Increased chemosensitivity via BRCA2-independent DNA damage in DSS1- and PCID2-depleted breast carcinomas.

Breast cancer, the most common malignancy among women, is closely associated with mutations in the tumor suppressor gene BRCA. DSS1, a component of the TRanscription-EXport-2 (TREX-2) complex involved in transcription and mRNA nuclear export, stabilizes BRCA2 expression. DSS1 is also related to poor prognosis in patients with breast cancer owing to the induction of chemoresistance. Recently, BRCA2 was shown to be associated with the TREX-2 component PCID2, which prevents DNA:RNA hybrid R-loop formation and transcription-coupled DNA damage. This study aimed to elucidate the involvement of these TREX-2 components and BRCA2 in the chemosensitivity of breast carcinomas. Our results showed that compared with that in normal breast tissues, DSS1 expression was upregulated in human breast carcinoma, whereas PCID2 expression was comparable between normal and malignant tissues. We then compared patient survival time among groups divided by high or low expressions of DSS1, BRCA2, and PCID2. Increased DSS1 expression was significantly correlated with poor prognosis in recurrence-free survival time, whereas no differences were detected in the high and low BRCA2 and PCID2 expression groups. We performed in vitro analyses, including propidium iodide nuclear staining, single-cell gel electrophoresis, and clonogenic survival assays, using breast carcinoma cell lines. The results confirmed that DSS1 depletion significantly increased chemosensitivity, whereas overexpression conferred chemoresistance to breast cancer cell lines; however, BRCA2 expression did not affect chemosensitivity. Similar to DSS1, PCID2 expression was also inversely correlated with chemosensitivity. These results strongly suggest that DSS1 and PCID2 depletion is closely associated with increased chemosensitivity via BRCA2-independent DNA damage. Together with the finding that DSS1 is not highly expressed in normal breast tissues, these results demonstrate that DSS1 depletion confers a druggable trait and may contribute to the development of novel chemotherapeutic strategies to treat DSS1-depleted breast carcinomas independent of BRCA2 mutations.

Development of a novel redirected T-cell-based adoptive immunotherapy targeting human telomerase reverse transcriptase for adult T-cell leukemia.

Although adult T-cell leukemia (ATL) has a poor prognosis, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) in some cases suggests that a cellular immune-mediated strategy can be effective. So far, however, no effective target for anti-ATL immunotherapy has been defined. Here we demonstrated for the first time that human telomerase reverse transcriptase (hTERT) is a promising therapeutic target for ATL, and we developed a novel redirected T-cell-based immunotherapy targeting hTERT. hTERT messenger RNA was produced abundantly in ATL tumor cells but not in steady-state normal cells. Rearranged human leukocyte antigen-A*24:02 (HLA-A*24:02) -restricted and hTERT461-469 nonameric peptide-specific T-cell receptor (TCR) α/ß genes were cloned from our previously established cytotoxic T lymphocyte clone (K3-1) and inserted into a novel retroviral TCR expression vector encoding small interfering RNAs for endogenous TCR genes in redirected T cells (hTERT-siTCR vector). Consequently, allogeneic or autologous gene-modified CD8(+) T cells prepared using the hTERT-siTCR vector successfully killed ATL tumor cells, but not normal cells including steady-state hematopoietic progenitors, in an HLA-A*24:02-restricted manner both in vitro and in vivo. Our experimental observations support the development of a novel hTERT-targeting redirected T-cell-based adoptive immunotherapy for ATL patients, especially those for whom suitable allo-HSCT donors are lacking.

Aurora kinase A-specific T-cell receptor gene transfer redirects T lymphocytes to display effective antileukemia reactivity.

Aurora kinase A (AURKA) is overexpressed in leukemias. Previously, we demonstrated that AURKA-specific CD8(+) T cells specifically and selectively lysed leukemia cells, indicating that AURKA is an excellent target for immunotherapy. In this study, we examined the feasibility of adoptive therapy using redirected T cells expressing an HLA-A*0201-restricted AURKA(207-215)-specific T-cell receptor (TCR). Retrovirally transduced T cells recognized relevant peptide-pulsed but not control target cells. Furthermore, TCR-redirected CD8(+) T cells lysed AURKA-overexpressing human leukemic cells in an HLA-A*0201-restricted manner, but did not kill HLA-A*0201(+) normal cells, including hematopoietic progenitors. In addition, AURKA(207-215)-specific TCR-transduced CD4(+) T cells displayed target-responsive Th1 cytokine production. Finally, AURKA(207-215)-specific TCR-transduced CD8(+) T cells displayed antileukemia efficacy in a xenograft mouse model. Collectively, these data demonstrate the feasibility of redirected T cell-based AURKA-specific immunotherapy for the treatment of human leukemia.

