathogens.

Christiane Habich, Volker Burkart 

Role of Toll-like receptor 4 (TLR4) in heat shock protein 60 

(hsp60)-mediated beta cell-directed immune reactivity in nonobese 

diabetic (NOD) mice 

Hsp60 expressed in beta cells is one of the putative autoantigens in the development of 

type 1 diabetes. Hsp60 shows receptor-mediated binding to macrophages and induces 

the release of beta cell damaging, inflammatory mediators (TNFα, IL6) from 

macrophages of NOD mice, a model of type 1 diabetes. The present study was designed 

to characterize the pro-inflammatory hsp60 signaling in macrophages and to analyze 

the role of TLR4 in these processes by the use of TLR4-deficient NOD mice. We 

investigated the activation of signaling proteins (NFkB, p42/p44, p38, JNKp46/p54) 

after hsp60 stimulation of macrophages from prediabetic and diabetic NOD/TLR4+/+, 

NOD/TLR4+/- and NOD/TLR4-/- mice. Hsp60 stimulated all investigated signaling 

proteins in prediabetic and diabetic NOD/TLR4+/+ mice. In NOD/TLR4+/- mice p38 

activation was weakly reduced (23-44%), whereas p42/p44 activation was strongly 

reduced (74-81%) independent of the diabetes stage. NFkB activation was continuously 

reduced with diabetes progression (38-99%). JNKp54 activation showed constant strong 

reduction (>90%), whereas JNKp46 was strongly reduced only in prediabetic mice 

(77%), but not in diabetic NOD/TLR4+/- mice (14%). In NOD/TLR4-/- mice activation 

of all investigated signaling proteins was constantly strongly reduced (NFkB >80%, p42/ 

p44 >90%, p38 77-87%, JNKp46/p54 77-96%). By the use of specific inhibitors, we 

analyzed the role of these signaling proteins in the production of beta cell damaging 

mediators TNFα and IL6 by macrophages from NOD/TLR4+/+ mice. Independent of 

diabetes progression, JNKp46/p54 and NFkB were found to be involved in hsp60mediated 

IL6 production. Furthermore, we could show that hsp60-mediated TNFα 

production from NOD/TLR4+/+ macrophages is regulated by p42/p44, p38 and NFkB. 

We conclude, that differential activation of TLR4-dependent signaling pathways by 

hsp60 may contribute to increased beta cell-directed immune reactivity observed at the 

time of diabetes manifestation.

Bernadette Pöllinger, Gurumoorthy Krishnamoorthy, Michael Bösl, Hans Lassmann, 

Andeas Holz, Hartmut Wekerle, Florian C. Kurschus* 

RR Mouse: Spontaneous relapsing remitting EAE in SJL/J mice 

expressing a MOG responsive T cell receptor 

Myelin oligodendrocyte glycoprotein (MOG) is regarded as major autoantigen in Multiple 

Sclerosis (MS) and its animal model • experimental autoimmune encephalomyelitis 

(EAE). Its anatomic localization on the outermost layer on central nervous system 

(CNS) myelin sheets suites it perfectly as target for auto-antibodies. We recently 

described a double transgenic C57BL/6 (H-2b ) mouse strain carrying a MOG specific T 

cell receptor along with a MOG specific immunoglobulin H chain (Krishnamoorthy G. et 

al., J Clin Invest. 2006;116:2385-2392). These mice develop spontaneous autoimmune 

disease affecting spinal cord and optic nerve, but sparing brain and cerebellum, thus 

resembling human opticospinal myelitis (Devic disease). 

In order to explore the possible effect of genetic factors on disease expression, we now 

report about new similar transgenic mouse lines, but on SJL/J (H-2s ) background. We 

created three T cell receptor (TCR) transgenic lines bearing CD4 cells specific for MOG92- 106 presented by I-AS . These lines differ in their penetrance of transgene expression on 

CD4 cells (TCR1640 , 70%; TCR1639 , 20%; TCR1586 , 5%). In contrast to the Devic model, 

single (TCR-) transgenic SJL/J mice spontaneously develop EAE at high frequency. 

Disease occurs in more than 75 % of TCR1640 but only in about 15 % of TCR1586 mice, 

if bred on SJL/J, but not on the B10.S background. Spontaneous EAE in many cases 

takes a relapsing-remitting course, with individual disease bouts affecting distinct CNS 

parts (cerebellum vs. spinal cord). Intriguingly TCR1640 and diseased TCR1586 SJL/J 

mice select and activate endogenous MOG-specific B cells to produce MOG reactive IgG1 

and IgG2ab autoantibodies. This new form of autoimmune B cell recruitment does not 

occur on the MOG knockout -or B10.S background and may therefore involve MOG 

exposure to the immune system during preclinical EAE lesions. 

The RR mouse is the animal model most faithfully recapitulating the most frequently 

occurring human MS variant. It will be of use studying issues including relapse 

generation, autoimmune T-B cell cooperation, and drug discovery.

Elisa Monzón-Casanova, Christian Söllner, Nico Westphal, Ingolf Berberich, Tohru 

Miyoshi-Akiyama, Takehiko Uchiyama, Ingrid Müller, Lutz* Walter, Thomas* Herrmann 

RT1Db2: Identification and first functional analysis of a new 

non-polymorphic MHC class II molecule 

The complete sequence of the rat MHC allowed the identification of a duplicated class II 

beta chain gene locus RT1-Db (RT1D: rat homologue of H2-E and HLA-DR). The 

duplicated gene is designated RT1-Db2, maps between the RT1-Db1 and RT1-Da genes 

and encompasses about 20 kb. RT1-Db2 had not been noticed and characterized so far 

and shows some interesting features. Compared to RT1-Db1, the exons that code for 

the cytoplasmic region are deleted (exon 5) and inactivated (exon 6). Yet, RT1-Db2 

shows a completely novel exon, which codes for the complete cytoplasmic region. Upon 

sequencing of several inbred rat strains, RT1-Db2 turned out to be monomorphic in the 

(usually) polymorphic exon 2. According to RT-PCR, RT1-Db2 is expressed in rat 

lymphocytes and ConA-stimulated lymphoblasts. To compare functional features of RT1- 

Db and RT1-Db2, RT-PCR products were cloned and co-expressed with RT1-Da in class 

II molecules negative mouse cells. Staining with the Da specific mAb OX-17 revealed 

surface expression for both Da comprising molecules, but expression of DaDb1 was 

consistently higher than of DaDb2. DaDb1-EYFP and DaDb2-EYFP fusion proteins 

differed in their intracellular localization and DaDb2 failed to form SDS-stable dimers, 

which may indicate a function for DaDb2 distinct from that of a restriction element in Tcell 

antigen recognition. Nevertheless, DaDb1 and DaDb2 presented the bacterial 

superantigens SEB and MAS similarly efficient. Interestingly, DaDb2 presented YPM 

(Yersinia pseudotuberculosis mitogen) at least 1000 fold better than the “classical“ 

RT1D (DaDb1). An analysis of Db1/Db2 chimeras allowed a preliminary localization of 

the regions of the Db2 molecule involved in high affinity-binding to YPM and decreased 

cell surface expression. 

*Shared senior authorship of TH and LW. 

Correspondence should be adressed to TH (herrmann-t@vim.uni-wuerzburg.de)

Julia Kzhyshkowska, Alexei Gratchev, Liis Krusell, Srinivas Mamidi, Gail Workman, E. 

Helene Sage, Sergij Goerdt 

Scavenger receptor stabilin-1 links endocytosis and 

intracellular sorting in alternatively activated macrophages 

The multifunctional scavenger receptor stabilin-1 is expressed on macrophages on 

placenta and adult tissues as well as on sinusoidal endothelial cells and macrophages in 

lymph nodes. In vitro induction of stabilin-1 on macrophages requires IL-4 and 

glucocorticoids. We showed that stabilin-1 mediates endocytosis of extracellular ligands 

acLDL, regulator of ECM-remodelling and cell adhesion SPARC, and hormone placental 

lactogen (PL). Both SPARC and acLDL are targeted via stabilin-1 for the degradation in 

lysosomes. In contrast, a portion of PL escapes degradation and is delivered into novel 

storage vesicles. These vesicles do not belong to classical endosomal/lysosomal system. 

However these vesicles communicate with trans-Golgi network (TGN). Stored PL can be 

secreted back to the extracellular space. Next, we found that stabilin-1 shuttles 

between TGN and endosomes. This trafficking pathway is mediated by GGAs, clathrin 

adaptors that interact with the DDSLL motif in the cytoplasmic tail of stabilin-1. Here 

stabilin-1 is involved in delivery of the novel chitinase-like protein SI-CLP from TGN to 

the endosomal/lysosomal compartment. 

SI-CLP was identified by us as a binding partner for stabilin-1 in yeast two-hybrid 

screening. SI-CLP contains a conservative lectin-type Glyco_18 domain, which lacks 

enzymatic activity, similarly to YKL-40 and Ym1/Ym2. YKL-40 and Ym1/Ym2 are 

secreted by macrophages, possess cytokine activity and are associated with Th2-type 

inflammations and allergies. We demonstrated that endogenous SI-CLP is 

overexpressed in human macrophages stimulated with IL-4 and dexamethasone and is 

transported via stabilin-1 from biosynthetic to the secretory pathway. High levels of SI- 

CLP were detected in bronchoalveolar lavage samples from patients with chronic 

bronchitis. 

We propose that alternatively-activated macrophages use stabilin-1 1) to coordinate 

ECM remodeling, angiogenesis, and tissue turnover via endocytosis and degradation of 

SPARC; 2) to regulate extracellular concentration of PL in placenta; 3) to regulate 

delivery of chitinase-like protein SI-CLP into the secretory pathway.

Tim Sparwasser, Andrea Hartl, Katharina Lahl, Heinz Fehrenbach, Holger Garn, Harald 

Renz, Hermann Wagner 

Selective depletion of Foxp3+ cells in DEREG mice allows 

functional analysis of regulatory T cells during experimental 

allergic airway inflammation 

Naturally occurring CD25+CD4+ regulatory T cells (Tregs) are currently intensively 

characterized because of their major importance in inhibiting the development of 

autoimmunity and allergy. Originally, CD4+ Tregs were identified exclusively by the 

constitutive expression of CD25, and many in vivo experiments have been performed 

using depleting antibodies directed against CD25. However, both the existence of CD25- 

Tregs, especially within peripheral tissues, as well as the expression of CD25 on 

activated conventional T cells, limits the interpretation of data obtained by the use of 

anti-CD25 depleting antibodies. The most specific Treg marker is the forkhead box 

transcription factor Foxp3, which has been shown to be expressed specifically in murine 

CD4+ Tregs. To address the question of the in vivo role of Tregs in asthma 

pathogenesis, we have generated bacterial artificial chromosome (BAC)-transgenic mice 

named DEREG (depletion of regulatory T cells), expressing a diphtheria toxin receptor 

(DTR) eGFP fusion protein under the control of the foxp3 locus allowing both detection 

and inducible depletion of Foxp3+ Tregs. Similar frequencies and total numbers of CD25 

+CD4+ Tregs, and comparable Foxp3 levels and suppressor function were observed 

between DEREG and WT mice. Histological and flow cytometry analysis of spleen, lymph 

node, and thymus revealed that the DTR-eGFP BAC transgene is specifically expressed 

in Foxp3+CD4+ T cells. The injection of diphtheria toxin led to at least 90% depletion of 

Foxp3+ Tregs and dramatically increased immune responses (DTH, OVA-immunization). 

Using a model of acute ovalbumin (OVA)-induced allergic airway inflammation we can 

demonstrate that depletion of Tregs during sensitization causes dramatically increased 

Th2 pathology. Since our model for the first time allows specific depletion of Tregs 

during acute immune responses without affecting activated effector T cells, our data 

obtained in DEREG mice will shed light on the key cellular players involved in the 

suppression of allergic airway inflammation.

Ulrike Schleicher, Jan Liese, Claudia Kurzmann, Christian Bogdan 

Selective requirement of Toll-like receptor 9 for the activation 

of natural killer cells in cutaneous and visceral leishmaniasis 

NK cells contribute to the control of intracellular parasites including Leishmania. In vitro 

studies suggested that the activation of NK cells depends on their interaction with 

plasmacytoid (pDC) or myeloid dendritic cells (mDC) that sense the pathogen and 

generate NK cell-stimulatory signals. Here, we investigated the cell types, pattern 

recognition receptors and cytokines that are required for NK cell activation during 

Leishmania infections in vivo. 

Toll-like receptor (TLR) 9 was indispensable for NK cell IFN-gamma production and 

cytotoxicity in both cutaneous (L. major) and visceral leishmaniasis (L. infantum). In 

vitro, plasmacytoid dendritic cells (pDCs) released both interferon (IFN)-alpha/beta and 

interleukin (IL)-12 in response to Leishmania promastigotes in a TLR9-dependent 

manner, although they did not internalize the parasites. In contrast, myeloid dendritic 

cells (mDCs) phagocytosed the parasites and produced high amounts of IL-12, but not 

IFN-alpha/beta, in the presence of TLR9. Studies with IL-12- or IFN-alpha/beta receptor 

(IFNAR)-knockout mice revealed that IFNAR-deficiency only slightly reduced the L. 

infantum-induced IFN-gamma production by NK cells, whereas NK cell cytotoxicity and 

IFN-gamma secretion was abolished in IL-12-deficient mice. The latter phenotype was 

also observed in mDC-depleted mice, whereas NK cell activation in response to L. 

infantum was maintained after depletion of pDCs. Intracellular cytokine staining showed 

that mDCs represent the source of IL-12 during the early phase of Leishmania infection. 

TLR9-deficient mice lacked IL-12 expression by mDCs after infection with L. major or L. 

infantum. Thus, NK cell priming in vivo in response to Leishmania parasites is strictly 

dependent on mDCs, which sense the pathogen via TLR9 and subsequently secrete IL- 

12 to stimulate the NK cells.

Ulrich Salzer, Jennifer Birmelin, Chiara Bacchelli, Torsten Witte, Ulrike Buchegger- 

Podbielski, Rita Rzepka, H Bobby Gaspar, Reinhold E Schmidt, Inga Melchers§, Bodo 

Grimbacher§ 

Sequence analysis of TNFRSF13b/TACI in patients with 

Systemic Lupus Erythematosus 

Background: BAFF and APRIL and their receptors BAFFR, BCMA and TACI are involved in 

the regulation of B cell homeostasis and differentiation. BAFF overexpression leads to 

systemic lupus erythematosus (SLE) like symptoms in mice and elevated BAFF levels 

have been observed in human SLE and mouse models for SLE. Furthermore, genetic 

inactivation of TACI in mice results in a SLE-like phenotype. 

Methods/Results: Based on our recent finding that TACI is mutated in patients with 

common variable immunodeficiency, of whom more than 30% suffer from autoimmune 

conditions, we analyzed TACI by heteroduplex analysis and subsequent sequencing in 

humans with SLE. 

Sequence analysis of TNFRSF13b/TACI in 119 unrelated SLE patients revealed four 

variants: R20C in exon 1, R72H in exon3, the silent variation c.327 G>A in exon 3, and 

A181E in exon 4. 

Conclusions: No significant association with any of these variants was found, when 

compared to the frequencies of the variants in a healthy control cohort. Furthermore the 

mutated alleles R20C and R72H did not segregate with the SLE phenotype in familial 

cases of SLE. Thus, our evaluation of the coding region of TNFRSF13b/TACI did not 

reveal any deleterious or disease associated mutations. 

Supported by EU SP23-CT-2005-006411; NIH/NIAID: USIDnet grant # NO1-A1-30070 

(B. G.), BMBF KN Rheuma C2.12 (T.W. and R.E.S), the BMBF KN Rheumatism 01 GI 

9949/C2.13 (I. M.). 

§contributed equally

Zeinab Abdullah, Tomo Saric, Hamid Kashkar, Nikola Baschuk, Benjamin 

Yazdanpanah, Bernd K. Fleischmann, Jürgen Hescheler, Martin Krönke, Olaf Utermöhlen 

Serpin-6 expression protects embryonic stem cells from lysis 

by antigen-specific CTL 

The immune response to embryonic stem (ES) cells is still poorly understood. In this 

study, we addressed the adaptive cellular immune response to undifferentiated and 

differentiated ES cells infected with lymphocytic choriomeningitis virus (LCMV), a 

vertically transmitted pathogen in mice and humans. In contrast to the prevailing view, 

we found that undifferentiated and differentiated murine ES cells express MHC class I 

molecules, although at low levels. When cocultured with LCMV-infected ES cells, 

syngeneic but not allogeneic LCMV-specific CTL secrete IFN-gamma. Strikingly, LCMVspecific 

CTL do not efficiently kill LCMV-infected ES cells. ES cells showed high-level 

expression of the serine protease inhibitor 6, an endogenous inhibitor of the CTLderived 

cytotoxic effector molecule granzyme B. Down-regulation of serpin-6 by RNA 

interference sensitized ES cells for CTL-induced cell death. The results of this study 

suggest that LCMV-infected murine ES cells present viral Ags and are recognized by 

LCMV-specific CTL in a MHC class I-restricted manner, yet resist CTL-mediated lysis 

through high-level expression of serine protease inhibitor 6

Michael Conzelmann, Michael Rieger, Michael Hess, Ulrich Strohhaecker, Axel Benner, 

Ute Hegenbart, Anthony D. Ho, Peter Dreger, Thomas Luft 

Serum cytokeratin-18 fragments as sensitive marker of 

epithelial apoptosis in intestinal and hepatic graft-versushost 

disease 

Graft-versus-host disease (GvHD) is a heterogenous group of syndromes with 

autoimmune-like characteristics that represents the main complication of allogeneic 

stem cell transplantation (SCT). However, diagnosis of GvHD and evaluation of response 

to immunosuppressive treatment is sometimes difficult. Since apoptosis is the 

histopathological hallmark in GvHD, we investigated whether the caspase-cleaved neoepitope 

of cytokeratin 18 fragments (CK18F) might be a serum marker for ongoing 

GvHD-induced target organ destruction. 

Serum CK18F kinetics was monitored by M30 antibody-based ELISA in 50 patients who 

fulfilled histopathological and/or clinical criteria diagnostic for GvHD. Both intestinal and 

hepatic GvHD were consistently associated with significant elevations of CK18F levels 

over baseline. Responses of GvHD to immunosuppressive therapy were paralleled by 

CK18F decreases, whereas resistant GvHD was characterized by persistent CK18F rises. 

In contrast, markers of T cell activation such as sCD25 responded to steroid therapy 

irrespective of clinical responses. 

Clinical conditions that might represent relevant differential diagnoses, such as toxic 

mucositis, non-complicated, infection-related diarrhea and veno-occlusive disease were 

not associated with CK18F elevations. 

In conclusion, CK18F monitoring provides a biomarker for quantitative assessment of 

GvHD-associated apoptotic activity in intestinal and hepatic GvHD which may help to 

distinguish active GvHD from GvHD-unrelated conditions with similar symptoms, and to 

monitor response to immunosuppressive treatment. Prospective studies are warranted 

to evaluate how CK18F may assist in diagnosis, grading, and treatment guidance of 

GvHD.

Pia Herzberger, Corinna Siegel, Christine Skerka, Volker Fingerle, Ulrike Schulte- 

Spechtel, Bettina Wilske, Volker Brade, Reinhard Wallich, Peter F. Zipfel, Peter Kraiczy 

Serum resistance of human pathogenic Borrelia spielmanii sp. 

nov. correlates with binding of complement regulators factor 

H and FHL-1 

B. spielmanii sp. nov. has recently been identified as a novel human pathogenic 

genospecies that cause Lyme disease in Europe. In order to elucidate immune evasion 

mechanisms of B. spielmanii as a means of evading the innate immune system we have 

compared the ability of isolates obtained from Lyme disease patients and tick isolate PC- 

Eq17 to escape from complement-mediated bacteriolysis. Applying a growth inhibition 

assay, we show that four B. spielmanii isolates, including PC-Eq17, are serum-resistant 

whereas a single isolate, PMew, was more sensitive to complement-mediated lysis. All 

isolates activate complement in vitro as demonstrated by covalent attachment of C3 , 

however, deposition of later activation products C6 and C5b-9 was restricted to the 

moderately serum-resistant isolate PMew and serum-sensitive B. garinii isolate G1. 

Furthermore, serum adsorption experiments revealed that all B. spielmanii isolates 

acquire the host alternative pathway regulators factor H and FHL-1 from human serum. 

Both complement regulators retain their factor I-mediated C3b inactivation activity 

when bound to spirochetes. In addition, two distinct factor H and FHL-1 binding 

proteins, BsCRASP-1 and BsCRASP-2, were identified that are approximately 23 to 25 

kDa in size. A further factor H-binding protein, BsCRASP-3, was exclusively found in the 

tick isolate PC-Eq17. In conclusion, this is the first report describing an immune evasion 

mechanism utilized by B. spielmanii sp. nov. and demonstrates capture of human 

immune regulators to resist complement-mediated killing. This work was funded by the 

Deutsche Forschungsgemeinschaft DFG, Project Kr3383/1-1 and Wa533/7-1.

Anette J. Bauer, Katharina M. Huster, Verena Labi, Roland M. Schmid, Dirk H. Busch, 

Andreas Villunger, Georg Häcker 

Several members of the mitochondrial apoptosis pathway 

control activated T cell death to terminate the T cell immune 

response 

Stimulation of APC by adjuvants is an important step in development of an effective 

immune response through the upregulation of costimulatory molecules and the release 

of cytokines. Previous work has shown that stimulation of dendritic cells by adjuvant 

can reduce cell death of activated T cells at the end of the response. Furthermore, it has 

been shown that this cell death requires the pro-apoptotic protein Bim and is blocked by 

Bcl-2 expression in T cells. In this study we investigated the interrelations of proteins 

implicated in the regulation of this activated T cell death. TLR ligands present during 

stimulation of spleen cell populations or in vivo prolonged the survival of activated T 

cells, an activity that was mimicked by DC-derived factors or the presence of IL-1, IL-7 

or IL-15. Bcl-2-transgenic T cells showed better survival than bim-/--cells, and DCderived 

factors increased the survival of bim-/--T cells, indicating a Bim-independent 

pathway. This pathway was found largely to be regulated by the pro-apoptotic Bcl-2 

protein Puma, possibly with a marginal contribution from Noxa. The NF-kappa B 

regulator Bcl-3 was involved upstream of the activity of Bim and had an anti-apoptotic 

effect. Bim and Puma appeared to be regulated by similar molecular activation 

pathways as DC-derived cytokines were able to delay the activation of both. Expression 

analysis showed a strong divergence of Bim expression and –activity, strongly 

suggesting post-translational regulation of Bim. Additional experiments examining 

function of antigen specific Bcl-2 transgenic T cells during Listeria infection in vivo show 

that Bcl-2 not only blocks death but also conserves function of T cells at the end of the 

immune reaction. Thus, apoptosis of activated T cells at the end of an acute immune 

response is a process regulated by members of the mitochondrial apoptosis pathway 

that is instrumental in switching off the immune response.

Jessica Prüßmeyer, Rory Koenen, Line Fraemohs, Christian Weber, Andreas Ludwig 

SHEDDING OF THE JUNCTIONAL ADHESION MOLECULE JAM-A 

BY THE DISINTEGRIN AND METALLOPROTEINASE ADAM17 

REGULATES NEUTROPHIL TRANSMIGRATION 

Junctional Adhesion Molecule-A (JAM-A) is a transmembrane adhesive protein of the 

IgG superfamily expressed at endothelial and epithelial junctions and on leukocytes. 

JAM-A participates in the organization of tight junctions and has been implicated in 

adhesion and transendothelial migration of neutrophils or mononuclear cells. Here we 

demonstrate that cultured endothelial cells as well as the cell lines HEK293 and ECV304 

release considerable amounts of soluble JAM-A ectodomain detectable as 35 kDa protein 

by Western Blotting. This release is enhanced by treatment with the phorbolester PMA 

and suppressed by metalloproteinase inhibitors. Both, enhancement or inhibition of 

release are associated with the downregulation or accumulation of surface expressed 

JAM-A, respectively, as demonstrated by FACS analysis. Specific inhibitors allowing to 

distinguish the disintgrin-like metalloproteinases ADAM10 and ADAM17 suggest that 

constitutive and PMA-induced release are preferentially mediated by proteolytic 

shedding via the activity of ADAM17. These findings were corroborated in loss of 

function and gain of function experiments by downregulation or overexpression of this 

protease. Functionally, treatment of endothelial cells with PMA or ADAM10/17 inhibitors 

was associated with suppressed IL-8-induced neutrophil transmigration. Pretreatment of 

neutrophils with soluble JAM-A considerably reduced transmigration, whereas 

pretreatment of endothelial cells or ECV304 cells slightly enhanced neutrophil 

transmigration. These data suggest a dual role of ADAM17-mediated JAM-A shedding: 

at the endothelial cell surface shedding would release a soluble antagonist interacting 

with adhesion molecules on neutrophils and thereby downregulate transmigration, 

whereas within the endothelial junctions shedding may be required to facilitate 

transmigration of neutrophils.

Oliver Frey, Lisa Bruns, Andreas Reichel, Lars Morawietz, Thomas Kamradt 

Short-term depletion of regulatory T cells converts selflimiting 

arthritis into a chronic disease 

We have established a new arthritis model, induced by systemic immunization of nontransgenic 

mice with the ubiquitous autoantigen glucose-6-phosphate isomerase. The 

disease is dependent on B and T cells and is normally self limiting, leading to resolution 

of inflammation between day 30-40 after disease induction. We hypothesized that 

naturally occurring regulatory T cell (Treg) might be important for the resolution of the 

disease and therefore investigated their role. 

Treg cells were depleted by intraperitoneal injection of an anti-CD25-antibody. Arthritis 

was induced by immunization of mice with recombinant G6PI. The severity of the 

disease was assessed clinically on 0-3 point score for each limb and by histology at the 

end of the experiments. The degree of Treg-depletion (FoxP3 staining) and cytokine 

production was assessed by flow cytometry. 

Injection of anti-CD25 antibody on days –11 and –8 before immunization resulted in a 

transient reduction of Treg, which returned to base-line levels on the clinical peak of the 

disease (d 12-15 after disease induction). This transient depletion profoundly affected 

the disease course, resulting in chronic-destructive arthritis over the whole follow-up 

period (80 days) with active inflammation and joint destruction, as evidenced by 

histological examination. This exacerbated arthritis was accompanied by increased 

numbers of IL-17-producing T cells and T cell proliferation. 

We conclude from our experiments that Treg do not actively participate in the resolution 

of arthritis, since the disease is still chronifies, when Treg cell number are fully 

normalized. Our data show that they act rather in early stages of disease development 

by controlling the extent of T cell activation.

Wenhan Li, Stefan Schultz, Nicolas Geis, Michael Kirschfink 

shRNA-mediated knockdown of CD59 sensitises tumour cells 

to complement attack and inhibits proliferation 

Complement-resistance of neoplastic cells, often caused by overexpression of one or 

more membrane complement regulatory proteins (mCRPs) is considered a main 

hindrance for an effective antibody-based immunotherapy of cancer. We have 

previously reported that inhibition or transient down-regulation of mCRPs sensitises 

tumour cells for complement-mediated cytotoxicity (CDC). 

We here describe the effect of stable knockdown of CD59 expression in prostate 

carcinoma cells. Du145 and PC-3 cells were transfected with a pSilencer 4.1CMV neo 

vector encoding hairpin siRNA targeting CD59 for stable down-regulation of CD59 

expression. After 4 weeks selection with geneticin, real-time PCR indicated that mRNA 

levels of CD59 were significantly reduced, going along with decreased CD59 protein 

levels on transfected cells. CD59 deficient prostate tumor cells became sensitive to 

complement-mediated cytolysis. Unexpectedly, we also observed a marked inhibition of 

proliferation of transfected tumor cells if compared to control shRNA-transfected cells . 

It appears that long-term down-regulation of CD59 expression by siRNA not only 

increases the sensitivity of prostate carcinoma cells to complement attack but may also 

exert a therapeutically relevant effect on cancer cell proliferation. 

-->

David Frommhold, Andreas Ludwig, M. Gabi Bixel, Alexander Zarbock, Annette G. 

Beck-Sickinger, Alma Zernecke, Christian Weber, Dietmar Vestweber, Klaus Ley, Markus 

Sperandio 

Sialyltransferase ST3Gal-IV interferes with chemokinedependent 

leukocyte adhesion during inflammation 

Recent in-vitro findings have demonstrated a role of sialylation for chemokine receptor 

binding to its ligand. This prompted us to investigate chemokine-induced leukocyte 

adhesion in inflamed post-capillary cremaster muscle venules of α2,3 sialyltransferase 

(ST3Gal-IV)-deficient mice. We found a marked reduction of leukocyte adhesion in 

unstimulated cremaster muscle venules upon injection of keratinocyte-derived 

chemokine (KC), which interacts with chemokine receptor CXCR2. In TNF-α-treated 

cremaster muscle venules where leukocyte adhesion depends on the overlapping 

function of CXCR2 and E-selectin, leukocyte adhesion was significantly decreased in 

ST3Gal-IV-/- mice in those experiments where E-selectin function was blocked. 

Additional in vitro assays revealed that KC-binding to CXCR-2 on isolated ST3Gal-IV-/- 

neutrophils was markedly reduced. Furthermore, KC-mediated adhesion of ST3Gal-IV-/- 

leukocytes at physiological flow conditions was significantly reduced as well as 

transendothelial migration of ST3Gal-IV-/- leukocytes in response to KC. In human 

neutrophils, enzymatic desialylation led to decreased binding of CXCR-2 ligands to 

CXCR-2 and diminished neutrophil degranulation in response to these chemokines. 

Taken together, our results provide substantial evidence that sialylation by ST3Gal-IV 

significantly contributes to CXCR2-mediated leukocyte functions and is required for 

leukocyte arrest and extravasation during inflammation in vivo.

Andrea Kießling, Juliane Ladhoff, Mir Farzin Mashreghi, Sabine Brösel, Elke 

Effenberger, Hans-Dieter Volk, Martina Seifert 

Silencing of IFNγ-receptor by siRNA inhibits MHC class II 

upregulation on endothelial cells 

Introduction: Immunogenicity of allogeneic endothelial cells (EC) is determined by their 

constitutive expression of Major Histocompatibility Complex (MHC) class I. In the 

setting of transplantation the proinflammatory cytokine interferon-γ (IFNγ) plays a 

pivotal role as a mediator of the rejection process by enhancing MHC class I and 

inducing MHC class II expression on EC. Silencing of IFNγ-receptor chain 1 (IFNγ-R1) by 

short interfering RNA (siRNA) might reduce the IFNγ-mediated MHC upregulation on the 

cellular graft, thereby diminishing allorecognition mechanisms. 

Methods: Primary rat aortic EC were transfected in vitro with own designed siRNA (1- 

100 nM) directed against rat IFNγ-R1 mRNA. Subsequently, cells were stimulated with 

10 ng/ml IFNγ for 24 hours. Expression levels of IFNγ-R1 and IFNγ-R2 mRNA were 

determined by quantitative RT-PCR. Surface expression patterns (e.g. MHC I, MHC II, 

VCAM-1) were analyzed flow cytometrically in comparison to control siRNA- and nontransfected 

cells. 

Results: IFNγ-R1 siRNA was capable to decrease mRNA expression level of IFNγ-R1, but 

had no effect on IFNγ-R2 expression. In particular, specific IFNγ-R1 siRNA treatment 

effectively reduced MHC class II induction after IFNγ stimulation to approximately 50 %, 

whereas control siRNA or non-transfected cells displayed high MHC class II levels. 

Application of 5 nM siRNA resulted in optimal inhibitory effects and simultaneously 

lowered activation of EC by the transfection process itself. 

Conclusions: Silencing of IFNγ-R1 by siRNA is a suitable tool to knock down the MHC 

class II expression on EC under IFNγ exposure. This might be a useful application for 

cell therapeutic treatment in an allogeneic transplantation setting.

Verena Boschert, Anja Krippner-Heidenreich, Ingo Grunwald, Marie Kühnle, Klaus 

Pfizenmaier, Peter Scheurich 

Single chain TNF, a covalently stabilized TNF derivate 

The cytokine tumor necrosis factor has a potential as an antitumoral therapeutic agent. 

Bioactive TNF forms a homotrimer which, at low concentrations, dissociates into nonfunctional 

monomers. The idea of single chain TNF (scTNF) is to covalently stabilize the 

trimer with two short linker peptides. This leads to a protein with a 6 fold higher affinity 

to its receptors (TNFR1 and TNFR2), full bioactivity and a higher stability in vitro and in 

vivo when compared to soluble TNF. Therefore, single chain TNF represents an 

interesting molecule for drug design. 

We have generated scTNFs selective for TNFR1 (scTNF[R1]3) and TNFR2 (scTNF[R2]3) 

by insertion of mutations at the receptor binding sites. Because of the covalent linkage 

of the TNF monomers it is also possible to produce scTNF derivates with mixed 

selectivities (scTNF R1[R2]2 and scTNF [R1]2R2). We are currently using these 

derivates to investigate the stoichiometry of ligand/receptor interaction.

Friedrich Koch-Nolte, Jan Reyelt, Janusz Wesolowski, Kristina Burkert, Nicole 

Schwarz, Felix Scheuplein, Friedrich Haag, Vanina Alzogaray, Fernando Goldbaum 

Single domain antibodies from llama effectively and 

specifically block an immunoregulatory T cell surface enzyme 

in vivo 

T cells express a flurry of ecto-enzymes that have their active sites exposed in the 

extracellular environment. These enzymes play important roles in cell trafficking, 

inflammation and apoptosis (1). The goal of our study was to develop a tool for blocking 

the function of a specific T cell ecto-enzyme in vivo. ART2.2 is a toxin-related ectoenzyme 

that transfers the ADP-ribose moiety from NAD onto other cell surface proteins. 

ART2.2 induces T cell death by activating the cytolytic P2X7 purinoceptor via ADPribosylation 

(2). We successfully generated ART2.2-blocking single domain antibodies 

from an immunized llama (3). The variable domain of heavy-chain antibodies (VHH 

domain) represents the smallest known antigen-binding unit generated by adaptive 

immune responses. Their long CDR3 endows VHH domains with the extraordinary 

capacity to extend into and block molecular clefts. Following intravenous injection, the 

ART2.2-specific VHH domainss effectively shut-off the enzymatic and cytotoxic activities 

of ART2.2 in lymphatic organs. This blockade was highly specific (blocking ART2.2 but 

not the related enzymes ART1 or ART2.1), rapid (within 15 minutes after injection) and 

reversible (24 hours after injection). Our findings constitute a proof of principle that 

opens up a new avenue for targeting T cell ecto-enymes in vivo. 

1) Salmi & Jalkanen Nat Rev Immunol 5: 760-771 (2005) 

2) Seman, et al. Immunity 19: 571-82 (2003) 

3) Koch-Nolte, et al. FASEB J in press (2007)

Andrej Mantei, Sascha Rutz, Ioanna Andreou, Martin Weber, Alexander Scheffold 

siRNA-mediated gene knock-down in primary mouse T cells 

RNA interference (RNAi)-mediated knock-down of target genes represents a powerful 

approach for functional genomics as well as therapeutic applications. However, for T 

lymphocytes, central regulators of immunity and immunopathologies, the application of 

RNAi is limited since both, the RNAi machinery itself as well as the transfection 

technologies to introduce siRNA into primary lymphocytes are inefficient. 

