Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity.

BACKGROUND: Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease. DESIGN AND METHODS: We analyzed in detail CD8(+) T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8(+) T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens. RESULTS: Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones. CONCLUSIONS: Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.

Proteosomal degradation of BCR/ABL protein can generate an HLA-A*0301-restricted peptide, but high-avidity T cells recognizing this leukemia-specific antigen were not demonstrated.

BACKGROUND AND OBJECTIVES: Cytotoxic T-lymphocytes (CTL) have been generated in vitro against chronic myeloid leukemia (CML)-associated BCR/ABL-specific peptides. We analyzed the existence of high-avidity T cells recognizing endogenously processed BCR/ABL-specific proteins. DESIGN AND METHODS: We performed binding studies of BCR/ABL-specific peptides, proteosomal digestion of BCR/ABL breakpoint overlapping protein, mass spectrometry of eluates from HLA-*0301-transduced K562 cells, and tried to isolate peptide-specific T-cells using tetramers. RESULTS: We confirmed the binding of the BCR/ABL-specific peptides KQSSKALQR to HLA-A*0301 and GFKQSSKAL to HLA-B*0801. Proteasomal digestion showed cleavage sites leading to KQSSKALQR but not to GFKQSSKAL. Using mass spectrometry KQSSKALQR could not be detected in the eluates from HLA-A*0301-transduced K562 cells. We attempted to induce BCR/ABL-specific CTL lines from 4 healthy donors using dendritic cells pulsed with KQSSKALQR and performed single cell sorting to isolate tetramer-positive T cells. None of 31 generated clones showed BCR/ABL-specific cytotoxicity. Isolation of tetramer-positive cells from peripheral blood of relapsed CML patients after allogeneic transplantation treated with donor lymphocyte infusion resulted in 38 T-cell clones which did not show peptide-specific cytotoxicity. INTERPRETATION AND CONCLUSIONS: We provide evidence that BCR/ABL protein processing can lead to KQSSKALQR peptide binding to HLA-A*0301. However, KQSSKALQR could not be detected in HLA-A*0301-transduced K562 cells, and KQSSKALQR could not be demonstrated to induce high-avidity BCR/ABL-specific CTL.

High-throughput characterization of 10 new minor histocompatibility antigens by whole genome association scanning.

Patients with malignant diseases can be effectively treated with allogeneic hematopoietic stem cell transplantation (allo-SCT). Polymorphic peptides presented in HLA molecules, the so-called minor histocompatibility antigens (MiHA), play a crucial role in antitumor immunity as targets for alloreactive donor T cells. Identification of multiple MiHAs is essential to understand and manipulate the development of clinical responses after allo-SCT. In this study, CD8+ T-cell clones were isolated from leukemia patients who entered complete remission after allo-SCT, and MiHA-specific T-cell clones were efficiently selected for analysis of recognition of a panel of EBV-transformed B cells positive for the HLA restriction elements of the selected T-cell clones. One million single nucleotide polymorphisms (SNP) were determined in the panel cell lines and investigated for matching with the T-cell recognition data by whole genome association scanning (WGAs). Significant association with 12 genomic regions was found, and detailed analysis of genes located within these genomic regions revealed SNP disparities encoding polymorphic peptides in 10 cases. Differential recognition of patient-type, but not donor-type, peptides validated the identification of these MiHAs. Using tetramers, distinct populations of MiHA-specific CD8+ T cells were detected, demonstrating that our WGAs strategy allows high-throughput discovery of relevant targets in antitumor immunity after allo-SCT.

Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that have emerged as a promising tool for clinical application. Further clinical interest has been raised by the observation that MSCs are immunoprivileged and, more important, display immunosuppressive capacities. These properties may be of therapeutic value in allogeneic transplantation to prevent graft rejection and to prevent and treat graft-versus-host disease. In the present study, we examined the in vivo immunomodulatory properties of MSCs in murine models of allogeneic bone marrow (BM) transplantation. Sublethally irradiated recipients received allogeneic BM with or without host or donor MSCs. The addition of host MSCs significantly enhanced the long-term engraftment associated with tolerance to host and donor antigens. However, the infusion of donor MSCs was associated with significantly increased rejection of allogeneic donor BM cells. Moreover, we showed that the injection of merely allogeneic donor MSCs in naive mice was sufficient to induce a memory T-cell response. Although the observed engraftment-promoting effects of host MSCs in vivo support the therapeutic potential of MSCs, our results also indicate that allogeneic MSCs are not intrinsically immunoprivileged and that under appropriate conditions, allogeneic MSCs induce a memory T-cell response resulting in rejection of an allogeneic stem cell graft.