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Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor–recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.

Abstract 4116 Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can mediate curative Graft-versus-Leukemia (GVL) reactivity although frequently at the cost of Graft-versus-Host Disease (GVHD). We previously illustrated that donor CD8 T lymphocytes recognizing HLA class-I restricted minor histocompatibility antigens (MiHAs) that are broadly expressed on tissues of the recipient cause GVL associated with GVHD, whereas T lymphocytes recognizing MiHAs selectively expressed on hematopoietic cells, including the malignant cells, can selectively mediate GVL without GVHD. Since in contrast to HLA class-I, expression of HLA class-II molecules is predominantly restricted to hematopoietic cells, we hypothesized that infused purified donor CD4 T lymphocytes may selectively recognize and eliminate hematopoietic cells from the recipient resulting in GVL without GVHD. We treated a patient with CML in blastic phase in remission after intensive chemotherapy with T cell depleted alloSCT from his HLA-identical sibling donor after myelo-ablative conditioning. After donor engraftment, recipient hematopoiesis reoccurred within 3 months to 90% of CD8 T lymphocytes, 13% of CD4 T lymphocytes and 5% of myelopoiesis. As part of a clinical trial, the patient was treated with 106/kg positively selected purified donor derived CD4 T lymphocytes resulting within 19 weeks in conversion into full donor chimerism in all hematopoietic cell lineages in the total absence of GVHD. To characterize the nature of this hematopoiesis restricted immune response, in vivo activated HLA-DR positive CD4 and CD8 T lymphocytes were clonally isolated by flowcytometric cell sorting at the time of the clinical response, expanded and tested for alloreactivity on patient and donor derived hematopoietic target cells using IFNγ ELISA. From the 204 expanding CD4 T lymphocyte clones 31 clones were alloreactive, whereas none of the 66 expanding CD8 T lymphocyte clones showed alloreactivity. To further identify the fine specificity of this hematopoiesis directed HLA class-II restricted immune response, target molecules of several T lymphocyte clones were molecularly characterized using whole genome association scanning. We first performed blocking studies with HLA class-II restricted monoclonal antibodies and identified HLA-DR to be the restriction molecule. Next, a large panel of third party EBV-LCLs was retrovirally transduced with each of the possible restriction molecules being HLA-DRB1*11:01, HLA-DRB1*15:01, HLA-DRB3*02:02 and HLA-DRB5*01:01. By comparing the recognition pattern of the transduced EBV-LCLs with the 1.1 million single nucleotide polymorphisms in each EBV-LCL, we identified 3 novel MiHAs. Synthesis and analysis of the patient and donor derived allelic peptide variants further confirmed the specificity of the MiHAs as LB-KHNYN-1K in the context of HLA-DRB5*01:01, LB-CTSB-1G in HLA-DRB1*11:01 and LB-ZDHHC13-1K in HLA-DRB1*15:01. Gene expression profiles of KHNYN (located on chromosome 14), CTSB (chromosome 8) and ZDHHC13 (chromosome 11) illustrated that the genes encoding these MiHAs were not only transcribed in hematopoietic cells, but also in other tissues including GVHD target tissues. These results further illustrated that the hematopoietic specificity of the CD4 T lymphocyte response was mainly defined by the restricted expression of the HLA-DR molecules on hematopoietic cells. We conclude that purified CD4 DLI can lead to GVL without GVHD by a selective HLA class-II restricted immune response against patient hematopoiesis. By molecular characterization of 3 novel HLA-DR restricted MiHAs we illustrated that the relative specificity of HLA class-II molecules on hematopoietic cells under non inflammatory conditions was probably responsible for this effect. Since HLA class-II is predominantly expressed on hematopoietic cells only, infusion of donor CD4 T lymphocytes under non inflammatory conditions after HLA identical alloSCT can result in efficient induction of GVL without the toxicity of GVHD. Disclosures: No relevant conflicts of interest to declare.

Although profound anti-leukemic immune responses can be induced with donor lymphocyte infusions in patients with relapsed or persistent leukemia after allogeneic stem cell transplantation, (late) relapses of the same disease develop regularly even in patients initially entering a complete remission. This suggests that a subpopulation of leukemic (precursor) cells with ultimate self-renewal capacity is capable of resisting T cell attack. We hypothesized that quiescent leukemic precursor cells can evade anti-leukemic therapy by their capacity to survive and persist in the presence of competent cytotoxic T cells. In addition, selectivity of cytotoxic T cells (CTLs) for target cells in active cell cycle in general may also explain why powerful immune responses directed against antigens that are broadly expressed on all tissues of the recipient, like the male-specific HY-antigens, do not necessarily result in severe damage to all tissues of the recipient. Therefore, we determined the efficacy of high affinity CTL clones directed against allo-HLA or minor histocompatibility antigens to kill normal and leukemic hematopoietic cells in dormancy and in active cell cycle, comprising normal and leukemic CD34+ precursor cells, normal B cells, T cells and monocytes, and activated B cells (EBV-LCL) and activated T cells (PHA blasts). Using a CFSE-based cytotoxicity assay allowing the analysis of susceptibility to lysis of specific cell types within a heterogeneous target cell population, we found that all activated target cells were very efficiently lysed, resulting in 60–90% lysis already after 4 hours of exposure to the CTL clones (E/T ratios 1/1–5/1). In contrast, target cells in relative dormancy including the non-proliferating CD34+ CML stem cell fraction, unmanipulated CD34 progenitor cells, and resting T and B cells were protected from CTL-induced cell death (0–20% lysis). Since normal expression of adhesion and HLA class I molecules was shown on these dormant cells, we investigated whether decreased avidity of the T cell/target cell interaction was underlying the poor susceptibility. Therefore, we artificially enhanced the avidity by exogenous loading of the target cells with saturating concentrations of the relevant peptide. This was sufficient to restore the sensitivity to levels comparable to activated target cells, suggesting that decreased avidity of the interaction between high affinity CTL and resting target cells plays a role in the resistance phenomenon. However, even after restoration of the high avidity interaction, a small population of (leukemic) target cells (0,1–10% of the total cell population) was capable of residing, suggesting that additional factors like resistance of quiescent target cells to one or more of the T cell effector mechanisms are involved. To analyze the influence of the sensitivity to T cell lysis of specific target cell types on the specificity of adoptive T cell therapy, we used non-hematopoietic target cells like mesenchymal stem cells and biliary epithelium cells as target cells. Alloreactive T cells showed also diminished capacity to lyse these target cells (10–20% lysis). The addition of inflammatory cytokines like TNF and interferons slightly increased the recognition. In conclusion, under steady state conditions potent allo immune responses may have limited activity against quiescent target cells. Therefore in order to cure the disease, specific activation strategies and/or prolonged persistence of specific T cells will be needed to achieve a potent anti-leukemic effect with controlled GVHD.