IL-21 can supplement suboptimal Lck-independent MAPK activation in a STAT-3-dependent manner in human CD8(+) T cells.

Although both MHC class II/CD8α double-knockout and CD8ß null mice show a defect in the development of MHC class I-restricted CD8(+) T cells in the thymus, they possess low numbers of high-avidity peripheral CTL with limited clonality and are able to contain acute and chronic infections. These in vivo data suggest that the CD8 coreceptor is not absolutely necessary for the generation of Ag-specific CTL. Lack of CD8 association causes partial TCR signaling because of the absence of CD8/Lck recruitment to the proximity of the MHC/TCR complex, resulting in suboptimal MAPK activation. Therefore, there should exist a signaling mechanism that can supplement partial TCR activation caused by the lack of CD8 association. In this human study, we have shown that CD8-independent stimulation of Ag-specific CTL previously primed in the presence of CD8 coligation, either in vivo or in vitro, induced severely impaired in vitro proliferation. When naive CD8(+) T cells were primed in the absence of CD8 binding and subsequently restimulated in the presence of CD8 coligation, the proliferation of Ag-specific CTL was also severely hampered. However, when CD8-independent T cell priming and restimulation were supplemented with IL-21, Ag-specific CD8(+) CTL expanded in two of six individuals tested. We found that IL-21 rescued partial MAPK activation in a STAT3- but not STAT1-dependent manner. These results suggest that CD8 coligation is critical for the expansion of postthymic peripheral Ag-specific CTL in humans. However, STAT3-mediated IL-21 signaling can supplement partial TCR signaling caused by the lack of CD8 association.

Induction of CD8 T-cell responses restricted to multiple HLA class I alleles in a cancer patient by immunization with a 20-mer NY-ESO-1f (NY-ESO-1 91-110) peptide.

Immunogenicity of a long 20-mer NY-ESO-1f peptide vaccine was evaluated in a lung cancer patient TK-f01, immunized with the peptide with Picibanil OK-432 and Montanide ISA-51. We showed that internalization of the peptide was necessary to present CD8 T-cell epitopes on APC, contrasting with the direct presentation of the short epitope. CD8 T-cell responses restricted to all five HLA class I alleles were induced in the patient after the peptide vaccination. Clonal analysis showed that B*35:01 and B*52:01-restricted CD8 T-cell responses were the two dominant responses. The minimal epitopes recognized by A*24:02, B*35:01, B*52:01 and C*12:02-restricted CD8 T-cell clones were defined and peptide/HLA tetramers were produced. NY-ESO-1 91-101 on A*24:02, NY-ESO-1 92-102 on B*35:01, NY-ESO-1 96-104 on B*52:01 and NY-ESO-1 96-104 on C*12:02 were new epitopes first defined in this study. Identification of the A*24:02 epitope is highly relevant for studying the Japanese population because of its high expression frequency (60%). High affinity CD8 T-cells recognizing tumor cells naturally expressing the epitopes and matched HLA were induced at a significant level. The findings suggest the usefulness of a long 20-mer NY-ESO-1f peptide harboring multiple CD8 T-cell epitopes as an NY-ESO-1 vaccine. Characterization of CD8 T-cell responses in immunomonitoring using peptide/HLA tetramers revealed that multiple CD8 T-cell responses comprised the dominant response.

Novel adoptive T-cell immunotherapy using a WT1-specific TCR vector encoding silencers for endogenous TCRs shows marked antileukemia reactivity and safety.

Adoptive T-cell therapy for malignancies using redirected T cells genetically engineered by tumor antigen-specific T-cell receptor (TCR) gene transfer is associated with mispairing between introduced and endogenous TCR chains with unknown specificity. Therefore, deterioration of antitumor reactivity and serious autoimmune reactivity are major concerns. To address this problem, we have recently established a novel retroviral vector system encoding siRNAs for endogenous TCR genes (siTCR vector). In this study, to test the clinical application of siTCR gene therapy for human leukemia, we examined in detail the efficacy and safety of WT1-siTCR-transduced T cells. Compared with conventional WT1-TCR (WT1-coTCR) gene-transduced T cells, these cells showed significant enhancement of antileukemia reactivity resulting from stronger expression of the introduced WT1-specific TCR with inhibition of endogenous TCRs. Notably, WT1-siTCR gene-transduced T cells were remarkably expandable after repetitive stimulation with WT1 peptide in vitro, without any deterioration of antigen specificity. WT1-siTCR gene-transduced T cells from leukemia patients successfully lysed autologous leukemia cells, but not normal hematopoietic progenitor cells. In a mouse xenograft model, adoptively transferred WT1-siTCR gene-transduced T cells exerted distinct antileukemia efficacy but did not inhibit human hematopoiesis. Our results suggest that gene-immunotherapy for leukemia using this WT1-siTCR system holds considerable promise.