Using modified electroporation (Nucleofection), we established siRNA transfection of 

primary murine T lymphocytes reaching about 90-100% transfection and knock-down 

efficiency with minimal impairment of cellular function. However, gene knockdown is 

only short-lived (24-48 hours) with conventional siRNA whereas persisting for up to 2 

weeks with chemically stabilized siRNA. Our data demonstrate that RNAi is fully 

functional in primary T lymphocytes but critically depends on siRNA stability within the 

cells. Targeting CD4 and the transcription factor GATA3 we show that this new approach 

allows functional gene analysis in primary T lymphocyte activation and differentiation in 

vitro as well as in vivo.

Uwe Koelsch, Burkhart Schraven, Luca Simeoni 

SIT and TRIM determine T-cell fate in the thymus 

Thymic selection is a tightly regulated developmental process essential for establishing 

central tolerance. Based upon experimental evidence, the intensity of TCR-mediated 

signaling is a key factor for determining cell fate in the thymus. It is widely accepted 

that low-intensity signals result in positive selection, whereas high-intensity signals turn 

on the negative selection program. Transmembrane adaptor proteins (TRAPs) have 

been demonstrated to be important regulators of T-cell activation. However, little is 

known on their role during T-cell development. We have previously shown that the 

transmembrane adaptor SIT regulates positive selection, whereas mice lacking the Tcell 

receptor interacting molecule (TRIM) showed normal T-cell development. As SIT 

and TRIM represent two related TRAPs strongly expressed in thymocytes, we have 

explored the possibility that they may share redundant functions during thymocyte 

selection. We found that SIT and TRIM cooperatively regulate TCR signaling potential, 

thus in turn influencing the outcomes of selection. Indeed, loss of both SIT and TRIM 

resulted in the upregulation of CD5, CD69 and TCRbeta expression, strong MAPK 

activation and enhanced positive selection. Moreover, by crossing SIT/TRIM doubledeficient 

mice onto transgenic mice carrying TCRs with different avidity/affinity, we 

found profound alterations of T-cell development. Unexpectedly, loss of SIT and TRIM 

resulted in a shift from non selection to positive selection whereas positive selection was 

converted to negative selection. In summary, we have demonstrated that SIT and TRIM 

have the striking ability to regulate cell fate of developing thymocytes, thus identifying 

TRAPs as essential regulators of central tolerance.

Anja Hänsel, Michael Meurer, Ernst Peter Rieber, Knut Schäkel 

Slan-dendritic cells (6-Sulfo LacNAc-expressing dendritic 

cells) in contrast to CD1c+ dendritic cells express high levels 

of functional TLR7- and TLR8- receptors 

The family of Toll like receptors (TLR) enables the recognition of specific microbial 

components widely expressed in bacteria, fungi, protozoa and viruses. Individual 

dendritic cell (DC) subtypes largely differ in their repertoire of expressed TLR which 

critically determines their functional role during the initiation of specific immune 

responses against individual pathogens. We previously described a highly 

proinflammatory type of myeloid DC called slanDC (6-Sulfo LacNAc-expressing dendritic 

cells) that produced particularly high levels of TNF-a as well as IL-12 in response to 

stimulation by the bacterial TLR4-ligand lipopolysaccharide. However, the exact 

expression pattern of TLR by slanDC remained unknown. Here we demonstrate that 

slanDC express high mRNA encoding for TLR2, 4, -5, -6, -7 and -8 as revealed by 

qualitative and quantitative RT-PCR. In contrast, CD1c+ myeloid DC express TLR3, but 

failed to express significant levels of TLR2, -4, -6 and -8, and plasmacytoid DC (pDC) 

expressed preferentially TLR7 and -9. In our functional studies we focussed on the 

relevance of the TLR7 and -8 expression by slanDC, whose natural ligands are single 

stranded viral DNA. When slanDC were stimulated with R848, a ligand of TLR7 and TLR8 

we observed a by far higher IL-12p40 and IL-12p70 production among slanDC 

compared to CD1c+ DC and pDC. Specific ligation of TLR7 (3M-001) and TLR8 (3M-002) 

revealed that slanDC, but not other DC subsets, can respond to both ligands with a 

strong production of IL-12 as well as TNF-a. The expression of functional TLR7 and TLR8 

by slanDC uncovered in this study may have practical implications when designing 

therapeutic vaccinations to induce prominent Th1-responses specific for tumor antigens.

Abudula Abulizi, Annika Grabbe, Markus Brechmann, Christian Polaschegg, Nadine 

Herrmann, Ingo Goldbeck, Kai Dittmann, Jürgen Wienands 

SLP-65 signal transduction requires SH2-mediated membrane 

anchoring and a kinase-independent adaptor function of Syk 

The SH2 domain-containing leukocyte adaptor protein of 65 kDa (SLP-65) is a substrate 

of activated tyrosine kinase Syk downstream of the B cell antigen receptor (BCR). A 

main function of SLP-65 is to orchestrate the assembly of Ca 2+ -mobilizing enzymes at 

the inner leaflet of the plasma membrane. However, the mechanism of SLP-65 

membrane anchoring remains an enigma. We now employed two genetic reconstitution 

systems to unravel structural requirements of SLP-65 for BCR-induced Ca 2+ 

mobilization and subsequent nuclear responses. First, mutational analysis of SLP-65 in 

DT40 B cells revealed that its C-terminal SH2 domain controls efficient tyrosine 

phosphorylation by the kinase Syk, plasma membrane recruitment, Ca 2+ flux as well as 

subsequent NFAT activation for the initiation of gene transcription. Second, restoring 

the Ca 2+ response in Jurkat T cell mutants with B cell signaling proteins uncovered a 

kinase-independent adaptor function of Syk that is required for linking phosphorylated 

SLP-65 to Ca 2+ mobilization. Hence, Syk is upstream as well as downstream of SLP-65. 

Moreover, the mechanism by which SLP-65 translocates the Ca 2+ initiation complex to 

the plasma membrane in B cells turned out to be fundamentally different to that utilized 

by the closely related SLP-76 adaptor in activated T cells.

Bernhard Reis, Tanja Scheikl, Norbert Huser, Bernhard Holzmann, Klaus Pfeffer, 

Sandra Beer 

SLy-an Orphan Adaptor Protein Displaying a Nonredundant 

Role in Lymphocyte Development and Activation 

Lymphocyte activation by antigen receptor triggering is an essential step in adaptive 

immunity which must be tightly regulated to mount an immune response towards a 

pathogen and to avoid autoimmune disorders. Major players in this complex network 

are adapter proteins containing characteristic SH2 or SH3 domains which are known to 

mediate protein protein interactions. 

SH3 and SAM domain containing protein expressed in lymphocytes (SLy) is a member 

of a distinct family of putative adapter and/or scaffold proteins highly conserved in 

mammals. SLy is exclusively expressed in lymphocytes and has been shown to be 

phosphorylated specifically upon antigen receptor engagement. 

To investigate the physiological functions of SLy, sly-mutant mice expressing a 

truncated protein lacking the nuclear localisation signal and the phosphorylation site 

(SLYD/D) have been generated by our group. SlyD/D mice exhibit reduced lymphoid 

organ sizes, diminished marginal zone B cell numbers and severely impaired antibody 

responses against T-dependent and -independent antigens. B and T cell proliferation is 

attenuated and T cell cytokine production is severely reduced. In vivo, survival of semiidentical 

cardiac allografts was substantially prolonged in Sly1D/D mice. However, 

global tyrosine and MAP Kinase phosphorylation, Ca2+ flux and transcription factor 

activation are normal. We show that SLy wild-type protein specifically shuttles in 

between nucleus and cytoplasm after antigen receptor activation. In contrast, SLymutant 

protein shuttling is impaired. The signal transduction pathway leading to the 

induced translocation of SLy protein and the role of the phosphorylation status and the 

nuclear localisation signal are further dissected. 

To further characterize the importance of SLy protein and to determine if the truncated 

form acts in a dominant negative way, we recently generated full knockout mice 

(SLy-/-). Preliminary data will be presented comparing the related phenotype of SLy-/- 

mice and SlyD/D mice, respectively.

Max von Holleben, Simone Klöter, Bernhard Reis, Klaus Pfeffer, Sandra Beer 

SLy2 represents a new member of a recently identified family 

of adapter proteins 

A novel putative adapter protein, SH3-lymphocyte protein 2 (SLy2), was found by virtue 

of its homology to the previously identified SLy1. The two proteins share over 80% 

sequence identity and contain three characteristic domains: an N-terminal nuclear 

localization signal (NLS), an SH3 domain, and a C-terminal SAM domain. SH3 and SAM 

domains are known to be involved in protein-protein interactions, which in addition to 

the absence of an enzymatically active domain, indicates an adapter function for both 

SLy1 and SLy2. 

Using a polyclonal serum against the N-terminal part we could demonstrate SLy2 

expression by western blotting in a broad range of tissues, including bone marrow, 

thymus, spleen, lung, brain, liver and ovary. Western Blot analysis of primary murine 

lymphocytes indicates an upregulation of SLy2 expression in B lymphocytes, after 

stimulation with maturation-/differentiation-, and proliferation-inducing agents, such as 

•CD-40, LPS, and IL-4. The high basal level of SLy2 in T lymphocytes did not increase 

upon stimulation by •CD3/•CD28. 

Preliminary expression-analyses have revealed an increased expression of SLy2 in 

human colon-carcinomas, which is in line with previous findings of Claudio and 

coworkers, reporting elevated expression-levels of SLy2 (HACS1) in different human 

myeloma cell-lines. In marked contrast to its potentially oncogenic role, SLy2 

overexpressing Jurkat or SVEC cells exhibit a defect in proliferation. 

Interestingly, several reports suggest a connection between an expressional 

deregulation of SLy2 in the brain and the appearance of Alzheimer’s disease in human 

patients. 

With the aim to unravel the impact of SLy2 function for murine development, immune 

responses and its role in the emergence of different malignancies, we are currently 

generating a SLy2 deficient mouse strain.

Bin Qu, Varsha Pattu, Eva C. Schwarz, Tanja Mayer, Jens Rettig, Markus Hoth 

SNARE proteins are involed in cytotoxic granule secretion at 

the immunological synapse. 

Cytotoxic T lymphocytes (CTLs) exert their cytotoxic activity through the polarized 

secretion of cytotoxic granules at the immunological synapse (IS). Soluble NSF 

attachment receptor (SNARE) proteins are well-known as the critical effectors of 

exocytosis of synaptic granules in neuronal cells. However, the function of SNARE 

proteins in exocytosis of cytotoxic granules in CTLs still remains unclear. To address this 

question, we established a method to generate human primary CTLs, which is based on 

the stimulation of isolated CD8+ T cells with anti-CD3/anti-CD28 antibody coated 

beads. The activation state of the CTLs was checked by the expression level of either 

CD25 (Interleukin-2 receptor) or CD62L (L-selectin). The cytotoxic capacity of the CTLs 

was confirmed by a cytotoxicity assay either on 96-well plates or on single cell level. In 

addition, we analyzed the expression pattern of SNARE proteins by conventional RT- 

PCR. We found that the expression profile of SNARE proteins is regulated upon CTL 

maturation. As we investigated by immunohistochemistry, some but not all SNARE 

proteins co-localized with cytotoxic granules at the IS. These results implicate that 

SNARE proteins are likely to play important roles in cytotoxic granules serection at the 

IS. Our data provide not only a defined method to generate human primary CTLs, but 

also an overview of the expression of SNARE proteins in CTLs, which will facilitate the 

analysis SNARE protein function in the immune system.

Simon Frank, Leif Sander, Stephan Bischoff, Axel Lorentz 

SNARE proteins in mature primary human mast cells 

Rationale: Mediator release of mast cells is a key process in allergic reactions. The 

events facilitating the fusion of granule and plasma membrane in the course of mast cell 

degranulation are not clear. SNARE (Soluble NSF Attachment Protein Receptors) 

proteins have been demonstrated to be involved in fusion of opposing membrane layers 

during exocytosis. Here, we analyzed expression of SNARE isoforms in human mast 

cells and which of them might be crucial for mast cell degranulation. Methods: Human 

mast cells were isolated and purified from surgical specimen of intestinal mucosa, using 

enzymatic digestion and MACS-technique. SNARE-protein expression was demonstrated 

employing RT-PCR and Western blot. Interaction of SNAREs was analyzed by 

immunofluorescence and immunoprecipitation. Results: Mature primary human mast 

cells express the tSNAREs (=target SNAREs) Stx-1B, Stx-2, Stx-3, Stx-4 and SNAP-23, 

but not SNAP-25, and the vSNAREs (=vesicular SNAREs) VAMP-3, VAMP-7, and VAMP- 

8. VAMP-2, which has been demonstrated to play a key role in eosinophil exocytosis, 

was only expressed at very low levels. SNAP-23 formed complexes with VAMP-7 and 

Syntaxin-4. Fluorescence microscopy revealed translocation of both VAMP-7 and VAMP- 

8 to the plasma membrane upon stimulation of the cells using 10-6 M iono/PMA. 

Conclusion: Human mast cells express a specific pattern of SNARE isoforms which are 

able to form stable complexes. SNAREs might play a major role in human mast cell 

exocytosis; inhibition of which could represent a novel therapeutical approach in 

treatment of allergic disorders.

Manfred Hönig, Ansgar Schulz, Catharina Schütz, Paul Fisch, Tatjana Kersten, Ulrich 

Pannicke, Markus Rojewski, Wilhelm Friedrich, Klaus Schwarz 

Somatic reversion of lymphocyte subpopulations after HSCT 

in a patient with JAK3 deficiency -Evidence for independent 

αβ- and γδ- T-lineage stem cells 

Spontaneous somatic reversions are reported for various immunodeficiencies (e.g. 

IL2RG, ADA, WAS, RAG1, Artemis, LAD1). We report on a 14 year old girl with SCID 

due to JAK3 deficiency, who was treated in infancy by HLA-haploidentical 

haematopoietic stem cell transplantation (HSCT) without conditioning. A functional 

donor T cell system was established. 10 years after HSCT, a small amount (10%) of 

autologous T cells was noted in addition to donor T cells in routine chimerism analysis. 

Reversions of both compound heterozygous point mutations (NM_000215: c.424C>T, 

c.1351C>T leading to NM_00026: p.[Arg142Cys]+[Arg451X] ) to wild type (wt) - 

sequence were found in this autologous T-cell population: &alpha&beta-TCR positive T 

cells (and NK cells) are reverted to wt-sequence in Exon4, whereas &gamma&delta-TCR 

positive T cells are reverted in the other affected allele (Exon9). The TCR-repertoires of 

these T-cell populations are oligoclonal. The reversions thus must have occurred before 

TCR-rearrangement but after the determination of an &alpha&beta- or &gamma&delta- 

T-cell fate. These findings strongly support a non-instructive model for this decision in Tcell 

development.

Kevin Thurley, Dorothea Busse, Thomas Höfer 

Spatiotemporal Cytokine Dynamics in T-Cell Regulation - 

A Mathematical Model of IL-2• •Diffusion and Regulatory T- 

Cells• (•Treg•) 

Cytokine signalling is crucial for understanding the immune system.• •Up to now,• 

•most knowledge has been based on ELISA studies,• •indicating extremely low• 

(•picomolar•) •concentrations of cytokines.• •A classic calculation from diffusion-limited 

reaction theory• (•Berg and Purcell,• •1972•) •shows that under such conditions,• •a 

signal would take hours to occur.• •This raises the question how,• •nevertheless,• 

•sensitive cytokine signalling in a noisy environment can be achieved.• 

A two-dimensional reaction-diffusion model brings new insights to this question.• •We 

consider a well-studied and physiologically important sample system:• •Activation of Thcells 

via Interleukin(IL•)•-2• •and immunosuppression by Treg.• •The model reflects 

experimentally proven features like autoactivation of Th-cells and low IL-2• 

•concentrations in the bulk phase.• •Three major• •results have been obtained so far:• 

•Firstly,• •the model predicts high local IL-2• •concentrations at secretion sites,• 

•which can possibly be verified experimentally with quantitative secretion assays.• 

•Thus,• •this property may serve to justify the model just as to explain effective signals 

at low cytokine concentrations.• •Secondly,• •we can show a paracrine signal over 

several cell diameters,• •confirming recent experimental results which suggest 

paracrine rather than autocrine signalling in T-cell activation (Long and Adler,• 

•2006•)•.• •And finally,• •immunosuppression by Treg may be explained by 

competitive IL-2• •uptake.• 

With the spatiotemporal modeling approach,• •we are able to predict the range of a 

cytokine signal in dependence of important system parameters.• •Apparently,• 

•inhomogeneities in the cytokine distribution are necessary to explain basic properties 

of the complex cytokine network.•

Ariel H. Achtman, Sven Golfier, Martin Lipp 

SPHINGOSINE-1-PHOSPHATE RECEPTOR-4 IS MAINLY 

EXPRESSED BY LYMPHOID CELLS 

Sphingosine-1-phosphate is a bioactive lipid involved in cell migration, survival and 

differentiation. It binds to a panel of receptors, called S1P1 to S1P5 , which differ in their 

expression patterns. At the mRNA level, it has been shown that S1P4 expression is 

restricted to lymphocyte-containing tissues. However, determining protein expression 

patterns for S1P4 has been hampered by the lack of sufficiently sensitive antibodies. 

Therefore, a reporter gene mouse was generated, in which the S1P4 gene is replaced by 

lacZ. The insertion and correct orientation of the gene were confirmed by Southern Blot 

and PCR. Back crossing onto the C57BL/6 background was initiated. Expression levels of 

the reporter protein β-galactosidase were low but detectable by luminometric analysis. β- 

Galactosidase expression was detected in the spleen, bone marrow, lymph nodes, 

thymus, peripheral blood and Peyer’s plaques. No expression was seen in the lung, 

brain, liver or heart. Further analysis revealed that S1P4 is expressed on white blood 

cells but not erythrocytes. In sorted cell populations, B cells and CD4 + and CD8 + T cells 

were the main cell populations carrying the gene. More detailed analysis of changes in 

S1P 4 expression during active immune responses is currently underway.

Shin-Young Na, Heike Eujen, Catherine Toben, Yi Cao, Anneliese Schimpl, Ralf Gold, 

Thomas Hünig 

Spontaneous development of CD8-mediated EAE in a new 

transgeneic mouse model 

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated central nervous 

system (CNS) autoimmune disease which widely used as an animal model of multiple 

sclerosis (MS). Previous studies have used immunization of rodents with myelin 

antigens in adjuvants to induce EAE, thereby focussing on pathogenic CD4 cells of the 

Th1 and Th17 types. More recently, however, also CD8 T-cells are increasingly receiving 

attention as major mediators of autoimmune attack in the human disease itself. 

We generated transgenic mice which express the model antigen ovalbumin (OVA) under 

an oligodendrocyte (ODC-)-specific MBP promoter. When we crossed these ODC-OVA 

transgenic mouse with either OT2 or OT1 transgenic mouse which express transgenic 

OVA-specific T-cell receptors (TCR) on CD4 and CD8 cells, respectively, we observed 

CD4 T cells are ignorant of OVA in ODC-OVA transgenic mice whereas OT1/ODC-OVA 

double transgenic mice develop fulminant EAE. Spontaneous development of severe EAE 

affected cerebellum, brainstem, optical nerve and spinal cord. Our initial data suggest 

that in this system, initial priming of OT-1 cells occurs within the brain, possibly by the 

OVA-expressing oligondenrocytes themselves. 

We also tested the ability of a mAb which recognises the OVA-8 peptide bound to the 

murine class I molecule H-2Kb (25-D1.16), for its ability to interfere with disease 

development and found that at high doses (>200µg/mouse), it was able to prevent 

spontaneous EAE development in OT1/ODC-OVA double transgenic mice.

Melinda Czéh, Gerald Willimsky, Thomas Kammertöns, Jehad Charo, Thomas 

Blankenstein 

Sporadic immunogenic cancer induces cytotoxic T lymphocyte 

unresponsiveness against MHC-mismatched targets 

Tumor-induced cytotoxic T lymphocyte (CTL) unresponsiveness against unrelated 

antigens has been described, but full MHC-mismatched CTL responses have not been 

found to be impaired. In a transgenic model of sporadic immunogenic cancer (LoxP-Tag 

mice) we investigated CTL responses against foreign peptide antigens and MHCallogeneic 

targets. Sporadic tumors in LoxP-Tag mice that express a strong tumorspecific 

transplantation rejection antigen (SV40 Tag) rapidly induce CTL tolerance, 

which is associated with enhanced serum TGF-β1 concentration and expansion of 

immature myeloid cells (Willimsky et al., manuscript in preparation). We show by in 

vivo kill assays that tumor bearing mice fail to develop LCMV-gp33-specific CTL 

responses. Furthermore, the tumors in H-2b LoxP-Tag mice induced complete CTL 

unresponsiveness against allogeneic H-2d antigens. Paradoxically, in most tumorbearing 

LoxP-Tag mice gp33-expressing or H2-d allogeneic tumor cells did not grew 

despite the CTL-unresponsiveness. The reason for this surprising discrepancy is 

currently being analyzed. Additionally, data on the function of NK cells in tumor-bearing 

LoxP-Tag mice will be presented.

Cornelia Beuter-Gunia, Daniel Degrandi, Sandra Beer, Klaus Pfeffer 

SSPII, a new IFN gamma upregulated secretory protein 

IFN gamma is a key regulator of a broad range of genes involved in innate immunity. 

By analyzing the transcriptome with Affymetrix Array Technology in IFNgamma/ 

TNFalpha stimulated Ana-1 macrophages a set of uncharacterized transcripts could be 

identified. 

One IFN gamma induced gene encodes for a 78 aa long protein. Sequence analysis of 

the transcript suggests that it contains an N-terminal leader sequence responsible for 

targeting to the secretory pathway. In accordance with this observation we could 

demonstrate that the His-tagged fusion protein is readily secreted into the cell culture 

supernatant after transfection of a His tagged cDNA expression construct in 293T cells. 

Real time PCR studies in primary IFN gamma stimulated bone marrow derived 

macrophage cells (BMDMO) showed induction of this gene transcript comparable to 

classic IFN gamma induced genes such as mGBP2 or iNOS. Moreover, stimulation of 

BMDMOs from IFNgR-/- mice indicated that this gene transcript can be directly 

upregulated by TLR ligands and IFN type I. Thus, we would suggest to call this protein 

sspii, i.e. short secreted protein induced by interferons. 

Further in vivo studies revealed that in organs of Listeria monocytogenes and 

Toxoplasma gondii infected mice sspii mRNA was upregulated following infection, which 

let us to the hypothesis, that sspii contributes to the immune defense against 

pathogens. 

Sspii is encoded on chromosome 19 of the mouse genome within 3 exons. Gene 

targeted ES cells carrying an inactivation of sspii could already be established. 

Further in vitro and in vivo experiments as well as the characterisation of a sspii knock 

out mouse line will hopefully contribute to the understanding of interferon mediated 

immune responses.


Stability and plasticity of IL-17 producing T cells 


Deutsches Rheumaforschungszentrum, Berlin 

Chariteplatz 1 

10117 Berlin 

Germany 

Tel +49-30-28460-600/667 

radbruch@drfz.de 

lexberg@drfz.de 

Classically, effector memory CD4 T cells are divided into distinct lineages on the basis of 

their cytokine production profile and the resulting effector function. Recently, a novel 

lineage of CD4 T cells has been described which is characterised by the production of IL- 

17. These cells have been termed Th17 cells. In several reports, this lineage has been 

allocated a role in autoimmune inflammation, therefore it is of importance to determine 

the stability of its expression and how it could be modulated to attenuate disease 

symptoms. 

Our aim is to analyse the induction and maintenance of a functional cytokine memory of 

Th17 cells. We are investigating the plasticity and stability of IL-17 memory expression 

in relation to the cytokine memory expression of the other lineages. 

Both Th1 and Th2 cells proved refractory to IL-17 induction and maintained their 

phenotype when stimulated under Th17 inducing conditions in the presence of TGF-beta 

and IL-6. To test the stability and plasticity of Th17 cells, we cultured Th17 cells under 

Th1 and Th2 inducing conditions and measured cytokine expression. The frequency of 

IL-17 expressing cells observed in one or four week old Th17 cells was reduced when 

restimulated for just one week in the presence of IL-12 or IL-4, although the 

reexpression of IL-17 was increasingly stabilized depending on the duration of 

polarization. The plastic nature of IL-17 expression in Th cells reveals that Th17 cells 

may need a certain environment in vivo to maintain IL-17 expression and are prone to 

regulation by both Th1 and Th2 cells.

Anita Correll, Andrea Tuettenberg, Christian Becker, Jürgen Knop, Helmut Jonuleit 

Stage-dependent quantification of regulatory T cells and 

antigen-specific T cell responses in melanoma patients 

Naturally occurring CD4 + CD25 + regulatory T cells (nTregs) are key players in the 

tolerance network. As immunosuppression seems to be one important way by which 

tumors succeed in immune escape, nTregs are assumed to play a crucial role in 

mechanisms contributing to the development and progression of cancer. 

Unlike the murine system, human nTregs cannot be distinguished from conventional T 

helper cells easily by markers like CD25 or Foxp3 since both are also expressed by CD4 

+ T helper cells upon activation. In this study, we used different marker combinations 

for nTreg phenotyping (CD4, CD25, CD127, Foxp3, HLA-DR) for quantitative analysis of 

human nTregs in the peripheral blood of stage II-IV melanoma patients. Using this 

experimental setting, we observed increased ratios of CD4 + CD25 + Foxp3 + HLA-DR 

+ CD127 low Tregs in peripheral blood of melanoma patients compared to healthy 

volunteers. Moreover, the relative ratios of nTregs increased with the progression of 

disease. Additionally, accumulation of nTregs in progressed melanoma patients also 

correlated with a general reduction of T cell responsiveness not only to different tumorassociated 

antigens but also to various recall antigens. These observations, the 

suppressed T cell reactivity in combination with increased nTreg ratios in patients with 

progressive melanoma possibly explain the disappointing success of immunotherapies in 

the patients.

Christiane Siewert, Sascha Cording, Markus M. Heimesaat, Oliver Liesenfeld, Stefan 

Bereswill, Christoph Loddenkemper, Gunnar Loh, Michael Blaut, Alf Hamann, Jochen 

Huehn 

Stay with friends: commensal microflora drives expansion of 

Foxp3+ Tregs in gut-associated lymphoid tissue 

Colitis models have provided compelling evidence for a protective role of Foxp3+CD25 

+CD4+ regulatory T cells (Treg) in intestinal homeostasis. Foxp3+ Tregs have been 

described as thymus-derived cells, however, more recent studies demonstrate a 

significant peripheral turnover. Here we investigated the proliferation of Foxp3+ Tregs 

in gut-associated lymphoid tissues (GALT) and whether this process is driven by 

commensal microflora. 

Germ-free and antibiotics-treated mice were analyzed for frequencies and numbers of 

Foxp3+CD4+ T cells. In addition, in vivo proliferation of Foxp3+ Tregs in different 

lymphoid compartments was determined by BrdU-incorporation. 

In germ-free mice we observed a decreased number of Foxp3+CD4+ T cells in the 

GALT. Similarly, depletion of the commensal microflora by antibiotic treatment led to a 

reduction of CD4+Foxp3+ cell numbers in the GALT, and, moreover, in spleen and 

peripheral lymph nodes. A significantly reduced frequency of cycling BrdU+Foxp3+ 

Tregs was found in the GALT. Interestingly, homeostatic proliferation was not 

compromised in mice deficient for TLR2, the main Toll-like receptor of Tregs. 

Stimuli from the commensal microflora of the gut contribute to the generation or 

maintenance of Tregs in the mucosal compartments and affect Treg numbers 

systemically. Lack of TLR2-mediated effects suggests that other signals, such as 

stimulation of Tregs by bacterial antigens, are driving Treg expansion in the mucosal 

system. These data show that microbial stimuli critically influence homeostasis of 

naturally occurring Foxp3+CD25+CD4+ Tregs and that these cells, which protect 

against intestinal inflammation, might not exclusively consist of self-reactive T cells.

Svetlana Karakhanova, Markus Schneider, Johannes Schenkel, Markus Munder 

Steroids modulate the T effector cell / TREG / dendritic cell 

balance during treatment of Graft-versus-Host Disease. 

Allogeneic stem-cell transplantation (allo-SCT) is a potentially curative treatment for 

various hematological malignancies. CD4+CD25++ regulatory T cells (TREG) regulate 

immune responses and Graft-versus Host Disease (GvHD) after allo-SCT. We have 

shown that the initial phase of GvHD correlates with a significant reduction of TREG in 

the peripheral blood, while at later stages and especially during intensified 

immunosuppressive therapy with steroids increased numbers of TREG appear in the 

peripheral blood. We also found a change in phenotype of highly purified, in vitro 

expanded CD4+CD25++ cells of patients during steroid medication. In contrast to 

expanded TREG from healthy donors or GvHD patients without steroid medication, CD4 

+CD25++ sorted cells from steroid-treated patients loose their TREG phenotype and 

function in expansion cultures. During in vitro activation, steroids induce an increased 

frequency of the CD4+CD25++ population in PBMC from healthy donors and lead to the 

acquisition of a TREG like phenotype in CD4+CD25- T cells. Also, CD4+CD25++ cells 

are less sensitive to steroid-induced cell death in comparison with conventional T cells. 

Finally, dexamethason treatment of different dendritic cell populations leads to the 

blockade of cytokine-induced maturation and consecutively to an impaired ability of the 

dendritic cells to prime immune responses in CD4+ cells. Our findings suggest that 

steroids can influence three major compartments of the immune response (T effector 

cells, TREG cells and dendritic cells) simultaneously. This has implications for the 

steroid-based treatment of GvHD after SCT.

Marcin Wlodarski, Lukasz Gondek, Zachary Nearman, Sandra Zwinger, Hans-Dieter 

Volk, Jaroslaw Maciejewski 

Strategies for quantitation and in-vivo tracking of clonal 

cytotoxic T-cell responses. 

Immune mechanisms are involved in the pathophysiology bone marrow failure 

syndromes. Hematopoietic inhibition can result from cytotoxic T cell (CTL) attack 

against normal hematopoiesis or reflect immune surveillance reaction directed against 

the dysplastic myeloid cell. We used clonally unique T-cell receptor (TCR) variable bchain 

(VB) CDR3 regions as markers of pathogenic CTL responses and show that while 

marrow failure syndromes are characterized by polyclonal expansions, overexpanded 

clones exist in these diseases and can be utilized in investigative setting. To test the 

applicability of clonotypic assays, we developed rational molecular methods for the 

detection of immunodominant clonotypes in blood and in historic marrow biopsies of 35 

aplastic anemia (AA), 37 myelodysplasia (MDS), and 21 paroxysmal nocturnal 

hemoglobinuria (PNH) patients, in whom specific CDR3 sequences and clonal sizes were 

determined. CTL expansions were detected in 81% and 97% of AA and MDS patients, 

respectively. In total, 81 immunodominant signature clonotypes were identified. Based 

on the sequence of immunodominant CDR3 clonotypes, we designed quantitative assays 

for monitoring corresponding clones, including clonotypic Taqman polymerase chain 

reaction (PCR)and clonotype-specific sequencing. No correlation was found between 

clonality and disease severity but in patients treated with immunosuppression, truly 

pathogenic clones were identified based on the decline that paralleled hematologic 

response. We conclude that immunodominant clonotypes associated with marrow failure 

may be used to monitor immunosuppressive therapy

Volker Teichgräber*, Surojit Sarkar*, Vandana Kalia, David Masopust, Laurie 

Harrington, Antonio Polley, Rafi Ahmed, E. John Wherry 

Strength of stimulus and clonal competition impact the rate of 

memory CD8 T cell differentiation 

The developmental pathways of long-lived memory CD8 T cells and the lineage 

relationship between memory T cell subsets remains controversial. While some 

studies indicate the two major memory T cell subsets, central memory (TCM) and 

effector memory (TEM), are related lineages others suggest that these subsets 

arise and are maintained independently of one another. In this study we have 

investigated this issue and examined the differentiation of memory CD8 T cell 

subsets by tracking the lineage relationships of both endogenous and TCR 

transgenic CD8 T cell responses after acute infection. Our data indicates that 

TCR transgenic as well as non-transgenic TEM differentiate into TCM in the 

absence of antigen. Moreover, the rate of memory CD8 T cell differentiation from 

TEM into the self-renewing and long-lived pool of TCM is influenced by signals 

received during priming including antigen levels, clonal competition and/or the 

duration of infection. While some TEM appear to not progress to TCM, the vast 

majority of TCM are derived from TEM. Thus, long-lasting, antigen-independent 

CD8 T cell memory results from progressive differentiation of memory CD8 T 

cells and the rate of memory T cell differentiation is governed by events occurring 

early during T cell priming. 

*These authors contributed equally

Sonja Meemboor, Eva Flenner, Alessandra Zingarelli, Martina Bessler, Wiltrud Kalka- 

Moll 

Streptococcus pneumoniae capsular polysaccharide-mediated 

CD4+ T cell-dependent immune response requires IL-6 

Abscess formation associated with intra-abdominal sepsis causes severe morbidity and 

can be fatal. Induction of abscesses requires the presence of CD4 + T cells. Zwitterionic 

polysaccharides (ZPS) represent a novel class of immunomodulatory bacterial antigens 

that stimulate CD4 + T cells in a MHC class II-dependent manner. The capsular 

polysaccharide Sp1 of Streptococcus pneumoniae serotype 1 is a ZPS model antigen 

and possesses a zwitterionic charge motif with free amino and carboxyl groups and 

promotes T cell-dependent intraperitoneal abscesses in an experimental murine model. 

Mice are administered Sp1 with sterile cecal content adjuvant (SCCA) which reflects the 

spillage of colonic contents that occurs during the onset of intra-abdominal sepsis in 

humans and aids the pro-inflammatory response. SCCA alone does not induce abscess 

formation. In this study we address the role of the pro- and anti-inflammatory acting 

cytokine IL-6 during Sp1-induced abscess formation. Macrophages are the most 

prevalent antigen-presenting cells in intraperitoneal lavage after intraperitoneal Sp1 

challenge and secrete significant amounts of IL-6 as shown by intracellular cytokine 

staining. Immunohistochemical analysis of the Sp1-induced abscesses reveal that IL-6 

secreting macrophages are incorporated in the abscess capsule. IL-6 induces a dosedependent 

CD4 + T lymphocytes migration as demonstrated in migration assays. We 

show that mice deficient of IL-6 fail to attract CD4 + T cells into the peritoneal cavity 

after Sp1 challenge and to hence develop abscesses. In addition, administration of a 

neutralizing Ab specific for IL-6 prevents abscess formation following Sp1 challenge. 

These data delineate the requirement of activation of antigen-presenting cells by ZPS 

and underscore the essential role of IL-6 in this disease process.

Daniel Schaefer, Anja Janysek, Jürgen Neumann, Norbert Koch 

Strong impact of invariant chain on surface expression of HLA- 

DQ 

Daniel Schaefer, Anja Janysek, Jürgen Neumann and Norbert Koch 

Division of Immunobiology, Institute of Genetics, University of Bonn 

MHC class II molecules are peptide receptors presenting peptides from extra cellular 

antigens on the cell surface of antigen-presenting cells. The peptide receptors are 

composed of alpha and beta subunits, which assemble in the ER with invariant chain 

(Ii). The HLA class II region encodes alpha and beta subunits of three different isotypes: 

DP, DQ and DR. In this study we examined the impact of Ii on intracellular transport 

and surface expression of HLA class II isotypes. We inspected various combinations of 

alpha and beta chain isotypes for transport and surface expression by employing 

western blotting, immunofluorescent staining and FACS-analysis. In contrast to DR and 

DP isotypes, surface expression of DQ heterodimers strictly depends on Ii.

