Identification of novel Lck‐derived T helper epitope long peptides applicable for HLA‐A2+ cancer patients as cancer vaccine

The present study attempted to identify T helper epitope long peptides capable of inducing cytotoxic T lymphocytes (CTL) from Lck antigen (p56^Lck), the src family tyrosine kinase, which is known to be aberrantly expressed in metastatic cancers cells, in order to develop a long peptide‐based cancer vaccine for HLA‐A2⁺ cancer patients. Based on the biding motif to the HLA‐DR and HLA‐A2 alleles, 94 peptides were prepared from the Lck antigen. These peptides were screened for their reactivity to immunoglobulin G (IgG) from plasma of cancer patients, followed by testing of their ability to induce both CD4⁺ and CD8⁺ T lymphocytes showing not only peptide‐specific IFN‐γ production but cytotoxicity against HLA‐A2⁺ cancer cells from peripheral blood mononuclear cells (PBMC) of HLA‐A2⁺ cancer patients. Among 94 peptides tested, the three T helper epitope long peptides and their inner CTL epitope short peptides with HLA‐A2 binding motifs were frequently recognized by IgG of cancer patients, and efficiently induced both CD4⁺ IFN‐γ⁺ and CD8⁺ IFN‐γ⁺ T lymphocytes. Patients' PBMC stimulated with these long peptides showed cytotoxicity against HLA‐A2⁺ Lck⁺ cancer cells in HLA‐class I and HLA‐class II dependent manners. These three peptides might be useful for long peptide‐based vaccines for HLA‐A2⁺cancer patients with Lck⁺ tumor cells.     

Identification of NY‐BR‐1‐specific CD4+ T cell epitopes using HLA‐transgenic mice

Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer‐related deaths among women. The differentiation antigen NY‐BR‐1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen‐specific CD4⁺ effector T cells, NY‐BR‐1 was screened for the presence of HLA‐restricted CD4⁺ T cell epitopes that could be included in immunological treatment approaches. Upon NY‐BR‐1‐specific DNA immunization of HLA‐transgenic mice and functional ex vivo analysis, a panel of NY‐BR‐1‐derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY‐BR‐1‐specific, HLA‐DRB1*0301– or HLA‐DRB1*0401‐restricted CD4⁺ T cell lines from splenocytes of peptide immunized HLA‐transgenic mice. Notably, all four CD4⁺ T cell lines recognized human HLA‐DR‐matched dendritic cells (DC) pulsed with lysates of NY‐BR‐1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4⁺ T cells specific for all four CD4⁺ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4⁺ T cell responses against the new epitopes are not deleted nor inactivated by self‐tolerance mechanisms. Our results present the first NY‐BR‐1‐specific HLA‐DRB1*0301– and HLA‐DRB1*0401‐restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer.     

Allorestricted T lymphocytes with a high avidity T‐cell receptor towards NY‐ESO‐1 have potent anti‐tumor activity

The cancer‐testis antigen NY‐ESO‐1 has been targeted as a tumor‐associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T‐cell‐based therapy is the induction of T cells capable of recognizing the NY‐ESO‐1‐expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY‐ESO‐1_157–165 epitope known to be naturally presented with HLA‐A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY‐ESO‐1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA‐A2‐positive, NY‐ESO‐1‐expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY‐ESO‐1‐specific T lymphocytes displayed TCRs with the highest avidity and best anti‐tumor recognition activity. TCRs derived from allorestricted, NY‐ESO‐1‐specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR‐transduced T cells for the treatment of NY‐ESO‐1‐expressing hematological malignancies and solid tumors. © 2009 UICC     

Development of a T‐cell receptor multimer with high avidity for detecting a naturally presented tumor‐associated antigen on osteosarcoma cells