A novel animal model of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in humanized mice.

EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH) is a rare yet devastating disorder caused by EBV infection in humans. However, the mechanism of this disease has yet to be elucidated because of a lack of appropriate animal models. Here, we used a human CD34(+) cell-transplanted humanized mouse model and reproduced pathologic conditions resembling EBV-HLH in humans. By 10 weeks postinfection, two-thirds of the infected mice died after exhibiting high and persistent viremia, leukocytosis, IFN-γ cytokinenemia, normocytic anemia, and thrombocytopenia. EBV-infected mice also showed systemic organ infiltration by activated CD8(+) T cells and prominent hemophagocytosis in BM, spleen, and liver. Notably, the level of EBV load in plasma correlated directly with both the activation frequency of CD8(+) T cells and the level of IFN-γ in plasma. Moreover, high levels of EBV-encoded small RNA1 were detected in plasma of infected mice, reflecting what has been observed in patients. These findings suggest that our EBV infection model mirrors virologic, hematologic, and immunopathologic aspects of EBV-HLH. Furthermore, in contrast to CD8(+) T cells, we found a significant decrease of natural killer cells, myeloid dendritic cells, and plasmacytoid dendritic cells in the spleens of infected mice, suggesting that the collapse of balanced immunity associates with the progression of EBV-HLH pathogenesis.

Mismatched human leukocyte antigen class II-restricted CD8⁺ cytotoxic T cells may mediate selective graft-versus-leukemia effects following allogeneic hematopoietic cell transplantation.

Partial human leukocyte antigen (HLA)-mismatched hematopoietic stem cell transplantation (HSCT) is often performed when an HLA-matched donor is not available. In these cases, CD8(+) or CD4(+) T cell responses are induced depending on the mismatched HLA class I or II allele(s). Herein, we report on an HLA-DRB1*08:03-restricted CD8(+) CTL clone, named CTL-1H8, isolated from a patient following an HLA-DR-mismatched HSCT from his brother. Lysis of a patient Epstein-Barr virus-transformed B cell line (B-LCL) by CTL-1H8 was inhibited after the addition of blocking antibodies against HLA-DR and CD8, whereas antibodies against pan-HLA class I or CD4 had no effect. The 1H8-CTL clone did not lyse the recipient dermal fibroblasts whose HLA-DRB1*08:03 expression was upregulated after 1 week cytokine treatment. Engraftment of HLA-DRB1*08:03-positive primary leukemic stem cells in non-obese diabetic/severe combined immunodeficient/γc-null (NOG) mice was completely inhibited by the in vitro preincubation of cells with CTL-1H8, suggesting that HLA-DRB1*08:03 is expressed on leukemic stem cells. Finally, analysis of the precursor frequency of CD8(+) CTL specific for recipient antigens in post-HSCT peripheral blood T cells revealed a significant fraction of the total donor CTL responses towards the individual mismatched HLA-DR antigen in two patients. These findings underscore unexpectedly significant CD8 T cell responses in the context of HLA class II.

Aurora-A kinase: a novel target of cellular immunotherapy for leukemia.

Aurora-A kinase (Aur-A) is a member of the serine/threonine kinase family that regulates the cell division process, and has recently been implicated in tumorigenesis. In this study, we identified an antigenic 9-amino-acid epitope (Aur-A(207-215): YLILEYAPL) derived from Aur-A capable of generating leukemia-reactive cytotoxic T lymphocytes (CTLs) in the context of HLA-A*0201. The synthetic peptide of this epitope appeared to be capable of binding to HLA-A*2402 as well as HLA-A*0201 molecules. Leukemia cell lines and freshly isolated leukemia cells, particularly chronic myelogenous leukemia (CML) cells, appeared to express Aur-A abundantly. Aur-A-specific CTLs were able to lyse human leukemia cell lines and freshly isolated leukemia cells, but not normal cells, in an HLA-A*0201-restricted manner. Importantly, Aur-A-specific CTLs were able to lyse CD34+ CML progenitor cells but did not show any cytotoxicity against normal CD34+ hematopoietic stem cells. The tetramer assay revealed that the Aur-A(207-215) epitope-specific CTL precursors are present in peripheral blood of HLA-A*0201-positive and HLA-A*2402-positive patients with leukemia, but not in healthy individuals. Our results indicate that cellular immunotherapy targeting Aur-A is a promising strategy for treatment of leukemia.

