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DOI： 10.1126/sciadv.ado6491

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DOI： 10.1080/2162402x.2024.2404665

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CD8+ T cells can recognize tumor antigens displayed by HLA class I molecules and eliminate tumor cells. Despite their low tumor mutation burden, immune checkpoint blockade (ICB) is often beneficial in patients with renal cell carcinoma (RCC). Here, using a proteogenomic approach, we directly and comprehensively explored the HLA class I-presenting peptidome of RCC tissues and demonstrated that the immunopeptidomes contain a small subset of peptides derived from human endogenous retroviruses (hERV). A comparison between tumor and normal kidney tissues revealed tumor-associated hERV antigens, one of which was immunogenic and recognized by host tumor-infiltrating lymphocytes (TIL). Stimulation with the hERV antigen induced reactive CD8+ T cells in healthy donor-derived (HD-derived) peripheral blood mononuclear cells (PBMC). These results highlight the presence of antitumor CD8+ T cell surveillance against hERV3895 antigens, suggesting their clinical applications in patients with RCC.

DOI： 10.1172/jci.insight.167712

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Combined chemoradiotherapy (CRT) and programmed cell death-ligand 1 (PD-L1) blockade is a new care standard for unresectable stage III non-small-cell lung cancer (NSCLC). Although this consolidation therapy has improved the overall survival of patients with NSCLC, the synergistic action mechanisms of CRT and immunotherapy on T cells remain unclear. In addition, there is a paucity of reliable biomarkers to predict clinical responses to therapy. In this study, we analyzed T-cell receptor (TCR) sequences in the peripheral blood of five patients with NSCLC. T-cell receptor analysis was undertaken before treatment, after CRT, and after PD-L1 blockade. Notably, we observed the expansion and alteration of the dominant T-cell clonotypes in all cases with a complete response. In contrast, neither expansion nor alteration of the TCR repertoire was observed in cases with progressive disease. T cell expansion was initiated after CRT and was further enhanced after PD-L1 blockade. Our findings suggest the systemic effect of CRT on circulating T cells in addition to the curative effect on limited tumor sites. Dynamic changes in circulating T-cell clonotypes could have a prognostic significance for combined CRT and PD-L1 blockade.

DOI： 10.1111/cas.15566

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CD8+ T cells recognize peptides displayed by HLA class I molecules and monitor intracellular peptide pools. It is known that the proteasome splices two short peptide fragments. Recent studies using mass spectrometry (MS) and bioinformatics analysis have suggested that proteasome-generated spliced peptides (PSPs) may account for a substantial proportion of HLA class I ligands. However, the authenticity of the PSPs identified using bioinformatics approaches remain ambiguous. In this study, we employed MS-based de novo sequencing to directly capture cryptic HLA ligands that were not templated in the genome. We identified two PSPs originating from the same protein in a human colorectal cancer line with microsatellite instability. Healthy donor-derived CD8+ T cells readily responded to the two PSPs, showing their natural HLA presentation and antigenicity. Experiments using minigene constructs demonstrated proteasome-dependent processing of two PSPs generated by standard and reverse cis splicing, respectively. Our results suggest a broader diversity of HLA class I Ag repertoires generated by proteasomal splicing, supporting the advantage of MS-based approaches for the comprehensive identification of PSPs.

DOI： 10.4049/jimmunol.2100717

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

MHC class I molecules display intracellular peptides on cell surfaces to enable immune surveillance under pathological conditions. The source of MHC class I antigens responsible for cancer protection is not fully understood. Here, we explored the MHC class I peptidome in mouse colon cancer cells using a proteogenomic approach. We showed that cryptic peptides derived from unconventional short open reading frames accounted for part of the MHC class I peptidome. Moreover, cancer growth was significantly prevented in mice immunized with a cocktail of synthesized cryptic peptides. Together, our data showed that the source of cancer antigens was not limited to fragments of consensus proteins. Cryptic antigens were displayed by MHC molecules and mediated anti-cancer effects, suggesting their therapeutic potential for cancer prevention.

DOI： 10.3390/immuno1040030

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CD8+ T cells recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathologic conditions such as cancer. Advances in proteogenomic analysis of HLA ligandomes have demonstrated that cells present a subset of cryptic peptides derived from noncoding regions of the genome; however, the roles of cryptic HLA ligands in tumor immunity remain unknown. In the current study, we comprehensively and quantitatively investigated the HLA class I ligandome of a set of human colorectal cancer and matched normal tissues, showing that cryptic translation products accounted for approximately 5% of the HLA class I ligandome. We also found that a peptide encoded by the long noncoding RNA (lncRNA) PVT1 was predominantly enriched in multiple colorectal cancer tissues. The PVT1 gene is located downstream of the MYC gene in the genome and is aberrantly overexpressed across a variety of cancers, reflecting its oncogenic property. The PVT1 peptide was recognized by patient CD8+ tumor-infiltrating lymphocytes, as well as peripheral blood mononuclear cells, suggesting the presence of patient immune surveillance. Our findings show that peptides can be translated from lncRNAs and presented by HLA class I and that cancer patient T cells are capable of sensing aberrations in noncoding regions of the genome.