For efficacy of peptide vaccination immunotherapy for patients with cancer, endogenous expression of the target peptide/human leukocyte antigen (HLA) on cancer cells is required. However, it is difficult to evaluate the expression status of a peptide/HLA complex because of the lack of a soluble T‐cell receptor (TCR) that reacts with tumor‐associated antigen (TAA) with high avidity. In the present study, we developed two soluble TCR‐multimers that were each directed to TAA, survivin‐2B (SVN‐2B) and PBF in the context of HLA‐A24 (SVN‐2B TCR‐multimer and PBF TCR‐multimer, respectively), from CTL clones that were established from peptide‐vaccinated patients. Both TCR multimers could recognize cognate peptide‐pulsed antigen‐presenting cells, C1R‐A24 cells, in a CD8‐independent method. Moreover, the PBF TCR‐multimer successfully recognized a PBF peptide naturally presented on HLA‐A24⁺PBF⁺ osteosarcoma cells. Taken together, the results indicated that a TCR‐multimer might be useful for detection of a TAA‐derived peptide presented by HLA in patients receiving immunotherapy.     

The shared tumor associated antigen cyclin‐A2 is recognized by high‐avidity T‐cells

Cyclin‐A2, a key cell cycle regulator, has been shown to be overexpressed in various types of malignancies with little expression in normal tissue. Such tumor‐associated genes potentially are useful targets for cancer immunotherapy. However, high‐avidity cyclin‐specific T cells are considered to be thymically deleted. We identified at least one nonameric HLA‐A*0201 binding cyclin‐A2 epitope by a reverse immunology approach. Using a highly efficient T‐cell expansion system that is based on CD40‐activated B (CD40‐B) cells as sole antigen‐presenting cells we successfully generated cyclin‐A2 specific CTL from HLA‐A*0201⁺ donors. Interestingly, high‐avidity cyclin‐A2 specific CTL lines, which recognized peptide‐pulsed and antigen expressing target cells, were indeed generated by stimulation with CD40‐B cells when pulsed with low concentrations of peptide, whereas CD40‐B cells pulsed at saturating concentrations could only induce low‐avidity CTL, which recognized peptide‐pulsed target cells only. One high‐avidity CTL line was subcloned and CTL clones, whose peptide concentration required for half‐maximal lysis were less than 1 nM, could lyse cyclin‐A2 expressing tumor cells. Taken together, cyclin A2 is an attractive candidate for immune intervention in a significant number of cancer patients and high‐avidity T cells can be readily generated using CD40‐B cells as antigen‐presenting cells. © 2009 UICC     

Novel soluble HLA‐A2/MELAN‐A complexes selectively stain a differentiation defective subpopulation of CD8+ T cells in patients with melanoma

Multimeric MHC I‐peptide complexes containing phycoerythrin‐streptavidin are widely used to detect and investigate antigen‐specific CD8+ (and CD4+) T cells. Because such reagents are heterogeneous, we compared their binding characteristics with those of monodisperse dimeric, tetrameric and octameric complexes containing linkers of variable length and flexibility on Melan‐A‐specific CD8+ T cell clones and peripheral blood mononuclear cells (PBMC) from HLA‐A*0201⁺ melanoma patients. Striking binding differences were observed for different defined A2/Melan‐A_26‐35 complexes on T cells depending on their differentiation stage. In particular, short dimeric but not octameric A2/Melan‐A_26‐35 complexes selectively and avidly stained incompletely differentiated effector‐memory T cells clones and populations expressing CD27 and CD28 and low levels of cytolytic mediators (granzymes and perforin). This subpopulation was found in PBMC from all six melanoma patients analyzed and proliferated on peptide stimulation with only modest phenotypic changes. By contrast influenza matrix_58‐66 ‐specific CD8+ PBMC from nine HLA‐A*0201⁺ healthy donors were efficiently stained by A2/Flu matrix_58‐61 multimers, but not dimer and upon peptide stimulation proliferated and differentiated from memory into effector T cells. Thus PBMC from melanoma patients contain a differentiation defective sub‐population of Melan‐A‐specific CD8+ T cells that can be selectively and efficiently stained by short dimeric A2/Melan‐ A_26‐35 complexes, which makes them directly accessible for longitudinal monitoring and further investigation.     

