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Abstracts J Immunother Volume 33, Number 8, October 2010 using delivery of DCs loaded with defined tumor antigens. These results support the clinical development of IL-23 gene-modified DCs in patients with cancer. High-Throughput Analysis of Plasmacytoid Dendritic Cells Reveals Differential Responses to Discrete Viral Entities Jaime M. Thomas*, Zoltan Pos*, Richard Y. Wang*, Ena Wang*, Paolo Lussow, Harvey J. Alter*, Francesco M. Marincola*. *Department of Transfusion Medicine, Infectious Diseases and Immunogenetics Section, Clinical Center-NIH; w Laboratory of Immunoregulation, Immunopathogenesis Section, National Institute of Allergy and Infectious Diseases-NIH, Bethesda, MD. Plasmacytoid dendritic cells (pDC) are key components of the innate immune system capable of synthesizing and rapidly releasing vast amounts of type 1 interferons (IFNs), particularly IFNa. Interaction between activated pDC and T cells in both the lymph nodes and the periphery, as well as the involvement of IFNa in the T-cell mediated control and/or therapy-induced rejection of various viral infections and cancers, makes pDC an attractive but still elusive target for immunotherapy. Here we investigated whether pDC, often regarded as mere IFN sources for the immune system, are able to discriminate between various functionally discrete stimuli, and to what extent this reflects differences in pDC responses other than IFNa release. To examine the ability and scope of pDC to differentially respond to various doses of intact and infectious HIV, HCV, and H1N1 viruses, whole genome gene expression analysis, ELISA, and flow cytometry, were applied to interrogate pDC responses at the transcriptional and protein level. Our data demonstrates that pDC differentially respond to various viral stimuli with significant changes in gene expression including those involved in pDC activation, migration, viral endocytosis, survival or apoptosis, CC and CXC chemokine secretion. In some cases, these genes were induced even at levels comparable to that of IFNa. Interestingly, we also found that depending on the viral entity and viral dose, induction of IFNa gene expression and the actual release of IFNa are not necessarily connected. In addition, our data suggests that under certain circumstances, the same virus can support pDC survival or induce apoptosis. Conclusion: A differential pDC response to different viral stimuli points to the possibility that live viruses or viral components could be exploited to modulate pDC behavior, especially in regards to IFNa and chemokine release in support of biological therapies to treat various cancers. In addition, the distinct pDC responses observed may provide insight into the varying clinical outcomes, including propensity for oncogenesis, resulting from exposure to the three different pathogenic viruses studied here. Clinically-Compatible 19F NMR Tracking of DC Vaccines In Vivo Julie Urban*, Brooke Helferw, Steven Thorne*, Eric Ahrensz, Amy Wesaw, Pawel Kalinski*. *Surgery, University of Pittsburgh School of Medicine; w Research and Development, Celsense, Inc.; zBiological Sciences, Carnegie Mellon University, Pittsburgh, PA. Dendritic cell (DC) vaccines are a valuable tool used in cancer immunotherapies to reset the patient’s immune system for enhanced anti-tumor activity. However, the effectiveness of therapeutic vaccines remains limited. The elimination of the antigen-carrying DC vaccines by pre-existing tumor-specific CTLs have been shown to limit the effectiveness of cancer vaccines in the therapeutic settings. While luminescence-based imaging methods can be used to non-invasively and semi-quantitatively evaluate the targeted delivery, migration and persistence of DC vaccines in mice and other small animals, to date there are few robust ways to perform similar studies in humans. Cell tracking of perfluorocarbon-labeled cells by 19F magnetic resonance imaging (MRI) provides a highly-specific signal to quantitatively assess in vivo migration and homing which may be used in clinical studies. CS-1000 is a self-delivering, perfluoropolyether (PFPE) emulsion designed for optimal MRI detection. Here, we tested for the ability to safely and effectively label a novel murine DC vaccine, while not affecting the DCs’ maturation status, IL12 production, lymph node-migratory capacity, and their ability to generate type-1 immune responses. In order to determine where the vaccinecarrying DCs are killed by the CTLs, we monitored the migration of DCs from the site of injection to the draining lymph nodes (dLN) using a semi-quantitative bioluminescence-based imaging and the more clinically-relevant and quantitative 19F MRI system. We have determined that PFPE labeling has no adverse side effects on murine DC function. Additionally, these two imaging techniques allowed us to measure the number of DCs at the site of injection (footpad) and dLN which enabled us to determine that vaccine-carrying DCs, when injected into mice with vaccine-specific CTLs, are eliminated at the site of injection without reaching the dLN. These studies