12th Congress of the European Hematology ... - Haematologica

successfully managed with GM-CSF discontinuation. The table summarises
the preliminary results (a final sample size of 15 patients is
planned). Conclusions. Our experience suggests that a large scale trial of
anti-idiotype vaccine (Id-KLH active immunotherapy) may be conducted
in Europe with the vaccine manufacturing taking place in the USA.
Clinical efficacy was not the main endpoint of the study, however, our
preliminary findings indicate a possible activity in disseminated MZL,
which might merit further evaluation
Table 1. Summary of preliminary results.
0419
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 IL2R GAMMA NULL MICE
E. Distler, C. Woelfel, S. Koehler, M. Nonn, N. Kaus, E. Schnuerer,
R.G. Meyer, T.C. Wehler, C. Huber, T. Woelfel, U.F. Hartwig, W. Herr
Johannes Gutenberg-University Mainz, MAINZ, Germany
Donor-derived CD8 + cytotoxic T lymphocytes (CTLs) eliminating host
leukemic cells mediate curative graft-versus-leukemia (GVL) reactions
after allogeneic hematopoietic stem cell transplantation (HSCT). The
leukemia-reactive CTLs recognize hematopoiesis-restricted minor histocompatibility
and leukemia-associated peptide antigens that are presented
by HLA class I molecules on recipient cells. Current in vitro techniques
for generating AML-reactive CTLs from unprimed healthy donors are of
relatively low efficiency and yield responder populations with unknown
biological significance. We established a new allogeneic mixed lymphocyte-leukemia
culture (MLLC) approach by stimulating donor CD8 + T
cells at comparably low numbers (i.e. 104 /well) with HLA class I-matched
primary AML blasts in 96-well microtiter plates. Using this so-called
mini-MLLC strategy, we aimed at creating multiple different responderstimulator
compositions in order to provide for the growth of leukemiareactive
CTL optimized culture conditions by chance. Before culture,
CD8 + T cells were immunomagnetically separated into CD62Lhigh + and
CD62Llow +/neg subsets enriched for naïve/central memory and effector
memory cells, respectively. The culture medium was supplemented with
interleukin (IL)-7, IL-12, and IL-15. On day 14, IL-12 was replaced by IL-
2. In 8 different related and unrelated donor/AML pairs with complete
HLA class I match, numerous CTL populations were isolated that specifically
lysed myeloid leukemias in association with various HLA-A, -B,
or -C alleles. These CTLs recognized neither lymphoblastoid B cell lines
of donor and patient origin nor primary B cell leukemias expressing the
corresponding HLA restriction element. CTLs expressed T cell receptors
of single V-β chain families, indicating their clonality. The vast majority
of CTL clones were obtained from mini-MLLCs initiated with
CD62Lhigh + cells. Using antigen-specific stimulation, multiple CTL populations
were amplified to 108-1010 cells within 6-8 weeks. We also investigated
the capability of mini-MLLC derived AML-reactive CTL clones
to inhibit the engraftment of human primary AML blasts in immunodeficient
nonobese diabetic/severe combined immune deficient IL-2R
common γ-chain deficient (NOD/SCID IL2R gamma null) mice. The
leukemic engraftment was specifically prevented if inoculated AML
blasts had been preincubated in vitro with AML-reactive CTLs, but not
with anti-melanoma control CTLs. Taken together, our results provide
12 th Congress of the European Hematology Association
the first evidence that CD8+ CTL clones raised from healthy donors
against AML blasts using primary in vitro stimulation might carry biologically
significant anti-leukemic activity. The efficient in vitro generation
and expansion of AML-reactive CTL clones from CD8+CD62L high + precursors
of healthy donors by the mini-MLLC approach allows several
potential applications. First, CTLs can be used within T cell-driven antigen
identification strategies to extend the panel of molecularly defined
AML antigens that are recognizable by T cells of healthy donors. Second,
because these CTLs can be readily isolated from the stem cell donor by
mini-MLLC prior to transplantation, they could be infused into AML
patients as a part of the stem-cell allograft, or early after transplantation
when the leukemia burden is low. Our current work focuses on the identification
of CTL-defined AML peptide epitopes using cDNA expression
cloning, and on the translation of the mini-MLLC approach into a
protocol that is compatible with good manufacturing practice guidelines.
0420
A COMPARATIVE ANALYSIS OF THE LEUKAEMIC POTENTIAL OF MATURE T CELLS VERSUS
HEMATOPOIETIC STEM CELLS
S.N. Newrzela, 1 Z. Li,2 C. Baum, 2 M. Hartmann, 1 S. Hartmann, 3
M.-L. Hansmann, 3 K. Cornils, 4 B. Fehse,4 M.D. Von Laer1 1 Georg-Speyer-Haus, FRANKFURT AM MAIN; 2 Hannover Medical School,
HANNOVER; 3 University of Frankfurt, FRANKFURT AM MAIN; 4 University
Medical Center Hamburg, HAMBURG, Germany
Background. After the report of two cases of leukaemia caused by insertional
mutagenesis of a retroviral vector in children with SCID, it became
clear that safety issues of therapeutic gene transfer must be addressed
more thoroughly. Aims. We wanted to analyse whether gene transfer
into mature T cells and haematopoietic stem cells bear the same risk of
generating T cell leukaemia through activation of specific T cell oncogenes,
such as LMO2, TCL1 and TrkA. Methods. To address this issue,
we used the Rag-1 mouse model, which allows long term analysis of
transplanted T cells and haematopoietic stem cells. We transduced
mature T cells and haematopoietic stem cells of C57BL/6 (Ly5.1) donor
mice with oncoretroviral vectors expressing LMO2, TCL1 and TrkA.
Results. Transduction efficacies of up to 70% were achieved for mature
T cells and approximately 90% for haematopoietic stem cells. After
transplantation into Rag-1-deficient recipients, stem cell transplanted
animals developed T cell lymphomas/leukemia for all investigated oncogenes
after characteristic incubation times, mostly of a CD8 + CD4 + double
positive phenotype. T cell lymphomas were characterised by gross
thymic mass, splenomegaly and heavily enlarged lymph nodes, although
none of the control- vector- transduced mice developed lymphoma/
leukaemia. LM PCR analysis revealed mono- or oligoclonality of the
tumours. T cell transplanted animals showed no signs of leukaemia
development so far. Summary and conclusions. Our results so far indicate
that mature T cells are less susceptible to transformation by known T cell
proto- oncogenes, but the studies are still ongoing.
haematologica/the hematology journal | 2007; 92(s1) | 155