Nana Ueffing, Eric Keil, Christian Freund, Ronald Kühne, Klaus Schulze-Osthoff, Ingo 

Schmitz 

Structural and mutational analyses of c-FLIP R reveal 

requirements for DISC-recruitment 

c-FLIP proteins are known as potent inhibitors of death receptor-mediated apoptosis by 

interfering with caspase-8 activation at the death-inducing signaling complex (DISC). 

Among the three human isoforms, c-FLIPlong (55 kDa), c-FLIPshort (26 kDa) and c-FLIPR (25 kDa), the latter one is only poorly characterized. Similar to procaspase-8 and -10 

and the viral FLIP protein MC159, FLIP proteins contain two rigidly associated death 

effector domains (DEDs) at their N-terminus that are thought to be important for DISC 

recruitment. However the precise molecular requirements for DISC formation are 

currently unknown. To further investigate the role of c-FLIPR in the regulation of death 

receptor pathways, we cloned mouse c-FLIPR from thymus by RT-PCR and found that it 

is the only short c-FLIP isoform expressed in mice. Molecular modeling showed that 

similar to FADD and MC159, c-FLIPR contains two prominent surface features in each 

DED important for protein-protein interactions, namely a hydrophobic patch and a 

charge triad (also called RxDL-motif). Interestingly, despite a high structural similarity 

of MC159 and c-FLIPR , point mutations in the charge triad, which abolish the antiapoptotic 

function of MC159, did not abrogate apoptosis inhibition by c-FLIPR . Instead, 

we show here that the hydrophobic patch in DED2 is critical for recruitment of c-FLIPR into the DISC. Thus, the DEDs of viral and cellular FLIPs interfere with apoptosis by 

divergent mechanisms, suggesting a surprisingly functional diversity of the DED motif. 

By the generation of different c-FLIPR deletion mutants we can show, however, that 

neither of the DEDs alone nor coexpression of both DEDs is sufficient to interact with 

the DISC or to protect from CD95-mediated apoptosis. Thus, also intra-molecular 

linkage and the tight package of both DEDs in c-FLIPR are essential for its anti-apoptotic 

capacities.

Björn Linke, Heiko Weyd, Lucie Dörner, Andrea Mahr, Peter H. Krammer 

Structural Requirements for Annexin 1 Mediated Suppression 

of Dendritic Cells 

Peripheral tolerance comprises several mechanisms to prevent autoimmune disease. To 

better understand molecular mechanisms of self-tolerance and nonresponsiveness 

against apoptotic “self” cells we generated monoclonal antibodies against apoptotic 

cells. We identified one monoclonal antibody which determined Annexin 1 as a new 

signalling molecule on the surface of human apoptotic cells. Coculture experiments of 

dendritic and apoptotic cells as well as incubation of dendritic cells with recombinant 

Annexin 1 lead to inhibition of DC maturation. 

The mechanism by which Annexin 1 mediates this effect remains to be studied further. 

In an attempt to identify the active site of Annexin 1 we generated different deletion 

constructs and mutants of Annexin 1. These proteins are analyzed for their capability to 

modulate maturation of DCs. Their effect on cytokine secretion and expression of DC 

surface markers including MHC and costimulatory molecules e.g. CD80 and CD86 and 

their influence on T cell proliferation in DC-T cell cocultures are studied. 

Furthermore, transwell experiments in our lab have shown that a direct cell contact 

between apoptotic and dendritic cells is required for the inhibition of DC maturation. By 

using photocrosslinking and immunoprecipitation we study the Annexin 1 receptor on 

the surface of DCs.

Nina Oberle, Nadine Eberhardt, Christine S Falk, Peter H Krammer, Elisabeth Suri- 

Payer 

Suppression Mechanisms of cytokine transcription in human 

CD4+CD25- T cells upon interaction with CD4+Foxp3+ 


CD4+CD25highFoxp3+ regulatory T cells (Treg) are critical mediators of peripheral self 

tolerance and immune homeostasis. Treg suppress proliferation and cytokine production 

by conventional T cells (Tcon). The exact mechanism of suppression, however, is still 

unknown. To gain a better understanding of Treg function, we investigated the kinetics 

of cytokine suppression in Tcon re-isolated from cocultures with pre-activated human 

Treg. Treg inhibited induction of Th1 cytokine mRNA as early as 1 hour after 

stimulation, whereas induction/suppression of Th2 cytokines was delayed to 10-15 

hours. We show that immediate cytokine mRNA suppression in Tcon was neither 

dependent on TGF-β / IL-10 or IL-2 consumption, nor on induction of the transcriptionalrepressor 

FOXP3 or other anergy-related genes (e.g. GRAIL, TOB, FOXJ1, ROG, ICER). 

In contrast, LAG-3, SOCS1 and SOCS3 mRNA were strongly upregulated in Tcon in the 

presence of Treg. However, protein analysis did not confirm a role for these proteins in 

early suppression. Thus, the identification of a fast inhibitory mechanism in Tcon 

induced by Treg constitutes an important step for future efforts to unravel the entire 

elusive suppressive mechanism.

Beatrix Schumak, Gerhard Wingender, Schwandt Timo, Frank Juengerkes, Thomas 

Tueting, Percy Knolle, Bernhard Holzmann, Andreas Limmer 

SUPPRESSION OF ADAPTIVE IMMUNE RESPONSES BY TOLL- 

LIKE RECEPTOR LIGANDS 

As bacterial infections are known to induce strong immune responses, immune 

activation by TLR-ligands demands effective control by the immune system; 

dysregulation can elicit extensive inflammatory reactions, causing immune damage, but 

also immune suppression. We observed that - in contrast to local application -systemic 

application of bacteria or bacterial CpG-rich DNA (CpG-ODN) in high concentrations 

inhibited the induction of adaptive immune responses. The suppressive effect was 

characterized by diminished CD8 T cell proliferation and reduced cytotoxicity in the 

spleen as well as a lack of CD4 mediated IgG antibody response. Experiments 

performed in splenectomized mice show that this CpG-mediated suppression is 

regulated in the spleen and several mechanisms are involved. Systemic CpG-ODN 

application leads in the spleen to induction of the enzyme indoleamine 2,3 dioxygenase 

(IDO), which depletes tryptophan that is necessary for T-cell proliferation. Furthermore, 

the production of interleukin 12 (IL-12) by CD8a positive dendritic cells in the spleen is 

also affected. These DCs are immune paralysed, as they are diminished in cell numbers 

and produce less IL-12, when they are re-stimulated in vivo. This phenomenon could 

have important implications in septic patients who suffer from long-term immune 

suppression and exhibit reduced levels of IL-12. This hypothesis is substantiated by 

CASP (colon ascendens stent peritonitis), a murine sepsis model. CASP performed 

before infection with adenovirus expressing ovalbumin (AdOVA) resulted also in 

suppressed adaptive immune responses against OVA. Analysis of bioluminescent 

luciferase expression (IVIS®200) as well as classical microbiological methods revealed 

that bacteria reached besides liver and lung the spleen in high numbers, where they 

induced IL-12 suppression and expression of IDO. This corresponded to subsequent 

induction of immune suppression.

Christiane A. Opitz, Tobias Lanz, Christian Lutz, Wolfgang Wick, Michael Platten 

Suppression of antigen-specific T cell immunity by 

mesenchymal stem cells: implications for stem cell therapy of 

autoimmune diseases. 

Mesenchymal stem cells (MSC) represent an attractive vehicle for the treatment of 

conditions associated with harmful T cell responses such as multiple sclerosis, as they 

not only home to sites of inflammation but also display unique suppressive properties 

on T cell immunity. Here we show that immunosuppression mediated by human MSC 

(hMSC) can be augmented by ligation of Toll-like receptors (TLR) expressed on hMSC. 

Activation of TLR3 and TLR4 on human MSC caused an upregulation of the tryptophandegrading 

enzyme indoleamine-2,3-dioxygenase (IDO). The upregulation of IDO by 

TLR3 activation was mediated in part by interferon-β (IFN-β) since IDO mRNA and 

kynurenine release were induced in response to IFN-β and since TLR3-mediated IDO 

upregulation and subsequent kynurenine release were suppressed by inhibition of the 

toll/IL-1 receptor domain-containing adapter inducing IFN-β (TRIF). Inhibition of IDO 

mitigated the immunosuppressive effect of TLR-activated MSC indicating that 

tryptophan catabolism is responsible for the immunosuppression induced by TLR 

activation. To evaluate the use of mesenchymal stem cells to suppress pathogenic 

antigen-specific T cell responses during autoimmune diseases we coincubated 

autologous MSC with T cells from mice carrying a transgenic T cell receptor that 

recognizes the immunodominant epitope of the myelin oligodendrocyte protein (MOG). 

MSC suppressed the proliferation of myelin-specific T cells after antigen-specific 

activation indicating that MSC may be active in suppressing pathogenic antigen-specific 

T cells responses during autoimmunity. Our data imply that MSC may be a novel 

immunomodulating approach to treat autoimmune disease such as multiple sclerosis.

Andrea Mahr 

Suppression of Dendritic Cell Activation by Annexin 1 

on the Surface of Apoptotic Cells 

Dendritic cells (DCs) are phagocytic cells that discriminate “non-self” from “self” 

structures, and consequently initiate immunity or tolerance. Pathogenic components, 

like bacteria-derived lipopolysaccharide (LPS), are recognized by Toll-Like Receptors 

(TLRs) on the surface of DCs, leading to DC activation and secretion of proinflammatory 

cytokines. This process can be inhibited by the uptake of apoptotic cells, the biggest 

source of “self”-material. Inhibition is probably mediated by tolerogenic signals on the 

surface of apoptotic cells, but the molecules relevant for this process are not yet well 

defined. A candidate protein is annexin 1, which is externalized by early apoptotic cells. 

In this study, it has been found that pre-incubation of DCs with apoptotic cells or 

purified recombinant annexin 1 before TLR stimulation could reduce the transcriptional 

upregulation and secretion of proinflammatory cytokines. 

Correspondingly, annexin 1 led to a substantial decrease of LPS-induced TLR signal 

transduction. 

The detailed analysis of annexin 1 function may lead to a more advanced understanding 

of the regulation of TLR signaling, and would give valuable insight into a potential 

mechanism of peripheral non-responsiveness.

Jens Haenig, Manfred B. Lutz 

Suppression of mature dendritic cell function by regulatory T 

cells in vivo is abrogated by CD40 licensing 

The priming of CD4+ effector T cells (Teff) in vivo is induced by mature dendritic cells 

(DC) and controlled by CD4+ CD25+ Foxp3+ regulatory T cells (Treg). It remains 

unclear however, how Teff priming versus Treg suppression are regulated during 

antigen presentation by DC in secondary lymphoid organs at the simultaneous presence 

of Teff and Treg. Here, we used an peptide-specific DO11.10 TCR-transgenic adoptive 

transfer model to follow the Teff priming kinetics and the mechanisms of suppression by 

Treg. Treg could not influence the early Teff expansion but limited and terminated the 

Teff response. DC-Treg cell contacts remained unaltered during suppression by Treg 

and led to a downregulation of the costimulatory molecules CD80, CD86, PD-L1 and PD- 

L2, but not MHC II, CD40, ICOS-L or CD70 from the mature DC surface. This effect was 

observed only after TNF or LPS maturation of DC but not after combined LPS-CD40 

stimulation. Together, our data indicate that Treg suppression against non-self antigens 

in vivo occurs delayed as compared to the Teff response, is mediated mainly through 

DC modulation, but is controlled by the type DC maturation.

Yu-Hwa Huang, Eva Tolosa, Heinz Wiendl 

Suppressive action of HLA-G-expressing T cells, a novel 

population of natural regulatory cells, is independent of 

antigen-presenting cell, facilitated by TCR-engagement, 

involves HLA-G-ILT-2 ligation and IL-10 

Regulatory T cells suppress harmful immune responses against foreign and self-antigens 

and play a key role in the mechanisms of autoimmunity. We recently identified a novel 

regulatory T cell population characterized by the expression of the immune-tolerogenic 

molecule HLA-G (Feger et al., 2007, Blood). “HLA-G T-regulatory cells” (HLA-G Tregs) 

exist in small, but sizeable quantities in peripheral blood under physiological conditions 

(0.1 to 4% of T cells). Our initial phenotypcial and functional characterization revealed 

that HLA-G Tregs are hypoproliferative, negative for FOXP3 and suppress autologous T 

cells in a non-contact-dependent manner. 

We here provide further data clarifying the mechanisms of suppression between HLA-G 

Tregs and CD4 T effector cells. CD4 HLA-G Tregs suppress proliferation of autologous 

polyclonal effector cells in the absence of antigen-presenting cells (APC), suggesting 

non-antigenic specifically suppression. This was demonstrated in an APC-free 

suppression system utilizing anti-CD3/28-beads as the stimulus for responder cells. 

Suppression was depending on the effector-responder ratio but independent from cellcell 

contact. Using a transwell system we could demonstrate that (i) TCR engagement 

facilitates CD4 HLA-G pos Treg - mediated suppression through the production of 

soluble HLA-G; (ii) Neutralization the engagement of HLA-G-ILT2 significantly reversed 

suppression; (iii) CD4 HLA-Gpos Treg produced IL-10 upon TCR stimulation; (iv) 

Blockage of the IL-10 receptor partially reversed suppression. In conclusion, CD4 HLA-G 

pos Treg exert suppression via a non-cell-cell contact dependent mechanism that 

involves HLA-G-ILT2 interaction as well as IL-10.

Kathrin Held, Elke Dauber, Michael Loos, Franz Petry 

Susceptibility of complement deficient mouse strains to 

systemic infection with Candida albicans 

C. albicans can activate all three pathways of the complement system leading to 

opsonization of the yeast cells, enhanced complement receptor mediated phagocytosis 

and infiltration of neutrophils due to C5a chemotactic activity. The aim of this study was 

to investigate the susceptibility to C. albicans infection in different complement deficient 

mouse strains. We compared mice with targeted disruption of the C1qa gene (C1q-/-) 

and a double knockout strain that lacks factor B and C2 (Bf/C2-/-). Mice were infected i. 

p. with 10 8 yeast cells of C. albicans SC5314 and monitored for mortality. Bf/C2-/- mice 

showed high mortality (90%) within the study period of 4 weeks. In contrast, mortality 

in C1q-/- mice was below 20% and all C57BL/6 control mice survived. Preliminary 

analysis of MBL-A and MBL-C double knockout mice suggest that they are more 

susceptible to infection than C1qa-/- mice but definitely more resistant than Bf/C2-/- 

mice. Kidneys of deceased mice were homogenized and the yeast load was estimated 

by culture. C.f.u. counts were approximately one log higher in Bf/C2-/- mice compared 

to C1qa-/- mice. PAS staining of kidney sections of Bf/C2-/- mice showed widespread 

germ tube formation confirming the high c.f.u. counts from cultured tissue 

homogenates. In C1qa-/- and MBL-A/C-/- mice germ tube formation was limited in size 

and number. In vitro binding studies demonstrated low efficiency of C1q binding but 

substantial binding of MBL-A and MBL-C. These results support the idea that total lack 

of complement activation in Bf/C2 double knock-out mice leads to uncontrolled tissue 

infection. Deficiency of classical pathway activation has only a low impact on host 

defence against C. albicans whereas the lectin pathway might contribute to the host 

defence against candidosis.

Liangping Li, Gerald Willimsky, Susanne Seitz, Yaxin Xu, Yongping Li, Lope Estevez 

Schwarz, Peter Schlag, Thomas Blankenstein 

SV40 LT-immortalized human primary normal and cancerous 

mammary epithelial cells are phenotypically similar but can 

be distinguished in selection medium and 3D culture 

Cellular immortalization is considered a hallmark of malignancy. Normal human 

mammary epithelial cells (NMECs) have two major in vitro growth restrictions, 

senescence and crisis. However, cancerous mammary epithelial cells (CMECs) that are 

thought to have passed growth barriers in vivo usually cannot be established long-term 

in vitro. Here we show that CMECs deprived of their natural environment and grown in 

conventional complete medium behave similar to NMECs, e.g. they stop producing 

telomerase and become senescent. Like NMECs, CMECs are rescued by SV40 large T 

(LT) from senescence but not from crisis. The telomere length of both NMEC-LT (N-LT) 

and CMEC-LT (C-LT) cells first shortens but later partially recovers after telomerase 

activation. Both cell types upregulate ErbB2 expression, acquire genetic changes, 

remain long-term dependent on LT and ErbB2 and are non-tumorigenic. Thus, CMECs 

are under a comparable in vitro selective pressure as NMECs despite their malignancy in 

vivo. This data demonstrate that most primary breast cancer cells are still unable to 

overcome the in vitro growth restrictions. Despite these similarities, N-LTs and C-LT 

cells cultured in selection medium show different growth in 2D/3D culture and in vivo.

Carmen Kroczek, Athanasia Avramidou, Stephan Feller, Christiane Lang, Hans-Martin 

Jäck, Dirk Mielenz 

Swiprosin-1 - molecular switch in B cell receptor signaling? 

B cell receptor (BCR) signals are essential for B cell differentiation, homeostasis and 

negative selection, which are regulated by strength and quality of BCR signals. 

Recently, we identified a novel adaptor protein, Swiprosin-1/EFhd2, in lipid rafts of B 

cell lines that undergo apoptosis after BCR stimulation. During murine B cell 

development, Swiprosin-1 exhibited highest expression in immature B cells of the bone 

marrow. Overexpression of Swiprosin-1 in the immature murine B cell line WEHI231 

enhanced spontaneous as well as BCR-induced apoptosis by lowering the BCR signal 

threshold. In contrast, shRNA-mediated down-regulation of Swiprosin-1 impaired 

specifically spontaneous and BCR-elicited apoptosis, but not BCR-induced G1 cell cycle 

arrest. Furthermore, Swiprosin-1 downregulation in WEHI231 cells led to increased IκBα 

phosphorylation and degradation after BCR stimulation whereas Erk phosphorylation 

was unchanged. Accordingly, Swiprosin-1 levels regulated net cell growth of WEHI231 

cell populations through reciprocal regulation of Bcl-xL-, but not Bim-levels, thereby 

controlling spontaneous apoptosis. Moreover Swiprosin-1 levels regulate proximal BCR 

signals: Downregulation of Swiprosin-1 prolonged total tyrosine phosphorylation, 

specifically tyrosine phosphorylation of a ~130 kDa protein, and diminished BCR 

induced calcium flux. We also observed co-clustering of a Swiprosin-1-EGFP fusion 

protein with the B-cell receptor in WEHI231 cells. Our data suggest that Swiprosin-1 

levels adjust BCR signaling thresholds in immature B cells. Hence, Swiprosin-1 may 

regulate life span and BCR signals in immature B cells by influencing the proximal BCR 

signaling pathway through as yet unidentified mechanisms.

Anke Osterloh, Ulrich Kalinke, Siegfried Weiß, Bernhard Fleischer, Minka Breloer 

SYNERGISTIC AND DIFFERENTIAL MODULATION OF IMMUNE 

RESPONSES BY HSP60 AND LPS 

Activation of professional antigen-presenting cells (APC) is a crucial step in the initiation 

of an efficient immune response. In this study we show that Hsp60 mediates immune 

stimulation by different mechanisms, dependent and independent of LPS. We have 

demonstrated earlier that both, Hsp60 and LPS, increase antigen-specific IFNgamma 

release in T cells. Here we show that in contrast to LPS Hsp60 induces IFNalpha 

production in professional APC. Neutralization of IFNalpha as well as the absence of 

functional IFNalpha/beta receptor on APC and T cells interfered with Hsp60-mediated 

IFNgamma secretion in antigen-dependent T cell activation, strongly suggesting that 

IFNalpha represents one factor contributing to Hsp60-specific immune stimulation. On 

the other hand, we show that Hsp60 bound to the cell surface of APC colocalizes with 

the LPS co-receptor CD14 and LPS binding sites. Hsp60 specifically binds bacterial LPS 

and both molecules synergistically enhanced IL-12p40 production in APC and 

IFNgamma release in antigen-dependent T cell activation. This effect was Hsp60-specific 

and dependent on LPS-binding by Hsp60. Furthermore, we show that Hsp60 exclusively 

binds to macrophages and DC but not to T or B lymphocytes and that both, T cell 

stimulation by Hsp60 as well as Hsp60/LPS complexes, strictly depends on the presence 

of professional APC and is not mediated by B cells. 

Taken together, our data support an extension of the concept of Hsp60 as an 

endogenous danger signal: Besides its function as a classical danger signal indicating 

unplanned tissue destruction to the innate immune system, in the incident of bacterial 

infection extracellular Hsp60 may bind LPS and facilitate microbe recognition by 

lowering the threshold of PAMP detection and enhancing TLR signaling.

Nousheen Zaidi, Timo Burster, Vinod Sommandas, Timo Herrmann, Bernhard O. 

Boehm, Wolfgang Voelter, Hubert Kalbacher 

Synthesis of novel cell penetrating aspartic proteases 

inhibitors as the targeted inhibitors of antigen processing 

Selective inhibition of enzymes involved in antigen processing such as cathepsin E and 

cathepsin D is a valuable tool for investigating the roles of these enzymes in the 

processing pathway. However, the aspartic protease inhibitors including the highly 

potent pepstatin A (PepA) are inefficiently transported across the cell membrane thus 

have a limited access to antigen processing compartments. Previously described 

mannose-pepstatin conjugates were efficiently taken up by the cells via receptor 

mediated uptake. But the cells that do not carry mannose receptors are not able to take 

up these conjugates efficiently. The aim of the present study was to synthesize new cell 

permeable aspartic protease inhibitors by conjugating pepstatin A with well-known cell 

penetrating peptides (CPPs). To achieve this the most frequently used CPPs namely, Tat 

(49-60), pAntp(43-58) (Penetratin) and 9-mer of L-arginine (R9), were synthesized on 

a trityl resin followed by coupling pepstatin A to the peptides as a complete molecule to 

the N-terminal amino group. The enzyme inhibition properties of these bioconjugates 

and their cellular uptake into dendritic cells, Boleths (EBV-transformed B cell line) and 

MCF7 (human breast cancer cell line) was studied. We found that the bioconjugate PepA- 

Penetratin (PepA-P) was the most efficient cell-permeable aspartic protease inhibitor in 

comparison to PepA. Moreover, we found that PepA-P efficiently inhibited the antigen 

processing in PMBCs (peripheral mononuclear blood cells), primary DCs (dendritic cells) 

and in primary B cells. Therefore, PepA-P can be used in studying the role of 

intracellular aspartic proteases in the MHC class II antigen processing pathway.

Edith Jasny, Martin Eisenblaetter, Tamara Visekruna, Klara Tenner-Racz, Christiane 

Stahl-Hennig, Andres Salazar, Ralph M. Steinman, Klaus Ueberla, Mariagrazia 

Uguccioni, Ralf Ignatius 

Synthetic double-stranded RNA, Poly ICLC, enhances the 

induction of cellular immune responses against protein 

antigens in rhesus macaques 

Background: Ligands for Toll-like receptors (TLRs) are promising adjuvants for the 

induction of humoral and cellular immune responses, as in the design of AIDS vaccines. 

Poly ICLC is a synthetic double-stranded RNA that should bind to TLR3 and has been 

stabilized against serum nucleases, which are present in the plasma of primates. 

Objectives: To study the effects of Poly ICLC on protein-specific immune responses of 

rhesus macaques in vivo and assess its effects on leukocyte subsets in vitro. 

Methods: Monkeys were immunized s.c. with keyhole limpet hemocyanin (KLH) together 

with or without Poly ICLC. Draining lymph nodes were removed after 18 h, and cellular 

and humoral immune responses were determined at several time points in standard 

assays. 

In vitro, human and monkey PBMCs or selected leukocyte subsets were incubated with 

Poly ICLC +/- Lipofectamine and cellular activation and cytokine secretion were 

monitored. 

Results: The application of Poly ICLC led to elevated serum levels of CXCL9 and 

CXCL10; both chemokines were also detected in draining lymph nodes by in situ 

hybridization. KLH-specific cellular immune responses were stronger in Poly ICLC coinjected 

animals than in controls and highest responses were observed in animals 

injected with 0.5 mg/kg Poly ICLC. Humoral immune responses to KLH were enhanced 

also by lower doses of Poly ICLC. The effect of Poly ICLC may not have been due to 

direct activation of dendritic cells (DCs) because rhesus macaque monocyte-derived DCs 

expressed little TLR3 as shown by flow cytometry. Likewise, we neither detected 

increased numbers of mature DCs in the draining lymph nodes following Poly ICLC 

injection nor could primary myeloid monkey DCs be activated by Poly ICLC in vitro. 

Instead, Poly ICLC did activate NK cells in PBMCs, as indicated by CD69 upregulation. In 

addition, Poly ICLC stimulated PBMCs secreted enhanced levels of CXCL9 and CXCL10. 

Conclusion: Poly ICLC has activity as an adjuvant for the induction of protein-specific 

cellular immune responses in monkeys.

Stefanie Frey, Christine D. Krempl, Stephan Ehl 

T cell dependent and T cell independent disease after 

infection with pneumonia virus of mice 

Infection of mice with pneumonia virus of mice (PVM) has been proposed as an 

excellent model to study the pathogenesis of human infection with the closely related 

respiratory syncytial virus (RSV). So far this model allowed interesting observations on 

pulmonary cytokine and chemokine responses, but poor lymphocyte recruitment, failure 

to detect dominant CTL epitopes and evidence for CTL suppression suggested that T 

cells play a minor role. We show that control of a sublethal infection with PVM strain 15 

in C57BL/6 mice was accompanied by a 100-fold increase in pulmonary CTL, 20% of 

which were specific for PVM. T cell deficient mice failed to eliminate PVM and became 

carriers in the absence of weight loss. CTL mediated virus control and weight loss were 

independent of IFN-γ or perforin. Mice with limited numbers of T cells could not achieve 

virus control without weight loss, indicating a tight balance between beneficial and 

detrimental effects of T cell responses to PVM. T cell deficiency slightly delayed but did 

not prevent mortality after high dose infection. These data demonstrate that both T cell 

dependent and T cell independent pathways contribute to PVM disease in a viral dosedependent 

fashion.

Annette I. Garbe, Jerome P. Jayasekera, Michael C. Carroll, Harald von Boehmer 

T cell vaccination with DEC-205-influenza hemagglutinin 

fusion antibodies 

Current influenza vaccines are known to induce antibodies to hemagglutinin (HA) and 

neuraminidase but less is known about their ability to induce cytolytic CD8 T cells to HA 

determinants. In the present study we have compared UV-inactivated influenza virus 

and HA delivered to dendritic cells by DEC-205 fusion antibodies in their ability to 

induce T cell memory. When analysing the generation of CD8 memory cells with a 

transgenic receptor for HA the DEC-205-HA fusion antibodies were found to be 

somewhat superior to UV-influenza virus. Moreover in wt BALB/c mice the fusion 

antibodies induced long term CD8 memory cells that developed into potent effector cells 

exhibiting cytolytic activity and IFN-gamma secretion after rechallenge with UVinactivated 

virus. On the other hand a single shot of DEC-205-HA fusion antibodies did 

not induce HA-specific antibodies whereas UV-inactivated virus did. Nevertheless the 

fusion-antibody vaccination increased HA-specific IgG2a, IgG2b and IgG1 antibodies if 

given prior to challenge with inactivated virus. Finally, in preliminary experiments a 

single dose of DEC-205-HA elicited protection against A/PR/8 virus. The results show 

that the HA-fusion antibody induces effective CD4 and CD8 T cell memory against 

influenza. This approach should be very useful for inducing T cell memory to cellinternal 

viral proteins of which only peptides are presented on the cell surface of 

infected cells and which are not subject to high mutation rates.

Christof Iking-Konert, Tim Vogl, Matthias Schneider, Konrad Andrassy, Gertrud Maria 

Hansch 

T-lymphocytes in patients with primary vasculitides: 

Expression of CD11b identifies activated T cells with the 

propensity to stimulate polymorphonuclear neutrophils 

Objetive 

To gain insight into the immune pathogenesis of primary, ANCA-associated vasculitides 

(AAV) T-lymphocytes of patients were analysed with regard to the expression of 

molecules indicative for current or previous activation. T-lymphocytes of patients with 

Wegener's granulomatosis (WG; n= 47), or microscopic polyangiitis (MPA; n=33) in 

remission were phenotypically and functionally analysed, and compared to Tlymphocytes 

of patients with active disease (n=10), and to cells of healthy donors. In 

vitro, T-cells of a similar phenotype were generated, and their interaction with 

polymorphonuclear neutrophils (PMN) was assessed. 

Results 

During active disease, a small, but conspicuous population of CD8+CD28+CD11b+ was 

found, which produced gamma interferon.Under immunosuppressive therapy, CD11b 

was exclusively seen on CD8+CD28- cells, the latter being more frequent in patients 

with long-lasting or severe disease. In vitro experiments confirmed that CD11b is upregulated 

by activated T-cells, concomitantly with synthesis of gamma interferon. 

During prolonged culture, CD11b remains on the surface, even when CD28 is lost, 

compatible with the notion that CD8+CD28+CD11b+ represent a transient phenotype in 

the course of T-cell activation. In vitro, the gamma interferon-producing T cells 

activated PMN to express CD64 and MHC class II molecules, thus generating the same 

PMN-phenotype as it is seen in patients with active ANCA-associated vasculitis. Of note 

is that a similar phenotype could be generated by supernatants of activated T-cells, or 

by gamma interferon alone, but not by antibodies to proteinase 3. 

In conclusion, CD11b expression identifies activation of a small population of CD8+Tcells 

during active primary vasculitis. By producing gamma interferon, these T-cells 

could activate PMN by a “cross talk” between PMN and T-cells, thus generating a longliving 

and potentially destructive PMN phenotpye.

Sven Meuth, Stefan Bittner, Ole Simon, Heinz Wiendl 

Tandem pore domain potassium channels TWIK-related acidsensitive 

K+ channel 1 (TASK1) and TASK3 modulate effector 

functions of T lymphocytes: a novel therapeutic target for Tcell 

mediated autoimmune diseases 

One decade ago, two-pore domain K+ channels (K2P), a new family of potassium 

channels was described and has since then attracted much attention due to their unique 

physiological properties. These potassium channels contribute to the resting membrane 

potential and a rising diversity of regulatory mechanisms (e.g. pH drop or O2 

deprivation) has been revealed. To date, little is known about their role on non-neuronal 

cells and under pathological conditions. 

We here investigate the role of the TWIK-related acid-sensitive potassium channels 1 

(TASK1) and TASK3 on human T lymphocytes for T cell functions in vitro as well as their 

pathogenic role in a model of multiple sclerosis (adoptive transfer experimental 

autoimmune encephalomyelitis, AT-EAE). TASK1 and TASK3 are expressed on human T 

lymphocytes as indicated by immunocytochemistry and western blotting procedures. 

Pharmacological treatment (bupivacaine, anandamide, spermine and ruthenium red) of 

isolated human CD3+ T cells revealed a dose dependent (~ 40%) reduction of an 

outward current in whole-cell patch-clamp recordings indicative of TASK channels. 

However, the same channel modulators significantly reduced IFNg production and 

decreased proliferation rate of T cells after CD3/CD28 bead stimulation. Kv1.3, an 

established potassium channel for T cell function, was used as control. 

In the next step we addressed the question whether the importance of TASK channels 

for T cell functions could as well be transferred into an in vivo model to demonstrate 

their pathogenetic relevance. AT-EAE was evoked by MBP-specific T lymphocytes and 

application of TASK channel inhibitors resulted in a delayed disease onset, milder 

disease course and earlier recovery. 

Taken together our data describe TASK channels as important mediators of T cell 

function in vitro and in a model of multiple sclerosis in vivo underlining their potential 

for immunotherapeutic strategies.

Kai Dittmann, Anja Uhmann, Ralf Dressel, Heidi Hahn, Jürgen Wienands 

Targeted inactivation of the Hh receptor Ptch abrogates 

lymphocyte development in mice 

The Hh/Ptch signaling system is known to control the development and neoplastic 

transformation of several cell types. However, the role of Hh/Ptch for the differentiation 

of B and T lymphocytes from hematopoietic stem cells (HSC) has not been assessed so 

far. To analyze the function of Hh/Ptch for lymphopoiesis in vivo, we have employed a 

genetically engineered mouse mutant in which the Ptch gene can be conditionally 

inactivated by virtue of the Cre/loxP recombination system. We show that targeted 

disruption of Ptch in the adult organism results in a dramatic specification and 

differentiation defect of the lymphoid lineage leading to rapid disappearance of newly 

generated B and T lymphocytes from peripheral lymphoid organs. The developmental 

block occurs at the level of the common lymphoid progenitor cell (CLP), which defines 

an early branching point of HSC differentiation and lineage commitment. In contrast to 

the lymphoid lineage, development of cell types of the myeloid lineage from common 

myeloid progenitors (CMP) appears normal. Our data identify Hh/Ptch-induced signaling 

as a key regulator for proper development of immunocompetent lymphocytes. Hence, 

the progression of tumors, which are initiated upon oncogenic Hh/Ptch mutations, may 

be further promoted due to impaired tumor surveillance by a compromised immune 

system.

Andreas Goldwich, Sabine Hahn, Sandra Schreiber, Ralf Wagner, Manfred Lutz, 

Eckhardt Kämpgen, Ulrich Schubert 

Targeting HIV-1 Gag into the DRiP-Pathway results in 

enhanced CD8 T cell activation 

The main source of peptides presented by the MHC-I pathway are proteins degraded via 

the ubiquitin proteasome pathway. Different protein substrate pools can be 

distinguished: first, short-lived defective ribosomal products (DRiPs) which are 

degraded in concert with or shortly after their synthesis, and second, functional proteins 

which are entering the standard protein live cycle. 

To analyze the contribution of these substrates to the generation of MHC- I-presented 

peptides, we established murine cell lines which express HIV-1 Gag variants harboring 

degron signals and the murine OVA-derived MHC-I model epitope SIINFEKL (SL). 

Although an HIV-1 Gag variant harboring an N-end degron (UbRGag) displayed wt half 

life, its inherent DRiP-rate was increased, resulting in enhanced MHC-I Ag presentation. 

Additionally, this increased presentation causes a better T cell stimulation of SL-specific 

B3Z hybridoma cells in vitro and adoptively transferred OT-1 T cells in vivo. 

Futhermore, enhanced numbers of SL-specific T cells in vivo were detected by IFN-γ 

ELISPOT after vaccination of naïve C57Bl6 mice in the case of the UbRGag variant. 

These results point out the importance of the DRiP-pathway in adaptive immunity and 

may be relevant for further vaccination studies against tumors or intracellular 

pathogens.

Stephan Schlickeiser, Katharina Tschimmel, Andreas Meisel, Christian Meisel, Inga 

Gebuhr, Uwe Pleyer, Hans-Dieter Volk, Birgit Sawitzki 

TARGETING N-GLYCOSYLATION AFFECTS APC FUNCTION IN 

VITRO AND IN VIVO 

Introduction: Transfer of tolerogenic DCs has become an attractive treatment 

alternative in transplantation and autoimmunity. Although distinct glycosylation patterns 

on tolerogenic immature DCs have been described, the impact of post-translational 

protein modification on DC function is still unexplored. Here we analyzed the effect of 

hypo-N-glycosylation on DC maturation and allo-stimulatory capacity in vitro and in 

vivo. 