DOI： 10.1158/2326-6066.CIR-20-0964

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Although CD8+ T cells recognize neoantigens that arise from somatic mutations in cancer, only a small fraction of nonsynonymous mutations give rise to clinically relevant neoantigens. In this study, HLA class I ligandomes of a panel of human colorectal cancer (CRC) and matched normal tissues were analyzed using mass spectrometry-based proteogenomic analysis. Neoantigen presentation was rare; however, the analysis detected a single neoantigen in a mismatch repair-deficient CRC (dMMR-CRC) tissue sample carrying 3967 nonsynonymous mutations, where abundant tumor-infiltrating lymphocytes (TILs) and inflamed gene expression status were observed in the tumor microenvironment (TME). Using the HLA class I ligandome data and gene expression profiles, a set of nonmutated tumor-associated antigen (TAA) candidates was concomitantly identified. Interestingly, CD8+ TILs predominantly recognized the detected neoantigen over the array of TAA candidates. Neoantigen-reactive CD8+ TILs showed PD-1 positivity and exhibited functional and specific responses. Moreover, T cell receptor (TCR) profiling identified the sequence of the neoantigen-reactive TCR clonotype and showed its expansion in the TME. Transduction of the sequenced TCR conferred neoantigen specificity and cytotoxicity to peripheral blood lymphocytes. The proteogenomic approach revealed the antigenic and reactive T cell landscape in dMMR-CRC, demonstrating the presence of an immunogenic neoantigen and its potential therapeutic applications.

DOI： 10.1172/jci.insight.146356

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CD8+ T cells are capable of recognizing mutation-derived neoantigens displayed by HLA class I molecules, thereby exhibiting the ability to distinguish between cancer and normal cells. However, accumulating evidence has shown that only a small fraction of nonsynonymous somatic mutations give rise to clinically relevant neoantigens. The properties of such neoantigens, which must be presented by HLA and immunogenic to induce a T-cell response, remain elusive. In this study, we explored the HLA class I ligandome of a human cancer cell line with microsatellite instability using a proteogenomic approach. The results demonstrated that neoantigens accounted for only 0.34% of the HLA class I ligandome, and most neoantigens were encoded by genes with abundant expression. Thereafter, T-cell responses were prioritized, and immunodominant neoantigens were defined using naive CD8+ T cells derived from healthy donors. AKF9, an immunogenic neoantigen with a mutation at a non-anchor position, formed a stable peptide-HLA complex. T-cell responses were analyzed against a panel of AKF9 variants with single amino-acid substitutions, in which mutations did not alter the high HLA-binding affinity and stability. The responses varied across individuals, demonstrating the impact of heterogeneous T-cell repertoires in this human cancer model. Moreover, responses were biased toward a variant group with large structural changes compared to the wild-type peptide. Thus, naive T-cell induction can be attributed to multiple determinants. Combining structural dissimilarity with gene-expression levels, HLA-binding affinity, and stability may further help prioritize the immunogenicity of non-anchor-type neoantigens.

DOI： 10.1080/2162402X.2020.1870062

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Host T cells infiltrate the cancer lesion and contribute to patient survival. T cells recognize antigen peptides displayed by the cancer cell human leukocyte antigen (HLA) system. Cancer antigens constitute an essential element of T-cell discrimination and play an indispensable role in anti-cancer responses. HLA ligandome analysis directly and comprehensively detects the peptides that are naturally presented by HLA of given cells, leading to discovery of cancer antigens. A proteogenomic approach, which combines conventional proteomics with genomic information, has further deciphered the landscape of the cancer HLA ligandome. Neoantigens that arise from somatic mutations are arguably the major type of peptides patient T cells recognize. Moreover, cancer cells present peptides derived from alleged noncoding regions, which also elicit T-cell responses thereby serving as cancer antigens. The diversity of newly discovered antigen sources implies that T cells are capable of sensing a variety of genomic aberrations in cancer.

DOI： 10.1111/pin.12841

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T cells recognize antigen peptides displayed by HLA molecules and specifically eliminate their target cells. Identification of responsible antigens as well as understanding the mechanism by which antigens are produced inside cells are equally crucial for cancer immunology. In this review, we introduce proteogenomics and its applications in cancer antigen research, which leverages mass spectrometry and next-generation sequencing. The approach comprehensively captures immunopeptidome displayed by HLA, revealing new classes of antigens, such as mutation-derived neoantigens, spliced peptides, and non-coding region derived peptides. These antigens may serve as therapeutic targets or biomarkers. Thus, proteogenomics is a promising approach for cancer antigen research and contributes to immunotherapy development.

DOI： 10.1080/25785826.2019.1640500

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Cytotoxic CD8+ T lymphocytes (CTLs) recognize peptides displayed by HLA class I molecules on cell surfaces, monitoring pathological conditions such as cancer. Difficulty in predicting HLA class I ligands is attributed to the complexity of the Ag processing pathway across the cytosol and the endoplasmic reticulum. By means of HLA ligandome analysis using mass spectrometry, we collected natural HLA class I ligands on a large scale and analyzed the source-protein sequences flanking the ligands. This comprehensive analysis revealed that the frequency of proline at amino acid positions 1-3 upstream of the ligands was selectively decreased. The depleted proline signature was the strongest among all the upstream and downstream profiles. Experiments using live cells demonstrated that the presence of proline at upstream positions 1-3 attenuated CTL responses against a model epitope. Other experiments, in which N-terminal-flanking Ag precursors were confined in the endoplasmic reticulum, demonstrated an inability to remove upstream prolines regardless of their positions, suggesting a need for synergistic action across cellular compartments for making the proline signature. Our results highlight, to our knowledge, a unique role and position of proline for inhibiting downstream epitope presentation, which provides a rule for defining natural peptide-HLA class I repertoire formation and CTL responses.

