RESUMO
CD8(+) T cells recognizing minor histocompatibility antigens (MiHA) on solid tumor cells may mediate effective graft-versus-tumor (GVT) reactivity after allogeneic stem cell transplantation (SCT). Previously, we identified 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 significant 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 effectively targeted by LRH-1-specific cytotoxic T lymphocytes under inflammatory 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-specific immunotherapy after SCT.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Sistema Hematopoético/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Neoplasias/imunologia , Receptores Purinérgicos P2/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA/fisiologia , Genótipo , Humanos , Imunoterapia/métodos , Molécula 1 de Adesão Intercelular/biossíntese , Microscopia de Fluorescência/métodos , Neoplasias/metabolismo , RNA Mensageiro/metabolismo , Receptores Purinérgicos P2X5 , Transplante de Células-Tronco , Fatores de Transcrição/fisiologia , Transplante HomólogoRESUMO
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.
Assuntos
Mutação da Fase de Leitura , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Polimorfismo Genético , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Linfócitos T Citotóxicos/citologia , Adulto , Sequência de Aminoácidos , Antígenos CD34/biossíntese , Sequência de Bases , Células da Medula Óssea/citologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Células Cultivadas , Cromo/metabolismo , Mapeamento Cromossômico , Cromossomos Humanos Par 17 , Proteínas de Ligação a DNA/genética , Epitopos/química , Feminino , Proteínas de Fusão bcr-abl/química , Ligação Genética , Marcadores Genéticos , Genótipo , Efeito Enxerto vs Leucemia , Antígenos HLA-B/química , Antígeno HLA-B7 , Haplótipos , Homozigoto , Humanos , Interferon gama/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/imunologia , Antígenos Comuns de Leucócito/química , Escore Lod , Masculino , Modelos Genéticos , Dados de Sequência Molecular , Neurônios/metabolismo , Linhagem , Peptídeos/química , Plasmídeos/metabolismo , Receptores Purinérgicos P2X5 , Recidiva , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante de Células-Tronco , Células-Tronco , Linfócitos T/citologia , Linfócitos T/imunologia , Fatores de Tempo , Fatores de Transcrição/genética , Transplante HomólogoRESUMO
Minor 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 CTL-mediated lysis in flow cytometry-based cytotoxicity assays. However, interferon-gamma 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.
Assuntos
Proteínas de Ligação a DNA/imunologia , Leucemia Linfoide/imunologia , Linfoma não Hodgkin/imunologia , Receptores Purinérgicos P2/biossíntese , Linfócitos T Citotóxicos/imunologia , Fatores de Transcrição/imunologia , Citotoxicidade Imunológica/imunologia , Humanos , Leucemia Linfoide/patologia , Linfoma não Hodgkin/patologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X5 , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células Tumorais CultivadasRESUMO
Minor histocompatibility antigens (mHAgs) constitute the target antigens of the T cell-mediated graft-versus-leukemia response after HLA-identical allogeneic stem cell transplantation (SCT). Several human mHAgs have been identified, but only a few are selectively expressed by hematopoietic cells representing potential targets for specific immunotherapy. Molecular approaches including cDNA library screening and genetic linkage analysis have been successfully applied to identify T cell-defined mHAgs, but each approach has its drawbacks which may lead to mis-identification of the mHAg of interest. We improved both molecular strategies to facilitate more robust identification of hematopoietic-restricted mHAgs. First, we adapted cDNA library cloning by using 293T cells with stable expression of the relevant MHC class I allele, CD80 and CD54. We demonstrated that cDNA library screening using this 293T expression system results in strong activation of cytotoxic T lymphocytes, which significantly contributes to improvement of the assay sensitivity. Second, we refined genetic linkage analysis using single nucleotide polymorphism (SNP) genotyping to narrow down the defined genetic region that holds the mHAg-encoding gene. We showed that SNP marker analysis provides additional information about the genetic position of the antigen-encoding gene. Application of these optimized molecular approaches will lead to more rapid and reliable molecular identification of hematopoietic-restricted mHAgs.
Assuntos
Ligação Genética , Ativação Linfocitária/genética , Antígenos de Histocompatibilidade Menor/genética , Polimorfismo de Nucleotídeo Único , Linfócitos T Citotóxicos/imunologia , Animais , Antígeno B7-1/genética , Células COS , Chlorocebus aethiops , Clonagem Molecular , Feminino , Frequência do Gene , Biblioteca Gênica , Antígenos HLA-B/genética , Antígeno HLA-B44 , Antígeno HLA-B7 , Humanos , Molécula 1 de Adesão Intercelular/genética , Interferon gama/biossíntese , Células K562 , Masculino , Antígenos de Histocompatibilidade Menor/análise , Linhagem , Reprodutibilidade dos Testes , TransfecçãoRESUMO
Tumor relapses in patients with precursor B-cell acute lymphoblastic leukemia (BALL) 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-gamma 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-cell-mediated antitumor immunity in patients.