HapMap scanning of novel human minor histocompatibility antigens.

Minor histocompatibility antigens (mHags) are molecular targets of allo-immunity associated with hematopoietic stem cell transplantation (HSCT) and involved in graft-versus-host disease, but they also have beneficial antitumor activity. mHags are typically defined by host SNPs that are not shared by the donor and are immunologically recognized by cytotoxic T cells isolated from post-HSCT patients. However, the number of molecularly identified mHags is still too small to allow prospective studies of their clinical importance in transplantation medicine, mostly due to the lack of an efficient method for isolation. Here we show that when combined with conventional immunologic assays, the large data set from the International HapMap Project can be directly used for genetic mapping of novel mHags. Based on the immunologically determined mHag status in HapMap panels, a target mHag locus can be uniquely mapped through whole genome association scanning taking advantage of the unprecedented resolution and power obtained with more than 3 000 000 markers. The feasibility of our approach could be supported by extensive simulations and further confirmed by actually isolating 2 novel mHags as well as 1 previously identified example. The HapMap data set represents an invaluable resource for investigating human variation, with obvious applications in genetic mapping of clinically relevant human traits.

Identification of a polymorphic gene, BCL2A1, encoding two novel hematopoietic lineage-specific minor histocompatibility antigens.

We report the identification of two novel minor histocompatibility antigens (mHAgs), encoded by two separate single nucleotide polymorphisms on a single gene, BCL2A1, and restricted by human histocompatibility leukocyte antigen (HLA)-A*2402 (the most common HLA-A allele in Japanese) and B*4403, respectively. Two cytotoxic T lymphocyte (CTL) clones specific for these mHAgs were first isolated from two distinct recipients after hematopoietic cell transplantation. Both clones lyse only normal and malignant cells within the hematopoietic lineage. To localize the gene encoding the mHAgs, two-point linkage analysis was performed on the CTL lytic patterns of restricting HLA-transfected B lymphoblastoid cell lines obtained from Centre d'Etude du Polymorphisme Humain. Both CTL clones showed a completely identical lytic pattern for 4 pedigrees and the gene was localized within a 3.6-cM interval of 15q24.3-25.1 region that encodes at least 46 genes. Of those, only BCL2A1 has been reported to be expressed in hematopoietic cells and possess three nonsynonymous nucleotide changes. Minigene transfection and epitope reconstitution assays with synthetic peptides identified both HLA-A*2402- and B*4403-restricted mHAg epitopes to be encoded by distinct polymorphisms within BCL2A1.

Correlation of high and decreased NY-ESO-1 immunity to spontaneous regression and subsequent recurrence in a lung cancer patient.

We show correlation between strong and decreased NY-ESO-1-specific immunity with spontaneous regression and subsequent recurrence, respectively, in a long-surviving patient with an NY-ESO-1-expressing lung adenocarcinoma. An integrated immune response consisting of IgG antibody, as well as CD4 and CD8 T cells, against NY-ESO-1 was observed at the time of spontaneous regression of multiple pleural metastases. After tumor dormancy for 3 years, the tumor started to progress. IgG antibody levels and the number of CD4 and CD8 T cells against NY-ESO-1 decreased, but were still detectable. On the other hand, the number of Foxp3+ CD25 high T regulatory cells gradually increased. The findings suggest the relevance of the NY-ESO-1 immune response and its regulation by Foxp3+ CD25 high T regulatory cells in the clinical course of this lung cancer patient.

Improving TCR affinity on 293T cells.

This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRß, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers. Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.

Impact of germinal center-associated nuclear protein polymorphisms on breast cancer risk and prognosis in a Japanese population.