verify that the novel CS-1000 can be used to safely monitor vaccine-carrying DCs in vivo. Identification of Four Conventional Dendritic Cell Subsets in Human Lymph Nodes: Phenotypic and Functional Analysis Rieneke van de Ven*, Mari van den Houtw, Jelle Lindenberg*, Sinead Lougheed*, Sybren Meijerz, Alfons van den Eertwegh*, Petrousjka van den Tolz, Rik Scheperw, Tanja de Gruijl*. Departments of *Medical Oncology; w Pathology; zSurgical Oncology, VU University Medical Center, Amsterdam, Netherlands. The extensive knowledge which has been gathered over the last decades on the phenotype, localization and specialized functions of Dendritic cell (DC) subsets in secondary lymphoid organs of mice, has unfortunately not led to effective in vivo DC-targeted immunotherapy strategies in humans. In order to take DC-targeted immunotherapy to the desired next level, more insight needs to be gained in the phenotype and functionality of DC subsets in human lymphoid tissue. Here, we report on the identification of four human conventional DC (cDC) subsets in skin-draining human lymph node (LN) material from early-stage melanoma patients. Two CD1a+ subsets were discerned, which were either CD11c_int DC-SIGN- Langerin+ or CD1a+ CD11c_hi DC-SIGN+ Langerin ± (based on intracellular Langerin and DC-SIGN staining). Phenotypically these subsets closely resembled skin-emigrated Langerhans cells and dermal DC, respectively. The other two CD1a-CD11c_hi subsets, which we consider to be more likely blood-derived LN-resident DC, were characterized as CD14- BDCA3+ CD103- and CD14 + BDCA3_int CD103+. The four subsets displayed a differential activation status based on the expression of various co-stimulatory/inhibitory molecules (CD40, CD80, CD86, CD83, B7H1, B7H4). Side-by-side analysis of the FACS-sorted subsets further revealed differences in allogeneic T cell stimulatory capacity, cytokine release profiles, and Thelperskewing ability. This report is the first to present data from extensive flowcytometric and functional analyses of cDC subsets in human LN, and provides a deeper understanding of DC in human LN as well as new leads for the development of human DCtargeted immunotherapies. Therapeutic Vaccination With Autologous mRNA Electroporated Dendritic Cells (DC) in Patients With Advanced Melanoma Sofie Wilgenhof*w, An M.T. Van Nuffelw, Daphné Benteynw, Jurgen Corthalsw, Carlo Heirmanw, Lauranne Pierretz, Arlette De Coninckz, Aude Bonehillw, Kris Thielemansw, Bart Neyns*. *Medical Oncology; zDermatology, UZ Brussel; w Laboratory of Molecular and Cellular Therapy & DC-bank, Vrije Universiteit Brussel, Brussels, Belgium. Background: mRNA electroporated autologous DC are under evaluation as therapeutic cancer vaccines. Potential advantages are the presentation of all tumor antigen confined epitopes by the patient’s own HLA-molecules and the improved immunostimulatory 880 | www.immunotherapy-journal.com r 2010 Lippincott Williams & Wilkins
J Immunother Volume 33, Number 8, October 2010 Abstracts capacity of the DC-formula. Combination of a therapeutic vaccine with cytokine therapy (IFN-a2b or IL-2) results in a synergistic anti-tumor effect. Methods: Immature DC (derived from peripheral blood monocytes obtained by leucapheresis and cultured for 6 days in IL-4/GM-CSF supplemented medium) were electroporated with synthetic mRNA encoding a fusion protein between MAGE-A1, -A3, -C2, Tyrosinase, MelanA/MART-1 or gp100, and DC-LAMP, and poly- I:poly-C12U or mRNA encoding TLR-4, CD70 and CD40L (TriMix). DC (12.5 10E6 DC/antigen) were administered by 4 to 6 ID-injections q2w, and q8w thereafter. IFN-a2b (5 MIU TIW) was initiated at progression, concomitant or following the 4th vaccine, respectively in cohort 1, 2, 3, and 4. Immune monitoring was performed by skin biopsy of delayed type IV hypersensitivity (DTH) reactions. Results: 70 melanoma pts were recruited between 06/ 0 05 and 06/ 0 09: 44 Male/26 Female; med age: 46 years (27 to 75); AJCC stage III: 30, IV-M1a: 8, -M1b: 6, -M1c: 26; WHO-PS 0: 46, 1: 19, 2: 5. A total of 466 DC-vaccines were administered (median/patient: 6, range 2 to 18). Vaccine related AE’s included: gr2 injection site reactions: all patients; gr2 fever/lethargy: 3 patients. Vaccinalantigen specific DTH infiltrating lymphocytes: 0/6 patients tested at vaccine initiation and in 12/21 (57.1%) pts after the 4th vaccine. After a mFU of 30 months, the mRFS for pts without evaluable disease (n = 30) is 23 months (95% CI: 11-34); 3 patients have died, mOS has not been reached. The tumor response among 40 patients with ED at baseline: 1 PR+14 SD [disease control rate (DCR): 38%] according to RECIST; 2 CR+2 PR, and 14 SD (DCR: 46%) according to immune-related response criteria (irRC). The 6-mth PFS (32%) and 1-year OS rate (57%) in patients with ED at baseline compares favorably with historical controls. Baseline WHO-PS was identified as an independent covariable for PFS and OS, -CRP for PFS, and -LDH for OS. In a landmark survivalanalysis from week 8, DCR by irRC was the strongest independent covariable for superior PFS and OS (P
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