Depletion of naive T cells from donor leukapheresis products (LPs) aims at the reduction of alloreactivity, while preserving memory T-cell reactivity (for example, to pathogens). This study established the immunomagnetic depletion procedure under clean room conditions using CD45RA beads and analyzed LPs of six donors for cell composition and functional immune responses. CD45RA depletion resulted in 3.4-4.7 log (median 4.4) reduction of CD45RA þ T cells, thereby eliminating naive and late effector T cells. B cells were also completely removed, whereas significant proportions of NK cells, monocytes and granulocytes persisted. CD45RA-depleted LPs contained effector and central memory CD4 þ and CD8 þ T cells that showed sustained IFN-g secretion to CMV, EBV, Aspergillus and Candida Ags. Alloreactivity was measured in MLRs between donors with complete HLA-mismatch. Alloreactive CD8 þ T cells were strongly reduced (median 41-log) upon CD45RA depletion, whereas alloreactive CD4 þ T cells persisted in significant numbers. In conclusion, clinical grade depletion of CD45RA þ naive T cells from donor LPs is feasible and highly efficient. The depleted products show sustained CD4 þ and CD8 þ T-cell reactivity to pathogens and effectively reduced CD8-mediated alloreactivity. Prophylactic and preemptive infusions after allogeneic SCT may improve T-cell reconstitution and pathogen-specific immunosurveillance, along with lower risk of inducing GVHD.

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The online version of this article has a Supplementary Appendix. BackgroundHLA mismatch antigens are major targets of alloreactive T cells in HLA-incompatible stem-cell transplantation, which can trigger severe graft-versus-host disease and reduce survival in transplant recipients. Our objective was to identify T-cell subsets with reduced in vitro reactivity to allogeneic HLA antigens. Design and MethodsWe sorted CD4 and CD8 T-cell subsets from peripheral blood by flow cytometry according to their expression of naive and memory markers CD45RA, CD45RO, CD62L, and CCR7. Subsets were defined by a single marker to facilitate future establishment of a clinical-grade procedure for reducing alloreactive T-cell precursors and graft-versus-host disease. T cells were stimulated in mixed lymphocyte reactions against HLA-deficient K562 cells transfected with single HLA-A/-B/-C/-DR/-DQ mismatch alleles. Alloreactivity was measured by interferon-γ spot production and cell proliferation. ResultsWe observed that allogeneic HLA-reactivity was preferentially derived from subsets enriched for naïve T cells rather than memory T cells in healthy donors, irrespective of the HLA mismatch allele. This separation was most efficient if CD45RA (versus other markers) was used for sorting. The numbers of allogeneic HLA-reactive effector cells were in median 7.2-fold and 16.6-fold lower in CD45RA neg memory CD8 and CD4 T cells than in entire CD8 and CD4 T cells, respectively. In contrast, proliferation of memory T cells in response to allogeneic HLA was more variably reduced (CD8) or equivalent (CD4) when compared to that of naïve T cells. We also demonstrated in HLA-matched donor-patient pairs that leukemia-reactive CD8 cytotoxic T-lymphocytes were mainly derived from subsets enriched for naïve T cells compared to memory T cells. ConclusionsMemory T-cell subsets of most healthy individuals showed decreased allogeneic HLA-reactivity, but lacked significant anti-leukemia responses in vitro. The clinical use of memory or naïve-depleted T cells might be beneficial for HLA-mismatched patients at high risk of graft-versushost disease and low risk of leukemia relapse. Preferred allografts are those which contain leukemia-reactive memory T cells. Alternatively, replenishment with leukemia-reactive T cells isolated from naïve subsets is desirable.Key words: alloreactivity, leukemia-reactive T cells, T-cell subsets, naïve T cells, immunotherapy. Citation: Distler E, Bloetz

Clinical tumor remissions after adoptive T-cell therapy are frequently not durable due to limited survival and homing of transfused tumor-reactive T cells, what can be mainly attributed to the long-term culture necessary for in vitro expansion. Here, we introduce an approach allowing the reliable in vitro generation of leukemia-reactive cytotoxic T lymphocytes (CTLs) from naive CD8+ T cells of healthy donors, leading to high cell numbers within a relatively short culture period. The protocol includes the stimulation of purified CD45RA+ CD8+ T cells with primary acute myeloid leukemia blasts of patient origin in HLA-class I-matched allogeneic mixed lymphocyte-leukemia cultures. The procedure allowed the isolation of a large diversity of HLA-A/-B/-C-restricted leukemia-reactive CTL clones and oligoclonal lines. CTLs showed reactivity to either leukemia blasts exclusively, or to leukemia blasts as well as patient-derived B lymphoblastoid-cell lines (LCLs). In contrast, LCLs of donor origin were not lysed. This reactivity pattern suggested that CTLs recognized leukemia-associated antigens or hematopoietic minor histocompatibility antigens. Consistent with this hypothesis, most CTLs did not react with patient-derived fibroblasts. The efficiency of the protocol could be further increased by addition of interleukin-21 during primary in vitro stimulation. Most importantly, leukemia-reactive CTLs retained the expression of early T-cell differentiation markers CD27, CD28, CD62L and CD127 for several weeks during culture. The effective in vitro expansion of leukemia-reactive CD8+ CTLs from naive CD45RA+ precursors of healthy donors can accelerate the molecular definition of candidate leukemia antigens and might be of potential use for the development of adoptive CTL therapy in leukemia.

Refractory or relapsed acute myeloid leukemia (AML) represents a frequent complication after allogeneic hematopoietic stem-cell transplantation (HSCT). We show herein that primary in vitro stimulation of CD45RA-selected CD4 T cells of stem-cell donors with 10/10 HLA-matched AML blasts results in expansion of cytolytic T-lymphocytes (CTL) that almost all recognize HLA-DPB1 mismatch alleles, which clinically occur in up to 80% of donor-patient pairs. Primary AML blasts were found to strongly express HLA-DPB1, whereas fibroblasts and keratinocytes used as surrogate target cells for graft-versus-host disease did express HLA-DPB1 only upon IFN-γ pre-treatment. Since patients' AML blasts are rarely available in clinical routine, we developed a protocol based on stimulation of donor-derived CD45RA-selected CD4 T cells with autologous dendritic cells electroporated with RNA encoding patients' HLA-DPB1 mismatch alleles. Short-term stimulated T cell-lines specifically lysed HLA-DPB1 mismatch-expressing AML blasts, but not fibroblasts and keratinocytes without IFN-γ pre-treatment. Notably, these CD4 CTL efficiently eliminated AML blasts upon adoptive transfer into leukemia-engrafted NSG mice. In conclusion, we show strong immunogenicity of HLA-DPB1 mismatch alleles in CD45RA-selected CD4 T cells of stem-cell donors and introduce a novel strategy to reliably generate HLA-DPB1-specific CD4 CTL that might be powerful cellular therapeutics in relapsed or refractory AML after HSCT.