Methods: BALB/c bone marrow-derived DCs were treated with the specific alpha-1,2mannosidase 

inhibitor kifunensine. After 24 h, DCs were matured with LPS, TNFa or anti- 

CD40mAb. DC N-glycosylation state (PHA binding), expression of MHCII and CD86 were 

analyzed by flow cytometry. Allo-stimulatory capacity of DCs was determined by 

analyzing the proliferation of co-cultured allogeneic T cells from naïve C57BL/6 mice. 

Capacity of hypo-N-glycosylated endogenous APC to elicit an effective anti-bacterial 

response was tested by injecting kifunensine in a mouse model of stroke (MCAO). 

Results: We detected a significantly enhanced PHA binding capacity in response to 

maturation stimuli. Hypo-N-glycosylation was accompanied by diminished surface 

expression of MHCII and CD86 prior and after maturation. LPS-induced activation of 

hypo-N-glycosylated DCs dramatically diminished their allo-stimulatory capacity as 

proliferation of co-cultured allogeneic T cells was completely abrogated. Control animals 

subjected to MCAO developed spontaneous systemic bacterial infections due to stroke 

induced immunodepression. Interestingly, injection of kifunensine dramatically 

enhanced APC deactivation, increased bacteremia and reduced overall survival rate 

from 75 to 37.5 %. In contrast Sham operated mice receiving kifunensine were not 

affected. 

Conclusions: Our results indicate that, depending on their maturation state, differences 

in protein N-glycosylation levels condition DCs to either stimulate or deactivate T cells. 

Thus, interference with APC N-glycosylation has high potential for tolerance induction in 

transplantation and autoimmune models.

Sebastian Temme, Anna Maria Eis-Hübinger, Peter Kuckenberg 

Targeting of the major histocompatibility complex class II 

pathway by herpes simplex virus type-1 glycoprotein B 

The HSV-1 encoded envelope protein gB mediates contact of the virus with the cell 

surface and initiates fusion with the plasma membrane. We discovered that gB is 

involved in an immune evasion strategy of HSV-1, targeting the MHC class II processing 

pathway, presumably to prevent presentation of viral peptides. Recently, we co-isolated 

complexes of HSV-1gB and MHC class II molecules from HSV-1 infected cells. In further 

studies the intracellular encounter of gB and MHC class II and their subcellular 

localisation was investigated. In transfected cells, invariant chain competes with gB for 

binding to MHC II subunits within the ER and largely prevents association of gB to MHC 

class II molecules. However, in the absence of invariant chain, gB and MHC class II 

subunits form aggregates that are retained in the ER. Despite the large excess of 

invariant chain, in gB-transfected cells some gB is found in association with MHC class 

II. Furthermore, the MHC II/gB complexes were not dectected on the cell surface. We 

conclude, that gB and MHC class II are transported separately and encounter after 

dissociation of invariant chain from MHC class II heterodimers, probably in endosomal 

compartments. Targeting of MHC II heterodimers by gB upon HSV-1 infection may 

silence a CD4+ T cell response.

Theron Johnson, Karsten Mahnke, Dirk Nettelbeck, Volker Storn, Alexander H. Enk 

Targeting of Tumor antigens to dendritic cells (DCs) using a 

novel single chain fragment variable 

DEC205 is a prototype receptor for antigen uptake, which is expressed specifically by 

DCs and increases antigen presentation by 500-fold over other presentation pathways 

such as pinocytosis. To utilize the antigen receptor to load DCs in vivo, we created novel 

single chain fragment variables (ScFv) specific for DEC205, using RT-PCR with 

degenerative primers on total RNA from a hybridoma cell line (HB290). The isolated 

variable heavy, and variable light regions were subcloned into an expression vector, 

fused to a His tag and c-myc tag, then expressed as a native protein in E. coli. The 

inclusion of the two tags allows for the isolation of the protein via a nickel-NTA column 

(His) and immunohistochemical detection (c-myc) via anti c-myc antibodies. In 

addition, we have created a ScFv anti-murine DEC205 fused to the melanoma tumor 

associated antigen gp100. In C57/Bl6 mice, initial immunohistochemical staining of 

CD11c+ lymph node cells showed positive staining of ScFv comparable to the mAb anti- 

DEC205. In further experiments, C57/Bl6 mice were vaccinated with the DEC205 ScFv 

in the footpad or left untreated. After 8 to 16 hours, lymph nodes were isolated and 

examined for DEC staining. Here, the ScFv vaccinated mice showed more c-myc 

positively stained cells in comparison to the untreated controls. The staining pattern is 

similar to immunohistochemical staining seen in mice injected with the mAb anti- 

DEC205. Thus, we can demonstrate that murine ScFv DEC205 can target to CD11c+ 

DCs in vivo. Further experiments using the ScFv DEC205-gp100 will show whether this 

targeting can result in induction of GP100 specific CD8+ T cells and therefore, may be a 

useful tool for the induction of an anti-melanoma response.

Tobias Seibel, Florian Schuetz, Christoph Domschke, Mariana Bucur, Christoph Sohn, 

Philipp Beckhove 

TGFβ and IFN-α regulate Type 1 T-Cell Responses in Primary 

Breast Carcinomas 

INTRODUCTION: 

App. 60% of breast cancer patients spontaneously develop tumor-reactive memory T 

cells in their bone marrow and immune infiltrates have been detected frequently in 

breast cancer lesions. We hypothesized that the local cytokine environment in the tumor 

may regulate antigen specific systemic immunity by influences on immature DCs (iDCs) 

during antigen uptake. 

METHODS: 

We assessed the presence of tumor antigen-reactive T cells in the bone marrow of 123 

primary operated breast cancer patients using ex vivo short term interferon-γ (IFN-γ) 

Elispot assay. Cytokine contents of TGFß1, IL-10, IL-4, PGE2, IFN-α and IL-12 in tumor 

lysates were analyzed using high sensitivity ELISA. Cytokine expression in the tumor 

tissue was visualized by immunfluorescence. The influence of tumor-derived cytokines 

on the capacity of iDCs to prime tumor specific T cell responses was analyzed by IFN-γ 

Elispot-assay after 7 days coculture in vitro of separated naïve T cells from breast 

cancer patients with tumor antigen pulsed autologous DCs pretreated with respective 

cytokines at concentrations present in tumor tissues. 

RESULTS: 

Tumor-specific T cell responses were detected in 45% of patients. These correlated with 

low amounts of TGFβ1 and elevated levels of IFN-α in corresponding tumor tissues. The 

majority (60%) of TGFß1 positive cells in the tumors belonged to a population of 

CD90low stroma fibroblast cells located at the interface between stroma and tumor 

cells. In vitro, pretreatment of iDCs with TGFß1 inhibited, and with IFN-α supported the 

priming of tumor-specific T cells. 

CONCLUSION: 

These results suggest that TGFβ and IFN-α in primary breast cancer tissue regulate the 

induction of systemic anti-tumor T cell responses by influencing functional properties of 

immature dendritic cells at the site of antigen encounter in the tumor tissue.

Guido Wabnitz, Philipp Lohneis, Yvonne Samstag 

The actin bundling protein L-plastin is important for LFA-1 

accumulation in the pSMAC of the immunological synapse 

Recognition of antigen on professional antigen-presenting cells (APC) by T-cells leads to 

a tremendous reorganisation of the actin cytoskeleton resulting in an ordered 

accumulation of activation and adhesion molecules at the interface between T-cells and 

APCs, termed the immunological synapse (IS). These processes are strongly dependent 

on costimulation. One possible candidate for linking costimulation to the reorganisation 

of the actin cytoskeleton is L-plastin. This actin-bundling protein is phosphorylated 

within 5 to 15 minutes after costimulation, but not following TCR engagement alone. 

Phosphorylation occurs exclusively on Ser-5. It is important for the appearance of the T 

cell activation molecules CD69 on the cell surface of costimulated PBT. 

Here we show that L-plastin enriches at the IS between untransformed human 

peripheral blood T-cells (PBT) and superantigen loaded APCs. There, it colocalizes with Factin. 

Time-lapse-video microscopy revealed that the relocalization of L-plastin occurs 

within the first seconds of T-cell/APC contact formation. Thus, it is faster than the 

phosphorylation of L-plastin. Given that a non-phosphorylatable L-plastin mutant (5A- 

LPL) similarly enriches at the IS as wildtype L-plastin, these data show that 

phosphorylation is not required for relocalization of L-plastin to the IS. In contrast, 

deletion of the actin binding sites of L-plastin abrogates its relocation to the IS. The 

same holds true if cells are preincubated with the F-actin destabilizing agent 

cytochalasin D at concentrations which do not drastically diminish the number of cellcell 

contacts. From these data we conclude that L-plastin relocalizes to the IS in an actin 

dependent - but phosphorylation independent - manner. The relevance of L-plastin for 

the IS was analyzed employing an siRNA approach. These experiments revealed that Lplastin 

is crucial for the accumulation of LFA-1 in the IS. Thus, L-plastin contributes to 

the maturation of the IS by enabling the formation of the pSMAC.

Anne Sappok, Anna S. Wenning, Melodie-Jo Wolfs, Tanja Mayer, Bettina Strauß, 

Markus Hoth, Eva C. Schwarz 

The activation and proliferation of primary human CD4+ Tcells 

following focal stimulation 

The immunological synapse (IS) is a highly ordered complex of molecules at the contact 

area between a T-cell and an antigen presenting cell (APC). The formation of the IS is a 

prerequisite for an efficient T-cell immune response. The interaction of T-cell receptor 

(TCR)/CD3 complexes with the major histocompatibility complex (MHC) on APC is 

central to IS formation. The activation of T-cells is characterized by a sustained Ca2+ 

influx through Ca2+ release activated Ca2+ (CRAC)/ORAI1 channels. To focally 

stimulate primary human CD4+ T-cells, we used anti-CD3- and anti-CD28-antibodies 

coated beads. To test if our antibody-coated bead-stimulation mimics the physiological 

stimulation with APC, we analyzed the effector status of our bead-stimulated CD4+ Tcells 

by the expression pattern of two surface proteins CD25 (interleukin-2 receptor) 

and CD62L (L-selectin). We observed up-regulation of CD25 and down-regulation of 

CD62L over two days, indicating T-cell activation. In addition, we observed a reorganization 

of the actin-cytoskeleton at the contact zone between the CD4+ T-cells 

and the antibody-coated beads, which is typical for the formation of an IS between a Tcell 

and an APC. We further analyzed IL-2 secretion and proliferation and their Ca2+dependence 

following bead-stimulation. To determine the doubling rate of the CD4+ Tcells 

under these conditions, we counted the cells over 13 days. Proliferation of CD4+ Tcells 

was preceded by an increase of the cells’ volume. From day 9 on, cell number 

decreased again, probably because apoptosis and necrosis exceeded proliferation. 

SiRNA technology (using Amaxa nucleofector) was established to down regulate several 

membrane proteins (e.g. TRPC3 and STIM1) in bead-stimulated CD4+ T-cells. Our 

results show that bead stimulation of primary human CD4+ T-cells together with siRNA 

technology can be used to analyze physiological functions of membrane proteins during 

T-cell activation.

Stefanie Kliche, Gael Menasche, Dennis Breitling, Mauro Togni, Xiaoqian Wang, Rico 

Pusch, Ana Kasirer-Friede, Theresia E. B. Stradal, Gary A. Koretzky, Burkhart Schraven 

THE ADAP/SKAP-55 SIGNALING MODULE REGULATES TCR- 

MEDIATED INTEGRIN ACTIVATION THROUGH PLASMA 

MEMBRANE TARGETING OF RIAM/RAP1 

Integrins, such as VLA-4 or LFA-1 (beta-1 or beta-2-integrins), play an important role 

during T-cell adhesion to the endothelium and also establishes and strengthens the 

contact between T-cells and APCs. However, on resting T-cells integrins are not 

constitutively adhesive. Rather, external stimuli (e.g. binding of antigen/MHC to the 

TCR) trigger the activation of LFA-1 or VLA-4 and enhance their avidity for their ligands 

ICAM-1, VCAM, or fibronectin (inside-out-signaling). The molecular basis for inside-outsignalling 

is not yet completely understood. Previously, it has been shown that two 

cytosolic adapter proteins, SKAP-55 (Src kinase-associated Phosphoprotein of 55 kDa) 

and ADAP (Adhesion and Degranulating promoting Adapter Protein), are critically 

involved in inside-out-signaling mechanism leading to activation of both LFA-1 and VLA- 

4. Moreover, we have shown that ADAP and SKAP-55 constitutively interact with each 

other and form a functional unit (the ADAP/SKAP-55 module) in T-cells. Structurefunction 

analysis further revealed that either the SH3 domain of SKAP-55 or the central 

proline-rich region of ADAP is absolutely mandatory to facilitate activation of integrins. 

We also show that Rap1-mediated activation of LFA-1 or VLA-4 is impaired in cells in 

which the interaction between ADAP and SKAP-55 has been interrupted. However, this 

is not due to an alteration of Rap1 GTPase activity but rather due to displacement of 

Rap1 from the plasma membrane (Kliche et al., 2006 MCB 26:7130-44). Recently, we 

identified RIAM (Rap1-GTP interacting adaptor molecule), a Rap1 effector protein, as a 

key component linking the ADAP/SKAP-55 module to the small GTPase Rap1 facilitating 

TCR-mediated integrin activation. We show that RIAM constitutively interacts with ADAP/ 

SKAP-55 module in Jurkat T-cells and primary T-cells. Collectively, our data suggest 

that the ADAP/SKAP-55/RIAM complex is required to target activated Rap1 to the 

plasma membrane facilitating TCR-mediated integrin activation (Menasche et al., MCB 

in press).

Stephanie Joachim, Joachim Storf, Norbert Pfeiffer, Franz Grus 

The Analysis of Antibody Patterns in Glaucoma Patients 

Purpose: In previous studies changes in antibody profiles against ocular antigens have 

been shown in sera of glaucoma patients. These findings suggest an autoimmune 

involvement in the pathogenesis of glaucoma at least in some patients. All studies could 

consistently demonstrate up- and down-regulations in immunoreactivities in glaucoma 

patients compared to healthy control subjects. However, all these studies have in 

common that they used crude protein extracts from retina or optic nerve. Therefore, it 

was the aim of this study to analyze the immunoreactivities in sera of glaucoma 

patients against the most important purified antigens identified in previous studies by 

customized protein micro-arrays. 

Methods: Sera of patients with primary open-angle glaucoma (n=50) and healthy 

controls (n=50) were included in this study. The protein arrays were prepared by 

spotting commercial available antigens onto special nitrocellulose-coated slides. Up to 

80 different antigens were used in each customized protein micro-array. The arrays 

were incubated with sera of patients (1:25, overnight) and the antibody-antigenreactions 

were visualized by chloronaphthol staining. After digitizing, the spot intensities 

were compared and analyzed by multivariate statistical techniques. 

Results: Using protein micro-arrays, we were able to detect immunoreactivities in sera 

of patients and healthy controls against purified antigens like heat shock protein 27, 60, 

and 70, and glial fibrillary acidic protein, glutathione-S-transferase, α-fodrin, and αcrystallin. 

The statistical analysis revealed a significant difference (P

Bettina Jux, Markus Frericks, Charlotte Esser 

The arylhydrocarbon receptor is a critical regulator of 

Langerhans cell maturation 

Langerhans cells (LC) are the professional antigen-processing cells of the epidermis. 

The transcription factor arylhydrocarbon receptor (AHR) controls expression of the 

xenobiotic metabolizing enzymes which degrade many low molecular weight chemicals 

(LMWC) present in the natural environment. Metabolic transformation of LMWC may 

result in their covalent protein-binding and use as haptens, and eventually allergic 

reactions, including contact dermatitis. We studied whether and how LC have strategies 

to avoid this risk. 

We found that the AHR is abundantly expressed in LC, but its signalling is impeded, as 

treatment with a known strong AHR ligand (2,3,7,8 tetrachlorodibenzo-dioxin) does not 

lead to gene induction. The concomitant high constitutive expression of the AHRrepressor 

in LC might explain this finding, and points to a relevant tolerogenic strategy 

of LC. We therefore investigated LC maturation and function in AHR-deficient mice. We 

found that (i) their LC express the AHR-repressor only weakly, (ii) AHR ko LC were 

arrested in maturation, i.e. they remained smaller and less granular, and did not 

upregulate expression of co-stimulatory molecules CD40, CD80, and CD24 after in vitro 

maturation, and (iii) their phagocytic capacity was affected. GM-CSF, a potential inducer 

of co-stimulatory molecules, is secreted in significantly lower amounts by AHR-ko 

epidermal cells (i.e. keratinocytes and LC). Our data suggest that the AHR has a critical 

role in maturation of LC and is part of their risk strategy against unwanted immune 

activation by potential skin allergens.

Sonja Ortler, Christoph Leder, Michel Mittelbronn, Alla Zozulya, Lieping Chen, Antje 

Kroner, Heinz Wiendl 

The B7-homologue 1 (B7-H1/PD-L1) contributes to 

confinement of immunopathological CNS damage: 

implications for Multiple sclerosis 

The coinhibitory B7-H1 critically influences adaptive immune responses both in the 

initiation and the effector phases of experimental autoimmune encephalomyelitis (EAE), 

an animal model of multiple sclerosis. To further understand the role of B7-H1/PD1 

pathway in the CNS autoinflammation, we analyzed the kinetics of adaptive and 

regulatory immune responses in MOG 35-55 induced EAE using B7-H1 -/- mice. Here, we 

show that B7-H1 -/- mice exhibit an accelerated disease onset and significantly 

exacerbated EAE severity following MOG immunization. This disease pattern was 

followed by significantly different kinetic of IFNγ secretion suggesting an involvement of 

this molecule in maintaining the survival of antigen-specific T cells during EAE. In 

accordance with EAE data, the numbers of T cells in the CNS were increased in B7-H1 -/- 

mice with significantly elevated frequency of infiltrating neuroantigen-reactive T cells. 

Additionally, we demonstrate that MOG-specific T cell presence in B7-H1 -/- mice are 

exclusively restricted to the CNS depending on the stage of the disease. B7-H1 

specifically expressed by antigen presenting cells (APC) had an inhibitory effect on MOGreactive 

T cells. Although B7-H1 had no influence on B cell responses, the absence of 

B7-H1 was associated with lower numbers of CD8 + CD28 - regulatory T cells. We also 

describe a strong upregulation of B7-H1 with highest immunoreactivity in areas of 

severe inflammation in human brain lesions during MS. 

Our findings demonstrate the critical importance of B7-H1 as an immune-inhibitory 

molecule capable of downregulating encephalitogenic T cell activation and expansion in 

the CNS thus contributing to the confinement of immunopathological damage.

Thomas Ebensen, Kai Schulze, Peggy Riese, Michael Morr, Carlos A. Guzmán 

The bacterial second messenger cdiGMP exhibits promising 

activity as mucosal adjuvant 

The development of mucosal adjuvants is still a critical need in vaccinology. In the 

present work we show that bis-(3′,5′)-cyclic dimeric guanosine monophosphate 

(cdiGMP), a second messenger that modulates cell surface properties of several microorganisms, 

exerts potent activity both as mucosal adjuvant. BALB/c mice were 

immunized by intranasal route with the model antigen beta-Galactosidase (beta-Gal) coadministered 

with cdiGMP. Animals receiving cdiGMP as adjuvant showed significantly 

higher anti-beta-Gal immunoglobulin G (IgG) titers in sera than controls (i.e. 512-fold; 

p

Martin Klemke, Elisabeth Kramer, Guido Wabnitz, Mathias Konstandin, Yvonne 

Samstag 

The chemokine SDF-1α induces dephosphorylation of the actin 

remodelling protein cofilin via a Gi/Ras/MEK pathway in 

primary human T cells 

The homeostatic and inflammatory migration of T cells is mediated via several 

chemokines which act on different G protein-coupled receptors (GPCR). CXCR4, the 

receptor for the chemokine SDF-1α, is expressed by most human T cell subpopulations. 

SDF-1α treatment activates multiple downstream signaling pathways, e.g. the MEK- and 

PI3K-pathways, which finally leads to reorganization of the actin cytoskeleton. However, 

the exact molecular mechanisms of CXCR4-mediated actin reorganization in primary 

human T cells are still unclear. The actin-binding protein cofilin is a central regulator of 

actin dynamics through its actin depolymerizing and severing activity. In resting human 

T cells, most of the cofilin molecules are inactivated through phosphorylation at Ser3. 

Here we show, that cofilin is dephosphorylated/activated upon treatment of primary 

human T cells with SDF-1α. This dephosphorylation is mediated via a Gi/Ras/MEK 

pathway, whereas the PI3K pathway is not involved. Interference with the cofilin 

dephosphorylation pathway by inhibition of MEK results in enhanced SDF-1α-induced 

actin polymerization and reduced velocity during SDF-1α mediated cell migration. These 

data imply that dephosphorylation/activation of cofilin through a Gi/Ras/MEK signaling 

pathway is necessary for the proper reorganization of the actin cytoskeleton during SDF- 

1α mediated migration of primary human T cells.

Birgit C Viertlboeck, Sonja Schweinsberg, Matthias A Hanczaruk, Ramona Schmitt, 

Louis du Pasquier, Friedrich W Herberg, Thomas W Göbel 

The chicken leukocyte receptor complex encodes a 

primordial, activating, high affinity IgY Fc receptor. 

Fc receptors are key players of the immune system that link the fine specificity of 

immunoglobulins and innate effector responses. Here we describe the first nonmammalian 

Fc gamma receptor, CHIR-AB1, a member of the leukocyte receptor 

complex, that binds IgY with high affinity with its single Ig domain. It is expressed on 

immature and mature B-lymphocytes, monocytes, macrophages and natural killer cells 

and harbours motifs of activating and inhibitory Fc receptors. In the absence of the 

common gamma chain, CHIR-AB1 can be expressed on B cells, but crosslinking does 

not induce intracellular calcium release. In contrast, cells expressing CHIR-AB1 and 

common gamma chain are triggered to release intracellular calcium upon stimulation 

with heat-aggregated IgY. CHIR-AB1 thus represents a novel primordial Fc receptor that 

combines features of different mammalian counterparts.

Matthias A. Hanczaruk, Thomas W. Göbel, Birgit C. Viertlboeck 

THE CHICKEN TRIGGERING RECEPTOR EXPRESSED ON 

MYELOID CELLS (TREM) 

Triggering receptors expressed on myeloid cells (TREM) represent a multigene family on 

human chromosome 6 with numerous members. TREM homologues have recently been 

identified on chicken chromosome 26 as members of the Immunoglobulin-like receptor 

family including activating and inhibitory receptors. In contrast to human TREMs, in 

chicken there is only one activating member, TREM-A1, characterized by one 

extracellular Ig domain and a positively charged transmembrane residue. There are two 

inhibitory members, TREM-B1 and TREM-B2, displaying cytoplasmatic ITIMs. Three 

different splice variants of TREM-B2 have been identified, one variant with only one Ig 

domain, one with two and one with four Ig domains, whereas there’s only one variant of 

TREMB-1 containing two Ig domains, apparently. 

Gene expression pattern for the various chicken TREM were analyzed by real-time RT- 

PCR and normalized on the housekeeping gene 18S RNA. By employing real-time RT- 

PCR with primers spanning Ig domain and transmembrane region of TREM-A1, we 

showed that TREM-A1 mRNA is highly expressed in brain, bone marrow and 

macrophages. We also found a variant of TREM-A1, mainly occurring in thrombocytes, 

by using primers specific for the single Ig-domain of TREM-A1 in real-time RT-PCR, 

indicating that there is a real splice-variant. High mRNA levels of TREM-B1 were 

detected in thrombocytes and bone marrow. The splice variant of TREM-B2 containing 

four Ig domains is highly expressed in macrophages and bone marrow, too. After 

stimulation of macrophages with LPS, APEC LPS, IFNγ and bacterial DNA the mRNAexpression 

of all three members, especially of TREM-A1, is downregulated. 

TREM-A1 shows high homology with human TREM2, whereas TREM-B1 is more similar 

to human TLT-1. Apparently there is no mammalian homologue for TREM-B2. 

We have recently generated a monoclonal antibody against TREM-A1, too, which 

stained virtually macrophages by flow cytometry, which will be used for further 

investigations. 

In summary some of the chicken TREMs share mammalian homologues implicating a 

conserved TREM system in the chicken, whereas others might be unique to the chicken.

Barbara Simm, Matthias Witt, Monika Braun, Barbara Mosetter, Rudolf Gruber, Dolores 

Schendel, Christine Falk 

The correlation of CD6 expression with infiltration of NK cells 

into synovial fluid of Arthritis patients. 

The repertoire of infiltrating NK cells into synovia of patients with inflammatory arthritis 

has been shown to be reversed with respect to the major NK subpopulations that are 

defined as CD16 + CD56 dim and CD16 - CD56 bright NK cells. We analyzed 14 paired 

samples of synovial fluid and peripheral blood from patients with various rheumatic 

diseases regarding the phenotypical and functional characteristics of the infiltrating NK 

cells. Therefore, we focussed on possible correlations of various NK markers with 

infiltration, on the cytotoxic capacitiy and on the recognition of ULBP4, a ligand for the 

activating NK receptor NKG2D. 

The phenotype of the synovial and peripheral lymphocytes was analysed by screening 

>20 markers. HLA and KIR typing was performed using the Luminex SSO-Typing 

Method. For the determination of the cytokine profiles, we applied Multiplex Bead Arrays 

of 50 analytes. The function of synovial NK cells was defined by CD107a-degranulation 

assays. ULBP4-transfectants of C1R were utilized to discriminate differences in NKG2Dmediated 

activation between peripheral and synovial NK cells. 

Our results confirmed the predominance of the CD56 bright CD16 - NK subpopulation in 

the synovial fluid. Amongst all 20 markers tested, CD6 showed the strongest correlation 

with this NK cell infiltration. In the periphery, CD6 expression on NK cells strongly 

correlates with CD16 + CD56 dim subgroup. Remarkably, almost all NK cells in the CD16 - 

subset infiltrating into the joints also are CD6 negative. Our results indicate that CD6 is 

differentially expressed on NK cell subsets and presents a novel NK cell marker for 

infiltration into synovial tissue. These infiltrating CD56 bright CD6 - NK cells display 

cytotoxic capacity and were additionally activated by ULBP4. The cytokine pattern of 

synovial fluid showed an increase of proinflammatory cytokines and chemokines that 

may contribute to the attraction of these CD6 - NK cells into tissues, in general.

Edgar Serfling, Rene Rost, Wen Chen, Sergei Chuvpilo, Eisaku Kondo 

The Crosstalk between NF-kB and NFATc1/aA, the Inducible 

Short Isoform of NFATc1, Protects B-Lymphocytes against 

Apoptosis 

Among the NFATc proteins NFATc1, c2 and c3 which are expressed in lymphocytes, the 

synthesis of NFATc1 is strongly induced upon immunoreceptor and co-receptor 

stimulation. This concerns mainly NFATc1/alphaA, a short isoform of NFATc1 which 

differs in both its N- and C-terminal peptides from other NFATc proteins. Whereas 

NFATc2 and c3 exert a pro-apoptotic activity, we show here that (i) inactivation of the 

Nfatc1 gene in chicken DT40 B cells results in a marked increase of B cell receptor and 

Ca++- induced apoptosis, whereas (ii) re-expression of NFATc1/alphaA in these cells 

leads to a delay in apoptosis induction which can be rescued by low doses of cyclosporin 

A. In addition to the induction of Bcl-6 and BAG-2 (Bcl-2 associated anthanogene 2), 

this is due to the NFATc1/alpphaA-mediated increase in PKC-theta• levels and further 

proteins controling NF-kappaB induction. Our data show that by enhancing NF-kappaB 

levels, NFATc1/alphaA renders lymphocytes resistant to apoptosis when it is expressed 

at high concentrations.

Martin Holdener, Edith Hintermann, Eric F. Johnson, Michael P. Manns, Matthias G. 

von Herrath, Urs Christen 

The CYP450 mouse model for autoimmune hepatitis: Breaking 

of self-tolerance in transgenic CYP2D6 and wildtype FVB-mice 

by viral infection 

The etiology of autoimmune hepatitis (AIH) is poorly understood although the major 

autoantigen, cytochrome P450 2D6 (CYP2D6), has been identified and immunodominant 

epitopes mapped. Therefore, we generated an animal model for human AIH using the 

natural autoantigen CYP2D6. We infected transgenic mice expressing human CYP2D6 in 

the liver (CYP-2D6 mice) with an Adenovirus-CYP2D6 vector (Ad-2D6) to break selftolerance. 

Surprisingly, upon infection with Ad-2D6 not only transgenic CYP2D6 mice 

but also wildtype FVB mice showed several persistent features characteristic for liver 

damage associated with AIH. These features included massive hepatic fibrosis, ‘fused’ 

liver lobules, disorganized architecture, cellular infiltrations and focal to confluent 

necrosis. Further, all Ad-2D6-infected mice generated high titers of anti-CYP2D6 

antibodies. Epitope mapping revealed that such anti-CYP2D6 antibodies predominantly 

recognized the core peptide sequence AQPPRD (CYP2D6 aa265-270), which is the 

immunodominant linear epitope recognized by LKM-1 antibodies from AIH patients. In 

contrast, mice infected with a control Adenovirus did neither develop liver damage nor 

generated anti-CYP2D6 antibodies. Interestingly the kinetics and severity of liver 

damage as well as antibody formation was enhanced in wildtype FVB mice compared to 

transgenic CYP2D6 mice. Our data indicate that the liver damage was reduced and 

delayed in transgenic CYP2D6 mice due to a certain degree of tolerance towards human 

CYP2D6 compared to wildtype FVBs, which do not express the human version of the 

2D6 isoenzyme. In wildtype FVB mice, due to the homology of the mouse isoenzymes of 

the CYP superfamily to human CYP2D6, autoimmune liver damage by Ad-2D6-infection 

was possibly induced via true ‘molecular mimicry’.

Tarvo Rajasalu, Wolfram Karges, Andreas Spyrantis, Andreas Wieland, Bernhard 

Böhm, Reinhold Schirmbeck 

The diabetogenic, insulin-specific CD8 T cell response primed 

in the experimental autoimmune diabetes model in RIP-B7.1 

mice 

Type 1 diabetes mellitus can result from the specific destruction of pancreatic beta cells 

by autoreactive T cells. As shown here, experimental autoimmune diabetes (EAD) is 

efficiently induced in RIP-B7.1 mice by preproinsulin (ppins)-encoding DNA vaccines. 

EAD develops in RIP-B7.1 mice within 3-4 weeks after a single immunization with ppinsencoding 

plasmid DNA. RIP-B7.1 mice develop insulitis, insulin deficiency and 

hyperglycemia after vaccination with plasmids encoding either murine ppins-I, murine 

ppins-II, or human hu-ppins. EAD induction critically depends on CD8 T cells and was 

independent of CD4 T cells. To be diabetogenic, ppins-specific CD8 T cells had to 

express IFNgamma. Neither expression of perforin, nor signaling through the type I 

interferon receptor are essential components of this pathogenic CD8 T cell phenotype. 

Using plasmids encoding truncated ppins variants, we show that EAD is only induced by 

DNA vaccines encoding the insulin A-chain. Diabetogenic CD8 T cells specifically 

recognize the Kb-restricted A12-21 epitope of the insulin A-chain. The RIP-B7.1 model 

hence represents an attractive model for the characterization of cellular and molecular 

events involved in the CD8 T cell- mediated immune pathogenesis of diabetes.

Stephan Thurau, Maria Diedrichs-Möhring, Christiane Hoffmann, Gerhild Wildner 

The distribution of uveitogenic GFP+ T cells in a rat model of 

uveitis 

Uveitis is an inflammatory autoimmune disease of the eye, mediated by CD4+ T cells 

specific for ocular antigens. In Lewis rats experimental autoimmune uveitis (EAU) can 

be induced by adoptive transfer of retina specific CD4+ T cells. In order to investigate 

the fate of uveitogenic T cells after ocular inflammation, during spontaneous relapses as 

well as during experimental reinduction of disease and after extended remission we 

injected GFP-transduced CD4+ T cell lines specific for the IRBP-peptide R14 in rats that 

developed uveitis 5-6 days later. At day 27 these rats were immunized with peptide R14 

in CFA and developed a uveitis relapse around day 35. In another experiment GFP+ CD4 

+ T cells specific for Ovalbumin (OVA) were adoptively transferred to Lewis rats. Nine 

days later rats were immunized with retinal peptide PDSAg in CFA to induce uveitis. 

Eyes, lymph nodes and spleens were collected at different time points. Six weeks after 

transfer GFP+ T cells of either specificity were observed in the retinas. In eyes with 

uveitis - either after spontaneous or induced relapse - the numbers of intraocular GFP+ 

cells increased slightly. Clusters of retina-specific GFP+ T cells were detectable, 

suggesting clonal expansion or focal attraction of new GFP+ cells invading from the 

periphery, whereas in clinically quiet eyes single GFP+ cells were distributed within the 

retina. OVA-specific T cells also migrated into the retina independently of EAU 

development, but never formed clusters in either active inflammation or quiescent 

stage. GFP+ cells were also found in peripheral lymph nodes (inguinal, mesenteric) and 

spleens more than 5 weeks after adoptive transfer. GFP+ cells were seen in the T cell 

areas of the spleen and in lymph nodes at any time during the observation period. 

Activated T cells of any specificity can invade the ocular tissues and remain viable within 

the eyes and lymphoid tissues for an extended time period without undergoing 

apoptosis. They are recruited to the eye from the periphery during a secondary disease 

course, but may also clonally expand in situ after antigen-specific stimulation.

Tobias Haas, Jochen Metzger, Frank Schmitz, Antje Heit, Eicke Latz, Hermann Wagner 

The DNA sugar backbone 2-deoxyribose determines Toll-like 

receptor 9 activation 

Toll-like receptor 9 (TLR9) is believed to represent a pattern recognition receptor 

specific for pathogen derived DNA, equipped with competence for “foreign-self” 

discrimination. CpG-dinucleotides, abundant in microbial DNA yet largely absent from 

mammalian DNA, are considered important attributes of “foreign”. Based on 

experiments using phosphorothioate (PS)-modified oligodeoxynucleotides (ODN) to 

obviate DNase-sensitivity and inefficient uptake of natural phosphodiester (PD) ODN, 

TLR9 has been found to require such CpG-motifs within ss PS ODN to become activated. 

We show that different rules apply for TLR9-mediated recognition of unmodified, natural 

DNA: PD ODN devoid of CpG, unlike PS ODN, activate TLR9 when translocated into 

TLR9-containing endosomes of immune cells. Determinant of this CpG-motif 

independent recognition is the PD 2-deoxyribose sugar backbone, which, as base-free 

homopolymer, binds to and basally activates TLR9. Random bases add to the 

stimulatory potential, even atypical bases like uracil are tolerated. Substitution of the 2deoxyribose 

sugar backbone with ribose, however, abrogates TLR9-binding and 

stimulation. In contrast, homopolymeric, base-free PS 2-deoxyribose is bound by TLR9 

with 100fold higher affinity than PD 2-deoxyribose and, surprisingly, has a potent 

competitive-inhibitory effect on ligand induced TLR9 activation, both in vitro and in vivo. 