DOI： 10.4049/jimmunol.1900029

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Immune surveillance of infected or tumor cells by CD8+ T cells requires that MHC class I molecules present a diverse repertoire of peptides on the cell surface. Even a few copies of individual peptides among this mixture are sufficient for recognition by the antigen receptors of appropriate CD8 T cells. Here we describe methods for biochemical analysis of the naturally processed peptides and their precursors in living cells. The peptides are fractionated using reverse phase high performance liquid chromatography and detected by the activation of CD8+ T cell hybridomas. The results provide information on the structure and amount of the peptides and yield insights into the mechanisms that generate the naturally processed peptides.

DOI： 10.1007/978-1-4939-9450-2_8

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Colorectal cancer consists of a small number of cancer stem cells (CSC) and many non-CSCs. Although rare in number, CSCs are a target for cancer therapy, because they survive conventional chemo- and radiotherapies and perpetuate tumor formation in vivo In this study, we conducted an HLA ligandome analysis to survey HLA-A24 peptides displayed by CSCs and non-CSCs of colorectal cancer. The analysis identified an antigen, ASB4, which was processed and presented by a CSC subset but not by non-CSCs. The ASB4 gene was expressed in CSCs of colorectal cancer, but not in cells that had differentiated into non-CSCs. Because ASB4 was not expressed by normal tissues, its peptide epitope elicited CD8+ cytotoxic T-cell (CTL) responses, which lysed CSCs of colorectal cancer and left non-CSCs intact. Therefore, ASB4 is a tumor-associated antigen that can elicit CTL responses specific to CSCs and can discriminate between two cellular subsets of colorectal cancer. Adoptively transferred CTLs specific for the CSC antigen ASB4 could infiltrate implanted colorectal cancer cell tumors and effectively prevented tumor growth in a mouse model. As the cancer cells implanted in these mice contained very few CSCs, the elimination of a CSC subset could be the condition necessary and sufficient to control tumor formation in vivo These results suggest that CTL-based immunotherapies against colorectal CSCs might be useful for preventing relapses. Cancer Immunol Res; 6(3); 358-69. ©2018 AACR.

DOI： 10.1158/2326-6066.CIR-17-0518

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Current cancer therapies target the bulk of the tumour, while a population of highly resistant tumour cells may be able to repopulate the tumour and metastasize to new sites. Cancer cells with such stem cell-like characteristics can be identified based on their phenotypical and/or functional features which may open up ways for their targeted elimination. In this review we discuss potential off-target effects of inhibiting cancer stem-cell self-renewal pathways on immune cells, and summarize some recent immunological studies specifically targeting cancer stem cells based on their unique antigen expression.

DOI： 10.1111/imm.12866

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DOI： 10.1080/2162402X.2016.1274476

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1080/2162402X.2017.1293214

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DOI： 10.1002/eji.201545835

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Classical MHC class I molecules open a window into the cell by presenting intracellular peptides (pMHC I) on the surface. The peptides are used for immune surveillance by circulating CD8+ T and NK cells to detect and eliminate infected or tumor cells. Not surprisingly, viruses and tumor cells have evolved immune evasion mechanisms to keep the window shades down and the cytotoxic cells oblivious to their presence. Here, we review counter mechanisms that nevertheless allow the immune system to detect and eliminate cells unable to properly process antigenic peptides in the endoplasmic reticulum.

DOI： 10.1016/j.coi.2013.11.006

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Effective CD8(+) T cell responses depend on presentation of a stable peptide repertoire by MHC class I (MHC I) molecules on the cell surface. The overall quality of peptide-MHC I complexes (pMHC I) is determined by poorly understood mechanisms that generate and load peptides with appropriate consensus motifs onto MHC I. In this article, we show that both tapasin (Tpn), a key component of the peptide loading complex, and the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP) are quintessential editors of distinct structural features of the peptide repertoire. We carried out reciprocal immunization of wild-type mice with cells from Tpn- or ERAAP-deficient mice. Specificity analysis of T cell responses showed that absence of Tpn or ERAAP independently altered the peptide repertoire by causing loss as well as gain of new pMHC I. Changes in amino acid sequences of MHC-bound peptides revealed that ERAAP and Tpn, respectively, defined the characteristic amino and carboxy termini of canonical MHC I peptides. Thus, the optimal pMHC I repertoire is produced by two distinct peptide editing steps in the endoplasmic reticulum.

