Abstract Book 2010 - CIMT Annual Meeting

054 Bloetz | Cellular therapy
Allo-reactivity to HLA class II mismatch alleles in CD4+
T-cell subsets
Andrea Blötz 1 , Eva Distler 1 , Elke Schnürer 1 , Simone Thomas 1 , Ugur Sahin 1 , Matthias Theobald
1 , Wolfgang Herr 1
1 Dept. of Medicine III – Hematology & Oncology, University Medical Center, Mainz, Germany
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In allogeneic hematopoietic stem cell transplan-
tation (allo-HSCT), allo-reactive T lymphocytes
of donor origin recognize antigens expressed on
patient-derived leukemia cells, thereby mediating
the beneficial graft-versus-leukemia effect. If alloantigens
are expressed on non-hematopoietic recipient
tissues, donor T cells also induce graft-versushost
disease (GvHD). Since HLA mismatch alleles
represent major targets of allo-reactive T lymphocytes,
patient and donor are usually matched for
the class I molecules HLA-A, -B, -C, and for the
class II molecules HLA-DRB1 and -DQB1, in order
do reduce the risk of GvHD. The HLA-DPB1 locus,
however, is still ignored in donor selection. Interestingly,
clinical studies have demonstrated that
disparities at HLA-DQB1 and distinct HLA-DPB1
alleles do not adversely affect the outcome of allo-
HSCT. Because HLA class II is predominantly expressed
on hematopoietic cells, allo-reactive CD4+
donor T cells recognizing HLA-DQB1 or permissive
HLA-DPB1 mismatch alleles may primarily target
patient leukemic and hematopoietic cells, while
sparing non-hematopoietic recipient tissues. In this
ongoing study we analyze the allo-reactive potential
of CD4+ T-lymphocyte subsets by stimulating
them in vitro against single HLA class II mismatch
alleles. For that purpose, CD4+ peripheral blood T
lymphocytes of healthy donors are sorted by flow
cytometry according to the expression or absence
of the differentiation markers CD45RA, CD45RO,
CD62L and CCR7. As standard antigen-presenting
cells for class II-mismatch stimulations, we use the
HLA-deficient cell line K562 upon electroporation
with in vitro transcribed RNA coding for the alpha
and beta chains of single HLA class II molecules
(HLA-DQ: DQA1-010201/DQB1-060201; HLA-DR:
DRA1-0101/DRB1-0701). This procedure results in
transient HLA expression for up to one week with
strongest staining intensity (>80% positive cells)
24h after electroporation. In addition, transfectants
do not lose HLA class II expression upon freezing,
thawing and irradiation cycles. In a first series of
experiments, sorted CD4 T-cell subsets of 3 healthy
donors were stimulated weekly with mismatched
K562/HLA-DR or -DQ transfectants, respectively,
and were tested for allo-reactivity 5 days after the
first (d12) and second (d19) restimulation in IFN-γ
ELISPOT assays. The strongest allo-HLA II reactivity
was detected in the CD45RApos and CD62Lpos
as well as in the CD45ROneg CD4+ subsets, in contrast
to the corresponding counterpart fractions,
confirming that allo-reactivity can be found mainly
in naive and central memory T cells. Alloreactivity
of the CCR7pos and CCR7neg cell fractions varied
considerably. In all cases, anti-HLA class II reactivity
of CD4 populations could be blocked by monoclonal
antibodies to the respective HLA class II
molecule used for stimulation. Proliferation did not
show clear differences between individual CD4+
T-cell subsets. In summary, we show herein that
the transient expression of HLA class II molecules
in K562 cells by RNA electroporation is a rapid and
efficient approach to detect and stimulate allo-HLA
class II reactive CD4+ T-cell responses. The strongest
class II mismatch reactivity was found in the
naive and central memory CD4+ T-cell subsets.
This approach based on K562 cells and HLA class
II RNA as „off-the-shelf” reagents allows to rapidly
generate CD4+ T cells with reactivity to single allo-
HLA-DQ/DP alleles, which may be of potential use
in adoptive immunotherapy of leukemias.