Identification of promiscuous HPV16‐derived T helper cell epitopes for therapeutic HPV vaccine design

Cervical carcinoma and several other human papillomavirus (HPV)‐induced malignancies are a global public health problem, thus novel treatment modalities are urgently needed. Immunotherapy is an attractive option for treatment of HPV infection and HPV‐mediated premalignant and malignant lesions. However, previous approaches—focusing on the induction of cytotoxic CD8+ T cells (CTLs)—have as yet not yielded clinical successes. Since CD4+ T cells have been shown to be crucial for the induction and maintenance of CTL responses, and more recently to be also important for direct anti‐tumor immunity, human leukocyte antigen (HLA) class II‐restricted epitopes are intensively investigated to improve the efficacy of peptide‐based HPV immunotherapy. We here present an approach to identify promiscuous HPV16‐derived CD4+ T helper epitopes, which are capable of inducing T cell immunity in a large proportion of the population. To this end, we combined HLA class II epitope prediction servers with in vitro immunological evaluation to identify HPV16 E2‐, E5‐, E6‐, and E7‐derived CD4+ T cell epitopes. Candidate selected HPV16‐derived epitopes were found to be restricted by up to nine HLA‐DR molecules. Furthermore, they were found to induce frequent and robust HPV16 peptide‐specific Th1 responses in healthy donors, as monitored by interferon (IFN)‐γ ELISPOT and cytokine secretion assays. Moreover, these selected peptides also induced specific IFN‐γ T cell responses in blood from HPV16+ CIN2/3 and cervical carcinoma patients. We thus conclude that the identified T helper epitopes are valuable candidates for the development of a comprehensive therapeutic HPV vaccine.     

Generation of NY-ESO-1-specific CD4+ and CD8+ T cells by a single peptide with dual MHC class I and class II specificities: a new strategy for vaccine design

The existence of overlapping CD8+ and CD4+ T-cell epitopes within certain tumor antigens provides an opportunity to test the hypothesis that relatively short peptides could be used to generate both CD8+ and CD4+ T cells against tumor. In this report, T-cell responses to a fragment of the tumor antigen NY-ESO-1 that contained an HLA-DP4-restricted helper T cell epitope as well as an HLA-A2-restricted cytotoxic T cell epitope were analyzed. One peptide, ESO:157-170 (SLLMWITQCFLPVF) was recognized by both NY-ESO-1-reactive CD8+ and CD4+ T-cell clones. Both CD4+ and CD8+ T cells were efficiently generated from the peripheral blood of multiple melanoma patients after in vitro stimulations using ESO:157-170. Dual-specific peptides containing both cytotoxic T-cell and helper T-cell epitopes may represent an attractive strategy of vaccine design aimed at generating tumor-reactive CD4+ and CD8+ T cells.     

A novel strategy for the discovery of MHC class II-restricted tumor antigens: identification of a melanotransferrin helper T-cell epitope

CD4+ helper T cells play a critical role in orchestrating host immune responses, including antitumor immunity. The limited availability of MHC class II-associated tumor antigens is still viewed as a major obstacle in the use of CD4+ T cells in cancer vaccines. Here, we describe a novel approach for the identification of MHC class II tumor-associated antigens (TAAs). By combining two-dimensional liquid chromatography and nanoelectrospray ionization tandem mass spectrometry, we developed a highly sensitive method for the detection of human leukocyte antigen (HLA)-DR-associated peptides of dendritic cells upon exposure to necrotic tumor cells. This approach led to the identification of a novel MHC class II-restricted TAA epitope derived from melanotransferrin. The epitope stimulated T cells derived from melanoma patients and healthy individuals and displayed promiscuity in HLA-DR restriction. Moreover, the same peptide was also presented by MHC class II-positive melanoma cells. This strategy may contribute to increase the number of tumor epitopes presented by MHC class II molecules and may support the development of more efficacious vaccines against cancer.     