DOI： 10.4049/jimmunol.1301043

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Immune surveillance of infected or tumor cells by CD8(+) T cells requires that MHC class I molecules present a diverse repertoire of peptides on the cell surface. Even a few copies of individual peptides among this mixture are sufficient for recognition by the antigen receptors of appropriate CD8(+) T cells. Here we describe a method for biochemical analysis of the naturally processed peptides in living cells. The peptide mixture in cell extracts is fractionated using reverse phase high performance liquid chromatography and detected by the activation of CD8(+) T cell hybridomas. The results provide information on the structure and amount of the peptides and yield insights into the mechanisms that generate the naturally processed peptides.

DOI： 10.1007/978-1-62703-218-6_15

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The MHC class I (MHC-I) molecules ferry a cargo of peptides to the cell surface as potential ligands for CD8(+) cytotoxic T cells. For nearly 20 years, the cargo has been described as a collection of short 8-9 mer peptides, whose length and sequences were believed to be primarily determined by the peptide-binding groove of MHC-I molecules. Yet the mechanisms for producing peptides of such optimal length and composition have remained unclear. In this study, using mass spectrometry, we determined the amino acid sequences of a large number of naturally processed peptides in mice lacking the endoplasmic reticulum aminopeptidase associated with Ag processing (ERAAP). We find that ERAAP-deficiency changed the oeuvre and caused a marked increase in the length of peptides normally presented by MHC-I. Furthermore, we observed similar changes in the length of viral peptides recognized by CD8(+) T cells in mouse CMV-infected ERAAP-deficient mice. In these mice, a distinct CD8(+) T cell population was elicited with specificity for an N-terminally extended epitope. Thus, the characteristic length, as well as the composition of MHC-I peptide cargo, is determined not only by the MHC-I peptide-binding groove but also by ERAAP proteolysis in the endoplasmic reticulum.

DOI： 10.4049/jimmunol.0903712

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Effective immune surveillance by CD8 T cells depends on the presentation of diverse peptides by MHC class I (pMHC I) molecules on the cell surface. The pMHC I repertoire is shaped in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with Ag processing (ERAAP). The ERAAP activity is required for producing peptides of appropriate length for generating optimal pMHC I. Paradoxically, ERAAP also inhibits generation of certain peptides such as the SVL9 (SSVVGVWYL) peptide encoded by the H13(a) histocompatibility gene and presented by D(b) MHC by an unknown mechanism. In this study, we show that the presentation of the SVL9-D(b) complex is inhibited when other peptides compete for binding D(b). Conversely, improving the binding of SVL9 peptide to D(b) suppresses the inhibition. Interestingly, the inhibitory effect of competitor peptides is observed only when ERAAP is expressed in the same cells. Thus, ERAAP, in concert with MHC I molecules, regulates the quality of processed peptides presented on the cell surface.

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Major histocompatibility complex (MHC) class I molecules present short, perfectly cleaved peptides on the cell surface for immune surveillance by CD8(+) T cells. The pathway for generating these peptides begins in the cytoplasm, and the peptide-MHC I (pMHC I) repertoire is finalized in the endoplasmic reticulum. Recent studies show that the peptides for MHC I are customized by the ER aminopeptidase associated with antigen processing and by dynamic interactions within the MHC peptide-loading complex. Failure to customize the pMHC I repertoire has profound immunological consequences.

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The major histocompatibility complex class I molecules display peptides (pMHC I) on the cell surface for immune surveillance by CD8(+) T cells. These peptides are generated by proteolysis of intracellular polypeptides by the proteasome in the cytoplasm and then in the endoplasmic reticulum (ER) by the ER aminopeptidase associated with antigen processing (ERAAP). To define the unknown mechanism of ERAAP function in vivo, we analyzed naturally processed peptides in cells with or without appropriate MHC I and ERAAP. In the absence of MHC I, ERAAP degraded the antigenic precursors in the ER. However, MHC I molecules could bind proteolytic intermediates and were essential for generation of the final peptide by ERAAP. Thus, ERAAP synergizes with MHC I to generate the final pMHC I repertoire.

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In this study, we first categorized nine squamous cell carcinoma (SCC) cell lines into two groups in terms of the expression of HLA-DR, -DP, and -DQ molecules. Subsequently, the expression of class II transactivator (CIITA) was studied in these cell lines, because it is widely accepted that the expression of MHC class II molecules is regulated by different types of CIITA transcripts that are initiated by distinct promoters. The majority of the SCC cell lines (six of nine) expressed HLA-DR molecules and CIITA promoter IV (pIV) transcripts in the presence of IFN-gamma. In contrast, three of the nine SCC cell lines were completely negative for class II molecules and all types of CIITA, suggesting epigenetic changes in the promoter region in these cells. Previously, methylation of CIITA pIV was reported to silence CIITA gene expression. We extensively studied the methylation status of CIITA pIV using a panel of 22 SCC cell lines. Remarkably, none of the SCC cell lines demonstrated hypermethylation at the site. In contrast, treatment with a histone deacetylation inhibitor in combination with IFN-gamma clearly restored the expression of the CIITA type IV gene in the HLA-DR-negative SCC cell lines, and the acetylation status of histone H3 examined by chromatin immunoprecipitation analysis was closely associated with the gene expression. Moreover, stable transfection of the CIITA gene into an HLA-DR-negative cell line restored constitutive expression of MHC class II molecules. Therefore, histone deacetylation, but not hypermethylation, modifies CIITA DNA and class II gene expression in SCC.