Adding random bases does not alter this dominant negative effect, whereas adding CpGmotifs 

reverses it and thus results in the well-known, robust TLR9-stimulatory activity 

of PS CpG ODN. These data show that the sugar backbone 2-deoxyribose determines 

TLR9-activation and explain the current misconception that CpG-motifs {interpreted as 

pathogen associated molecular patterns (PAMPs)} are crucial for DNA-recognition by 

TLR9. We propose that TLR9 is a receptor for natural DNA with no inherent ability to 

discriminate “foreign” from “self”. Expressed within endosomes, TLR9 is not exposed to 

extracellular “self”-DNA yet poised to recognise DNA from pathogenic invaders.

Dieter Kube, Diana Pinkert, Nils Schoof, Frederike von Bonin, Jennifer Theiss, Fred 

Schaper, Johannes Bode, Martina Vockerodt 

The EBV oncoprotein Latent Membrane Protein 1 affects the 

expression of suppressors of cytokine signaling (SOCS) in 

transformed B lymphocytes 

Constitutive activation of signal transducer and activator of transcription 1 and 3 

(STAT1, STAT3) is a distinctive feature of Epstein-Barr virus (EBV) infected B cells 

characterized by the latency program III. The expression of STAT1 and the activation of 

STAT3 in these cells can be modulated by the latent membrane protein 1 (LMP1), a 

protein with known oncogenic properties. The mechanism of activation and the 

functional responses are still unclear. The expression of full length LMP1 is responsible 

for an enhanced STAT1 protein expression in Burkitt lymphoma cells (BL). STAT3 is 

tyrosine phosphorylated without changes in STAT3 protein levels. A specific pattern of 

Janus kinases (Jak) is activated by EBV, in part by LMP1 mediated activation of 

respective cytokines. In LMP1 expressing BL cells suppressor of cytokine signaling 

(SOCS) 3 mRNA is expressed involving transcriptional and posttranscriptional 

mechanisms. The SOCS3 mRNA half life is regulated by LMP1 mediated p38/SAPK2 

activation. Jak3 is involved in SOCS3 expression in BL cells as shown by specific 

inhibitors. LMP1 activates SOCS3 promoter elements. Overexpression of SOCS3 

prevents to a defined extend the activation of STAT3 by cytokines. SOCS1 mRNA is 

expressed in all analysed cells independent from EBV. Our results support the role of 

LMP1 in modulating cytokine signaling (supported by DFG Ku 954/7-2 and the 

Graduiertenkolleg 1034).

Dong Wang, Melanie Drenker, Thomas Werfel, Miriam Wittmann 

The epidermal inflammatory response in lupus erythematosus 

shows an autocrine amplification loop. Major role of 

TNF&alpha and IL-18. 

Cutaneous manifestations belong to the most common clinical features of lupus 

erythematosus (LE). Tissue resident cells are increasingly being recognised as active 

cells in the pathophysiology of various autoimmune diseases. Evidence is accumulating 

that keratinocytes play an important role in regulating and maintaining the pathology in 

cutaneous LE. It was the aim of this study to analyse differences in the inflammatory 

response of keratinocytes from patients suffering from LE as compared to healthy 

controls. We analysed keratinocytes outgrown from epidermal stem cells from the outer 

root sheet of the hair follicle and could determine marked differences of keratinocytes in 

response to proinflamatory mediators. Main findings by flow cytometry and ELISA 

include that keratinocytes from LE patients express higher IL-18R on their cell surface in 

response to TNF&alpha or IFN&gamma stimulation. Furthermore in response to IL-18 

these cells produced large amounts of TNF&alpha but failed to express the cellprotective 

IL-12p40. IL-12 has been shown to protect keratinocytes from UV induced 

apoptosis. We found that keratinocytes derived from LE patients are as well protected 

as normal keratinocytes by IL-12 but not IL-18. In contrary, keratinocytes from LE 

patients were more prone to die upon exposure to IL-18. Our results demonstrate an 

intrinsic difference in the inflammatory response of keratinocytes and suggest an 

autocrine positive feedback loop involving TNF&alpha, IL-18 and IL-12 family members. 

These results point to a potential benefit of a local anti-TNF&alpha or anti-IL-18 therapy 

for cutaneous lupus manifestations.

Andreas Vilcinskas, Boran Altincicek 

The greater wax moth Galleria mellonella as a mini-host 

model for human pathogens and as a reservoir for novel 

peptide antibiotics 

Insects attract increasing attention as mini-host models for human pathogens to 

analyse infection and immunity at cellular and molecular level. Lacking adaptive 

immunity, insects are particularly suitable models to elucidate interaction between 

virulence factors and the innate immune system. Particularly one lepidopteran, the 

greater wax moth Galleria mellonella, has recently been emerged as a powerful model 

to study pathogenesis of prominent bacteria and yeasts causing severe diseases in 

humans (Chamilos et al. 2007, Lancet Infect Dis.7, 42-55). G. mellonella larvae provide 

a number of advantages, for example, they can be reared at temperatures to which 

human pathogens are adapted and which are essential for the synthesis of their 

virulence factors among which thermolysin-like metalloproteinases play a predominant 

role. The latter were discovered to mediate sensing of infection and activation of innate 

immune responses in G. mellonella in the sense of the danger model introduced by Poly 

Matzinger (Altincicek et al. 2007, Infect Immun 75, 175-183). 

In addition, combined transcriptomic and proteomic analyses of innate immunity in G. 

mellonella led to the identification of a diverse array of antimicrobial peptides among 

which the first and to date only inhibitor of microbial metalloproteinases from animals 

was discovered. This insect metalloproteinase inhibitor (IMPI) specifically inhibits toxic 

enzymes which are responsible for a broad spectrum of pathological symptoms during 

infectious diseases in humans, and which have been recognized as promising targets for 

the rational design of second generation antibiotics (Wedde et al. 2007, Biol Chem 388, 

119-127). Presently, we investigate the 3D-structure of the IMPI as a template for the 

design of novel peptide antibiotics.

Leslie Elsner, Vijayakumar Muppala, Mathias Gehrmann, Jingky Lozano, Heike 

Bickeböller, Thomas Herrmann, Lutz Walter, Frauke Alves, Gabriele Multhoff, Ralf 

Dressel 

The heat shock protein HSP70 promotes NK cell activity 

against tumors which express inducible NKG2D ligands 

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous 

danger signal which can increase the immunogenicity of tumors and induce CTL 

responses. We show here that HSP70 also activates NK cells which recognize stressinducible 

NKG2D ligands on tumor cells. 

Tumor size and the rate of metastases derived from HSP70-overexpressing human 

melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in 

SCID/beige mice which lack additionally functional NK cells. NK cells derived from SCID 

mice with HSP70-overexpressing tumors showed in vitro an augmented cytotoxicity 

against cells which expressed NKG2D ligands, such as MHC class I chain-related (MIC) A 

and B molecules. MICA/B expression was found to be induced in the tumors. 

Interestingly, a counter selection was observed against the expression of MICA/B in the 

HSP70-overexpressing melanomas compared to control melanomas in SCID, but not in 

SCID/beige mice. In accordance with these observations, HSP70-containing exosomes 

from HSP70-overexpressing tumor cells in contrast to HSP70-negative exosomes from 

control tumor cells were able to activate mouse NK cells in vitro to kill YAC-1 cells which 

express NKG2D ligands or human melanoma cells in which MICA/B expression was 

induced by pharmacological means. Recombinant HSP70 had similar effects on the NK 

cells as tumor cell-derived HSP70-positive exosomes. 

The synergistic activity of two stress-inducible danger signals, HSP70 and MICA/B, 

apparently leads in this model to enhanced activation of NK cells and results in a 

reduced tumor growth and completely suppresses metastatic disease. 

This work was supported by grants from the DFG (DR 394/2, GRK 1034) and the EU 

(MRTN-CT-2004-512253; TRANS-NET).

Aysefa Doganci, Petra Scholtes, Joachim Maxeiner, Roman Karwot, Edgar Schmitt, 

Hans A. Lehr, I.Cheng Ho, Susetta Finotto 

The IL-2R alpha and IL-2R beta chains differentially regulated 

allergic airway inflammation in an experimental in vivo model 

To assess the role of IL-2 signal transduction in experimental asthma, we compared the 

effect of intranasal blockade of the IL-2Rα or β-chains. In our studies, local blockade of 

the IL-2Rβ-chain, led to an amelioration of airway resistance and inflammation in OVA 

sensitized and challenged mice. Moreover, both treatements led to a marked reduction 

of Th2 type cytokines IL-4, IL-5 while IL-13 was inhibited specifically by the blockade of 

the IL-2Rβ-chain. Consistent with these findings, blockade of the IL-2Rβ-chain, as 

opposed to blockade of the IL-2Rα chain, led to a suppression of CD4+ T cells 

proliferation in the local draining lymph nodes where immunosuppressive CD4+CD25+ 

T regulatory cells were downregulated specifically only by the IL-2Rα blockade. Taken 

together these results suggest that local blockade of the IL-2Rβ-chain during the 

antigen challenge phase could be a successful therapy in allergic disease by rescuing 

the T regulatory cells in the local draining lymph nodes where they regulate the 

development of pathogenic Th2 cells in this experimental model of asthma. 

This work is supported by SFB 548 Grand.

Christine S. Falk, Dominik ter Meer, Iris Bigalke, Alexander Buchner, Barbara 

Mosetter, Barbara Simm, Monika Braun, Dusan Prevalsek, Dolores Schendel, Hans- 

Jochem Kolb 

The Impact of HLA-C and KIR Genetics on Survival of AML 

versus ALL Patients following Haploidentical Bone Marrow / 

Stem Cell Transplantation. 

Introduction: Recent evidence suggests that alloreactive NK cells in haploidentical BMT/ 

SCT may provide graft-versus-leukemia (GVL) effects. In particular, the constellation of 

KIR-positive donor NK cells in absence of inhibitory HLA-C ligands in the patient may 

provide advantages for AML patients. Currently, clinical protocols attempt to benefit 

from NK alloreactivity by selecting KIR/HLA-C incompatible donors. Materials and 

Methods: Since 1996, more than 100 patients with leukemic malignancies have been 

transplanted with haploidentical bone marrow at day 0 followed by G-CSF-mobilized T 

cell-depleted peripheral blood stem cells (PBSC) at day 6 under immune suppression 

(ATG, MTX, CyA). PBSC derived from 37 donors were characterized with respect to NK 

and T cell subpopulations prior to and after T cell depletion. Functionality was 

determined by cell CD107a degranulation and cytokine secretion against K562 and 

some haploidentical recipient cells. HLA-C and KIR genes of 80 patient/donor 

combinations were typed by the Luminex-based SSO method. Results and Conclusions: 

Although complete haploidentical bone marrow was transplanted at day 0, no 

enhancement of acute GVHD compared to HLA-identical matched unrelated BMT was 

observed in 98 haploidentical transplanted patients – independently from HLA-C or KIR 

constellations. Complete chimerism was established early post transplantation. In 

general, T cell-depleted PBSC contained more NK cells than CD34+ stem cells 

demonstrating that high numbers of alloreactive NK cells do not correlate with aGVHD. 

Functional analyses of NK subpopulations in PBSC prior to and after T cell depletion 

revealed i) differences in cytokine secretion among particular donor/recipient 

constellations, ii) weak cytotoxic activity against K562, iii) changes in the composition of 

KIR-positive and KIR-negative NK subpopulations, vi.) Most importantly, the 

combination of HLA-C and KIR haplotypes revealed different constellations for AML vs. 

ALL patients that correlate with significantly increased propability of survival.

Kristina Kunert, Constanze Schönemann, Hans-Dieter Volk, Ruth Neuhaus, Andreas 

Pascher, Claudia Wenzel, Peter Neuhaus, Johann Pratschke, Katja Kotsch 

The impact of KIR/HLA ligand incompatibility on acute 

rejection episodes after orthotopic liver transplantation 

Killer-cell immunoglobulin-like receptors (KIRs) are expressed on natural killer cells 

(NK) and subpopulations of T cells of the memory phenotype. They encode both, 

inhibitory and activating receptors within the leukocyte receptor complex (LRC) located 

on chromosome 19. Several studies reported that incompatibility between donor KIR 

receptors and their corresponding HLA ligands in the recipient thus leading to 

alloreactivity of NK cells reduces the extent of Graft versus Host Disease (GvHD) and 

minimizes the risk of relapse following bone marrow transplantation. Our own studies 

demonstrated the influence of KIR/HLA-ligand incompatibility on the development of 

acute cellular rejection following renal transplantation. Recently it has been shown that 

particularly HLA-C epitope disparity between donors and recipients influences 

posttransplant liver outcome (Bishara et al., Human Immunol 2005). Because NK cells 

infiltrate liver allografts very early after transplantation (Obara et al., Am J Transplant, 

2005), we examined the relevance of KIR/HLA-ligand incompatibility in acute rejection 

following orthotopic liver transplantation. Analysis considered respective matches and 

mismatches between KIR receptors of the recipients and corresponding ligands of donor 

grafts. 28 patients of the collective showed one or more biopsy proven acute rejection 

episodes within the first week following liver transplantation. The control group included 

57 patients with stable graft function. By analyzing the HLA genotype of the donor we 

observed a significant higher percentage of patients rejecting their allograft who 

received a donor graft positive for the Bw4 epitope (p=0.006), the ligand for the 

inhibitory receptor KIR3DL1. Furthermore we detected significantly more recipients who 

received grafts displaying additional MHC molecules which are not recognized as “self” 

by the patient in the rejection group (p=0.015). In contrast to our initial hypothesis to 

uncover a higher percentage of KIR/HLA ligand incompatibilities within the rejection 

group, frequencies of disparities were reduced in patients suffering from acute rejection, 

especially in the case for the inhibitory receptors KIR2DL2, KIR3DL1 and the activating 

receptors KIR2DS1 and KIR2DS2. In summary a more comprehensive receptor-ligand 

interaction is necessary to understand how KIR-HLA genotypes contribute to transplant 

outcome but our data suggest an impact of KIR/HLA-ligand compatibility on graft 

rejection in the early period after liver transplantation.

Mauro Togni, Dirk Reinhold, Burkhart Schraven, Aneegret Reinhold 

The importance of adhesion and migration of dendritic cells 

during the induction of immune reaction: A role for the 

adaptor protein SKAP-HOM 

SKAP55 (Scr kinase-associated phosphoprotein of 55 kD) and the related protein SKAP- 

HOM are adaptor molecules involved in inside-out signalling. In contrast to SKAP55, 

which is exclusively expressed in T cells, SKAP-HOM is an ubiquitous adaptor protein. 

Previous studies have shown that in the absence of SKAP-HOM antigen receptor 

triggered integrin-mediated adhesion is severely impaired in B cells but not in T cells. In 

vivo, the loss of SKAP-HOM results in a less severe clinical course of experimental 

autoimmune encephalomyelitis (EAE) following immunization of mice with the 

encephalitogenic peptide of MOG (Myelin Oligodendrocyte Glycoprotein). Since SKAP- 

HOM is highly expressed also in dendritic cell (DC), this prompted us to study its role in 

DC biology. Here we show that the absence of SKAP-HOM does not affect the generation 

of DCs in the steady state as well as the organ colonization by DCs. In addition, the in 

vivo and in vitro maturation of DC is normal. In contrast, in FITC painting experiments, 

migration of DC from the skin to the draining lymph nodes is enhanced in the absence 

of SKAP-HOM. In vitro, the spontaneous and the CCL19-induced chemotaxis are also 

increased in DC lacking SKAP-HOM. Moreover, adhesion of mature DCs to extracellular 

matrix (fibronectin) was found to be reduced in the absence of SKAP-HOM. 

Furthermore, the conjugate formation in vitro between peptide-loaded DC and antigenspecific 

T cells was delayed. As a possible consequence, the in vivo activation of specific 

T cells after adoptive transfer experiments resulted to be reduced. In conclusion, our 

data suggest that SKAP-HOM plays a role in modulating the adhesion of different cell 

types upon activation.

Rachid Marhaba, Mario Vitacolonna, Dagmar Hildebrand, Michal Baniyash, Pia 

Freyschmidt-Paul, Margot Zöller 

The importance of myeloid suppressor cells in the regulation 

of autoimmune effector cells by a chronic allergic eczema 

Induction of a chronic allergic eczema is a most efficient therapeutic for alopecia areata 

(AA). We had noted a reduction in regulatory T cells (Treg) during AA induction and 

wondered, whether Treg may become recruited or expand during repeated skin 

sensitization or whether additional regulatory cells account for hair regrowth. 

AA could not be cured by the transfer of CD4 + CD25 high from mice repeatedly treated 

with a contact sensitizer. This obviously is a consequence of a dominance of freshly 

activated as compared to regulatory CD4 + CD25 + T cells. Instead, a population of Gr-1 

+ CD11b + cells was significantly increased in skin and spleen of AA mice repeatedly 

treated with a contact sensitizer. Gr-1 + CD11b + spleen cells mostly expressed CD31. 

Expression of several proinflammatory cytokines, the IFNγR and the TNFRI was 

increased. Particularly in the skin, Gr-1 + cells expressed several chemokines and CCR8 

at an high level. Gr-1 + CD11b + cells most potently suppressed AA effector cell 

proliferation. When co-cultured with CD4 + or CD8 + cells from AA mice, the Gr-1 + CD11b 

+ cells secreted high levels of NO. However, possibly due to high level Bcl-2 protein 

expression in AA T cells, apoptosis induction remained unaltered. Instead, ζ chain 

expression was strongly downregulated, which was accompanied by a decrease in 

ZAP70 and ERK1/2 phosphorylation. 

Thus, a chronic allergic eczema supports expansion and activation of myeloid 

suppressor cells that, via ζ-chain downregulation contribute to autoreactive T cell 

silencing.

Michal Smida, Sabine Lindquist, Anita Posevitz-Fejfar, Diana Karitkina, Ramnik Xavier, 

Brian Seed, Burkhart Schraven, Jonathan A. Lindquist 

The importance of PAG in regulating proximal signaling 

The phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) is a 

transmembrane adaptor protein that negatively regulates the activity of Src family 

kinases by recruiting the cytosolic C-terminal Src kinase (Csk) to the plasma 

membrane. Csk phosphorylates the negative regulatory tyrosine conserved within the Cterminus 

of all Src family kinases. Thus, given the potentially important role of PAG as a 

negative regulatory molecule, it was predicted that PAG-deficiency should have severe 

effects; a simple knock-out was expected to be embryonic lethal and deletion in early 

thymocytes was predicted to lead to TCR-independent T-cell development and 

autoimmunity, as had previously been shown for Csk. However, PAG/Cbp-deficient mice 

were recently published and appear to be perfectly normal; our investigation of PAGdeficient 

mice clearly shows that Src kinase activity is enhanced in newborn mice, as 

one would expect, and that a compensatory mechanism has developed by 3 months of 

age. Therefore, it appears that a redundant mechanism exists to ensure the Src kinases 

do not go unregulated and that a simple knock-out is not the best strategy to 

investigate PAG function. 

In addition to negatively regulating Src kinase activity, we have identified PAG as a 

negative regulator of Ras via its ability to recruit RasGAP. Additionally, we have shown 

that the displacement of PAG from the lipid rafts enhances both Src kinase activation 

and chemokine-induced migration. Since both the Src and Ras families are important for 

regulating cellular activation and oncogenesis, alterations in PAG expression or 

localization could be a contributing factor in cancer and autoimmunity.

Lilli Podola, Hans Jürgen Ahr, Hans-Werner Vohr 

THE INDUCTION PHASE OF ALLERGIC ASTHMA INVESTIGATED 

BY A SHORT-TERM MODEL IN BROWN NORWAY RATS 

Dermal and bronchial sensitization represents an increasing clinical problem. Particularly 

low-molecular weight molecules like isocyanides contribute to the development of 

occupational respiratory lung disease. 

Today pre-clinical studies for hazard identification of respiratory sensitisation are 

performed by long lasting and labour extensive inhalation studies which are currently 

based on biphasic study protocols, induction and challenge. As there are no validated 

protocols up to now exposure time and quantity vary in respect to the experimental 

design. 

The goal of our studies is to tap initiating mechanisms from short-term treatments as a 

tool for hazard identification. Therefore, our investigations concentrated upon the 

induction period of experimental asthma. Female Brown Norway rats were used after 

intra-tracheal application of test substances like TMA in a 4 day model. After application 

on two consecutive days evaluation of different parameters in the lung and draining 

lymph nodes were performed. To avoid pronounced influence by unspecific inflammation 

concentrations of applicants were used at the threshold level of irritation as measured 

by cytotoxic reactions in the lung. We focussed on cellular events and their regulatory 

mechanisms rather than the critical analysis of breathing pattern, i.e. functional activity. 

Surprisingly, cell counts of eosinophils, one end point very simple to determine in BALF, 

turned out to be a reliable end point. This holds true for the detection of asthmato-genic 

substances as well as the irritant SDS. However from the data obtained so far different 

kinetics of parameters seem to indicate distinct disease pattern. 

To further analyse specific activation special emphasis was put on the regulation of T 

cell responses in the draining lymph nodes and lungs followed by in vitro studies of 

specific cell proliferation and multiplex cytokine pattern. 

It should be possible to use this model to investigate the influence of routes of 

administration on the development of allergic asthma.

Lucie Dörner, Heiko Weyd, Andrea Mahr, Björn Linke, Dagmar Riess, Christine S. Falk, 

Peter H. Krammer 

The influence of apoptotic cells and annexin 1 on peripheral 

tolerance 

Peripheral tolerance comprises several mechanisms that ensure the elimination or 

silencing of potentially auto-reactive T cells that have escaped thymic selection. 

Peripheral T cells are silenced when they encounter self-antigen presented by dendritic 

cells (DC). One important source of auto-antigens are apoptotic cells that have been 

engulfed by DC. It has, however, not been fully understood which signals on apoptotic 

cells determine the non-reactivity against self. Analysing the surface of apoptotic cells 

we identified the cytosolic protein annexin 1 as an potential signal recognized by DC. 

In order to investigate the role of annexin 1, we established an ex vivo coculture system 

of primary murine DC and apoptotic spleen cells or recombinant annexin 1. DC that 

were cultured in the presence of apoptotic spleen cells or recombinant annexin 1, 

respectively, showed a reduced LPS-induced secretion of the pro-inflammatory 

cytokines TNF, IL-1β and IL-12. The secretion of other cytokines was not altered. 

Furthermore, recombinant annexin 1 reduced the LPS-induced surface expression of the 

costimulatory molecules CD86 and CD40 while the expression of MHC-II remained 

constant. Adding syngeneic OT-II-T cells, we observed a reduced T-cell stimulation as 

measured by IFN-γ secretion. Taken together, the data suggest that annexin 1 acts as 

an anti-inflammatory signal on the surface of apoptotic cells and might contribute to 

peripheral tolerance.

Shafaqat Ali, Michael Huber, Christian Kollewe, Werner Falk, Michael U. Martin 

The Interleukin-1 Receptor Accessory Protein is essential for 

Interleukin-33 induced activation of T cells and mast cells 

Interleukin-33 (IL-33) is a novel member of the IL-1 family of cytokines. It binds to the 

IL-33 receptor alpha chain, formerly known as ST2/T1, which is a member of the IL-1 

receptor family (Schmitz et al. Immunity 2005, 23, 479-490). Here we show that lack of 

the Interleukin-1 receptor accessory protein (IL-1RAcP) abrogates responses to IL-33 in 

the mouse thymoma clone EL-4 D6/76. Reconstitution with full length IL-1RAcP, but not 

with a C-terminally truncated version lacking the TIR-domain, is sufficient to restore 

responsiveness to IL-33. Blocking of murine IL-1RAcP with the neutralizing antibody 

4C5 inhibits response of mouse thymoma cells and bone marrow-derived mouse mast 

cells to IL-33 with respect to cytokine production. IL-33 activates IL-1 receptor 

associated kinase (IRAK-1), the stress kinases p38 and c-Jun N-terminal kinase, and 

the NF-kappaB pathway in an IL-1RAcP dependent manner. IL-33 is able to induce the 

production and release of pro-inflammatory cytokines in transiently transfected cells 

and bone marrow-derived mast cells, indicating that IL-33 may have a proinflammatory 

potential (in the innate response) like its relatives IL-1 and IL-18 besides 

of its Th2-skewing properties in the adaptive response described previously. The 

interaction of IL-1RAcP and IL-33Ralpha-chain is dependent on IL-33 as demonstrated 

in co-immunoprecipitation assays. 

We conclude that IL-1RAcP is required as co-receptor in the signalling IL-33 receptor 

complex. Thus IL-1RAcP is utilized by more than one beta-chain of the IL-1 receptor 

family and may resemble a common beta-chain of the IL-1 receptor family.

Manuela Hessmann, Dominik Rueckerl, Toshiyuki Takai, Stefan Ehlers, Christoph 

Hoelscher 

The ITAM-bearing adapter protein DAP12 modulates 

inflammatory immune responses after Mycobacterium 

tuberculosis infection 

Activation of the innate immune system leads to the release of pro-inflammatory 

cytokines and is considered to be a prerequisite for driving a protective adaptive 

immune response after infection. However, inflammatory immune responses may also 

lead to tissue destruction and regulatory mechanisms are necessary to prevent 

uncontrolled inflammation. Hence, activating and inhibitory host factors are required in 

order to balance protection and immunopathology. When associated with different 

receptors, the ITAM-containing signalling adaptor DAP12 (DNAX activating protein of 12 

kDa) has been shown to affect inflammatory immune responses. 

To analyse the influence of DAP12 on cell-mediated immune responses during 

mycobacterial infection we infected DAP12-deficient (KO) mice with Mycobacterium 

tuberculosis. In contrast to wildtype mice, infection of DAP12 KO animals resulted in 

increased inflammatory immune response. In the absence of DAP12, the expansion of 

activated T cells was enhanced accompanied with elevated antigen-specific effector 

functions. On the cellular level, we could show that DAP12 KO macrophages 

overproduce pro-inflammatory cytokines in response to M. tuberculosis infection. 

In summary, we assume DAP12-dependent pathways in macrophages to modulate 

inflammatory immune responses during mycobacterial infection. These signalling 

pathway may represent novel target in order to mitigate chronic inflammation in human 

tuberculosis.

Veronika Lukacs-Kornek, Sven Burgdorf, Sabine Specht, Miroslaw Kornek, Christian 

Kurts 

The kidney – renal LN system contributes to cross-tolerance 

against innocuous soluble antigen 

Soluble antigens devoid of inflammatory stimuli, derived for example from self-serum or 

food proteins, induce T cell tolerance in the spleen. Here we describe an additional role 

of the kidney – rLN system in tolerogenic presentation of such circulating antigens. 

Protein below albumin molecular weight constitutively passed the kidney glomerular 

filter and was concentrated in the tubular compartment. Enriched filtrated antigen was 

endocytosed by kidney dendritic cells (kDC). Simultaneously, it was transported cellindependently 

within two minutes to DC resident in rLNs. These DC phenotypically 

differed from kDC carrying filtrated antigen, and used a different mechanism, MRmediated 

endocytosis, to internalize antigen. They activated specific CD8+ T cells, 

which subsequently proliferated without producing effector cytokines or developing 

cytotoxic activity, and showed a curtailed life-span. Such tolerogenic T cell activation 

was independent of steady-state migratory kidney DC because nephrectomy after 

antigen injection did not change the T cell response. These findings demonstrate that 

the kidney dispatches concentrated blood-borne antigens to the rLNs, where they can 

be captured by resident DC, resulting in tolerogenic CD8+ T cell activation. This 

mechanism may contribute to T cell cross-tolerance against innocuous circulating 

protein antigens.

Gennadiy Zelinskyy, Sandra Balkow, Simone Schimmer, Tanja Werner, Markus M. 

Simon, Ulf Dittmer 

The level of Friend retrovirus replication determines the 

cytolytic pathway of CD8+ T cell-mediated pathogen control 

Cytotoxic T-cells (CTL) play a central role in the control of viral infections. Their antiviral 

activity can be mediated by at least two cytotoxic pathways namely the granule 

exocytosis pathway, involving perforin and granzymes, and the Fas-FasL pathway. 

However, the viral factor(s) that influence the selection of one or the other pathway for 

pathogen control are elusive. Here we investigate the role of viral replication levels on 

the induction and activation of CTL, including their effector potential, during acute 

Friend Murine Leukemia Virus (F-MuLV) infection. F-MuLV inoculation results in a lowlevel 

infection of adult C57BL/6 mice that is enhanced about 500-fold upon co-infection 

with the Spleen Focus Forming Virus (SFFV). Both the low- and high-level F-MuLV 

infection generated CD8+ effector T cells that were essential for the control of viral 

replication. However, the low-level infection induced CD8+ T cells expressing solely 

FasL but not the cytotoxic molecules granzyme A and B, whereas the high-level 

infection resulted in induction of CD8+ effector T cells secreting molecules of the 

granule exocytosis pathway. By using knockout mouse strains deficient in one or the 

other cytotoxic pathway we found that low-level viral replication was controlled by CTL 

that expressed FasL but control of high-level viral replication required perforin and 

granzymes. Additional studies, in which F-MuLV replication was enhanced 

experimentally in the absence of SFFV co-infection supported the notion that only the 

replication level of F-MuLV was the critical factor that determined the differential 

expression of cytotoxic molecules by CD8+ T cells and the pathway of CTL cytotoxicity.

Iryna Prots, Alla Skapenko, Jörg Wendler, Stefan Mattyasovszky, Clarisse Yone´, 

Bernd Spriewald, Harald Burkhard, Joachim R Kalden, Peter E Lipsky, Hendrik Schulze- 

Koops 

The loss-of-function IL4R single nucleotide polymorphism 

I50V is associated with early erosive rheumatoid arthritis 

IL4 and its receptor, IL4R, are major regulators of T helper cell differentiation and are 

believed to play an important role in the pathogenesis of rheumatoid arthritis (RA), 

which is characterized by unbalanced Th1-mediated inflammation. To evaluate the role 

of IL4R single nucleotide polymorphisms (SNPs) in RA pathogenesis, we analyzed the 

association of two IL4R SNPs, I50V and Q551R in the IL-4-binding and STAT6-binding 

domains, respectively with RA susceptibility and severity. A well-characterized cohort of 

471 RA patients and 371 age and sex matched healthy controls were genotyped by 

sequence specific PCR for their alleles at I50V and Q551R SNPs. The patients were 

stratified according to radiological outcome at two years disease duration into an 

erosive and a non-erosive group to assess disease progression as early bone erosions 

and joint destructions are most characteristic for severe RA. Allele frequencies, 

genotype frequencies and haplotype frequencies defined by the two IL4R SNPs were 

similar between RA patients and healthy controls. In marked contrast, significant 

differences between the erosive and the non-erosive group of patients were observed in 

the distribution of I50V genotypes. The V50 allele was strongly associated with early 

aggressive erosive disease, as 68.1% of the V50 homozygous patients compared to 

only 37% of the I50 homozygous patients had developed bone erosions two years after 

disease onset (OR 3.86, p < 0.0001). To delinate a possible mechanism by which IL-4R 

SNPs might modulate disease progression in RA, we analyzed the effect of IL-4 on the 

function of T cells of healthy individuals expressing different IL-4R I50V alleles. The V50 

allele significantly reduced the response of CD4 T cells to IL-4, as assessed by STAT6 

phosphorylation, GATA3 expression and downmodulation of the IL-12 receptor ?2 chain. 

Furthermore, the IL-4-mediated inhibition of Th1 and increase of Th2 cell differentiation 

was significantly diminished in V50 homozygous CD4 T cells. Our data identify the lossof-function 

I50V IL4R SNP as a novel genetic marker in RA with high predictive value for 

early joint destruction. Moreover, the data also provide a first intriguing molecular 

mechanism of an altered immune response that results in increased tissue destruction 

in a human autoimmune disease.

Petra Hoffmann, Tina J. Boeld, Ruediger Eder, Jochen Huehn, Stefan Floess, Udo 

Baron, Sven Olek, Reinhard Andreesen, Matthias Edinger 

The Naive CD45RA+, But Not The CD127- Subpopulation of 

Human CD4+CD25high T Cells Homogeneously Maintains 

Regulatory T Cell Characteristics Upon In Vitro Expansion 

The adoptive transfer of CD4+CD25high regulatory T (Treg) cells is envisaged as a 

promising strategy for the treatment of autoimmune diseases and the promotion of 

immunological tolerance after allogeneic organ or stem cell transplantation. For such 

clinical applications, efficient in vitro expansion protocols for Treg cells are required. We 

previously demonstrated that strong co-stimulation provided by cross-linked CD3 and 

CD28 antibodies together with high-dose IL-2 resulted in a more than 3-log polyclonal 

expansion of highly suppressive Treg cells (Blood 104:895; 2004). To avoid the 

outgrowth of potentially auto- or alloreactive effector T cells in such cultures, isolation 

of highly purified Treg cells from human PBMC is of crucial importance. Lack of IL-7 

receptor (CD127) expression has recently been proposed as a useful tool for the 

discrimination of Treg cells from activated conventional T cells. Here we analyzed FACSpurified 

CD127low/neg CD4+CD25+ T cells after 2 to 3 weeks of in vitro expansion. For 

all phenotypic and functional characteristics examined, such as CD25, CCR7, CD62L, 

CTLA-4 and FOXP3 expression, production of pro- and anti-inflammatory cytokines or 

methylation status of the foxp3 gene locus, CD127low/neg cells displayed a 

heterogeneous profile after in vitro expansion. This was mainly due to the presence of 

CD45RO+FOXP3+ memory type Treg cells in the starting population, as further purified 

CD45RA+CD127low/negCD4+CD25+ T cells developed into a homogeneous population 

that retained all characteristics of natural Treg cells after in vitro expansion. Similar 

results were obtained by us before with naive CD4+CD25high T cells isolated without 

consideration of CD127 as a separate sort criterion (Blood 108:4260; 2006). Based on 

these findings, we suggest that isolation of CD127low/neg CD4+CD25+ T cells is 

insufficient for the generation of pure Treg cell products, but that isolation and 

expansion of CD45RA+ naive CD4+CD25high T cells represents a promising strategy for 

adoptive cell therapies.

Sandra Düber, Martin Hafner, Elias Hobeika, Michael Reth, Ari Waisman, Karsten 

Kretschmer, Siegfried Weiss 

The origin of B-1a cells: Analysis in mice with inducible B cell 

development 

The origin of B-1a cells, which are suggested to act as a ‘first line of defense’ against 

mucosal and systemic pathogens, is still controversially discussed. It is unclear, whether 

these cells are only generated during fetal and neonatal life, or whether the adult bone 

marrow can also give rise to such B cells. Most of the previous results leading to the 

controversy were derived from transgenic mice or adoptive transfer experiments. But 

now we are able to investigate the origin of B-1a cells under more physiological 

conditions. For this we have generated mice with inducible B cell development. 

Switching on B cell development in adult mice should definitively answer the question 

about the involvement of the adult bone marrow in the generation of B-1a cells. 

To make B cell development inducible the recombination activating gene 1 (Rag1) was 

chosen as target. The coding sequence of Rag1 was initially inverted for gene 

inactivation resulting in a block of B (as well as T) cell development. Flanking this region 

with loxP sites in opposite orientation made it possible to revert it by expression of Cre 

recombinase resulting in restoration of the transcription unit. The temporal control of 

Cre expression was realized by crossing-in the MerCreMer mouse. These mice express 

the tamoxifen-inducible MerCreMer recombinase under the control of the B cell specific 

mb-1 promoter. 