BACKGROUND: Germinal center-associated nuclear protein (GANP) is a phosphoprotein involved in mRNA export and regulation of DNA recombination. Although GANP expression in human breast cancer tissue is associated with breast cancer prognosis, the association between the genetic background of GANP and susceptibility and prognosis of breast cancer is unclear. METHODS: We selected 694 breast cancer cases and 1376 age- and menopausal status-matched non-cancer controls from the Hospitable-based Epidemiologic Research Program, conducted at Aichi Cancer Center between 2001 and 2005. We evaluated the impact of two polymorphisms at the GANP locus (rs2839178 and rs11702450) on the susceptibility and prognosis of breast cancer. Reference alleles were defined as the A allele for rs2839178 and G allele for rs11702450. RESULTS: The GG genotype of rs2839178 was statistically significantly associated with breast cancer risk (odds ratio [OR] 0.48, 95% confidence interval [CI] 0.30-0.76, P = 0.002). In prognostic analysis, compared to those with AA genotype of rs2839178, patients with AG or GG genotypes had longer disease-free survival (DFS) (hazard ratio [HR] 0.71, 95% CI 0.49-1.04 and HR 0.42, 95% CI 013-1.42, respectively, P for trend = 0.04). eQTL analysis indicated that association with rs2839178 can be explained by the effect of rs2839173 on expression of GANP/MCM3AP. CONCLUSIONS: The G allele of rs2839178 at the GANP locus was significantly associated with reduced breast cancer risk and longer DFS in breast cancer patients, showing a consistent direction in the association between susceptibility and clinical outcome. GANP is, therefore, important for the occurrence and progression of sporadic breast cancer.

The DNA demethylating agent 5-aza-2'-deoxycytidine activates NY-ESO-1 antigenicity in orthotopic human glioma.

Cancer/testis antigens (CTAs) are considered to be suitable targets for the immunotherapy of human malignancies. It has been demonstrated that in a variety of tumors, the expression of certain CTAs is activated via the demethylation of their promoter CpG islands. In our study, we have shown that while the composite expression of 13 CTAs in 30 human glioma specimens and newly established cell lines from the Japanese population was nearly imperceptible, the DNA-demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) markedly reactivated CTA expression in glioma cells but not in normal human cells. We quantified the diminished methylation status of NY-ESO-1-one of the most immunogenic CTAs-following 5-aza-CdR treatment by using a novel Pyrosequencing technology and methylation-specific PCR. Microarray analysis revealed that 5-aza-CdR is capable of signaling the immune system, particularly, human leukocyte antigen (HLA) class I upregulation. (51)Cr-release cytotoxicity assays and cold target inhibition assays using NY-ESO-1-specific cytotoxic T lymphocyte (CTL) lines demonstrated the presentation of de novo NY-ESO-1 antigenic peptides on the cell surfaces. In an orthotopic xenograft model, the systemic administration of 5-aza-CdR resulted in a significant volume reduction of the transplanted tumors and prolonged the survival of the animals after the adoptive transfer of NY-ESO-1-specific CTLs. These results suggested that 5-aza-CdR induces the expression of epigenetically silenced CTAs in poorly immunogenic gliomas and thereby presents a new strategy for tumor immunotherapy targeting 5-aza-CdR-induced CTAs.

Epstein-Barr virus nuclear antigen 1-specific CD4+ T cells directly kill Epstein-Barr virus-carrying natural killer and T cells.

Epstein-Barr virus (EBV) nuclear antigen (EBNA)1 is expressed in every EBV-infected cell, regardless of the state of EBV infection. Although EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies, it is less clear whether EBNA1-specific CD4(+) T cells can act as direct effectors. Herein, we investigated the ability of CD4(+) T-cell clones induced with overlapping peptides covering the C-terminal region of EBNA1, and identified minimal epitopes and their restricted major histocompatibility complex class II molecules. Of these, a novel epitope, EYHQEGGPD, was found to be presented by DRB1*0401, 0403 and 0406. Five CD4(+) T-cell clones recognized endogenously processed and presented antigens on EBV-transformed lymphoblastoid cell lines (LCL) and one example proved capable of killing EBV-carrying natural killer (NK) and T-cell lines derived from patients with chronic active EBV infection (CAEBV). Identification of minimal epitopes facilitates design of peptide-based vaccines and our data suggest that EBNA1-specific CD4(+) T cells may play roles as direct effectors for immunotherapy targeting EBV-carrying NK and T-cell malignancies.

Effective induction of anti-tumor immune responses with oligomannose-coated liposome targeting to intraperitoneal phagocytic cells.