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T cells recognize peptides presented by human leukocyte antigen molecules on the cell surface, enabling the immune surveillance of pathological conditions such as cancer. Cancer-testis (CT) antigens are promising targets for cancer immunotherapy because of their restricted expression in normal tissues. In this study, we performed antigen screening of T cell receptors isolated from tumor-infiltrating lymphocytes (TILs) in acral melanoma, using cDNA expression cloning and identified a novel CT antigenic epitope encoded by MAGE-A6 with a single nucleotide polymorphism (SNP). This SNP conferred immunogenicity to the epitope, eliciting a robust immune response against tumor cells. While antigen identification has increasingly relied on reverse immunology approaches using reference sequences that do not contain SNPs, forward immunology approaches, such as cDNA expression cloning, directly identify antigens recognized by T cells exhibiting immune responses, enabling the detection of SNP-derived epitopes. Furthermore, in hot tumors such as acral melanoma that are characterized by a low tumor mutational burden, but high TIL infiltration, TILs predominantly respond to shared antigens with high immunogenicity. These findings underscore the utility of forward immunology in antigen discovery and highlight the potential of SNP-dependent tumor antigens in cancer immunotherapy.

DOI： 10.1080/2162402X.2025.2528110

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Despite advancements in the treatment of oral cancer, cancer survival rates remain low, highlighting the need for new therapeutic strategies targeting cancer stem-like cells. Cancer stem-like cells are a small population of cancer cells within tumors that drive recurrence and metastasis. They are often resistant to conventional treatments. Immunotherapy has shown promise against cancer stem-like cells, particularly with the use of cytotoxic T lymphocytes targeting specific markers. Survivin, an apoptosis protein inhibitor, is overexpressed in several malignancies, including oral cancer, and is associated with tumor recurrence and reduced survival. Survivin-2B-specific cytotoxic T lymphocytes were produced and evaluated for their ability to target CD44+ (cancer stem-like cells) and CD44- cells (non-cancer stem-like cells), respectively, from oral cancer cell lines (HSC-2 and HSC-3, respectively). Quantitative polymerase chain reaction (qPCR) analysis confirmed similar survivin-2B expression in both cell types. Cytotoxic T lymphocyte assays revealed the effective lysis of both cancer stem-like cells and CD44- cell populations, supporting the potential of survivin-2B-specific cytotoxic T lymphocytes to overcome cancer stem-like cell-associated resistance. These findings suggest that survivin-2B peptide vaccines are effective in preventing cancer relapse by targeting cancer stem-like cells, with future directions aimed at developing multipeptide "cocktail" vaccines to reduce the risk of immune evasion.

DOI： 10.1111/eos.70019

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In many malignancies, an increased number of tumor-infiltrating lymphocytes (TILs) is recognized as a favorable prognostic factor, with exceptions such as renal cell carcinoma. However, the clinical significance of TIL size remains unclear. T-cell activation by mitogens increases cell size, partly via c-myc expression, suggesting that larger T cells may be more activated. We hypothesized that TIL size might be prognostically relevant in cancer patients. Here, we examined the relationship between the size and number of tumor-infiltrating CD8 + T cells and patient prognosis in 96 cases of esophageal squamous cell carcinoma (ESCC). We employed artificial intelligence (AI) analysis to quantify the mean size of intratumoral CD8+ T cells in each sample. Patients were then divided into "Large" and "Small" CD8+ T cell groups according to the median T-cell size. Similarly, we classified cases into "High" and "Low" groups based on CD8 + T-cell numbers. We found that patients in the Large CD8+ T cell group had significantly better overall survival than those in the Small CD8+ T cell group by a univariate analysis (p = 0.039), but the difference did not reach statistical significance in a multivariate analysis (p = 0.054). Patients in the High CD8 + T cell group had better outcomes than those in the Low CD8+ T cell group. There was no significant correlation between CD8+ T cell size and count, and their combination (Large/High) identified a subgroup of patients with the most favorable prognosis. Our findings suggest that CD8+ T cell size could serve as an independent prognostic marker in ESCC.

DOI： 10.1038/s41598-025-10885-3

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The development of effective immunotherapies for solid tumors remains a significant challenge. In previous studies, we identified PVT1, a long non-coding RNA, with the peptide HF10 derived from PVT1, presented by HLA-A24. This study aims to develop a single-chain variable fragment (scFv) that specifically recognizes the HLA-A24/HF10 complex (HF10 scFv) and to evaluate its specificity, reactivity, and therapeutic potential as part of a T cell engaging bispecific antibody (HF10xCD3) in vitro and in vivo. Using a scFv phage display library, we screened for scFv clones targeting the HLA-A24/HF10 peptide complex. The selected HF10 scFv was engineered into an IgG1 format (HF10-hIgG1), which demonstrated high affinity (KD = 2.18 × 10⁻⁸ M) and specific detection of the HLA-A24/HF10 complex on HLA-A24( +)/PVT1( +) tumor cell lines. Furthermore, HF10 scFv was incorporated into a T cell engaging bispecific antibody (HF10xCD3), which induced cytotoxicity in these tumor cell lines. In a mouse xenograft model, HF10xCD3 administration exhibited significant anti-tumor activity. In conclusion, HF10xCD3 represents a promising candidate for immunotherapy targeting solid tumors.