Oral administration of tamoxifen to the mice leads to a wave of newly generated B cells 

and allows their analysis in the periphery. Experiments in which the mice were induced 

at an age of 8 weeks clearly show that B-1a cells are present in the peritoneum. Also if 

older mice were used for the induction (5 months old) B-1a cells can be detected in 

similar numbers. A certain proportion of these cells shows phosphatidyl choline (PtC)specificity, 

a typical property of B-1a cells.

Ulrich Steinhoff, Alexander Visekruna, Anjo Kroesen 

The proteasome controls NF-κB-mediated inflammation in IBD 

patients 

Proteasomes are the central proteolytic machinery of cells involved in degradation of 

proteins and generation of MHC class I epitopes. Additionally they play a central role in 

regulation of inflammatory processes mediated by NF-κB. We analyzed the structure 

and function of 20S proteasomes in normal and inflamed intestinal tissues from Crohn’s 

disease (CD)and ulcerative colitis (UC) patients. 

Our results show tissue-specific differences in proteasome composition as previously 

reported in mice. 

However, we found marked differences in proteasome composition between CD and UC 

patients. Inflammation affected the proteasome composition only in CD patients with 

strong expression of immunosubunits in the inflamed tissues. 

Immunoproteasomes isolated from CD patients degraded IκBα and processed p105 

much faster than constitutive proteasomes from UC patients. Immunohistochemical 

analysis confirmed that the amount of activated NF-κB correlated with the expression of 

immunoprotesasomes. 

This demonstrates that the mechanism of inflammation differs between UC and CD 

patients and that proteasomes are crucially involved in Th1-mediated inflammation.

Sandra Kraemer, Regina Krohn, Hongqi Lue, Manfred Dewor, Christian Weber, Jürgen 

Bernhagen 

The pseudo-ELR motif of MIF is required for its functions as 

CXC arrest chemokine 

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that 

plays a pivotal role in acute and chronic inflammatory diseases such as septic shock, 

and atherosclerosis. We have recently demonstrated that MIF, contrary to its historic 

and eponymous name, is a non-cognate ligand of the chemokine receptor CXCR2. MIF 

binds to CXCR2 with nanomolar affinity and induces leukocyte arrest and chemotaxis 

through this receptor (Bernhagen et al., Nat. Med. 13, 2007). CXCR2 signaling induced 

by cognate ligands, such as IL-8, requires an N-terminal Glu-Leu-Arg (ELR) motif. The 

MIF monomer not only displays a striking architectural homology to the IL-8 dimer, but 

we also noticed that MIF has an ELR-like 3D motif, which we tentatively term pseudo- 

ELR motif. It is formed by the two non-adjacent residues Arg11 (R11) and Asp44 (D44), 

which reside in an ELR-like spacing in exposed neighboring loops, imitating the ELRmotif. 

While it is tempting to speculate that the pseudo-ELR motif conveys binding and 

activity specificity comparable to what is observed for classical ELR+ CXC chemokines, 

no functional data has been available. We have thus generated MIF mutants with 

residues R11 and D44 mutated to address the question whether the pseudo-ELR motif is 

functionally required for an interaction with CXCR2 and for MIF’s chemokine-like 

activities. We have cloned the mutants R11A-MIF, D44A-MIF, and the double mutant 

R11A-D44A-MIF. Although mutagenesis involved alterations in charge and isoelectric 

point, the mutants were expressed and purified by an almost identical procedure as that 

established for wildtype MIF. Spectroscopic analysis by far-UV circular dichroism 

confirmed that the mutants exhibited an overall structural integrity. However, R11A-MIF 

showed an increase in β-sheet structure, indicating that the pseudo-ELR site constitutes 

a sensitive conformational region of the molecule. We will report on the functional 

relevance of this motif for the leukocyte recruitment activity of MIF. Intriguingly, initial 

experiments suggest that mutating R11/D44 markedly interferes with CXCR2dependent 

monocyte arrest on human aortic endothelial cells in flow induced by MIF.

Nicole Dietrich, Nelson Gekara, Siegfried Weiss 

The regulatory mechanisms inviolved in iNOS, TNF-&alpha, 

and IL-6 induction during host defence 

One of the most important hallmarks of pathogen recognition by host cells is the 

secretion of inflammatory mediators. Depending on several factors, like the site of 

secretion and their relative concentrations, such mediators play two distinct roles in the 

disease state, one protective and the other injurious. Toll-like receptors (TLRs) play a 

central role in the inflammatory responses triggered by various pathogens. But even 

after an area of intense research focus, the signalling pathways via which the various 

TLRs induce different inflammatory responses are still not fully understood. TNF-&alpha, 

IL-6, and nitric oxide (NO) play critical roles in many of the anti-microbial defenses as 

well as in harmful disease such as septic shock and other injurious chronic inflammatory 

conditions. The present study was carried out with the aim to ellucidate the downstream 

signalling pathways via which TLR2, TLR3 and TLR4 agonists (MALP-2, Poly IC and LPS 

respectively) induce TNF-&alpha, IL-6 and NO responses. Using various pharmacological 

inhibitors for p38, JNK, ERK, PI3K, PKC and NF-kB signalling pathways (SB203580, 

SP600125, PD098059, LY294002, staurosporine and BAY11-7085 respectively), we 

show that the induction of these mediators involves overlapping as well as distinct 

signalling events. We show that all the tested inhibitors could block the induction of TNF- 

&alpha by the above agonists, but only the PI3K, PKC and JNK inhibitors could block IL- 

6 and NO responses. Interestingly the p38 pathway had an enhancing effect on IL-6 and 

NO production. Further analysis of macrophages deficient in IFNAR (Type I interferon 

receptor) suggest a role for Type I interferons in the induction of NO and IL-6 but not 

TNF-&alpha.

Uwe Kolsch, Christina Weber, Caroline Ritter, Anna Viehbahn, Thomas Brune 

THE REPRODUCTIVITY OF MICE IS INDEPENDENT OF A MHC-I 

HAPLOTYPE MISMATCH BETWEEN THE MOTHER AND THE 

FETUS 

Introduction: Recent investigations have shown fetal survival depending upon 

maternal TH2-switch and existence of regulatory T-cells. Up to now it is unclear if fetal 

survival depends on maternal TH1/TH2 basal immune responses and on MHC-recognition 

and MHC-haplotyp, factors influencing MHC - T cell interaction. Methods: To address 

the question whether different types of basal immune responses influence 

reproductivity, inbred mouse strains with different TH1/TH2-reactivities and different 

MHC haplotypes (CBA, Balb/C, C57BL/6, DBA1) were crossed to F3 generation. TH1/TH2 imbalances were measured by CD69-upregulation in mixed lymphocyte cultures (MLC) 

from parental animals. Results: CD8+-lymphocytes from TH1 reacting strains are 

activated to a higher extent when compared to lymphocytes of TH2 reacting strains. In 

contrast immune responses between MHC I-haplotype homozygous F2 and F3-animals 

are balanced. Numbers of progeny were significantly reduced when male TH2-reacting mice were crossed with female TH1-reacting mice compared to breedings from female 

TH2-reacting mice crossed with male TH1-reacting mice. The latter was comparable to 

F1-hybrid breeding pairs and breeding pairs of MHC I-haplotype homozygous F2 mice. 

There was no difference in progeny numbers when MHC I-haplotype homozygous F2 

mice with total or no MHC mismatch were crossed. Discussion: TH2 reactivity is 

advantageous for pregnancy outcome but TH2 switching is independent of MHC Ihaplotype. 

Breeding of genetically non identical mice positively influences pregnancy 

outcome independently of the MHC I-haplotype.

Andrey Bogdanov, Tatyana Rybakova, Nikolay Belyaev 

The role of alpha-fetoprotein in induction of immunological 

tolerance to tumor growth: direct and indirect breaking of 

tumor immunosurveillance 

It was shown, alpha-fetoprotein (AFP) is immunoregulatory agent provided the 

immunologic tolerance development during pregnancy. AFP induced disorders of certain 

cell-mediated immunity effector reactions in vitro and immunosuppression in AFPoverproducing 

transgenic mice. We investigated implication of AFP in down regulation of 

immunological resistance to cancerous tumor. Our results demonstrated that AFP 

preexposure promotes more intensive (almost in three times) growth of primary tumor 

nodule and dissemination of tumor. The latent periods of appearance of primary tumor 

nodule as well as first metastatic protuberances were shorter that in control animals. 

Study of cell-mediated immunity presented number of considerable impairments. 

Spontaneous and IL-12-induced productions of IFN-γ and TNF-α from NK cells and •D8+ 

T cells, as well secretion of IFN-γ from NKT cells and TNF-β from •D8+ T cells were 

extensively inhibited as compared with control animals at the same period. Similar 

situation observed during cytolytic activity study of NK-, NKT- and •D8+ T cells as well 

as PHA-induced proliferation of NKT- and •D8+ T cells. Isolated dendritic cells from AFPinjected 

animals showed weaker antigen-presenting ability and secretion of IL-12, IL- 

15, IL-18 and IL-23. Investigation of effector •D4+ T cells demonstrated higher Th2 

cells prevalence on Th1 cells in AFP-injected animals that control animals at the same 

period or tumor development. In conclusion, numbers of functionally active Th3- and 

Treg1 cells as well as suppressor activity and numbers of TGF-β1- and IL-10-produced 

hematopoietic stem sells were higher in spleen of AFP-injected animals. All these 

immunological disorders were found at very early stages of tumor growth in AFPinjected 

animals only. Moreover, discovered changes of cell-mediated immunity 

displayed before appearance of palpable primary tumor nodule in AFP-injected animals. 

Thus, AFP induced both direct and/or indirect editing of cell-mediated immunity that 

lead to suppression of immune attack against tumor and in the end to extensive tumor 

invasion.

Filiz Demircik, Ari Waisman 

The role of B cells and other antigen presenting cells in 

infections 

To clarify the role of B cells in infection models like Leishmania major, murine 

Cytomegalovirus (MCMV) and influenza, we are using three different mouse strains. The 

JHT mice are B cells deficient; the IgG1i strain has B cells producing and secreting IgG1 

antibodies but no other antibody isotypes; and the IgMi mice, which can produce IgM as 

the B cell receptor but have no antibodies in the sera. In these mouse strains we are 

looking for differences in the T cell repertoire that is established upon infection. 

Furthermore we want to deplete B cells and thereby look at their role in infection. To 

deplete B cells at distinct time points during, before or after clearing of the infection, we 

will establish a CD19-Cre x iDTR mouse. In this developed novel iDTR system, the 

diphtheria toxin receptor (DTR) can be expressed on the surface of cells following a Cremediated 

deletion of a STOP cassette. In the iDTR mice, lineage ablation is achieved by 

injecting diphtheria toxin (DT) into mice expressing the iDTR as well as a tissue specific 

Cre transgene. Finally we plan to create a new mouse with the DTR downstream of the 

CD11b promoter and then together with lineage specific Cre mouse strains we will be 

able to achieve a more specific ablation, especially for antigen presenting cells and be 

able, for example, to specifically delete cells that express CD11b and CD11c or CD11b 

and LysM. This system will allow us to specifically ablate sub-populations of dendritic 

cells, macrophages or B cells (B-1 cells) and study their role in different infectious 

systems.

Sonja Reißig, Ari Waisman 

The Role of CYLD in T cell Development and Regulation 

CYLD is a tumour suppressor gene known to play an important role in NF-kappa B 

signaling. 

To analyse the function of CYLD in vivo we used the CYLD ex7/8 mouse strain, which is 

characterized by loss of the full-length transcript and overexpression of a short splice 

variant of the cyld gene. Here we show the role of CYLD in T cell development and T cell 

regulation by analysing CYLD ex7/8 mice and comparing them to wildtype mice. 

In CYLD ex7/8 mice the overexpression of the small transcript of CYLD resulted in 

splenomegaly and lymphadenopathy. Additionally, the B cell population in spleen and 

lymphnodes is increased at the expense of T cells. 

Analysis of T cell development in CYLD ex7/8 mice revealed a reduction of total numbers 

of thymocytes compared to control mice. Additionally, a partial developmental block at 

the DN1 stage was observed. In order to investigate the contribution of CYLD in positive 

and negative selection in the thymus in vivo, the HY-TCR transgene (HYtg) was crossed 

to CYLD ex7/8 mice. The analysis of CYLD ex7/8 HYtg males revealed an increase in CD4 

+ CD8 + DP as well as in CD8 SP thymocytes, suggesting a less pronounced negative 

selection in CYLD mutant mice compared to HYtg control mice. The analysis of 

CYLD ex7/8 HYtg females indicated a role of CYLD also in positive selection, since a 

developmental block in the transition of CD4 low CD8 + TCR high to CD4 + CD8 + DP 

thymocytes was observed. The investigation of peripheral lymphoid T cell populations in 

CYLD ex7/8 mice indicated an increased CD4 population at the expense of CD8 T cells. In 

addition, an increase in number and proportion of Tregs in CYLD ex7/8 mice was 

observed, most likely responsible for the perceived increase in CD4 T cells. 

The analysis of CYLD in NF-kappa B signaling demonstrated an increased activation of 

the NF-kappa B pathway in stimulated CYLD ex7/8 T cells. In addition, the expression of 

important tumour related genes in CYLD ex7/8 T cells was investigated and revealed an 

upregulation of different tumour promoting genes. 

Taken together, these results indicated an important role of the alternatively spliced 

transcript of CYLD ex7/8 in T cell development as well as in NF-kappa B signaling of T 

cells.

Fabia T.L. Rocha, Rüdiger Arnold, Renate Rutz, Michael Kirschfink 

The role of FLIP Long in C5a-modulated spontaneous apoptosis 

of neutrophils 

Neutrophils (PMN) are the most abundant type of immune cell in the circulation, and 

one of their key characteristics is their short life span (8-20 hours). In the absence of 

survival factors they undergo spontaneous apoptosis, a process critical to resolution of 

inflammation. The exact influence of the complement system in neutrophil apoptosis is 

still obscure. C5a has been described to delay PMN apoptosis through the Bad-mediated 

signaling pathway in mitochondria (intrinsic pathway), however, its possible impact on 

the extrinsic death receptor pathway in PMN apoptosis is still unknown. 

A possible involvement of C5a in the modulation of spontaneous PMN apoptosis was 

investigated by analysis of the extrinsic pathway regulator cellular FLIPLong (c-FLIPL ). 

Neutrophils were isolated from 20 healthy adult donors. FACS analysis of annexin V 

binding was carried out to evaluate apoptosis. Activity of caspases 9, 8, and 3 was 

measured by substrate cleavage using AFC fluorescence. c-FLIPL and β-actin were 

quantified using LightCycler-RT-PCR. For all experiments, neutrophils were analysed 

after 1 hour or 21 hours of incubation with or without C5a. After 21 hours of incubation 

we observed a significant delay in spontaneous apoptosis upon C5a treatment, however 

only in a subgroup of donors (10/20, p < 0.005). Delay in spontaneous PMN apoptosis 

of these individuals went along with a significant up regulation of the regulator c-FLIPL (p < 0,05), but not with reduced caspase 8 activity. 

These findings suggest that - in contrast to previous reports - C5a acts as a PMN 

survival factor only under certain conditions, which appear to involve the regulatory 

activity of c-FLIP. In our study we demonstrated for the first time the expression of 

FLIPL in neutrophils and its upregulation in cases where C5a delayed neutrophil 

apoptosis. 

-->

Denise Bogdanski, Thomas M. Frangen, Christian Schinkel, Gert Muhr, Manfred Köller 

The role of IL-17 in the post traumatic phase of polytrauma 

patients. 

Interleukin-17 (IL-17) is produced by a T-cell subpopulation (Th17/Th17). IL-17 as a 

central mediator in inflammatory processes connects T-cell stimulation with neutrophil 

mobilization. The role of IL-17 in the immune dysfunction after servere injuries is not 

clarified. In a study with 71 patients (55 men, 16 women) systemic IL-17 and IL-6 

concentrations of polytraumatized patients were daily analyzed by ELISA. The majority 

of the patients (94%) showed no systemic IL-17 or the IL-17 concentrations were in the 

range of healthy volunteers. Only in 6% of the patients was an increase in systemic IL- 

17 values detected. To identify the possible role of IL-17 in the post traumatic phase 

the patients were divided in two groups. Group A (47 men, 15 women) consists of 

patients with IL-17 concentrations in the range of healthy volunteers and group B (8 

men, 1 woman) consists of patients with elevated IL-17 concentrations. In group B 

systemic IL-6 (median 41 pg/ml, range 3-709 pg/ml) correlated with systemic IL-17 (75 

pg/ml, range 0.1-1822 pg/ml, r=0.258, p≤0.01). The patients in group B showed no 

conformity in injury patterns, ISS, age, outcome, intensive care periode or clinical 

complications. 

After a period of four years we were able to obtain a blood sample from a now 

recovered patient who formerly belonged to the high IL-17 group. Now, the systemic IL- 

17 level was below the detection limit, but stimulation of PBMCs obtained from this 

patient showed elevated numbers of IL-17 producing cells as determined by ELISpot 

and flow cytometry. It has to be proven by further studies whether highly increased 

systemic IL-17 concentrations detected in distinct patients were related to a genetic 

predisposition of these subjects.

Inna Lavrik, Alexander Golks, Dirk Brenner, Julia Hoffmann, Carina Pforr, Peter 

Krammer 

The role of new c-FLIP proteins in life and death of the T cell. 

C-FLIP, also known as FLAME-1/I-FLICE/CASPER/CASH/MRIT/CLARP/Usurpin, is a welldescribed 

inhibitor of death receptor-mediated apoptosis. Until present only two 

isoforms of c-FLIP: c-FLIPL and c-FLIPS, were found to be expressed at the protein 

level. In our studies, we found two new protein products of c–FLIP: p25-FLIP, which 

turned out to be the third c-FLIP isoform, c-FLIPR; and p22-FLIP, which was shown to 

be a c-FLIP N-terminal cleavage product. Both new c-FLIP proteins block caspase-8 

activation at the DISC and death receptor-induced apoptosis. In addition, p22-FLIP was 

shown to play a prominent role in NF-kappa B activation via interaction with the IKK 

complex. Recently, we developed new insight into the mechanism of c-FLIP-mediated 

NF-kappa B induction. Our new findings and the role of c-FLIP proteins in life and death 

of T cells are going to be discussed.

Eva Gueckel, Silke Meister, Mario Zaiss, Sankar Ghosh, Reinhard E. Voll 

The role of NF-κB for the development and function of 

naturally occurring regulatory T cells 

Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (nTregs) develop in the 

thymus and represent a mature T cell subpopulation capable of inhibiting the 

proliferation and cytokine release of effector T cells. nTregs are involved in preventing 

autoimmune diseases and graft rejection; however, they may also prevent 

immunological tumour killing. Deletion of different components in the NF-κB signalling 

pathway using knockout-mouse models showed a strong decrease in the nTreg cell 

number. However, it is still unclear if NF-κB is important for the development and 

differentiation or survival of nTregs. In addition, the role of NF-κB for the 

immunoregulatory effector functions of nTregs is not yet clearly understood. To address 

these questions we used thymocyte-specific expression of either an inhibitor of NF-κB 

(IκBα-Tg) or a constitutively active mutant of IKKβ (IKKβ-Tg) in transgenic mice. 

Thymocytes from 4-/6-week-old IκBα transgenic mice showed a significant decrease in 

the relative and absolute numbers of nTregs. Unexpectedly, the analysis of thymocytes 

from IKKβ Tg mice, displaying increased NF-κB activity, also showed a trend towards 

decreased nTreg cell numbers. In the periphery the absolute numbers of nTregs did not 

show any marked differences in both transgenic mouse lines. Finally, sorted nTregs 

from IKKβ Tg mice were less potent in suppressing the proliferation of effector T cells 

than those from C57BL/6 mice. These data indicate that the NF-κB activity modulates 

nTreg cell development as well as their function. These findings have important 

implications for new therapeutical strategies targeting autoimmune diseases, allergies 

and chronic infections.

Zoran V. Popovic, Roger Sandhoff, Tjeerd P. Sijmonsma, Eva Kiss, Sylvia Kaden, Edgar 

Tone, Hermann-Josef Gröne 

The role of sulfatide SM4s in apoptotic cell uptake and 

macrophage phenotype alteration 

Sulfoglycolipids are present on the surface of a variety of cells. Sulfatide (SM4s) is 

increased in renal, colon, lung, and ovarian cancer and is a hallmark of adverse 

prognosis. Macrophages significantly contribute to the inflammatory infiltrate in 

malignancies. We postulated that SM4s can modulate macrophage function through an 

influence on apoptotic cell clearance at the cancer site. The effect of SM4s on the 

uptake of apoptotic particles by macrophages, macrophage cytokine profile and receptor 

expression were investigated. Murine peritoneal macrophages were incubated with 

either SM4s-loaded or SM4s-free Colon26 (murine colon cancer) or Renca (murine renal 

cancer) apoptotic cells. Presence of SM4s on apoptotic cells was confirmed by 

immunocytochemistry and transmission electron microscopy. Phagocytosis and receptor 

expression were analyzed by FACS. Uptake of apoptotic cells was confirmed by confocal 

and electron microscopy. Cytokines were measured by ELISA. Presence of SM4s in 

apoptotic cell membrane increased the number of apoptotic cells in the macrophage 

phagosomal compartment up to 3 fold. Scavenger receptors and the sulfate group of 

SM4s were the key factors for the increase in apoptotic cell clearance. Enhanced uptake 

of SM4-covered apoptotic cells led to an increase in P-selectin expression by 

macrophages (up to 40%, p

Marko Janke, Michael Peine, Thordis Hohnstein, Lars Morawietz, Alexander Scheffold 

THE ROLE OF TH1 AND TH17 T CELLS IN INFLAMMATORY 

REACTIONS IN VIVO 

Introduction: 

The role of Interferon-γ (IFN-γ) produced by Th1 cells in vivo is controversially 

discussed. While Th1 cells can induce inflammatory reactions, like delayed type 

hypersensitivity (DTH), neutralisation of the prototypic Th1 cytokine IFN-γ leads to an 

even more pronounced DTH or a more severe inflammatory arthritis. Recently, IL-17 

produced by Th17 cells was shown to act highly pro-inflammatory in inflammatory 

autoimmune diseases, like collagen-induced arthritis. Therefore, our aim was to analyse 

the role of Th1 vs Th17 cells in inflammatory reactions using DTH or the ovalbumininduced 

arthritis model (OIA). In this model, arthritis is induced by intra-articular (i.a.) 

injection of ovalbumin into Ova-immunised mice. 

Results: 

We show that IFN-γ as well as IL-17 is produced to equal amounts by ex vivo isolated 

antigen-specific CD4+ T cells taken from arthritic mice. Interestingly, in anti-IL-17 

treated mice arthritis was significantly ameliorated. In contrast, the transfer of in vitro 

generated Th17 cells did not enhance arthritis symptoms, while Th1 cells did enhance 

arthritis. Similarly, Th17 cells only evoked a mild DTH reaction in sharp contrast to Th1 

cells, which induced strong and long-lasting DTH. This difference was probably caused 

by different migratory capacity into inflammatory sites, since Th1 but not Th17 cells 

migrated into the inflammatory foot and the draining lymph node. 

Conclusion: 

In vitro generated Th1 cells exert the expected pro-inflammatory function in vivo. In 

contrast, while IL-17 seems to increase OIA severity, in vitro generated Th17 cells failed 

to enhance or evoke inflammatory reactions, like arthritis or DTH. Thus, IL-17 

producing cells generated by currently described in vitro protocols do not possess the 

full functional potential of there in vivo counterparts. It will be important to determine 

the missing parameters required for full Th17 functional activity, in addition to secretion 

of IL-17.

Jan Liese, Ulrike Schleicher, Christian Bogdan 

The role of TLR9 signalling in murine cutaneous leishmaniasis 

In most strains of mice subcutaneous infections with Leishmania (L.) major lead to selfhealing 

skin lesions. A prominent CD4+ T-helper cell (Th)-based interferon (IFN)gamma 

response is associated with the ultimate resolution of the disease. The innate 

immune respose against the pathogen is characterised by the expression of inducible 

nitric oxide synthase (iNOS) and by the rapid induction of NK cell cytotoxicity and IFNgamma 

production in the draining lymph node (LN). Mice deficient for the adaptor 

molecule MyD88 are unable to control Leishmania parasites in vivo, indicating the 

requirement of Toll-like receptor signalling for a protective immune reponse. TLR9, 

which uses MyD88 for signalling, is located in the endosome, where it might interact 

with intracellular L. major. Therefore, we investigated the immune response against 

Leishmania in TLR9 deficient (TLR9-/-) C57BL/6 mice. 

In the LN of L. major-infected TLR9-/- mice NK cell IFN-gamma expression and 

cytotoxicity were strongly reduced as compared to wild type controls. In vitro, L. major 

promastigotes as well as L. major DNA induced interleukin (IL)-12 in bone-marrow 

derived myeloid dendritic cells (DCs) in a TLR9-dependent manner. IL-12p35-/- mice 

were deficient for NK cell activation after L. major infection, suggesting that L. major 

induces IL-12 via TLR9 in DCs which in turn triggers early NK cell activity in the LN. 

TLR9-/- mice exhibited more severe dermal lesions during the acute phase of the 

infection compared to wild type mice, but ultimately controlled the infection. TLR9-/- 

and wild type mice showed no difference of the mRNA levels of IFN-gamma, IL-12p35, 

IL-12p40 or iNOS in the skin or LN. However, TLR9-/- mice transiently expressed higher 

levels of IL-4, IL-13, and arginase 1 mRNA, indicating an enhanced Th2 cytokine 

production. 

Thus, TLR9-/- mice have a deficient innate immune response to L. major, which is 

associated with an aggravated clinical course of infection, but does not influence the 

ultimate outcome of infection.

Paul Gutwein, Anja Schramme, Liliana Schäfer, Hermann-Josef Gröne, Mohamed 

Sadek, Ingeborg Hauser, Josef Pfeilschifter 

The role of transmembrane and released CXCL16 during 

inflammatory processes in the kidney 

Inflammatory cell infiltrates are a hallmark of chronic kidney diseases (CKDs). 

Understanding the molecular mechanism that regulate renal leukocyte recruitment 

suggest chemokines and chemokine receptors as new targets for specific 

pharmacological intervention. In vitro we have analyzed the expression, localization and 

cleavage of CXCL16 in human renal mesangial cells. We can show that CXCL16 

expression is found in the nucleus, in lysosomes and in the endoplasmatic reticulum 

whereas no colocalization of CXCL16 with the Golgi stacks could be observed. 

Interestingly, when we stimulated the cells with INF-g, IL1b or LPS we could induce the 

expression of CXCL16. Additionally the application of IFN-g resulted in increased 

amounts of soluble CXCL16 in the supernatant of the cells. By using RNA interference 

we could inhibit ADAM10 and ADAM17 very efficiently and we clearly can show that both 

metalloproteinases are involved in the constitutive as well as in the inducible release of 

CXCL16 from mesangial cells. Interestingly the expression of endogenous CXCL16 was 

not influenced by the knockdown of ADAM10 or ADAM17 suggesting that both 

metalloproteinases may play important function in the transport of CXCL16. 

In vivo we found CXCL16 expression in the healthy kidney mainly in podocytes and 

distal tubular cells. Furthermore increased amounts of CXCL16 were found in the 

glomerulus of kidney transplanted patients. Importantly CXCR6 expressing immune 

cells were found in high numbers in the inflamed kidney of transplanted patients. 

Analyzing urine samples of patients with inflammatory kidney diseases, we found 

increased amounts of soluble CXCL16 compared to healthy volunteers. Taken together 

this data suggest an important participation of the chemokine CXCL16 and its receptor 

CXCR6 in the recruitment of leukocytes into the kidney during inflammatory processes.

Kai Schulze, Thomas Ebensen, Karina Watzke, Michael Morr, Carlos Guzman 

The second messenger 5′GMP is a promising adjuvant for 

vaccine development 

Vaccination by the mucosal route usually requires the use of specific adjuvants in order 

to stimulate efficient immune responses. Unfortunately, there are only a few molecules 

exhibiting this property, and, up to now, there is no candidate licensed for human use. 

Thus, there is an urgent need to develop new adjuvants. In the present work we 

demonstrate that 5′ guanosine monophosphate (5´GMP), a second messenger which 

modulates the cell surface properties of several microorganisms, acts as a potent 

mucosal adjuvant. Co-administration of 5´GMP with the model antigen ß-galactosidase 

(β-gal) to BALB/c mice by intranasal route resulted in 2,300-fold increased ß-galspecific 

serum IgG titers respect to what observed in animals immunized with β-gal 

alone. Strong antigen-specific secretory IgA responses were also measured in lung 

lavages (p=0.003). The analysis of both the major antigen-specific IgG isotypes in sera 

and the cytokines secreted by splenocytes restimulated ex vivo with β-gal suggested 

that a mixed Th1/Th2-response pattern was promoted in vaccinated mice. The obtained 

results suggest that 5´GMP constitutes a promising candidate adjuvant for the 

establishment of immune prophylactic interventions.

Matthias Eberl, David Vermijlen, Francesco Dieli, Nadia Caccamo, Serena Meraviglia, 

Giuseppe Cicero, Andrew Roberts, Bernhard Moser, Hassan Jomaa, Adrian Hayday 

The unexpected functional plasticity of human γδ T cells: 

implications for immunotherapy and beyond 

Human peripheral blood Vγ9/Vδ2 T cells expand extensively in many infections, in 

reaction toward the microbial metabolite HMB-PP. This 'transitional' response of γδ T 

cells, occurring temporally between the rapid innate and slower adaptive response, is 

widely viewed as pro-inflammatory and/or cytolytic. However, under the influence of an 

appropriate microenvironment, Vγ9/Vδ2 T cells readily assume features conventionally 

associated with Th1 (IFN-γ, TNF-α), Th2 (IL-4, GATA3), and follicular B helper TFH cells 

(CXCL13, IL-21R), respectively, as well as with professional antigen-presenting cells 

(HLA-DR, CD86). Such broad plasticity emphasizes the capacity of human γδ T cells to 

influence profoundly the nature of the immune response to different challenges, and is 

of relevance for ongoing clinical applications. Among the molecular signatures identified, 

tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), for instance, was only 

expressed by pro-inflammatory, Th1-like, but not by Th2-like or TFH-like Vγ9/Vδ2 T 

cells. This could be directly corroborated in vivo in a phase I trial to promote non-MHCrestricted 

anti-tumour efficacy in metastatic hormone-refractory prostate cancer, using 

the Vγ9/Vδ2 T cell agonist zoledronate. Most patients treated with zoledronate in 

combination with low-dose IL-2, but conversely only 2 out of 9 treated with zoledronate 

alone, displayed a long-term shift of γδ T cells toward an activated effector/memory-like 

state (TEM ), producing IFN-γ and perforin. These patients also maintained serum TRAIL 

levels. Most importantly, γδ TEM cell numbers showed a correlation with declining 

prostate-specific antigen levels and objective clinical outcomes. γδ T cell phenotypes and 

serum TRAIL may thus constitute novel biomarkers of prognosis in metastatic cancer 

immunotherapy. On a final note, our data also imply that while co-delivery of IL-2 

appears crucial for optimal cytotoxicity, the considered use of other cytokines such as IL- 

21 in carcinoma trials might potentially lead to an (unwanted or wanted) diversion of 

the immune system away from a pro-inflammatory response.

Rebekka Geiger, Antonio Lanzavecchia, Federica Sallusto 

THE “T CELL AMPLIFICATION METHOD” AS A NEW TOOL TO 

ANALYZE THE REPERTOIRE OF HUMAN T CELLS 

By using T cell receptor (TCR) gene amplification and subsequent sequencing it was 

shown that circulating human T cells contain 2.5x107 distinct TCRs in the naïve pool 

and 2x105 TCRs in the memory pool. These molecular approaches, while showing TCR 

heterogeneity, cannot establish a direct relationship between TCR sequence and antigenspecificity. 

We are developing a new method to analyze the antigen-specific repertoire 

of human T cells. Highly pure naïve CD4+ T cells were sorted from peripheral blood 

mononuclear cells (PBMC’s) of several healthy donors on the basis of CD45RA and CCR7 

expression and stimulated polyclonally in limiting dilution (1,000 cells/well) by PHA, 

irradiated feeder cells and IL-2. For comparison, memory T cells (CD45RA-) were also 

isolated from the same donors and stimulated in limiting dilution (400 cells/wells) under 

the same conditions. After a ~1000-5000-fold amplification, each line was stimulated 

with autologous monocytes and a panel of naïve and recall antigens. Proliferation was 

measured using 3H-Thymidine incorporation as read-out. Using this method we 

assessed frequencies of T cells specific for different antigens, e.g. KLH, Anthrax PA, TT, 

DerpI and PPD. The data obtained so far indicate that frequencies of antigen-specific T 

cells in the naïve pool are much higher than previously thought, whereas the 

frequencies of antigen-specific T cells in the memory pool vary with the antigenic 

experience of the individual.

Stefanie Thiele, Andreas Pahl 

Therapy of chronic airway diseases with nucleic acids 

Asthma is a chronic inflammatory disease of the airways characterized by airway 

obstruction, epithelial damage and airway hyperresponsiveness. Inflammatory cells like 

eosinophils, macrophages and neutrophils are capable of releasing cytokines, proteases, 

reactive oxygen species and lipid mediators that contributes to the pathogenesis of 

asthma. Chronic obstructive pulmonary disease (COPD) is a disease state characterized 

by a progressive, irreversible limitation of airflow associated with an abnormal 

inflammatory response to noxious particles or gases. 

Aim of the project is the development of a nucleic acid based therapy for these kinds of 

chronic airway diseases. First different knock-down strategies will be evaluated in vitro. 

In parallel the optimal route of application and in vivo delivery method is investigated. 

To this end transgenic GFP-mice will be used. This allows the monitoring of cell specific 

knock-down effects. Finally genes relevant for chronic airway diseases will be knocked 

down in vivo. Various animal models of chronic airway diseases are established in the 

laboratory. The effect of the gene specific knock-down will be analysed for various 

pathological features in these animal models.

Arvind Batra, Markus M. Heimesaat, Stefan Bereswill, Jeannette Pietsch, Thorsten 

Stroh, Rainer Glauben, Inka Fedke, Martin Zeitz, Britta Siegmund 

TLR – expression in cells from adipose tissue – a contribution 

to chronic inflammation? 

Hypertrophy of mesenteric fat is a characteristic finding in Crohn’s disease. However, 

the underlying mechanisms and the contribution to the disease itself are still unknown. 

In addition to its endocrine role, adipose tissue is the producer of various proinflammatory 

mediators like leptin, IL-6 and TNF-α. We recently demonstrated leptindependent 

expression and function of Toll-like receptors (TLR) in murine preadipocytes 

and adipocytes (Batra et al., Am J Pathol, in press). To transfer these findings to 

humans TLR1 to -10 expression and function was analyzed in preadipocyte cell lines, 

established from human mesenteric fat, by RT-PCR and TLR-specific stimulation with 

subsequent evaluation of cytokine production. To further determine whether in vivo a 

direct interaction of cells from adipose tissue and bacterial TLR-ligands can occur, 

bacterial translocation was monitored in acute and chronic DSS-induced colitis in WT 

and MyD88 +/- mice by analysis for the presence of life bacteria via culture. 

