Minor histocompatibility antigens (mHAgs) constitute the targets of the graft-versus-leukemia response after HLA-identical allogeneic stem cell transplantation. Here, we have used genetic linkage analysis to identify a novel mHAg, designated lymphoid-restricted histocompatibility antigen-1 (LRH-1), which is encoded by the P2X5 gene and elicited an allogeneic CTL response in a patient with chronic myeloid leukemia after donor lymphocyte infusion. We demonstrate that immunogenicity for LRH-1 is due to differential protein expression in recipient and donor cells as a consequence of a homozygous frameshift polymorphism in the donor. Tetramer analysis showed that emergence of LRH-1-specific CD8 + cytotoxic T cells in peripheral blood and bone marrow correlated with complete remission of chronic myeloid leukemia. Furthermore, the restricted expression of LRH-1 in hematopoietic cells including leukemic CD34 + progenitor cells provides evidence of a role for LRH-1-specific CD8 + cytotoxic T cells in selective graft-versus-leukemia reactivity in the absence of severe graft-versus-host disease. These findings illustrate that the P2X5-encoded mHAg LRH-1 could be an attractive target for specific immunotherapy to treat hematological malignancies recurring after allogeneic stem cell transplantation.

SummaryMinor histocompatibility antigens (MiHA) selectively expressed by haematopoietic cells are attractive targets for specific immunotherapy after allogeneic stem cell transplantation (SCT). Previously, we described LRH-1 as a haematopoietic-lineage restricted MiHA that is capable of eliciting an allogeneic cytotoxic T-lymphocyte (CTL) response after SCT and donor lymphocyte infusion. Importantly, the gene encoding LRH-1, P2X5, is not expressed in prominent graft-versus-host-disease target tissues such as skin, liver and gut. Here, we investigate whether LRH-1-specific immunotherapy may be exploited for the treatment of lymphoid malignancies. We examined P2X5 mRNA expression in a large panel of patient samples and cell lines from different types of lymphoid malignancies by real-time quantitative reverse transcription polymerase chain reaction. P2X5 mRNA was highly expressed in malignant cells from all stages of lymphoid development. Furthermore, all LRH-1-positive lymphoid tumour cell lines were susceptible to LRH-1 CTLmediated lysis in flow cytometry-based cytotoxicity assays. However, interferon-c production was low or absent after stimulation with some cell lines, possibly due to differences in activation thresholds for CTL effector functions. Importantly, primary cells from patients with lymphoid malignancies were effectively lysed by LRH-1-specific CTL. These findings indicate that MiHA LRH-1 is a potential therapeutic target for cellular immunotherapy of lymphoid malignancies.

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The results of our study seem to indicate a better sensitivity for the Respifinder. Analysis of patient samples is necessary to evaluate the clinical performance.

Tumor relapses in patients with precursor B-cell acute lymphoblastic leukemia (B-ALL) occur frequently after primary treatment. Therefore, development of additional treatment modalities to eliminate residual tumor cells is needed. Active immunotherapy using dendritic cells (DCs) loaded with tumor-associated antigens is a promising approach to induce specific T-cell immunity in patients with cancer. In previous studies, we described HB-1 as a B-cell lineage-specific antigen that is recognized by donor-derived cytotoxic T lymphocytes (CTLs) on allogeneic B-ALL tumor cells. Here, we investigated the potential use of the HB-1 antigen as an autologous T-cell vaccine target. To determine whether HB-1-specific CTL precursors are present within the T-cell repertoire, we induced expansion of CD8 ؉ T cells using mature monocyte-derived DCs pulsed with the previously identified HB-1.B44 antigenic peptide. In 6 of 8 donors, CD8 ؉ CTL lines have been generated that exert cytotoxicity against target cells exogenously pulsed with peptide or endogenously expressing the HB-1 antigen. From one of these HB-1-specific T-cell lines, we isolated a CD8 ؉ CTL that produces interferon-␥ on stimulation with B-ALL tumor cells. Interestingly, the HB-1 antigen also induced CD4 ؉ T-helper responses on activation with protein-loaded mature monocyte-derived DCs. We identified 2 novel epitopes recognized in the context of HLA-DR4 and HLA-DR11 with the use of HB-1-specific CD4 ؉ T-cell clones generated from different donors. These present data, that HB-1 induces both helper and cytotoxic T-cell responses, indicate that the HB-1 antigen is a candidate target to induce T-cellmediated antitumor immunity in patients.

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Allogeneic hematopoietic stem cell transplantation (HSCT) is a potent treatment for patients with hematological malignancies. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived cytotoxic T lymphocytes (CTL) eliminate malignant cells of the recipient. Minor histocompatibility antigens (MiHA) are the major targets of the GVT response, and expansion of MiHA-specific CTL has been shown to coincide with tumor remission following SCT. Unfortunately, GVT response is often accompanied by graft-versus-host disease (GVHD) causing severe damage to skin, liver and gut. Therefore, it would be highly beneficial to direct GVT immunity to MiHA that are selectively expressed by “malignant” hematopoietic cells. Recently, we identified a novel lymphoid lineage-restricted MiHA, designated LRH-1, which is derived from a frame shift polymorphism in the P2X5 purinergic receptor protein (J. Clin. Invest.2005:115:3506–3516). Here, we examined by real-time quantitative RT-PCR mRNA expression of P2X5 in patient samples and cell lines from numerous lymphoid malignancies. We observed that P2X5 mRNA is highly expressed in tumor cells from all stages of lymphoid development. Furthermore, we demonstrated that LRH-1−specific CTL efficiently lyse LRH-1+ tumor cells and cell lines of lymphoid origin. These findings illustrate that LRH-1 is an attractive target for specific immunotherapy after allogeneic HSCT. A potentially efficient strategy is the application of MiHA-loaded dendritic cells (DC) to boost MiHA-specific T cell responses already primed in vivo after HSCT. To facilitate efficient presentation of LRH-1 by DC, we have optimized RNA electroporation of DC using in vitro-transcribed mRNA. We observed that RNA electroporation of mature DC resulted in a high yield of viable cells, high expression of co-stimulatory molecules, no loss of migratory capacity towards lymph node-specific chemokines, and high protein expression of the introduced antigens. Furthermore, we demonstrated that RNA-electroporated DC display long-lasting peptide presentation to LRH-1− specific CTL and induce proliferation of LRH-1− specific effector-memory CTL ex vivo. These data will be of importance in designing LRH-1− based immunotherapy in transplanted patients with lymphoid malignancies.

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