Detection of spontaneous CD4+ T-cell responses in melanoma patients against a tyrosinase-related protein-2-derived epitope identified in HLA-DRB1*0301 transgenic mice.

PURPOSE: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell-based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified. Whereas a number of HLA class I-presented TRP-2-derived epitopes are known, information about HLA class II-presented antigenic ligands recognized by CD4+ T helper (Th) cells is limited. EXPERIMENTAL DESIGN: The search for TRP-2-derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1*0301 ligands in combination with peptide and protein immunizations of HLA-DRB1*0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in human T-cell cultures. RESULTS: This strategy led to the characterization of TRP-2(60-74) as an HLA-DRB1*0301-restricted Th epitope. Importantly, TRP-2(60-74)-reactive human CD4+ Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1*03+ melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-2(60-74)-reactive T cells, suggesting that these T cells were already activated in vivo. CONCLUSION: Peptide TRP-2(60-74) might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients.     

T cell recognition of HLA‐A2 restricted tumor antigens is impaired by the oncogene HER2

The HER2 oncogene is frequently over‐expressed in human cancers and a promising target for immune therapy. Previous studies have shown that over‐expression of mouse or rat HER2 leads to markedly reduced levels of major histocompatibility complex (MHC) class I and molecules of the antigen processing and presentation machinery (APM), thus resulting in a phenotype promoting tumor escape from the immune system. Our study focuses on analyzing the effect of HER2 on MHC class I antigen presentation and sensitivity to tumor‐antigen specific cytotoxic T lymphocytes (CTLs) in HLA‐A2.1⁺ melanoma cell lines. We demonstrate significant inverse correlations both between the expression of HER2 and total MHC class I surface expression as well as between HER2 and HLA‐A2. A significant reduction of HLA‐A2 levels was found when melanoma and carcinoma cell lines were transfected with a human HER2 gene. A signaling‐competent HER2 molecule was crucial for the observed HLA‐A2 down‐regulation, as transfectants expressing high levels of HER2 mutated in the tyrosine signaling domain did not show altered HLA‐A2 expression. Importantly, the human melanoma cell line EST049 demonstrated reduced HER2 and melanoma antigen‐specific recognition by CTLs upon HER2 transfection. In addition, high expression of HER2 prevented both IFN‐γ mediated HLA‐A2 up‐regulation and improved recognition by HLA‐A2‐restricted CTLs in treated cells. Moreover, key APM molecules were down‐regulated by HER2. These findings implicate that HER2 over‐expressing tumors may be more prone to escape from HLA‐A2 restricted CTLs suggesting that immunotherapy approaches inducing an integrated humoral, cellular and innate immune response would be most effective.     

Circulating MELAN-A/MART-1 specific cytolytic T lymphocyte precursors in HLA-A2+ melanoma patients have a memory phenotype

Melan-A/MART-1 is a melanoma differentiation antigen that is recognized by a high proportion of cytolytic T lymphocyte (CTL) clones derived from human leukocyte antigen (HLA)-A2⁺ melanoma patients. Whereas peptide Melan-A/MART-1_27–35 was originally defined as the immunodominant CTL epitope, we have previously reported that peptide Melan-A/MART-1_26–35 was recognized more efficiently by the majority of tumor-reactive CTL clones. As demonstrated here, CTL populations generated from blood lymphocytes of either melanoma patients or healthy individuals after in vitro stimulation with peptide Melan-A/MART-1_26–35 killed specifically HLA-A2⁺ Melan-A⁺ allogeneic melanoma cells, thus suggesting their potential use in adoptive immunotherapy. We characterized the surface phenotype of the circulating CTL precursors (CTLp), which respond to in vitro stimulation with peptide Melan-A/MART-1_26–35. In melanoma patients, these CTLp predominantly expressed the CD45RO memory marker. In contrast, they were mainly, although not exclusively, found in the CD45RA subpopulation of CD8⁺ T cells in healthy individuals. The demonstration that Melan-A/MART-1-specific CTLp in peripheral blood lymphocytes from HLA-A2⁺ patients with metastatic melanoma express a memory phenotype provides direct evidence that in vivo priming of this antigen may occur during tumor progression. Int. J. Cancer 78:699–706, 1998. © 1998 Wiley-Liss, Inc.     