In concordance with the murine system, in human preadipocyte cell lines TLR mRNA is 

expressed and cells respond to TLR-specific stimulation as evaluated by IL-6 production. 

In acute DSS-induced colitis no bacterial translocation was observed. In chronic DSSinduced 

colitis bacterial translocation into various abdominal tissues including fat 

occurred. Interestingly, bacterial translocation was more common in the MyD88 +/- mice 

with alterations in TLR-signaling. Our data provide strong evidence that functional TLR 

are expressed in preadipocytes and adipocytes across species. Suggesting that bacteria 

translocating to the mesenteric fat during intestinal inflammations such as Crohn’s 

disease can directly activate preadipocytes and adipocytes and thus might contribute to 

the mesenteric hypertrophy observed.

Linda Diehl, Anna Schurich, Regina Grochtmann, Silke Hegenbarth, Lieping Chen, 

Percy Knolle 

Tolerogenic maturation of LSEC after cognate T cell 

interaction promotes 

B7-H1-dependent CD8 + T cell tolerance 

Liver sinusoidal endothelial cells are unique organ-resident antigen presenting cells 

capable of cross-presentation and subsequent tolerisation of naive CD8 + T cells. Here, 

we investigated the molecular mechanisms underlying this tolerance induction in naive 

CD8 + T cells. MHC class I-restricted antigen-presentation by LSEC led to initial 

stimulation of naive CD8 + T cells, which upregulated CD69, CD25, CD44 and PD-1 and 

proliferated similar to dendritic cell activated CD8 + T cells. Importantly, cognate 

interaction with naive CD8 + T cells triggered increased expression of co-inhibitory B7- 

H1 but not co-stimulatory CD80/86 molecules exclusively on LSEC but not DC. This 

matured phenotype of B7-H1 high CD80/86 low was critical for induction of CD8 + T cell 

tolerance by LSEC: B7-H1 deficient LSEC, that failed to interact with PD-1 on stimulated 

T cells, were incapable of inducing CD8 + T cell tolerance. Moreover, increased 

costimulation via CD28 interfered with tolerance induction, indicating that the noninducible 

low expression levels of CD80/86 on LSEC supported B7-H1-dependent 

tolerance induction. LSEC-tolerized CD8 + T cells had a distinctive phenotype from naive 

and activated T cells with CD25 low , CD44 high , CD62L high . They also expressed the 

homeostatic cytokine receptors CD127, CD122 and high levels of Bcl-2, indicating 

survival rather than deletion of tolerant CD8 + T cells. Importantly, upon adoptive 

transfer into congenic animals tolerized CD8 + T cells failed to show specific cytotoxicity 

in vivo. Conclusion: Cognate interaction of LSEC with nave CD8 + T cells elicits a unique 

tolerogenic maturation of LSEC and permissiveness of T cells for tolerogenic signals 

demonstrating that LSEC-induced tolerance is an active and dynamic process.

Elke Gülden, Seiji Imamura, Masaru Ihira, Christiane Habich, Hubert Kolb, Volker 

Burkart 

Toll-like receptor 4: A key regulator of beta-cell directed 

autoimmunity in a mouse model of type 1 diabetes. 

Type 1 (insulin-dependent) diabetes (T1D) is caused by the destruction of autologous 

insulin producing pancreatic beta cells by innate and adaptive immune mechanisms. 

Recent investigations implicate that the progression of autoimmune reactivity is 

controlled by Toll-like receptor 4 (tlr4), originally described as the mammalian receptor 

for lipopolysaccharide (LPS). To investigate the role of tlr4 in the pathogenesis of insulindeficiency 

diabetes, we backcrossed the tlr4 defect of the C57BL/10ScCr mouse on the 

background of the non-obese diabetic (NOD) mouse, a model of human T1D. We found 

that female NOD mice with a homozygous tlr4 defect (tlr4-/-), showed a significant 

acceleration of diabetes development (p

Marina Scheler, Manuela Brenk, Helene Wilms, Thomas Bieber, Dagmar von Bubnoff 

Toll-like receptor ligands, and not T cell signals, are strong 

inducers for Indoleamine-2,3-dioxygenase (IDO),and lead to 

T cell suppression 

Immature dendritic cells (DCs) express receptors for inflammatory chemokines and 

migrate to sites of inflammation. Inflammatory cytokines as well as classes of microbial 

molecules (pathogen-associated molecular patterns, PAMP) trigger maturation and drive 

migration of DCs into the T cell areas of the draining lymph nodes. Stimuli derived from 

activated T cells such as IFN-γ and CD40L further impact on DC activation and survival. 

The outcome of the DC-T cell interaction can be productive priming or tolerance. 

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades the essential aminoacid 

tryptophan from the environment and leads to T cell tolerance. IDO can be induced 

in antigen-presenting cells upon inflammatory stimuli. IDO is thought to be important to 

prevent autoimmunity and allergy, but IDO can also facilitate chronic inflammation 

through induction of pathogen persistence. Using quantitative assays to monitor IDO 

expression and enzyme activity, we show that DC maturation renders these cells 

putatively capable of inducing tolerance via IDO. The strongest IDO activity was not 

seen in DCs stimulated with the best-established IDO-inducing protocol (i.e. with IFN-γ), 

but during stimulation by strongly maturing TLR ligands such as highly purified LPS 

(TLR4 ligand) or poly I:C (TLR3 ligand), suggesting T cell independent and autonomous 

IDO induction through pathogens in the innate immune system. Further, we show a 

strong upregulation of IDO in human DCs in skin ulcera associated with severe 

pathogen load. Together, these results can be viewed as an immediate and T-cell 

independent mechanism to ensure control of immune effector functions aiming to 

counter-regulate the adaptive T cell response.

Hans-Heinrich Oberg, Jan Lenke, Susann Beetz, Norbert Reiling, Dieter Kabelitz, 

Daniela Wesch 

Toll-like receptor-2 ligands act on Treg to abolish suppression 

of CD4 + CD25 - responder T cell proliferation and IL-2 

production 

CD4 + CD25 high regulatory T cells (Treg) control cellular immune responses and maintain 

peripheral tolerance. Treg suppress proliferation of CD4 + CD25 - responder T cells, which 

can be overcome indirectly by stimulation of Toll-like receptors (TLR) on antigenpresenting 

cells. Thus, TLRs function as molecular sensors that guide innate immune 

responses and are also involved in the regulation of adaptive immune responses. We 

and others have recently shown that TLRs are also expressed in human T lymphocytes. 

In this study we investigated whether TLR2 ligands have a similarly crucial role in 

regulating human Treg as has been described for TLR5 and TLR8 ligands. We used a 

feeder cell-free suppression assay to examine the influence of TLR ligands in the 

absence of antigen-presenting cells. We observed that TLR2 ligands such as bacterial 

lipopeptides Pam2CSK4, Pam3CSK4 and FSL-1 did not exert any effect by themselves 

but enhanced the anti-CD3/anti-CD28-stimulated proliferation of CD4 + CD25 - T cells, 

whereas proliferation of Tregs was not affected. Moreover, lipopeptide-induced reversal 

of Treg suppression did not seem to be due to a costimulatory effect on CD4 + CD25 - T 

cells because only the preincubation of Treg but not of CD4 + CD25 - T cells with either 

Pam2CSK4, Pam3CSK4 or FSL-1 nearly completely restored the suppression. The IL-2 

content in supernatant of stimulated CD4 + CD25 - T cells was diminished in the presence 

of Treg and was restored when Treg were preincubated with TLR2 ligands. In ongoing 

investigations we examine the influence of TLR2 ligands on p27 kip as a marker for cell 

cycle arrest. Our results provide the first evidence that lipopeptides are able to 

antagonize human Treg activity and thus could play a critical role in regulating Treg and 

T cells activation. 

This work was supported by the DFG (SFB415, project A15)

Elena Babich, Ata-Ur Rasheed, Hans-Peter Rahn, Martin Lipp, Gerd Müller 

Tracking follicular B helper T cell differentiation and function 

by gene expression analysis 

T cell-dependent immune responses are associated with the development of germinal 

centers in which antigen-specific B cells differentiate into memory B cells and highaffinity 

antibody-secreting plasma cells. The germinal center reaction critically depends 

on the presence of follicular B helper T (TFH) cells, a specialized subset of CD4 T cells 

providing B cell help. TFH cells have been initially identified by expression of the 

chemokine receptor CXCR5, which guides these cells into follicles, and their ability to 

efficiently induce immunoglobulin secretion by germinal center B cells. In addition, TFH 

cells control affinity maturation and isotype switching of germinal center B cells. We 

have recently shown that the differential expression of CXCR5 and the co-stimulatory 

molecule ICOS defines subsets of tonsillar CD4 T cells that differ in their stimulatory 

capacity, proliferative capacity and susceptibility to apoptosis. Our results suggest that 

in humans follicular B helper T cell activity is confined to CXCR5(hi)ICOS(hi) CD4 T 

cells. To more precisely define the differentiation pathway and molecular mechanisms 

mediating essential physiological and pathophysiological functions of TFH cells we have 

now extended our gene expression analysis and functional characterization of CD4 T cell 

subsets. In this connection we are particularly interested in better understanding the 

relationship of CXCR5 expressing CD4 T cells in the periphery and in lymphoid tissues as 

well as the role of TFH cells in the pathophysiology of chronic inflammatory and 

autoimmune diseases.

Ana Teles, Catharina Thuere, Milan Popovic, Anne Schumacher, Paul Wafula, Hans- 

Dieter Volk, Ana Zenclussen 

Trafficking of regulatory T cells during pregnancy: Expression 

profile of Treg-related chemokine and chemokine receptor at 

the maternal-fetal interface. 

Regulatory T cells (Treg) are shown to have an important role in the maintenance of 

fetus tolerance along pregnancy. The mechanisms leading to the recruitment of these 

cells at the fetal-maternal interface are still unknown, but like in other systems, 

chemokines are likely to be involved. 

Given the existent concordance between the expression of chemokines in the uterus/ 

placenta and their receptors in Treg, we selected several receptor-ligand pairs that may 

be involved in the recruitment of these cells into the fetal-maternal tissues along 

pregnancy. By real time RT-PCR we investigated the expression patterns of the selected 

chemokine ligands in different phases of pregnancy (day 0, 2, 5, 8, 10 or 12 of 

pregnancy) in uterus, decidua and placenta from normal pregnant C57/BL6-mated BALB/ 

c mice. Levels of Treg were analyzed by flow cytometry and by quantifying foxp3 

mRNA. 

Pregnant animals showed a gradual increase in the % of Treg in blood, spleen and 

decidua along pregnancy as well as increased foxp3 mRNA levels at the fetal-maternal 

interface. Regarding chemokines, our data suggest differential patterns of expression at 

different time points. We observed a gradual up-regulation of CXCL12 in placenta 

beginning on day 12 and of CCL21 in decidua beginning on day 10 of pregnancy. 

CXCL12 was further found to be increased in decidual tissue with a peak on the 5th day 

of pregnancy (implantation). For the chemokines CXCL13 and CCL20 no specific pattern 

of expression could be found. 

These results suggest different chemokines being responsible for the trafficking of Treg 

at different pregnancy stages.

Katharina Gropp, Michael Reuter, Gerhard D. Wieland, Christine Skerka, Peter F. Zipfel 

Transcription factors EGR-2 and NFkappaB induce chemokine 

synthesis in macrophages challenged with the human 

pathogenic yeast Candida albicans 

Candida albicans is the major human opportunistic fungal pathogen that can cause lifethreatening 

systemic infections. Macrophages represent the first cellular level for 

recognition of this human pathogenic yeast and recognize yeast cells by Toll Like 

Receptors (TLRs) and induce immune response by synthesis of cytokines. 

Recently we described that EGRs (early growth response protein) interact with the 

nuclear factor NFkappaB and strongly activate cytokine gene expression. To investigate 

whether Candida albicans induces in macrophages the EGR and NFkappaB transcription 

factors we determined the induction and also the transcriptional function of EGR and 

NFkappaB in macrophages triggered by Candida albicans. RT-RNA analysis revealed 

strong induction of EGR-2 and NFkappaB in macrophages exposed to the yeast- and 

hyphae-form of Candida albicans. However, whereas the yeast-form of Candida albicans 

induces the synthesis of pro-inflammatory cytokines like MIP-1alpha and MIP-1beta, the 

hyphae-form leads to the induction of anti-inflammatory cytokines like IL-10. The use of 

defined signal transduction inhibitors (PD 98059 and BAY 11-7082) showed that the 

induction of cytokines MIP-1alpha and MIP-1beta in response to Candida albicans is 

transmitted via the ERK- und NFkappaB-pathway. With transfection assays and confocal 

microscopy we demonstrate that the synthesis of chemokines MIP-1alpha and MIP- 

1beta in macrophages in response to Candida albicans is mediated by synergistic 

activity of EGR-2 and NFkappaB.

Yuriy Shebzukhov, Sergei Grivennikov, Andrey Kruglov, Chang-Yi Cui, Ina Wagner, 

Anna Litvin, Hans-Joachim Mollenkopf, David Schlessinger, Dmitry Kuprash, Sergei 

Nedospasov 

Transcriptional analysis of lymphoid organs from mice with 

conditional inactivation of TNF and LT 

Tumor necrosis factor (TNF) alpha and lymphotoxins (LT) are important cytokines 

involved in development of immune system, autoimmunity and host defense against 

infections. Using mice with conditional inactivation of TNF-alpha or LT-alpha in T cells, B 

cells and macrophages/neutrophils we are dissecting physiological role of these 

cytokines produced by different types of leukocytes. To investigate underlying molecular 

mechanisms and signaling pathways we perform gene array analysis followed by 

Northern blotting and/or quantitative RT-PCR. We found that in lymphoid organs TNFalpha 

derived from different cell types can activate distinct sets of genes, including 

chemokines and adhesion molecules. Overall T and B cell derived TNF-alpha control 

distinct subsets of genes in the spleen and lymph nodes. Some of TNF-alpha dependent 

genes are controlled solely by B cells derived cytokine. 

Earlier we found that different in vivo functions of TNF-alpha and LT-alpha are mediated 

by distinct types of leukocytes, now we demonstrated that these functions might be 

controlled by different downstream genes.

Monika Lindemann, Alexandra Schumann, Melanie Fiedler, Camino Valentin-Gamazo, 

Dietmar Knop, Christoph E. Broelsch, Michael Roggendorf, Hans Grosse-Wilde 

Transfer of adoptive immunity to hepatitis B virus by liver 

transplantation* 

Liver transplantation is often the ultimate option of therapy for chronically hepatitis B 

virus (HBV) infected patients. Despite antiviral therapy reinfection is still a problem. 

Adoptive transfer of HBV immunity with the liver after vaccination of living liver donors 

could be a new approach to prevent reinfection in the recipients. The period in which to 

achieve HBV immunity in donors is usually short (1-2 months). In this study we 

vaccinated living liver donors in a short time immunization protocol (4 injections in two 

weeks intervals) using Hepimmune (Berna Biotech), a recombinant vaccine that 

contains the PreS1, PreS2, and S proteins of HBV. Humoral (anti-HBs titer) and cellular 

(proliferation assay) immune responses were examined prior to each immunization, pre 

and post transplantation. So far 14 patients received a liver from an immunized living 

donor. In 4 recipients adoptive immune transfer was observed. One HBV naive recipient 

developed humoral and cellular immune responses post transplantation (anti-HBs of 

1800 IU/l and stimulation index of 4.9). Two further naive recipients showed cellular 

immune responses (stimulation indices of 2.7 and 7.1) and in one chronically HBV 

infected recipient specific immune responses were measurable at day 5 (anti-HBs of 120 

IU/l) and vigorously increased thereafter (anti-HBs of 58000 IU/l and stimulation index 

of 3.1). This finding most likely reflects the boosting of HBV specific immunity by HBs 

antigen persisting in the host. Taken together, we were able to show that HBV specific 

humoral and cellular immunity can be transferred to recipients with the liver graft. 

Thereby, the reinfection in chronically HBV infected recipients could be prevented. 

*This study was partially supported by the DFG (KFO 117).

Annette Busch, Thomas Quast, Waldemar Kolanus, Percy Knolle, Andreas Limmer 

Transfer of T cell surface molecules to Dendritic cells upon T 

cell priming involving two distinct mechanisms differing in 

quality and time 

The adaptive immune response is initiated in secondary lymphoid organs by contact 

between antigen-presenting cells (APCs) and antigen-specific CD4+ T cells. This 

activation is characterized by multiple antigen recognition events that comprise the 

interaction of both cell types through various molecules. In addition to the exchange of 

costimulatory signals and cytokines the cells transfer cell surface molecules. To date 

this is reported only as a unidirectional process of transfer from APC surface molecules 

to the T cell. Here we describe for the first time the transfer of human and murine T cell 

surface receptors to the APC. This transfer occurs in two phases that differ in quality 

and time. The first set of molecules is transferred rapidly after T–APC contact, is cell– 

cell contact-dependent, bound in an acid wash-resistant form to the cell surface and 

resembles a mechanism described as trogocytosis, whereupon the APC actively gnaws 

membrane fragments from the T cell membrane. The second set of T cell molecules is 

exchanged after T cell activation (>16h), is non-covalently bound (acid wash sensitive) 

and is transferred in a cell–cell contact-independent mode, resembling molecule 

exchange via exosomes. The transferred molecules may have an important role in APC 

conditioning and immunoregulation since they include the T cell receptor (TCR), CD3 

and costimulatory molecules. Murine dendritic cells (DCs) loaded with T cell surface 

molecules from ovalbumin (OVA)-specific CD4+ T cells by antigen-specific interaction 

were less efficient in priming naïve CD4+ T cells of the same specificity. The great 

amount of transgenic TCR on the DC surface may be responsible for the reduced 

activation of the T cells by masking the antigen-bearing MHC molecules for access of 

following antigen-specific CD4+ T cells. These findings may indicate an important role of 

transferred T cell molecules to APC in the intraclonal competition of T cells for APC 

access.

Jacqueline Moebius, Michael Basler, Marcus Groettrup 

TRANSGENIC MICE REVEAL A NOVEL ROLE FOR LMP7 IN TCR 

TRIGGERED EXPANSION OF T CELLS 

During an ongoing immune response three cytokine-inducible proteasome-subunits, 

LMP2 (β1i), LMP7 (β5i) and Mecl-1 (β2i), are incorporated into newly synthesized 

proteasomes instead of their corresponding constitutive subunits Delta (β1), MB1 (β5) 

and MC14 (β2) to provide the cells with a more diverse antigen processing machinery. 

Accumulating evidences point to an additional, so far uncharacterised role of 

immunoproteasome subunits: In adoptive transfer experiments immunoproteasome 

deficient T cells are unable to compete against wild-type T cells of the recipient during 

an antiviral immune response. After ruling out rejection phenomena being responsible 

for these observations we are currently investigating how immunoproteasomes can 

control T cell expansion in terms of influencing homing, proliferation or apoptosis of 

cells. 

In a first approach we wanted to determine the fate of the virus-specific T cells in these 

experiments and therefore crossed LMP7 gene targeted mice with P14 mice (T cell 

receptor transgenic for the Lymphocytic Choriomeningitis Virus epitope GP33). Again, 

40 hours after transfer we found about ten times less LMP7 -/- /P14 T cells in the spleens 

of recipient mice than P14 T cells in the control animals. In vitro stimulation of these 

cells with their appropriate peptide (GP33), presented on gamma-irradiated spleen cells, 

leads to a profound hyper proliferation of the LMP7 -/- /P14 T cells compared with the P14 

wild-type cells. Currently, we are testing the propensity of LMP7 -/- T cells to undergo 

apoptosis after TCR triggering and we compare T cell differentiation markers of LMP7 

deficient and proficient T cells. We hypothesize that immunoproteasomes serve a 

function e.g. in the processing of a factor needed for antigen driven T cell expansion 

that can not be fulfilled by constitutive proteasomes.

Volker Storn, Karsten Mahnke, Sonja Schallenberg, Theron S. Johnson, Tanja Bedke, 

Natalio Garbi, Bernd Arnold, Günter J. Hämmerling, Alexander H. Enk 

Treatment of established RMA-Tag tumors via DC directed 

targeting of tumor antigens is further improved by depletion 

of regulatory T cells. 

Tumor antigens, chemically coupled to the antiDEC205 antibody, can target dendritic 

cells in vivo and were successfully applied in murine preventive tumor vaccination 

studies. In this study our aim was to test whether antiDEC205-protein conjugates have 

the potential to reduce the tumor growth of already established tumors. For this end we 

coupled the tumor antigen Tag to antiDEC205 and treated RMA-Tag tumor-bearing mice 

with these conjugates. When the tumors had reached an average diameter of 5 mm the 

mice were injected with antiDEC205-Tag conjugate or Tag protein, plus adjuvants in 5day 

intervals. Although tumor growth was initially slowed down after injection of the 

antiDEC205-Tag conjugates as compared to Tag-treated mice, it resumed two to three 

weeks after the first vaccination. Analysis of the immune response initially revealed a 

strong induction of tumor specific CD8+ T-cells in antiDEC205-Tag treated mice. 

However, when tumor growth resumed increased numbers of CD4+CD25+FoxP3+ 

regulatory T cells (Tregs) in the tumor draining lymph node were noticeable. Thus, to 

assess the suppressive effects of Tregs during tumor therapy, anti-CD25 antibodies 

were used to deplete Tregs from tumor bearing animals during anti-tumor treatment. 

By concomitant injection of antiDEC205-Tag conjugates we could cure up to 40% of the 

tumor bearing mice and tumors were rejected. Furthermore, the survival of tumor 

bearing mice of the autochthonous tumor model RIP-Tag5 could be prolonged for up to 

ten weeks after treatment with antiDEC205-Tag conjugates compared to untreated, or 

Tag-treated mice respectively. In aggregate our data indicate that depletion of CD25+ 

Treg during cancer therapy bolsters the effectiveness of DC based anti-tumor therapies 

and that antiDEC205 targeted vaccination improves treatment of autochthonous 

tumors.

German Salamov, Rupert Holms, Wolfgang G. Bessler, Ravshan Ataullakhanov 

Treatment of Hepatitis C Virus Infection with Human Ezrin 

Peptide One (HEP1) in HIV Infected Patients 

This report shows the therapeutic benefit of HEP1 (Human Ezrin Peptide 324-337; 

TEKKRRETVEREKE) monotherapy of HCV infection in HIV infected patients in 2 clinical 

studies. In the Pilot Study I, 16 of 18 patients responded well to the treatment with 

significant reductions of HCV viral load and a normalization of serum liver enzymes. In 8 

of 18 patients, HCV RNA became undetectable, and 3 of 8 interferon/ribavirin treatment 

failure patients showed undetectable HCV load following HEP1 treatment. In the second 

study, 8 of 10 patients responded well to the treatment with a pronounced reduction of 

the HCV viral load and a normalization of serum liver enzymes. 3 of 15 patients (20%) 

showed an undetectable viral load 30 days after the end of a 30 day course of HEP1 

treatment. In both studies, all genotypes of HCV were sensitive to HEP1 treatment. 

Analysis of the combined data from both studies showed the overall efficacy of HEP1 

therapy: in 37 HCV+HIV patients, HEP1 therapy gave the following results: 10 of 37 

(27%) HCV+HIV patients showed a reduction of viral load between -7 log (- 

10,000,000x) and -3 log (-1,000x); 4 of 37 (11%) a reduction of -3 log (-1,000x); 6 of 

37 (16%) a reduction of -2 log (-100x); 11 of 37 (30%) a reduction of -1 log (-10x); 6 

of 37 (16%) a reduction of less than -1 log (-10x); 0 of 37 (0%) had an increase in viral 

load, and the average reduction in viral load for all 37 patients was -2 log (-100x). No 

adverse reactions or side effects were detected and the improving CD4/CD8 ratio 

showed that the therapy had no negative impact on the immunological status. Thus, 

oral HEP1 therapy matches the efficacy results for injectable peginterferon/oral ribavirin 

therapy with the advantages of more rapid action and less side effects. HEP1 therapy 

should be used in patients where either peginterferon/ribavirin therapy fails or is 

contraindicated.

Claudia Stühler, Sarah Lurati, Manik Chatterjee, Ralf C. Bargou, Hermann Einsele, Max 

S. Topp 

Treatment of T lymphocytes with Hsp90 inhibitors selectively 

kills activated cells while preserving viability and 

functionality of resting cells 

Graft-versus-host disease (GvHD) is a major cause of morbidity and mortality in 

patients with hematological malignancies undergoing allogeneic hematopoietic stem cell 

transplantation (HSCT). Current treatment of GvHD utilizes immunosuppressive 

regimens which put the patient at a high risk of opportunistic infections. Therefore 

selective approaches for specific eradication of GvHD-specific T-cells are highly 

warranted. Inhibition of heat shock protein 90 (Hsp90) could be one approach, as 

Hsp90 aids in the maturation and stabilization of a wide variety of client proteins, some 

of which are integral components of signal transduction pathways of activated T-cells. 

In the first set of experiments selective upregulation of both Hsp90 subunits in activated 

primary T-cells was confirmed and shown to be impeded by application of the Hsp90 

inhibitors Geldanamycin and 17-DMAG as well as with siRNA. Addition of Hsp90 inhibitor 

to either mitogen-activated T-cells or T-cells activated by allogeneic stimulator cells lead 

to preferential eradication of activated cell populations. The preferential killing of only 

activated cells could be shown by stimulation of 17-DMAG pretreated mixed lymphocyte 

cultures with either autologous APC presenting viral antigens or rechallenge with the 

same allogeneic dendritic cells. Whereas virus-specific T-cells could readily be activated, 

no activation could be detected in 17-DMAG pretreated T-cell cultures exposed for a 

second time to the allogeneic stimulators. Untreated cultures on the other hand 

responded to both stimuli. To verify which crucial signalling pathways of activated Tcells 

are abrogated in the presence of Hsp90 inhibitors further western blot experiments 

were performed. 

This experimental data demonstrate that Hsp90 inhibitors can selectively remove 

alloreactive T lymphocytes upon activation without compromising the viability and 

functionality of the remaining lymphocyte populations including the virus-specific 

immune response.

Kai Zanzinger, Carola Schellack, Gernot Geginat, Adelheid Cerwenka 

TREM-1 in infection and cancer 

Cells of the innate immune system play an important role in the first line of defense 

against bacteria and viruses. Their activation is controlled by different receptor families 

including the Ig superfamily with both inhibitory and activating isoforms. Activating 

isoforms deliver stimulatory signals by their association with transmembrane adapter 

proteins bearing Immunoreceptor Tyrosine-based Activation Motifs (ITAM). DAP12 is 

such an adapter protein that associates with several receptor molecules such as the 

Triggering Receptor Expressed on Myeloid cells-1 (TREM-1). 

In human and mice, TREM-1 is highly expressed on granulocytes. However, little is 

known about the modulation of its expression on monocyte subpopulations in mice. 

Blood of naïve mice comprises two subsets of monocytes: ‘resident’ (F4/80 + Gr-1 - CD62L - 

CX3CR1 hi ) and ‘inflammatory’ monocytes (F4/80 + Gr-1 + CD62L + CX3CR1 lo ). In our study, 

we analyzed TREM-1 expression on these monocyte subsets upon stimulation with Tolllike 

receptor ligands, infection with L. monocytogenes and in different cancer models. 

We show that in naïve mice TREM-1 is only detectable on ‘resident’ but not on 

‘inflammatory’ monocytes. In contrast, TREM-1 is upregulated on the F4/80 + Gr-1 + 

‘inflammatory’ monocyte subpopulation upon application of Toll-like receptor ligands 

and in L. monocytogenes infection. In addition, also in different tumor models the 

‘inflammatory’ monocyte subpopulation upregulates TREM-1. Interestingly, this 

population in tumor-bearing mice is phenotypical and functionally identical to recently 

described myeloid-derived suppressor cells (MDSC). 

The future objectives of this project are to identify the factors that regulate TREM-1 on 

this monocyte subpopulation in cancer.

Anna-Maria Herr, Olivia Sövegjarto, Lutz Walter 

TRIM proteins: Novel candidates for HIV resistance? 

Evolutionary processes of host-pathogen interaction considerably shape the genomes of 

both organisms, favouring the development of resistance genes (host) and of immune 

escape mechanisms (pathogen). Notably, retroviruses impact the evolution of the 

vertebrate immune system. Retroviruses encounter dominant postentry restrictions in 

cells of particular species. Human immunodeficiency virus (HIV)-1 for example is 

blocked by TRIM5α, a tripartite motif (TRIM) protein, in cells of Old World monkeys. 

Rhesus monkey TRIM5α more potently blocks HIV-1 infection than human TRIM5α. 

Recently, dozens of novel TRIM proteins have been identified. Although most of their 

physiological roles are not yet clear, preliminary evidence suggests a rapid evolution 

and adaptation to antiviral activity of some of the TRIM proteins. 

In this work, the evolution of the TRIM genes is studied in the primate lineage. We 

focused on TRIM genes with comparable domain-structure as TRIM5α (RING, B-box 2, 

coiled coil and B30.2 domain). By comparing the rates of synonymous (dS) and 

nonsynonymous (dN) substitutions between human and rhesus macaque TRIM genes 

we found differentially conserved TRIM genes. In contrast to the more conserved 

TRIM10 with only a few nonsynonymous substitutions between human and rhesus 

macaque, TRIM22 shows signs of positive selection. Rhesus TRIM22 is 91% identical to 

human TRIM22 in amino acid sequence. Interestingly we found the B30.2 of TRIM22 

less conserved (dN/dS=1,24) than the tripartite motif (dN/dS=0,61). Stremlau et al. 

(2004) showed, that the B30.2 domain of TRIM5α is responsible for restriction of HIV in 

rhesus macaques. With respect to these findings our data suggest that TRIM22 and 

other TRIM genes could also be involved in evolutionary processes of host-pathogen 

interaction and may open up novel perspectives in antiviral therapies.

Christiane Desel, Stefan H.E. Kaufmann 

Tuberculosis: Efficacy of DNA vaccines encoding dormancyassociated 

antigens 

It is estimated that one third of the world’s population is latently infected with 

Mycobacterium tuberculosis, the causative agent of tuberculosis. Of those 2 billion 

people 5-10% will develop active disease during their lifetime. The infection claims 2 

million lives every year and 9 million new cases are reported annually. 

M. tuberculosis has a striking capacity to evade the host’s immune system and to 

prevent its elimination. As a response to stress caused by a vigorous immune response 

the bacteria induce a dormancy programme enabling survival within host macrophages. 

But as soon as the immune surveillance fails, the dormant bacteria will be resuscitated 

leading to active tuberculosis. The only available vaccine against tuberculosis is 

Mycobacterium bovis BCG, one of the safest live vaccines known. However its protective 

effect against pulmonary tuberculosis in adults is debatable and protection wanes with 

time. Not only better pre-exposure vaccines to prevent infection in the first place but 

also post-exposure vaccines for those individuals harbouring dormant bacteria are 

urgently needed. 

It is known that mycobacteria express different antigens during the dormant state as 

compared to actively replicating ones and also the immune mechanisms to control 

primary and latent infection differ. Post-exposure vaccines will have to be designed 

based on antigens upregulated or solely expressed during dormancy. Dormancy is 

linked to hypoxic conditions within the granuloma and surrounding tissue and the 

dormancy survival regulator DosR has been identified as the primary factor which 

mediates the genetic response to reduced oxygen levels as well as exposure to nitric 

oxide. Here we examine the immunogenicity and efficacy of DNA vaccines based on 

DosR-regulated antigens in murine models of persistent and latent Mycobacterium 

tuberculosis infection.

Jenny Pahne, Subramanya Hegde, Nadine Schröer, Sigrun Smola-Hess 

Tumor cell-derived interleukin-6 skews myeloid dendritic 

cells from an immunostimulatory to a pro-tumorigenic 

phenotype 

Persistent infection with human papillomaviruses can lead to cervical intraepithelial 

neoplasia, which after years may further progress to cancer. Development of malignant 

disease is not the direct consequence of infection but the result of a complex interplay 

between the transformed cells and their microenvironment. During progression to 

malignancy a pronounced shift from TH1 to TH2 cytokines is observed. As a 

consequence, cytotoxic T cell responses necessary to eliminate the tumor are impaired. 

Currently, the mechanism, how cervical neoplastic cells contribute to this immunological 

shift is not known. 

Here we show that supernatants of cervical carcinoma cells suppress up-regulation of 

CCR7 and production of interleukin-12 (IL-12) in myeloid dendritic cells, which is 

important for mounting a TH1 response. Signalling pathways regulating CCR7 and IL-12 

expression were investigated. Supernatants of neoplastic cells strongly inhibited nuclear 

factor-kappaB (NF-kappaB) binding activity in dendritic cells, which is central to their 

immunostimulatory function. Corresponding to the expression pattern in vivo, cervical 

carcinoma cells did not produce IL-10 in vitro, but the pro-inflammatory cytokine IL-6. 

Neutralization of IL-6 in cervical cancer cell supernatants restored IL-12 production and 

NF-kappaB binding activity in dendritic cells. Of note, not all functions of dendritic cells 

were suppressed by the neoplastic cells. Despite inhibition of NF-kappaB binding 

activity, tumor cell-derived IL-6 up-regulated the pro-angiogenic and pro-tumorigenic 

matrix-metalloproteinase MMP-9 in dendritic cells. 

In summary, our data demonstrate that tumor cell-derived IL-6 skews dendritic cells 

from an immunostimulatory to a pro-tumorigenic phenotype.

Gerald Willimsky, Melinda Czéh, Christoph Loddenkemper, Johanna Gellermann, 

Harald Stein, Peter Wust, Thomas Blankenstein 

Tumor immunogenicity, not tumor burden is the primary 

cause of general cytotoxic T lymphocyte unresponsiveness 

Cancer is sporadic in nature, characterized by an initial clonal oncogenic event and a 

usually long latency. When and how sporadic cancer subverts the immune system is 

unknown. Here we show in a model of sporadic immunogenic cancer that tumor-specific 

tolerance closely coincides with first tumor antigen recognition by B cells. During the 

subsequent long latency period until tumors progress the mice develop general 

cytotoxic T lymphocyte (CTL) unresponsiveness, which is associated with high TGF- 

&beta1 levels and expansion of immature myeloid cells (iMC). In contrast, in mice with 

large non-immunogenic tumors, unrelated CTL responses are undiminished. 

Interestingly in these tumor-bearing mice iMCs are most dramatically expanded, but 

TGF-&beta1 serum levels are normal. We conclude that premalignant immunogenic 

lesions induce tolerance and therefore tumor latency is unlikely caused by CTL control. 

A persistent immunogenic tumor antigen causes general CTL unresponsiveness but 

tumor burden and iMC per se do not mediate immune suppression.

Christoph D. Brenner, Susan King, Imke Wolters, Georg Bornkamm, Martin Röcken, 

Ralph Mocikat 

Tumour control by natural killer cells in a spontaneous mouse 

lymphoma model 

We have previously shown that downregulation of MHC class I provides a “danger” 

signal that alerts the immune system resulting in target cell rejection and T-cell 

memory. Injection of MHC class I-low target cells activated natural killer (NK) cells 

thereby instructing dendritic cells to express IL-12 and to prime cytotoxic T 

lymphocytes (CTL). This “cross-priming” could explain the rapid induction of T-cell 

responses against viruses but left unanswered the question as to why this NK-cell help 

is apparently not effective enough to convey protective CTL responses against MHC 

class I-modulated autochthonous tumours. In the present study we used c-myctransgenic 

mice, which spontaneously develop B-cell lymphomas by the age of 12 

weeks, and studied the impact of tumour progression on the tumour reactivity of NK 

cells. Activation markers of NK cells were elevated after onset of tumour growth and 

clearly correlated with MHC class I downregulation of the developing tumours. 