DOI： 10.1007/s00262-025-03976-7

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Inducing antigen peptide-specific cytotoxic T cells is challenging, partly due to the difficulty of maintaining the quality of antigen-presenting cells, such as dendritic cells. Consequently, artificial antigen-presenting cells (aAPCs) derived from the erythroleukemia cell line K562 have been employed for T cell stimulation. K562-based aAPCs can be utilized for both non-specific and antigen-specific T cell stimulation. Antigen peptide-pulsed aAPCs are commonly used to stimulate T cells with known specific antigenic peptides, which require identifying antigenic peptides from cognate antigen proteins. Therefore, antigen gene-overexpressing aAPCs might be useful for detecting unknown antigenic peptides. In this study, we evaluated the efficacy of cytotoxic T lymphocyte (CTL) induction using antigen gene-overexpressing aAPCs. To enhance antigen presentation efficiency, we assessed the signal peptide (SP) fused with MHC class I trafficking signal (MITD) sequences (SP-MITD). SP-MITD, fused with epitopes from the neoantigen AKF9 and the viral antigen CMVpp65, was transduced into aAPCs. We compared the CTL induction ability of peptide-pulsed aAPCs, only mini-gene-overexpressing aAPCs [SP-MITD (-)], and mini-gene fused with SP-MITD overexpressing aAPCs [SP-MITD (+)]. The SP-MITD (+) gene-overexpressing aAPCs exhibited the highest CTL induction efficiency compared to both peptide-pulsed and SP-MITD (-) gene-overexpressing aAPCs. These findings suggest that antigen gene-fused with SP-MITD transduced aAPCs are highly effective for inducing CTLs specific to both known and unknown antigenic peptides.

DOI： 10.1016/j.bbrep.2025.101946

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Adoptive T cell therapy, using T cell receptor-engineered T (TCR-T) cells and chimeric antigen receptor T (CAR-T) cells, is a potent immunotherapy option. Bladder cancer is a prevalent urological malignancy, particularly in cases of muscle invasion and metastasis, for which systemic therapy is crucial. Immunotherapy utilizing immune checkpoint blockade has been approved for bladder cancer treatment. The antitumor effect of an immune checkpoint blockade based on cytotoxic T cells (CTLs) and the patient's immune status is essential. The chemotherapeutic drug cisplatin (CDDP) is a key drug in bladder cancer treatment. However, it has been shown to suppress T cells, making combination therapy with CDDP and immunotherapy difficult. To address this, we developed TCR-T cells specific for bladder cancer cells. In previous studies, we found that the tumor-associated antigen CLSPN is overexpressed in CDDP-resistant bladder cancer cells and that the antigenic peptide HLA-A*02:01/CLSPN1254-1262, encoded by CLSPN, could be targeted by a CTL clone. The TCR was cloned from the HLA-A*02:01/CLSPN1254-1262 specific CTL clone yc3. We also designed a codon-optimized TCR sequence using GeneArt® GeneOptimizer® (Opt TCR) and compared the TCR-T cells using the original TCR sequence (Ori TCR-T cells) and the codon-optimized TCR sequence (Opt TCR-T cells). Opt TCR-T cells exhibited higher TCR transduction efficiency, higher TCR expression levels, higher avidity, and greater cytotoxicity than did Ori TCR-T cells. These results suggest that HLA-A*02:01/CLSPN1254-1262 specific Opt TCR-T cells are promising candidates for CDDP combination therapy.

DOI： 10.1080/21645515.2024.2414542

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The application of deep learning algorithms to predict the molecular profiles of various cancers from digital images of hematoxylin and eosin (H&E)-stained slides has been reported in recent years, mainly for gastric and colon cancers. In this study, we investigated the potential use of H&E-stained endometrial cancer slide images to predict the associated mismatch repair (MMR) status. H&E-stained slide images were collected from 127 cases of the primary lesion of endometrial cancer. After digitization using a Nanozoomer virtual slide scanner (Hamamatsu Photonics), we segmented the scanned images into 5397 tiles of 512 × 512 pixels. The MMR proteins (PMS2, MSH6) were immunohistochemically stained, classified into MMR proficient/deficient, and annotated for each case and tile. We trained several neural networks, including convolutional and attention-based networks, using tiles annotated with the MMR status. Among the tested networks, ResNet50 exhibited the highest area under the receiver operating characteristic curve (AUROC) of 0.91 for predicting the MMR status. The constructed prediction algorithm may be applicable to other molecular profiles and useful for pre-screening before implementing other, more costly genetic profiling tests.