Identification of a new HLA-A(*)0201-restricted T-cell epitope from the tyrosinase-related protein 2 (TRP2) melanoma antigen

For the development of peptide-based immunotherapies, the identification of additional tumor antigens and T-cell epitopes is required. Because HLA-A(*)0201 is the most common allele in Caucasians, who represent the majority of patients with melanomas, 6 peptides carrying an HLA-A(*)0201 motif were synthesized from tyrosinase-related protein-2 (TRP2) melanoma antigen and tested for binding affinity to the HLA allele using processing-defective T2 cells. These peptides were then pulsed onto autologous dendritic cells and used to stimulate in vitro CD8(+)-enriched T cells isolated from peripheral blood of HLA-A(*)02(+) healthy donors or melanoma patients for the induction of specific cytotoxic T lymphocytes (CTLs). One peptide, TRP2(288-296) (SLDDYNHLV), the best HLA-A(*)0201 binder, elicited specific CTLs from 1 of 4 patients and 3 of 4 healthy donors. The induced CTLs from the patient and from 1 donor efficiently recognized HLA-A(*)02(+) TRP2(+) melanomas as well as COS-7 cells expressing HLA-A(*)0201 and TRP2 in an HLA class I-restricted manner, as assessed by cytokine production and direct cytolysis. The remaining 2 CTL lines derived from 2 donors displayed low T-cell receptor avidity, which could lyse melanoma cells in the presence of exogenous peptide. Since TRP2 is an antigen expressed in most melanomas, identification of the TRP2/HLA-A(*)0201 peptide SLDDYNHLV may facilitate the design of present peptide-based immunotherapies for the treatment of a large fraction of melanoma patients.     

Tax is a potential molecular target for immunotherapy of adult T‐cell leukemia/lymphoma

We expanded CTL specific for Tax (a human T‐lymphotropic virus type‐1‐encoded gene product) in vitro from PBMC of several adult T‐cell leukemia/lymphoma (ATL) patients, and document its potential significance as a target for ATL immunotherapy. Tax‐specific CTL responses against tumor cells were restricted by Tax‐expression and the appropriate human leukocyte antigen (HLA) type. Tax‐specific CTL recognized HLA/Tax‐peptide complexes on autologous ATL cells, even when their Tax expression was so low that it could only be detected by RT‐PCR but not by flow cytometry. Recognition resulted in interferon gamma (IFN‐γ) production and target cell lysis. This would be the first report that Tax‐specific CTL from ATL patients specifically recognized and killed autologous tumor cells that expressed Tax. The Tax‐specific CTL responded to as little as 0.01 pM of the corresponding peptide, indicating that their T‐cell receptor avidity was much higher than that of any other CTL recognizing viral or other tumor antigens. This is presumably the reason why the Tax‐specific CTL recognized and killed autologous ATL cells despite their very low Tax expression. In addition, cell cycle analyses and experiments with primary ATL cell‐bearing mice demonstrated that ATL cells present at the site of active cell proliferation, such as in the tumor masses, expressed substantial amounts of Tax, but it was minimally expressed by the tumor cells in a quiescent state, such as in the blood. The present study not only provides a strong rationale for exploiting Tax as a possible target for ATL immunotherapy but also contributes to our understanding of the immunopathogenesis of ATL.     

