Key Points• GVHD after HLA-DPB1-mismatched CD41 DLI after TCD-alloSCT is mediated by allo-reactive HLA-DPB1-directed CD41 T cells.• Viral infections after TCDalloSCT can induce HLA class II on nonhematopoietic tissues, making them targets for CD41 T cells in GVHD.CD81 T cell-depleted (TCD) donor lymphocyte infusion (DLI) after TCD allogeneic hematopoietic stem cell transplantation (alloSCT) has been associated with a reduced risk of graft-versus-host disease (GVHD) while preserving conversion to donor hematopoiesis and antitumor immunity, providing a rationale for exploring CD41 T cell-based immunotherapy for hematologic malignancies. Here, we analyzed the clinical course and specificity of T cell immune responses in 2 patients with acute myeloid leukemia (AML) who converted to full-donor chimerism but developed severe acute GVHD after prophylactic CD41 DLI after 10/10-HLA-matched, but HLA-DPB1-mismatched TCD-alloSCT. Clonal analysis of activated T cells isolated during GVHD demonstrated allo-reactivity exerted by CD41 T cells directed against patient-mismatched HLA-DPB1 molecules on hematopoietic cells and skin-derived fibroblasts only when cultured under inflammatory conditions. At the time of CD41 DLI, both patients contained residual patient-derived T cells, including cytomegalovirus (CMV)-specific T cells as a result of CMV reactivations.Once activated by CMV antigens, these CMV-specific T cells could stimulate HLA-DPB1-specific CD41 T cells, which in turn could target nonhematopoietic tissues in GVHD. In conclusion, our data demonstrate that GVHD after HLA-DPB1-mismatched CD41 DLI can be mediated by allo-reactive HLA-DPB1-directed CD41 T cells and that ongoing viral infections inducing HLA class II expression on nonhematopoietic cells may increase the likelihood of GVHD development. This trial is registered at http://www.controlled-trials.com/ISRCTN51398568/LUMC as #51398568. (Blood. 2013;122(11):1963-1973 IntroductionIn allogeneic hematopoietic stem cell transplantation (alloSCT), T-cell depletion of the graft efficiently prevents the occurrence of severe acute graft-versus-host disease [GVHD]) 1,2 but also adversely affects posttransplant antitumor and antipathogen immunity.1-3 Early intervention with donor lymphocyte infusion (DLI) after T cell-depleted (TCD)-alloSCT may prevent relapse of the malignancy and improve immune reconstitution against pathogens but is frequently associated with reintroduction of GVHD. 4,5 Therefore, exploration of treatment strategies to improve the balance between graft-versus-leukemia (GVL) reactivity and antipathogen immunity and GVHD is warranted.To minimize the risk of GVHD, patients are preferably transplanted with stem cell grafts from HLA-matched sibling or unrelated donors (URD).6 HLA matching is generally performed for HLA-A, -B, -C, -DRB1, and -DQB1 alleles (10/10 match) but not for HLA-DPB1. Therefore, 70% to 80% of URD alloSCT are HLA-DPB1-mismatched. 6,7 In contrast to HLA class I, constitutive expression of HLA class II molecules is mainly confined to normal...

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T cells recognizing tumor-associated Ags such as Wilms tumor protein (WT1) are thought to exert potent antitumor reactivity. However, no consistent high-avidity T cell responses have been demonstrated in vaccination studies with WT1 as target in cancer immunotherapy. The aim of this study was to investigate the possible role of negative thymic selection on the avidity and specificity of T cells directed against self-antigens. T cell clones directed against the HLA-A*0201–binding WT1126–134 peptide were generated from both HLA-A*02–positive (self-HLA–restricted) and HLA-A*02–negative [nonself (allogeneic) HLA [allo-HLA]-restricted] individuals by direct ex vivo isolation using tetramers or after in vitro priming and selection. The functional avidity and specificity of these T cell clones was analyzed in-depth. Self-HLA–restricted WT1-specific clones only recognized WT1126–134 with low avidities. In contrast, allo-HLA–restricted WT1 clones exhibited profound functional reactivity against a multitude of HLA-A*02–positive targets, even in the absence of exogenously loaded WT1 peptide, indicative of Ag-binding promiscuity. To characterize this potential promiscuity, reactivity of the T cell clones against 400 randomly selected HLA-A*0201–binding peptides was investigated. The self-HLA–restricted WT1-specific T cell clones only recognized the WT1 peptide. In contrast, the allo-HLA–restricted WT1-reactive clones recognized besides WT1 various other HLA-A*0201–binding peptides. In conclusion, allogeneic HLA-A*02–restricted WT1-specific T cells isolated from mismatched donors may be more tumor-reactive than their autologous counterparts but can show specific off-target promiscuity of potential clinical importance. As a result of this, administration of WT1-specific T cells generated from HLA-mismatched donors should be performed with appropriate precautions against potential off-target effects.