DOI： 10.3390/cancers16101810

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Immunotherapy using immune checkpoint inhibitors (ICIs) has shown superior efficacy compared with conventional chemotherapy in certain cancer types, establishing immunotherapy as the fourth standard treatment alongside surgical intervention, chemotherapy, and radiotherapy. In cancer immunotherapy employing ICIs, CD8-positive cytotoxic T lymphocytes are recognized as the primary effector cells. For effective clinical outcomes, it is essential that the targeted cancer cells express HLA class I molecules to present antigenic peptides derived from the tumor. However, cancer cells utilize various mechanisms to downregulate or lose HLA class I molecules from their surface, resulting in evasion from immune surveillance. Correlations between prognosis and the integrity of HLA class I molecules expressed by cancer cells have been consistently found across different types of cancer. This paper provides an overview of the regulatory mechanisms of HLA class I molecules and their role in cancer immunotherapy, with a particular emphasis on the significance of utilizing pathological tissues to evaluate HLA class I molecules expressed in cancer cells.

DOI： 10.1111/tan.15472

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BACKGROUND/AIM: Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. MATERIALS AND METHODS: Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. RESULTS: Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. CONCLUSION: High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

DOI： 10.21873/anticanres.16989

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PURPOSE: Although immune checkpoint inhibitors (ICIs), together with cytotoxic chemotherapy (chemoimmunotherapy), have been adapted for the initial treatment of extensive-disease small-cell lung cancer (ED-SCLC), they have achieved limited success. In ED-SCLC, a subtype of SCLC, the expression of immune-related molecules and clinical data are not well understood in relation to ICI treatment efficiency. METHODS: We examined lung biopsy specimens from patients diagnosed with ED-SCLC treated with chemoimmunotherapy or chemotherapy. SCLC subtype, expression of HLA class I, and infiltration of CD8-positive cells were examined using immunohistochemistry (IHC). Subsequently, the association between clinical factors, IHC results, and progression-free survival or overall survival was assessed. RESULTS: Most of the cases showed the achaete-scute homolog 1 (ASCL1) subtype. Among the 75 SCLC cases, 29 expressed high levels of HLA class I, while 46 showed low levels or a negative result; 33 patients were characterized as CD8-high, whereas 42 were CD8-low. In the chemoimmunotherapy cohort, multivariate analysis revealed a correlation between CD8-high and improved survival. Specifically, patients in the CD8-high group of the chemoimmunotherapy cohort experienced enhanced survival compared to those in the chemotherapy cohort, which was attributed to ICI addition. IHC subtype analysis demonstrated a survival advantage in the SCLC-I and SCLC-A groups when ICI was combined with chemotherapy compared to chemotherapy alone. CONCLUSION: Our study highlights the predictive value of IHC-classified subtypes and CD8-positive cell infiltration in estimating outcomes for patients with ED-SCLC treated with chemoimmunotherapy as a first-line therapy. These findings have practical implications for daily clinical assessments and treatment decisions.

DOI： 10.1007/s00432-024-05652-2

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The recognition by cytotoxic T cells (CTLs) is essential for the clearance of SARS-CoV-2 virus-infected cells. Several viral proteins have been described to be recognized by CTLs. Among them, the spike (S) protein is one of the immunogenic proteins. The S protein acts as a ligand for its receptors, and several mutants with different affinities for its cognate receptors have been reported, and certain mutations in the S protein, such as L452R and Y453F, have been found to inhibit the HLA-A24-restricted CTL response. In this study, we conducted a screening of candidate peptides derived from the S protein, specifically targeting those carrying the HLA-A24 binding motif. Among these peptides, we discovered that NF9 (NYNYLYRLF) represents an immunogenic epitope. CTL clones specific to the NF9 peptide were successfully established. These CTL clones exhibited the ability to recognize endogenously expressed NF9 peptide. Interestingly, the CTL clone demonstrated cross-reactivity with the Y453F peptide (NYNYLFRLF) but not with the L452R peptide (NYNYRYRLF). The CTL clone was able to identify the endogenously expressed Y453F mutant peptide. These findings imply that the NF9-specific CTL clone possesses the capability to recognize and respond to the Y453F mutant peptide.

DOI： 10.1080/25785826.2024.2304363

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We previously identified papillomavirus binding factor (PBF) as an osteosarcoma antigen recognized by an autologous cytotoxic T lymphocyte clone. Vaccination with PBF-derived peptide presented by HLA-A24 (PBF peptide) elicited strong immune responses. In the present study, we generated T cell receptor-engineered T cells (TCR-T cells) directed against the PBF peptide (PBF TCR-T cells). PBF TCR was successfully transduced into T cells and detected using HLA-A*24:02/PBF peptide tetramer. PBF TCR-T cells generated from a healthy donor were highly expanded and recognized T2-A24 cells pulsed with PBF peptide, HLA-A24+ 293T cells transfected with PBF cDNA, and sarcoma cell lines. To establish an adoptive cell therapy model, we modified the PBF TCR by replacing both α and β constant regions with those of mice (hybrid PBF TCR). Hybrid PBF TCR-T cells also showed reactivity against T2-A24 cells pulsed with PBF peptide and to HLA-A24+ 293T cells transfected with various lengths of PBF cDNA including the PBF peptide sequence. Subsequently, we generated target cell lines highly expressing PBF (MFH03-PBF [short] epitope [+]) containing PBF peptide with in vivo tumorigenicity. Hybrid PBF TCR-T cells exhibited antitumor effects compared with mock T cells in NSG mice xenografted with MFH03-PBF (short) epitope (+) cells. CD45+ T cells significantly infiltrated xenografted tumors only in the hybrid PBF TCR T cell group and most of these cells were CD8-positive. CD8+ T cells also showed Ki-67 expression and surrounded the CD8-negative tumor cells expressing Ki-67. These findings suggest that PBF TCR-T cell therapy might be a candidate immunotherapy for sarcoma highly expressing PBF.