Nonetheless, NK cells isolated from tumour-bearing animals were not capable of lysing 

tumour cells in vitro, and this was associated with reduced expression of several surface 

markers such as CD49b. Most interestingly, tumour growth could be delayed if NK cells 

received additional stimuli in vivo by treating mice with NK-cell-activating reagents. This 

indicates that NK cells indeed play a role in controlling growth of spontaneous 

lymphoma.

Nicolas Sabarth, Louise Chamberlain, John Tite, Sara Brett, Craigen Jenny 

Two-sided effect of T cell help in tolerance breaking and 

immunodominance 

To develop a successful cancer vaccine it is necessary to break immunological tolerance 

to self-antigens expressed on tumour cells. Broken immunological tolerance results in a 

functional CD8 T cell response, the development of which is known to be dependent on 

provision of CD4 help. In the transgenic RIP OVALOW mouse model which is 

hyporesponsive, ergo immunological tolerant, to ovalbumin, DNA vaccination with 

ovalbumin in combination with GM-CSF and the TLR7 agonist Imiquimod provided a 

strong synergistic adjuvant effect on CD4 cells resulting in broken tolerance in both the 

CD4 and CD8 compartments. Compared to wildtype mice RIP OVALOW mice had CD8 T 

cells of lower avidity, but functional CD8 T cell responses were evident by induction of 

tumour protection and correlating autoimmunity which served as a surrogate of efficient 

target cell killing. Heterologous CD4 help has been shown to improve functional CD8 

responses and we confirmed that inclusion of the T helper epitope PADRE enhanced CD8 

responses compared to self antigen alone both in RIP OVALOW mice and their wildtype 

counterpart although no autoimmunity was induced. Addition of GM-CSF and Imiquimod 

however resulted in dominance of the PADRE response over the ovalbumin specific CD4 

responses in both mouse strains. The effect of the PADRE on the CD8 T cell response 

was depended on the state of tolerance of the animals: PADRE specific CD4 help 

compensated for the lack of ovalbumin specific CD4 help and the CD8 T cell response 

was little affected in wildtype mice. In contrast, in tolerant RIP OVALOW mice the 

combination of PADRE and GM-CSF & Imiquimod decreased ovalbumin specific CD8 

numbers and function, abrogating diabetes development. Only the limited use of PADRE 

together with adjuvant enhanced DNA immunisation provided some benefit evident by 

accelerated autoimmunity in RIP OVALOW mice, i.e. target cell killing. 

Hence, Imiquimod/GM-CSF enhanced DNA immunisation has good potential as 

therapeutic cancer vaccine but the use of heterologous T cell help has to be handled 

with care and needs further investigation.

Christian Wahl, Petra Bochtler, Reinhold Schirmbeck, Jörg Reimann 

Type I IFN-producing CD4 Valpha14i NKT cells facilitate 

priming of IL-10-producing CD8 T cells by hepatocytes 

Upon entering the liver CD8 T cells encounter large numbers of NKT cells patrolling the 

hepatocyte (HC) surface facing the perisinusoidal space. We asked whether hepatic NKT 

cells modulate the priming of CD8 T cells by HC. Hepatic (alpha-galactosyl-ceramideloaded 

CD1d dimer binding) NKT cells produce predominantly IL-4 when stimulated with 

glycolipid-presenting HC but predominantly IFN-gamma when stimulated with glycolipidpresenting 

dendritic cells. These NKT cells prime naive CD8 T cells to a (K(b)-presented) 

peptide ligand if they simultaneously recognize a CD1d-binding glycolipid presented to 

them on the surface of the responding CD8 T cells that they prime. No IL-10-producing 

CD8 T cells are detected if these T cells are primed by either HC or NKT cells. In 

contrast, IL-10 is produced by HC-primed CD8 T cells if IFN-beta-producing NKT cells 

are coactivated by the same HC presenting a glycolipid (in the context of CD1d) and an 

antigenic peptide (in the context of K(b)). Hence, IL-10-producing CD8 T cells are 

generated in a type I IFN-dependent manner if the three cell types (CD8 T cells, NKT 

cells, and ligand-presenting HC) specifically and closely interact. IL-10-producing CD8 T 

cells generated under these conditions down-modulate IL-2 (and proliferative) 

responses of naive CD4 or CD8 T cells primed by DC. If in close proximity, NKT cells can 

thus locally modulate the phenotype of CD8 T cells during their priming by HC thereby 

limiting the local activation of proinflammatory immune effector cells and protecting the 

liver against immune injury.

Markus Krumbholz, Hans Faber, Florian Steinmeyer, Lisa-Ann Hoffmann, Hannah 

Pellkofer, Tania Kümpfel, Tobias Derfuß, Camelia Ionescu, Michaela Starck, Thomas 

Giese, Gunther Hartmann, Christian Hafner, Reinhard Hohlfeld, Edgar Meinl 

Type I interferon therapy increases systemic BAFF expression 

Type I interferons (IFN-I) play a key role both in the physiological immune response 

after e.g. viral infection and in autoimmune diseases like SLE. Besides effects on T cells, 

effects on the B cell system are increasingly recognized. We asked whether IFN-I 

regulate the BAFF/APRIL system, which is crucial for the normal B cell physiology, and 

may also drive autoimmune diseases and malignancies. 

We report that systemic BAFF expression was elevated in MS patients treated with IFN- 

&beta, but not in untreated patients. BAFF transcription in vivo in monocytes and 

granulocytes correlated with that of MxA, a typical IFN-&beta regulated gene. Further in 

vitro analysis confirmed that IFN-&beta concentrations reached in vivo in treated MS 

patients induce BAFF in these immune cell subsets and also in tissue resident cells 

(fibroblasts and astrocytes). In contrast to BAFF, APRIL was only slightly induced by IFN- 

&beta. The hybrid transcript TWE-PRIL was expressed several orders of magnitude 

lower in vivo. The receptors BCMA, TACI, BARF-R were not regulated by IFN-&beta. 

The systemic induction of BAFF by type I IFNs shed light on a mechanism for the 

physiological interplay of innate and adaptive immunity at the level of B cells, on 

complex immunomodulatory effects of IFN-&beta treatment in MS patients, on how type 

I IFN therapy may lead to the known induction of autoantibodies or even clinical 

autoimmunity as a side effect, and on how type I IFNs may aggrevate autoimmunity in 

e.g. SLE.

Susanne Rauchmann, Karin Knieke, Marion Rudolph, Holger Hoff, Monika Brunner- 

Weinzierl 

Ubiquitin-ligases in co-stimulation of T-lymphocytes 

Many aspects of cellular function are regulated by ubiquitination. One aspect of Tlymphocyte 

function for which the contribution of ubiquitin-mediated regulation is not 

fully investigated is T cell activation mediated by co-stimulatory receptors. While it has 

been shown that CD28 mediated co-stimulatory signals down-regulate the activity of 

the ubiquitin-ligase Cbl-b the role of the closely related receptor CD152 (CTLA-4) in the 

regulation of ubiquitination is not known. Here we can show that in CD152 deficient Thelper 

cells the expression levels of several ubiquitin-ligases are drastically decreased. 

The diminished activity of the RING-domain containing ubiquitin-ligases c-Cbl and Cbl-b, 

the HECT-type ligases NEDD4 and Itch as well as Grail could explain the hyperactive 

phenotype displayed by CD152 deficient CD4+ T cells. This phenotype is less severe in 

TCR-transgenic CD152 deficient T-helper cells that corresponds with less drastic 

differences in the expression of ubiquitin-ligases. Different expression levels of c-Cbl, 

Cbl-b and Itch before onset as well as at late time-points after stimulation might point 

at an important role of ubiquitin-ligases for the induction of a resting phenotype. Future 

work will be necessary to analyse the different contribution of positive and negative costimulatory 

receptors for the induction of ubiquitin-ligase activity.

Christine Wolff, Peter Härle, Johannes Wildmann, Hugo O. Besedovsky, Rainer H. 

Straub, Adriana del Rey 

Uncoupling of the HPA axis and the sympathetic nervous 

system in rheumatoid arthritis is linked to marked changes of 

norepinephrine levels in the hypothalamus 

Introduction: 

Uncoupling of the hypothalamic-piuitary-adrenal (HPA) axis and the sympathetic 

nervous system (SNS) has been demonstrated in rheumatoid arthritis. This is evident 

by an overactivity of the SNS and a relative low activity of the HPA axis. However, the 

reasons for this phenomenon and the consequences for the inflammatory disease are 

not known. 

Aim of the study: 

To study the time course of norepinephrine content in the hypothalamus during the 

course of collagen type II – induced arthritis in the rat. 

Material and methods: 

Female Dark Agouti (DA) rats were immunized with type II collagen to induce arthritis. 

Animals were killed and samples were taken on day 5, 14, 28, 40 and 55. 

Measurements of norepinephrine (NE) of brain samples (hypothalamus, pons, medulla 

oblongata) were done via HPLC. To measure plasma NE and corticosterone we applied 

radioimmunoassay. 

Results: 

We could not find any significant differences in the neurotransmitter levels in the 

medulla oblongata and in the pons between controls and arthritic animals. Importantly, 

on day 5, the levels of NE in the hypothalamus of rats with arthritis were lower than in 

controls . Levels of NE subsequently increased and were significantly higher in arthritic 

animals on day 40 and 55. Blood levels of NE in rats with arthritis were significantly 

higher than in controls already at beginning of symptomatic arthritis. Furthermore, we 

found a significant increase of plasma corticosterone in arthritic animals on day 5 

followed by a reduction to baseline levels from day 14 onwards. 

Conclusion: 

For the first time, this study demonstrated uncoupling of the HPA axis and the SNS in 

animal model of arthritis. Future experiments are designed to modulate hypothalamic 

NE content in order to modulate peripheral inflammation.

Sonja Textor, Rosita Accardi, Matthias Dürst, Massimo Tommasino, Lutz Gissmann, 

Adelheid Cerwenka 

Up-regulated expression of activating NK cell receptor ligands 

in human cervical cancer 

NK cell activation is mediated by a delicate balance of signals received by activating and 

inhibiting receptors. Certain activating receptors recognize ligands, which are induced 

upon cellular stress like transformation or viral infection. Inhibitory signals are delivered 

to NK cells through interaction with self MHC class I or related molecules. Expression of 

these ligands is modulated by certain viruses and on tumors rendering the infected or 

transformed cells more or less susceptible to NK cell attack. 

The aim of our study was to evaluate the impact of the two major transforming 

oncogenes of Human Papilloma Viruses (HPV), E6 and E7, on the regulation of NK cell 

receptor ligand expression. First, we analysed the expression of NK cell receptor ligands 

on keratinocytes after transduction with a retroviral vector system carrying HPV16-E6 

and/or -E7 by flow cytometry. Comparing HPV16-E6E7 expressing keratinocytes with 

the corresponding parental cells, we detected up-regulation of MICA, a ligand for the 

activating NK cell receptor NKG2D. Expression of other ligands, like CD155, a ligand for 

the activating NK cell receptor DNAM-1, remained unchanged. 

Since high-risk HPVs, like HPV16, are the etiological agents of cervical cancer (CxCa), 

biopsies of CxCa and prelesions were stained for NK cell receptor ligands and NK cells 

by immuno-histochemistry in situ. Tumor-infiltrating NK cells (CD56+) were found in all 

cervical cancer stages. High expression of the activating NK cell receptor ligands, MICA 

and CD155, was only observed in CxCa specimens, whereas precursor lesions showed 

no MICA expression und moderate CD155 expression. Some CxCa biopsies showed low 

MHC class I expression in combination with high expression of activating NK cell 

receptor ligands, making CxCa an appealing target for NK cell- based immunotherapy.

Felix Lasitschka, Jutta Schröder-Braunstein, Thomas Giese, Carolin Reiser, Bernd 

Sido, Stefan C. Meuer 

Uptake of Cystine in T-Lymphocytes following CD2 and CD3 

stimulation 

Background: 

Glutathione (GSH) is the most abundant nonprotein thiol involved in the maintenance of 

the cellular redox state. In this capacity it influences T-Lymphocyte responsiveness to 

various stimuli and is a prerequisite for cell cycle progression following CD2 and CD3 

stimulation. 

While it is known that macrophages can provide T-Lymphocytes with cysteine, the 

limiting substrate for GSH synthesis, by intracellularly reducing cystine to cysteine, we 

now investigated the possibility of the direct uptake of cystine by T-Lymphocytes after 

stimulation. 

Methods: 

Uptake of radiolabelled cystine in cultured T-Lymphocytes following CD2 and CD3 

stimulation over the timecourse of 72 hours was measured in the presence or absence 

of different inhibitors of system xc-, a spe¬cific transporter for cystine. Proliferation was 

measured via uptake of [3H]-Thymidine , viability of cells was investigated through PI/ 

Annexin V staining. xCT expression on T-Lymphocytes was analysed by flow cytometry . 

Results: 

T-Lymphocytes show a markedly increased uptake of cystine over the timecourse of 72 

hours following CD2 and CD3 stimulation. Inhibitors of system xc-, Glutamate, 

Quisqualate and Homocysteate, reduced the uptake of cystine and the prolifera¬tive 

potential of stimulated cells without affecting their viability. The percentage of xCT 

posi¬tive cells as determined by flow cytometry increased over time as well. 

Conclusion: 

CD2 and CD3 stimulation of T-Lymphocytes induces a hitherto unknown uptake of 

cystine, which can be reduced by specific inhibitors of system xc-. Thus, system xc- 

contributes in part to the redox regulated reactivity towards CD2 and CD3. 

Further investigations regarding cystine uptake in human lamina propria T-Lymphocytes 

(LPT) after stimulation, which show a lower response towards CD3 stimulation, but a 

preserved CD2 proliferation compared to autologuos peripheral blood T-Lymphocytes 

are required to test, whether the uptake of cystine by LPT plays a role in mucosal redox 

regulation.

Meike Winter, Roel Schins, Irmgard Förster 

Uptake of nanoparticles in the food by gut-associated 


Nanoparticles are defined as particles with a diameter smaller than 0.1µm. They are 

constituents of many foods including dairy creamers, ketchup and chewing gum. The 

regular intake of inorganic particles by humans is estimated as 10 12 particles per day. 

Since nanoparticles are already known to induce severe inflammatory reactions and 

cancer in the lung, and contribute to the establishment of cardiovascular diseases, there 

is an urgent need for further studies on the toxic potential of nanoparticles in the 

intestine. It has been suggested that a low nanoparticle diet might be associated with a 

better prognosis of inflammatory bowel disease (IBD), even though contradicting data 

exist. In ex vivo studies an amplifying effect of ultrafine titanium dioxide on LPSinduced 

gut inflammation could be demonstrated. We are presently analysing the 

uptake of orally administered nanoparticles in cells of the gut associated immune 

system, especially in dendritic cells, which are potent inducers of immunoreactions and 

thought to be involved in the pathology of IBD. In in vitro studies we could already 

show that murine bone marrow derived dendritic cells display differentiation defects if 

generated in the presence of nanoparticles. Differentiation was less efficient and a 

reduction of cell size could be observed in the presence of ultrafine titanium dioxide. 

When generated in the presence of ultrafine carbon black the effects were even more 

pronounced. Thus, the number of dendritic cells was reduced, cells were smaller and the 

density of MHC- and costimulatory molecules on the cell surface was reduced. If 

ultrafine carbon black was added to immature dendritic cells, MHC-class-II was 

upregulated, suggesting a stimulatory effect of ultrafine carbon black on dendritic cell 

maturation. 

In the future we aim to investigate the effect of nanoparticles on the manifestation of 

disease in different mouse models for acute and chronic IBD.

Sascha Barabas, Tanja Bauer, Regina Gary, Petra Lindner, Juha Lindner, Wolfgang 

Jilg, Hans Wolf, Ludwig Deml 

Urea-mediated cross-presentation of soluble Epstein-Barr 

Virus BZLF1 

Soluble extracellular polypeptides are almost exclusively delivered to the exogenous 

MHC class II presentation pathway of antigen-presenting cells (APC), thus diminishing 

their capacity to stimulate CD8+ cytotoxic T lymphocytes (CTL). Here, we describe the 

urea-mediated translocation of soluble Epstein-Barr virus (EBV) immediate early protein 

BZLF1 to the endogenous cytosolic pathway of MHC I-dependent polypeptide 

presentation. 

APC pulsed with urea-treated BZLF1 (uBZLF1) efficiently reactivate BZLF1-specific CTL 

and T helper cells from HLA-matched EBV-seropositive donors. uBZLF1 shows strong 

attachment to the cell membrane before uptake by clathrin-mediated endocytosis and 

degradation at the proteasome for cross-presentation. Dendritic cells, monocytes but 

also B cells are able to mediate MHC I-restricted epitope presentation from uBZLF1. This 

strategy considerably promotes reactivation and propagation of protein-specific CTL. 

The procedure described here has potential for use in evolving improved strategies for 

protein-specific monitoring and immunotherapy of viral infections.

Emmanuel Prodhomme, Claude Muller 

Vaccination against Tobacco Specific N-nitrosamines: 

Synthesis of a new NNK hapten for the induction of specific 

antibodies. 

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most abundant 

and potent pro-carcinogens in tobacco smoke. In order to induce a strong and 

substained antibody response against NNK we developed a functionalized derivative that 

closely mimicked its structural features in particular the pyridyloxobutyl moiety, the 

adjacent ketone and the N-nitrosamino-group. This hapten was conjugated via a linker 

to the highly immunogenic diphteria toxoid licensed as a vaccine in humans to induce 

polyclonal and monoclonal antibodies. 

Both monoclonal and polyclonal antibodies reacted strongly with NNK and NNAL and to 

a lesser extend with some of the metabolites of NNK. Interestingly, the mAbs did not 

react with the metabolites of the detoxification pathways such as NNK-N-Oxide and 

NNAL-N-Oxide. Therefore such antibodies detect NNK and NNAL and may have the 

potential to modulate their redistribution in vivo, perhaps reducing some detrimental 

effects of smoking.

Carsten Alt, Travis Harrison, Akihiro Matsukawa, Charles Litterst, Jon Mirsalis, Annalisa 

D'Andrea 

Vaginal cytokine production as a biomarker for the safety 

evaluation of microbicide-induced vaginal irritation 

Vaginal microbicides may help to reduce the risk of infection with HIV or other sexually 

transmitted diseases. For safety evaluation of novel vaginal microbicides, vaginal 

irritation is generally assessed in rabbits by histopathologic analysis of the gastrouterine 

organs after 10 days of treatment. This evaluation requires the sacrifice of the animals. 

Minimally invasive approaches to measure the vaginal irritation caused by novel 

microbicides would offer a more desirable alternative. 

We measured the vaginal irritation caused by the spermicides benzalkonium chloride 

(BZK) and nonoxynol-9 (N-9). We assessed not only the histopathological changes, but 

also: (a) the cytokine amounts in vaginal tissue lysates and in cotton swab samples 

collected from the vaginal surface, and (b) the inflammatory cells in cytobrush samples 

collected from the vaginal surface. 

After 6 days of treatment with N-9, a significant increase in histopathological changes 

was observed. This effect was transient, being no longer detectable after 10 days of 

treatment with N-9. Simultaneously processed vaginal tissues contained significantly 

increased amounts of IL-8 and IL-1β, but only after 10 days of treatment with BZK. 

Swab samples from the vaginal surface contained significantly elevated levels of MCP-1, 

IL-8, and IL-1β, in some cases as early as 3 days after treatment began. Increased 

numbers of inflammatory cells were detectable, possibly attracted by increased 

amounts of the inflammatory chemokines MCP-1 and IL-8 on the vaginal surface. 

These results suggest that measurement of IL-8, IL-1β, MCP-1, and inflammatory cells 

on the vaginal surface may offer a noninvasive method to assist in developing novel 

vaginal microbicides.

Beatrice Bolinger, Philippe Krebs, Engeler Daniel, Ying Hua Tian, Elke Scandella, 

Simone Miller, Nicola P. Restifo, Pierre-Alain Clavien, Burkhard Ludewig 

Vascular endothelial cells presenting minor histocompatibility 

antigen do neither activate nor tolerize CD8+ T cells 

Endothelial cells (EC) are located at a unique position between circulating lymphocytes 

and peripheral tissues and thereby participate in the recruitment of T lymphocytes from 

the bloodstream to the sites of infection and inflammation. After transplantation of 

vascularized allogeneic tissues, allopresentation by EC may contribute to allograft 

rejection. However, the interaction between T cells and EC presenting a minor 

histocompatibility antigen is still not fully understood. Using a transgenic mouse model 

with β-galactosidase (βgal) expression confined to the vascular endothelium (Tie2-LacZ 

mice) and mice with CD8+ T cells expressing a βgal-specific T cell receptor (Bg1 mice), 

the role of EC during cognate interaction with CD8+ T cells was assessed in vivo. 

Adoptive transfer of TCR transgenic Bg1 cells into naïve Tie2-LacZ recipients lead to 

activation and proliferation of Bg1 CD8+ T cells. Generation of bone marrow chimeric 

mice using mice which exhibit a mutated H2-Kb-molecule (bm1 mice) and impaired 

presentation of Kb-restricted peptides, revealed that activation of βgal-specific CD8+ T 

cells in Tie2-LacZ mice is mediated by bone marrow derived cells. In vivo depletion of 

dendritic cells (DC) revealed that activation of CD8+ T cells in this setting depended 

exclusively on cross-presentation by DC. Likewise, tolerization of TCR tg Bg1 cells was 

not dependent on Ag presentation by vascular EC. The finding that the βgal antigen 

expressed by vascular EC remains immunologically ignored by CTL in Tie2-LacZ mice 

could be reproduced using heterotopic heart and orthotopic liver transplantation of Tie2- 

LacZ organs into naïve C57BL/6 mice.Taken together, our results suggest that 

presentation of a minor histocompatibility antigen by vascular EC leads neither to 

activation nor to proliferation of specific CD8+ T cells.

Michaela Kern, Kai Scholz, Ulrich Kalinke, Joachim Schultze, Ulrich Koszinowski, 

Gunther Hartmann, Percy A. Knolle 

Viral infection but not pattern recognition induces functional 

maturation of tolerogenic liver endothelial cells leading to 

induction of effector CD8 T cells 

Liver sinusoidal endothelial cells (LSEC) contribute to the tolerogenic function of the 

liver by mediating CD4 and CD8 T cell tolerance towards soluble antigens. Here, we 

address the question whether tolerogenic LSEC also have sentinel function and are 

susceptible to functional maturation similar to dendritic cells that gain immunogenic 

properties upon appropriate maturation stimuli. LSEC constitutively expressed Toll like 

receptors 2, 3, 4, 7, 9 and were stimulated by ligands to membrane-bound TLRs as well 

as cytoplasmic helicases. Neither TLR nor helicase-mediated stimulation, however, 

changed the tolerogenic LSEC function revealing a fundamental difference to dendritic 

cells that undergo functional maturation upon similar stimuli. LSEC were susceptible to 

infection with murine cytomegalovirus (MCMV), but controlled viral immune escape and 

replication through early type I IFN expression. Importantly, infection of LSEC with 

recombinant MCMV-OVA prevented induction of CD8 T cell tolerance and led to 

generation of OVA-specific effector CD8 T cells. Virus-induced functional maturation of 

LSEC occurred in a cell-autonomous fashion, i.e. the tolerogenic phenotype of 

uninfected LSEC was not altered in coculture with infected LSEC. No single mechanism 

was identified that caused functional maturation. However, expression profiling of virusinfected 

vs TLR-stimulated LSEC revealed a complex gene expression signature. Our 

study demonstrates that ligation of PRR in itself is not sufficient to achieve functional 

maturation in organ resident APC. Unraveling the mechanisms responsible for such 

maturation will be important for our understanding of local immune regulation.

Dennis Lindau, Dagmar Sigurdardottir, Evelyna Derhovanessian, Thomas Leyhe, 

Graham Pawelec, Stefan Stevanovic, Lars Stoltze 

Visualizing Abeta-specific cytotoxic T cells with HLA tetramers 

Brains of individuals with Alzheimer´s disease (AD) are characterized by the formation 

of neurofibrillary tangels in neurons, the accumulation of amyloid-beta (Abeta) into 

senile plaques and a prominent activation of local inflammation. Because genetic 

approaches point to Abeta as a major pathologic mediator in AD, reducing its levels in 

the brain is seen as a promising disease-modifying therapy. However, a clinical trial of 

Abeta immunization in AD patients led to meningoencephalitis in 6 % of treated 

individuals and was discontinued. These side effects were most likely induced by CNSinfiltrating 

lymphocytes. In contrast new therapeutic interventions showed a decrease in 

the amyloid plaque burden of an AD mouse model after immunization with glatiramer 

acetate, which most likely is T cell mediated. To this end we have identified three 

different Abeta-derived epitopes recognized by human cytotoxic T cells (CTL) in the 

context of HLA-A*0201. In vitro expanded Abeta-specific CTLs are functional in terms of 

cytoxicity, showing high specifity without cross-reactivity and normal peptide avidity. 

Combining cell surface phenotyping by HLA tetramers with functional assays, we have 

obtained a detailed picture of Abeta-specific CTLs in healthy young and elderly donors 

as well as AD patients. The frequent detection in peripheral blood especially in the 

elderly groups of this auto-antigen-specific lymphocyte population will be discussed in 

relation to disease course and possible clinical use.

Patricia Bach, Elisabeth Kamphuis, Bernhard Odermatt, Gerd Sutter, Christian J. 

Buchholz, Ulrich Kalinke 

VSV-G Displaying Retrovirus-Like Particles Induce a Type I 

IFN Receptor Dependent Switch to Neutralizing IgG Antibodies 

Vesicular stomatitis virus (VSV) infection rapidly induces IFN-α/β that confers initial 

survival, whereas long-term protection is mediated by neutralizing IgG responses. Since 

co-administration of IFN-α/β can enhance antibody responses against soluble antigens, 

we addressed whether virus-induced IFN-α/β also had an impact on the induction of 

neutralizing antibody responses. To this end, we generated apathogenic retrovirus-like 

particles (VLP) displaying the VSV glycoprotein (VLP-VSV). Reminiscent of live VSV, VLP- 

VSV induced VSV neutralizing IgM responses that switched to IgG in a T help-dependent 

manner. In type I IFN receptor deficient (IFNAR-/-) mice, VLP-VSV injection elicited 

neutralizing IgM, whereas the IgG switch was absent. The lack of subclass switch was 

associated with a reduced germinal center reaction. Conditional knockout mice with a 

lymphocyte-specific IFNAR ablation showed normal antibody responses against VLP- 

VSV, as well as against live VSV. Thus, IFNAR triggering critically promoted the T helpdependent 

subclass switch of virus neutralizing antibody responses against VLP-VSV. 

Interestingly, in the context of VLP-VSV as well as VSV immunization, IFNAR triggering 

of B lymphocytes did not play a critical role.

Jessica Nickel, Birgit Löer, Reinhard Bauer, Roland Bornheim, Elisabeth Kremmer, 

Michael Hoch, Waldemar Kolanus 

Wech, a novel intracellular regulator of integrin-dependent 

cell adhesion 

Wech is a member of the RBCC/TRIM family of cytoplasmic multidomain proteins, which 

bears conserved protein motifs such as a BBox zinc-finger domain, a coiled coil domain 

and a carboxyterminal NHL domain. It was very recently linked to integrin-mediated 

adhesion regulation in Drosophila. Embryos deficient for wech have very similar 

phenotypes to integrin and talin null embryos, including muscle detachment from the 

body wall. It has been shown that wech is essential for the link between integrins and 

the cytoskeleton in the Drosophila epidermal muscle attachment site, by connecting ILK 

and talin to the integrin. Single copy genes of wech orthologs (also named LIN41) are 

found in mice and humans. We generated an antibody against human and murine wech 

and found that the murine wech protein is expressed in z-discs of adult muscles, where 

wech colocalizes with ILK and talin. We also detected a partial colocalization with β1integrins. 

SiRNA mediated knockdown of wech in the human keratinocyte line HaCat 

and in Hela cells results in a strong loss of productive anchorage and subsequent 

detachment of the cells. This indicates that wech is a positive regulator of cell adhesion 

in these cells. As already shown for ILK and talin, wech is essential for directional 

migration, as shown by wound healing experiments. We went on to analyze wech 

functions in immune cells and surprisingly found that wech is a negative regulator of cell 

adhesion in Jurkat E6 cells. SiRNA-mediated knock-down of wech resulted in a marked 

increase of Jurkat adhesion to ICAM-1. These results suggest a differential function of 

the wech protein in regulating β1- or β2-integrins. Together, our studies provide 

evidence that wech is a novel regulator of integrin-dependent cell adhesion in multiple 

cell types.

Hamid Kashkar, Jens Michael Seeger, Andreas Hombach, Anke Deggerich, Benjamin 

Yazdanpanah, Olaf Utermöhlen, Gerd Heimlich, Hinrich Abken, Martin Krönke 

XIAP targeting sensitizes Hodgkin Lymphoma cells for 

cytolytic T cell attack 

The immunosurveillance of Hodgkin's Lymphoma (HL) by cytotoxic T lymphocytes (CTL) 

is insufficient and the clinical experience with adoptive transfer of CTLs is limited. We 

have previously reported that defects in mitochondrial apoptotic pathways and elevated 

XIAP-expression confer resistance to different apoptotic stimuli in HL cells. Here we 

aimed to develop molecular strategies to overcome the resistance of HL cells against 

CTL-mediated killing via granzyme B (grzB). In HL cells grzB-induced mitochondrial 

release of pro-apoptotic Smac is blocked, which results in complete abrogation of 

cytotoxicity mediated by CTLs. Cytosolic expression of recombinant mature Smac 

enhanced caspase activity induced by grzB and restored the apoptotic response of HL 

cells. Similarly, down-regulation of XIAP by RNA interference markedly enhanced the 

susceptibility of HL cells for CTL-mediated cytotoxicity. XIAP gene knock-down 

sensitized HL cells for killing by antigen-specific CTLs redirected by grafting with a 

chimeric anti-CD30scFv-CD3zeta immunoreceptor. The results suggest that XIAP 

targeting by Smac agonists or XIAP-siRNA can be used as a synergistic strategy for 

cellular immunotherapy of Hodgkin´s lymphoma.

Isabel Koch, Reinhard Hoffmann 

Yersinia virulence protein YopP inhibits NK cell cytokine 

production by blocking host cell IL-12 signaling. 

Yersinia enterocolitica causes acute gastrointestinal disease and evades the host´s 

immune response by injecting, via a type III protein secretion system, anti host effector 

proteins into the host cell’s cytoplasm. These effector proteins (Yersinia outer proteins, 

Yops) interfere with host cell signal transduction; in particular, YopP inhibits NFκB and 

MAPK pathways. 

NK cells are an important early source of IFN-γ before the onset of adaptive immunity. 

To evaluate whether Y. enterocolitica could directly modulate NK cell function, we 

isolated 2B4 positive NK cells from spleens of C57BL/6 mice, infected them with highly 

virulent Y. enterocolitica in vitro, and evaluated NK cell IFN-γ production. Infection with 

wild-type Y. enterocolitica (translocating all six Yop effector proteins) markedly reduced 

IFN-γ production induced by IL-12, IL-12+IL-18, and agonistic α-NKG2D antibody in 

both naïve and IL-2 stimulatied NK cells. Evaluation of mutant Yersinia strains identified 

YopP as an important mediator of NK cell disarmament: YopP not only inhibited 

phosphorylation of p38 in response to stimulation with IL-12+IL-18, but also in 

response to IL-12 alone. Strikingly, YopP, which has not previously been shown to 

interfere with JAK-STAT signal transduction, inhibits tyrosine phosphorylation of STAT-4 

in response to IL-12 or IL-12+IL-18 stimulation. 

To demonstrate the relevance of these results in vivo, we isolated NK cells from mice 

infected with wild-type or YopP deficient Y. enterocolitica. We could show that NK cells 

from wild-type infected mice produce lower amounts of IFN-γ protein after restimulation 

with IL-12+IL-18. To the best of our knowledge, this is the first report of a 

bacterial pathogen directly targeting NK cells for the suppression of an effective immune 

response.

Hajo Haase, Lothar Rink 

Zinc ions act as a signal in leukocyte activation 

Cytosolic alterations of calcium ion concentrations are an integral part of intracellular 

signal transduction. Similar functions have been hypothesized for other metal ions, in 

particular zinc ions (Zn2+), but this still awaits experimental verification. Zn2+ is 

important for a range of cellular functions, especially in the immune system. Among 

other modulatory effects, it influences the formation and secretion of pro-inflammatory 

cytokines, including tumor necrosis factor (TNF)-alpha. However, it remains to be 

shown that these effects are due to a physiological intracellular signaling system 

involving Zn2+, rather than being coincidental. Here we demonstrate that an increase 

of intracellular zinc ion concentration occurs upon stimulation of human leukocytes, and 

that this increase is required for signal transduction. Treatment of whole blood with 

Escherichia coli, lipopolysaccharide, or Pam3CSK4, which trigger so-called pattern 

recognition receptors, led to an increase in intracellular Zn2+, predominantly in 

monocytes. Chelation of Zn2+ with the membrane permeable zinc-specific chelator 

TPEN (N,N,N’,N’-tetrakis-(2-pyridyl-methyl)ethylenediamine) completely blocked the 

increase and the E. coli-induced activation of p38 MAPK and Erk1/2, and abrogated the 

release of TNF-alpha, demonstrating that intracellular labile Zn2+ involved in signaling. 

This function of Zn2+ might not be limited to the immune system and pattern 

recognition receptors, but might be a generalized second signaling system based on 

intracellular fluctuations of metal ion concentrations, acting parallel to Ca2+.

Laura Kahmann, Sabine Warmuth, Birgit Plümäkers, Axel M. Gressner, Marco Malavolta, 

Eugenio Mocchegiani, Lothar Rink, Peter Uciechowski 

Zinc supplementation in the elderly reduces spontaneous 

inflammatory cytokine release and restores T cell functions 

Advanced aging is accompanied by low grade inflammatory activity that includes 

increased levels of pro-inflammatory cytokines. On the other hand, aging is also 

associated with mild zinc deficiency. Both conditions contribute to decreased immune 

functions resulting in increased susceptibility to autoimmune diseases, infections and 

malignancies. The aim of this study was to give more insight into what extent low grade 

inflammation is caused by zinc deficient status. Here we report the effect of improved 

intracellular zinc status on low grade inflammatory activity in 19 healthy elderly 

subjects. Our experiments show that adjustment of labile zinc by moderate zinc 

supplementation reduces spontaneous cytokine release such as IL-6 and defects in 

termination of inflammatory activity. Zinc supplementation results in reduced amounts 

of unspecific pre-activated T cells and leads to improved T cell response upon mitogenic 

stimulation shown by significantly increased IFN-γ, TNF-α and IL-10 release. Therefore, 

besides other anti-inflammatory drugs, zinc does not suppress, but improves immune 

reaction upon pathogen invasion. These results suggest that mild zinc deficient healthy 

elderly subjects might benefit from moderate zinc supplementation due to improved 

immune responses leading to reduced incidences of infections and autoimmune diseases.

Abstracts (complete list) - Wissenschaft Online

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