The online version of this article has a Supplementary Appendix. BackgroundOne of the major challenges in allogeneic stem cell transplantation is to find a balance between the harmful induction of graft-versus-host disease and the beneficial graft-versus-leukemia and pathogen-specific immune responses. Adoptive transfer of in-vitro generated donor T cells with specific anti-leukemic or pathogen-specific activity may be effective. However, in many cases this requires the in-vitro priming and expansion of antigen-specific precursor T cells from the naïve donor T-cell repertoire. Design and MethodsAntigen-specific CD8 T cells were generated by co-culture of CD45RO-depleted, regulatory T cell-depleted donor peripheral blood mononuclear cells with autologous peptide-loaded dendritic cells, followed by two re-stimulations with peptide-loaded autologous monocytes. Responding T cells were isolated based on CD137 expression and further purified using peptide/major histocompatibility complex tetramers. ResultsUsing this method we were able to reproducibly generate functionally high avidity T cells directed against multiple viral antigens and minor histocompatibility antigens from the naïve T-cell repertoire of seronegative, minor histocompatibility antigen-negative donors. Furthermore, we demonstrated that reduction of the regulatory T-cell frequency by depletion of CD45RO + responder cells resulted in improved priming and expansion of antigen-specific precursor T cells. ConclusionsIn conclusion, we present a robust method for the in-vitro induction and isolation of antigenspecific T cells from the naïve repertoire. We demonstrate that the likelihood of successful generation of primary immune responses is determined by a delicate balance between the numbers of antigen-specific precursor T cells and the numbers and activation state of regulatory T cells locally at the site of priming of the immune response.Key words: primary immune responses, regulatory T cells, CMV, minor histocompatibility anti gens, immunotherapy. tory T cells. Haematologica 2011;96(8):1204-1212. doi:10.3324/haematol.2010 This is an open-access paper. Citation: Jedema I, van de Meent M, Pots J, Kester MGD, van der Beek MT, and Falkenburg JHF. Successful generation of primary virus-specific and anti-tumor T cell responses from the naïve donor T-cell repertoire is determined by the balance between antigen-specific precursor T cells and regula Successful generation of primary virus-specific and anti-tumor T-cell responses from the naïve donor T-cell repertoire is determined by the balance between antigen-specific precursor T cells and regulatory T cells

Purpose: The poor immunogenicity of most leukemias and the lack of specificity of the donor Tcells limit the in vivo effectiveness of conventional donor lymphocyte infusions in many patients suffering from persistent or recurrent leukemia after allogeneic stem cell transplantation. These limitations may be overcome by the adoptive transfer of in vitro generated leukemia-reactive T cells. Although the potential clinical efficacy of this approach has been shown previously, lack of reproducibility of the procedure and the inability to show persistence and survival of the transferred T cells hampered further clinical application. The purpose of this study was to develop a new, broadly applicable strategy for the efficient generation and isolation of leukemia-reactive Tcells with a better probability to survive and expand in vivo. Experimental Design: Myeloid and B-cell leukemias were modified into professional immunogenic antigen-presenting cells, and used to stimulate HLA-matched donor T cells. After two stimulations, responding donorTcells were isolated based on their secretion of IFN-g and tested for their capacity to recognize and kill the primary leukemia. Results: Using one universal stimulation and isolation protocol for various forms of leukemia, T-cell populations containing high frequencies of leukemia-reactive T cells could reproducibly be generated and early isolated under mild stimulatory conditions. Isolated Tcells still had high proliferative potential and their reactivity seemed to be restricted to cells of the patient's hematopoiesis. Conclusion: We here show a new robust procedure for the generation and isolation of leukemiareactiveTcells for adoptive transfer.