We recently established a novel drug delivery system (DDS) using oligomannose-coated liposomes (OMLs) which are probably taken up by macrophages (Mvarphi) to carry anti-cancer drugs to milky spots known as preferential metastatic sites of gastric cancers [Y. Ikehara, T. Niwa, L. Biao, S.K. Ikehara, N. Ohashi, T. Kobayashi, Y. Shimizu, N. Kojima, H. Nakanishi, A carbohydrate recognition-based drug delivery and controlled release system using intraperitoneal macrophages as a cellular vehicle, Cancer Res. 66 (2006) 8740-8748]. In the present study, we applied this intraperitoneal DDS for systemic cancer immunotherapy employing ovalbumin (OVA) as a model antigen. The cells taking up the OMLs containing FITC-OVA injected into the peritoneal cavity were predominantly Mvarphi, as they showed adhesive characteristics and expressed F4/80 and CD11b almost exclusively. The phagocytic cells also took up bare OVA directly to the same extent as OML-enclosed OVA (OML-OVA), as it is a highly mannosilated protein. The phagocytic cells taking up OML-OVA, however, could activate OVA-specific CD8+ (from OT-I: H-2Kb/OVA257-264-specific) and CD4+ (from OT-II: H-2Ab/OVA323-339-specific) T cells much more effectively in vitro than those taking up bare OVA. Furthermore, only the mice pre-immunized with OML-OVA rejected E.G7-OVA (OVA-transfected EL4) but not EL4. These results indicate that the OMLs can also be used as an effective antigen delivery system for cancer immunotherapy activating both CTL and Th subsets.

Aberrant expression of BCL2A1-restricted minor histocompatibility antigens in melanoma cells: application for allogeneic transplantation.

It has been shown that allogeneic hematopoietic stem cell transplantation (HSCT) can be one of the therapeutic options for patients with metastatic solid tumors, such as renal cancer. However, the development of relatively severe GVHD seems to be necessary to achieve tumor regression in the current setting. Thus, it is crucial to identify minor histocompatibility antigens (mHags) only expressed in tumor cells but not GVHD target organs. In this study, we examined whether three mHags: ACC-1 and ACC-2 encoded by BCL2A1, and HA-1 encoded by HMHA1, could serve as such targets for melanoma. Real-time PCR and immunohistochemical analysis revealed that the expression of both BCL2A1and HMHA1 in melanoma cell lines and primary melanoma cells was comparable to that of hematopoietic cells. Indeed, melanoma cell lines were efficiently lysed by cytotoxic T lymphocytes specific for ACC-1, ACC-2, and HA-1. Our data suggest that targeting mHags encoded not only by HMHA1, whose aberrant expression in solid tumors has been reported, but also BCL2A1 may bring about beneficial selective graft-versus-tumor effects in a population of melanoma patients for whom these mHags are applicable.

Identification of a human leukocyte antigen-A24-restricted T-cell epitope derived from interleukin-13 receptor alpha2 chain, a glioma-associated antigen.

OBJECT: The human leukocyte antigen-A24 (HLA-A24) allele is highly expressed in Asians. This allele is expressed in 60% of the Japanese population and in a significant number of people of other ethnicities. The interleukin-13 type alpha2 receptor (IL-13Ralpha2) has been shown to be a glioma-specific antigen, and is abundantly expressed in a majority of high-grade astrocytomas. In this study, the authors first investigated the suitability of IL-13Ralpha2 as a target antigen of malignant glioma cells, and then identified a potential HLA-A24-restricted peptide derived from IL-13Ralpha2. METHODS: The expression of IL-13Ralpha2 in glioma tissues was examined by reverse transcription-polymerase chain reaction analysis. To identify the desired epitope, the authors selected 5 candidate peptides from IL-13Ralpha2 that were predicted to bind to HLA-A24. The lytic activity of cytotoxic T lymphocytes (CTLs) induced by peptide-pulsed dendritic cells was analyzed against various glioma cell lines and freshly isolated human glioma cells. RESULTS: In a series of glioma tissues obtained in 29 patients, the authors found that > 50% of high-grade gliomas expressed IL-13Ralpha2. Of the 5 peptides tested, P174 (WYEGLDHAL) was found to be the most useful for the induction of HLA-A24-restricted and IL-13Ralpha2-specific CTLs. A CTL line induced by P174 also showed antigen-specific cytotoxicity to surgically removed glioma cells depending on their level of expression of IL-13Ralpha2 and HLA-A24. CONCLUSIONS: Interleukin-13Ralpha2 is a glioma-specific antigen, and the immunogenic peptide P174 may contribute to a peptide-based immunotherapy against malignant glioma cells expressing HLA-A24.