Simultaneous generation of CD8+ and CD4+ melanoma-reactive T cells by retroviral-mediated transfer of a single T-cell receptor

Adoptive immunotherapy of cancer requires the generation of large numbers of tumor antigen-reactive T cells for transfer into cancer patients. Genes encoding tumor antigen-specific T-cell receptors can be introduced into primary human T cells by retroviral mediated gene transfer as a potential method of providing any patient with a source of autologous tumor-reactive T cells. A T-cell receptor-specific for a class I MHC (HLA-A2)-restricted epitope of the melanoma antigen tyrosinase was isolated from a CD4(+) tumor-infiltrating lymphocyte (TIL 1383I) and introduced into normal human peripheral blood lymphocytes by retroviral transduction. T-cell receptor-transduced T cells secreted various cytokines when cocultured with tyrosinase peptide-loaded antigen-presenting cells as well as melanoma cells in an HLA-A2-restricted manner, and could also lyse target cells. Furthermore, T-cell clones isolated from these cultures showed both CD8(+) and CD4(+) transduced T cells could recognize HLA-A2(+) melanoma cells, giving us the possibility of engineering class I MHC-restricted effector and T helper cells against melanoma. The ability to confer class I MHC-restricted tumor cell recognition to CD4(+) T cells makes the TIL 1383I TCR an attractive candidate for T-cell receptor gene transfer-based immunotherapy.     

T cells raised against allogeneic HLA-A2/CD20 kill primary follicular lymphoma and acute lymphoblastic leukemia cells

T cells mediating a graft‐versus‐leukemia/lymphoma effects without causing graft‐versus‐host disease would greatly improve the safety and applicability of hematopoietic stem cell transplantation. We recently demonstrated that highly peptide‐ and HLA‐specific T cells can readily be generated against allogeneic HLA‐A*02:01 in complex with a peptide from the B cell‐restricted protein CD20. Here, we show that such CD20‐specific T cells can easily be induced from naïve precursors in cord blood, demonstrating that they do not represent cross‐reactive memory cells. The cells displayed high avidity and mediated potent cytotoxic effects on cells from patients with the CD20^pos B cell malignancies follicular lymphoma (FL) and acute lymphoblastic leukemia (ALL). However, the cytotoxicity was consistently lower for cells from two of the ALL patients. The ALL cells that were less efficiently killed did not display lower surface expression of CD20 or HLA‐A*02:01, or mutations in the CD20 sequence. Peptide pulsing fully restored the levels of cytotoxicity, indicating that they are indeed susceptible to T cell‐mediated killing. Adoptive transfer of CD20‐specific T cells to an HLA‐A*02:01^pos patient requires an HLA‐A*02:01^neg, but otherwise HLA identical, donor. A search clarified that donors meeting these criteria can be readily identified even for patients with rare haplotypes. The results bear further promise for the clinical utility of CD20‐specific T cells in B cell malignancies.     

Identification of HLA‐A2‐ and A24‐restricted T‐cell epitopes derived from SOX6 expressed in glioma stem cells for immunotherapy

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma‐bearing mice. In this study, we attempted to identify SOX6‐derived peptides as specific targets for effective and safe T‐cell‐mediated immunotherapy targeting SOX6‐positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)‐A*2402 (A24)‐restricted peptides, RFENLGPQL (SOX6₅₀₄) and PYYEEQARL (SOX6₆₂₈) or the HLA‐A*0201 (A2)‐restricted peptide, ALFGDQDTV (SOX6₄₄₇) was capable of inducing SOX6 peptide‐specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I‐restricted and an antigen‐dependent manner. Furthermore, peptide vaccines of SOX6₆₂₈, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H‐2^d, induced CTLs specific for SOX6₆₂₈ in H‐2^d mice. Normal autologous cells from mice, in which SOX6‐specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA‐A24 or ‐A2 positive glioma patients and should be considered as a promising strategy for safe and effective T‐cell‐based immunotherapy of patients with gliomas.