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.

CD8 + T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate eVective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identiWed LRH-1 as a hematopoietic-restricted MiHA encoded by the P2X5 gene. Here, we report that LRH-1 is aberrantly expressed on solid tumor cells. P2X5 mRNA expression is demonstrated in a signiWcant portion of solid tumor cell lines, including renal cell carcinoma (RCC), melanoma, colorectal carcinoma, brain cancer and breast cancer. Importantly, P2X5 gene expression was also detected in a subset of primary solid tumor specimens derived from RCC, brain cancer and breast cancer patients. Furthermore, P2X5 expressing solid tumor cells can be eVectively targeted by LRH-1-speciWc cytotoxic T lymphocytes under inXammatory conditions. The expression of HLA-B7 and CD54 on tumor cells increases upon cytokine stimulation resulting in improved T cell activation as observed by higher levels of degranulation and enhanced tumor cell lysis. Overall, hematopoietic-restricted MiHA LRH-1 is aberrantly expressed on solid tumor cells and may be used as target in GVT-speciWc immunotherapy after SCT.

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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.

Allogeneic stem cell transplantation (SCT) in combination with donor lymphocyte infusion (DLI) is an experimental treatment for patients with metastatic solid tumors. The therapeutic efficacy is attributed to the graft-versus-tumor (GVT) response during which donor-derived T cells eliminate malignant cells via recognition of minor histocompatibility antigens (MiHA). To reduce accompanying GVHD, it is crucial to identify MiHA which are selectively expressed on hematopoietic cells and solid tumor cells. Previously, we identified a hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest.2005:115:3506–3516). Here, we report that LRH-1, in addition to its hematopoietic cell-restricted expression, is aberrantly expressed on epithelial tumor cell lines. We observed that P2X5 mRNA is significantly expressed in 14 out of 42 (33%) solid tumor cell lines tested by real-time quantitative RT-PCR. We detected P2X5 transcripts in 3 out of 11 renal cell carcinoma cell lines, 2 out of 4 melanoma cell lines, 3 out of 7 colorectal carcinoma cell lines, 4 out of 10 brain tumor cell lines and 2 out of 10 breast cancer cell lines. To determine whether P2X5 mRNA expression in solid tumor cell lines results in susceptibility to lysis by LRH-1-specific CTL, we performed flow cytometry-based cytotoxicity assays using P2X5-expressing tumor cell lines. Based on LRH-1 genotyping analysis, we selected six solid tumor cell lines for the cytotoxicity studies. Remarkably, LRH-1-specific CTL efficiently lysed and inhibited the growth of DAOY brain tumor cells up to 3 days of co-culture. The renal cell carcinoma cell lines SKRC-33 and SKRC-18 and the melanoma cell line BLM were also susceptible to LRH-1 CTL-mediated lysis, although less effectively. However, pre-incubation of these tumor cell lines with IFNγ and TNFα significantly increased the susceptibility to LRH-1-specific CTL and resulted in complete target cell lysis. Furthermore, these cytokine-stimulated cell lines induced higher levels of CTL degranulation as determined by CD107a staining. No cytotoxicity was observed against LRH-1-negative FM3 melanoma and SKRC-24 renal cell carcinoma cell lines. These findings illustrate that the GVT reactivity observed in solid tumors after allogeneic SCT may be selectively enhanced by LRH-1-specific immunotherapy.

Allogeneic stem cell transplantation (SCT) is a specialized form of immunotherapy for treating patients with hematological malignancies. The curative potential 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. Recently, we identified a novel hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene (J. Clin. Invest.2005:115:3506–3516). Interestingly, tetramer analysis showed a direct association between in vivo expansion of LRH-1-specific CD8+ T cells and the disappearance of Bcr-Abl positive tumor cells in the CML patient from whom LRH-1-specific CTL was originally isolated. In addition, we detected in vivo expansion of LRH-1-specific CTL in an AML patient who was in clinical remission without GVHD. Furthermore, we demonstrated that P2X5 mRNA is significantly expressed in leukemic CD34+ progenitor cells from most CML as well as AML patients. These findings indicate a role for LRH-1 in inducing GVT immunity against myeloid leukemic progenitor cells. Here, we investigated the ex vivo responsiveness of myeloid leukemic CD34+ progenitor cells to LRH-1-specific CTL. First, we addressed this question using the CD34+ KG1 cell line which is positive for LRH-1. By using a CFSE-based survival assay we demonstrated that KG1 cells stably transfected with HLA-B7 could be efficiently lysed by LRH-1-specific CTL. Next, we determined responsiveness of purified CD34+ progenitor cells from HLA-B7+ CML patients to LRH-1 CTL-mediated killing. In the CFSE-based survival assay as well as a hematopoietic progenitor cell inhibition assay we showed that LRH-1-specific CTL efficiently recognize and kill CD34+ progenitor cells from LRH-1+ CML patients. In contrast, LRH-1-specific CTL did not inhibit the proliferation of CD34+ progenitor cells from LRH-1- CML patients. These findings illustrate that the P2X5-encoded LRH-1 antigen is an attractive target for adequate eradication of myeloid leukemic progenitor cells after allogeneic SCT.