DOI： 10.1111/cas.15967

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BACKGROUND/AIM: Clear cell carcinoma is a prevalent histological type of ovarian cancer in East Asia, particularly in Japan, known for its resistance to chemotherapeutic agents and poor prognosis. ARID1A gene mutations, commonly found in ovarian clear cell carcinoma (OCCC), contribute to its pathogenesis. Recent data revealed that the ARID1A mutation is related to better outcomes of cancer immunotherapy. Thus, this study aimed to investigate the immunotherapy treatment susceptibility of OCCC bearing ARID1A mutations. MATERIALS AND METHODS: Expression of ARID1A was analyzed using western blotting in ovarian cancer cell lines. OCCC cell lines JHOC-9 and RMG-V were engineered to overexpress NY-ESO-1, HLA-A*02:01, and ARID1A. Sensitivity to chemotherapy and T cell receptor-transduced T (TCR-T) cells specific for NY-ESO-1 was assessed in ARID1A-restored cells compared to ARID1A-deficient wild-type cells. RESULTS: JHOC-9 cells and RMG-V cells showed no expression of ARID1A protein. Overexpression of ARID1A in JHOC-9 and RMG-V cells did not impact sensitivity to gemcitabine. While ARID1A overexpression decreased sensitivity to cisplatin in RMG-V cells, it had no such effect in JHOC-9 cells. ARID1A overexpression reduced the reactivity of NY-ESO-1-specific TCR-T cells, as observed by the IFNγ ESLIPOT assay. CONCLUSION: Cancer immunotherapy is an effective approach to target ARID1A-deficient clear cell carcinoma of the ovary.

DOI： 10.21873/cgp.20460

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Publishing type：Research paper (scientific journal)   Publisher：Japanese Society for Lymphoreticular Tissue Research  

DOI： 10.3960/jslrt.23044

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Eribulin inhibits microtubule polymerization and improves the overall survival of patients with recurrent metastatic breast cancer. A subgroup analysis revealed a low neutrophil to lymphocyte ratio (NLR) (<3) to be a prognostic factor of eribulin treatment. We thus hypothesized that eribulin might be related to the immune response for breast cancer cells and we analyzed the effects of eribulin on the immune system. Immunohistochemical staining revealed that human leukocyte antigen (HLA) class I expression was increased in clinical samples after eribulin treatment. In vitro assays revealed that eribulin treatment increased HLA class I expression in breast cancer line cells. RNA-sequencing demonstrated that eribulin treatment increased the expression of the NOD-like family CARD domain-containing 5 (NLRC5), a master regulator of HLA class I expression. Eribulin treatment increased the NY-ESO-1-specific T-cell receptor (TCR) transduced T (TCR-T) cell response for New York oesophageal squamous cell carcinoma 1 (NY-ESO-1) overexpressed breast cancer cells. The eribulin and TCR-T combined therapy model revealed that eribulin and immunotherapy using TCR-T cells has a synergistic effect. In summary, eribulin increases the expression of HLA class 1 via HLA class 1 transactivatior NLRC5 and eribulin combination with immunotherapy can be effective for the treatment of breast cancer.

DOI： 10.1111/cas.15986

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The mortality rate of oral cancer has not improved over the past three decades despite remarkable advances in cancer therapies. Oral cancers contain a subpopulation of cancer stem cells (CSCs) that share characteristics associated with normal stem cells, including self-renewal and multi-differentiation potential. CSCs are tumorigenic, play a critical role in cancer infiltration, recurrence, and distant metastasis, and significantly contribute to drug resistance to current therapeutic strategies, including immunotherapy. Cytotoxic CD8+ T lymphocytes (CTLs) are key immune cells that effectively recognize peptide antigens presented by the major histocompatibility complex class I molecules. Increasing evidence suggests that cancer antigen-specific targeting by CTLs effectively regulates CSCs that drive cancer progression. In this study, we utilized data from public domains and performed various bioassays on human oral squamous cell carcinoma clinical samples and cell lines, including HSC-2 and HSC-3, to investigate the potential role of olfactory receptor family 7 subfamily C member 1 (OR7C1), a seven transmembrane G-protein-coupled olfactory receptor that is also expressed in nonolfactory tissues and was previously reported as a novel marker and target of colon cancer initiating cell-targeted immunotherapy, in CSC-targeted treatment against oral cancer. We found that the OR7C1 gene was expressed only in oral CSCs, and that CTLs reacted with human leukocyte antigen-A24-restricted OR7C1 oral CSC-specific peptides. Taken together, our findings suggest that OR7C1 represents a novel target for potent CSC-targeted immunotherapy in oral cancer.

DOI： 10.1111/cas.15873

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Immune checkpoint inhibitors (ICIs) for various types of malignancy, including non-small-cell lung cancer, have improved prognosis in some cases. Granuloma formation after ICI administration suggests a tumor antigen-specific cytotoxic T cell response with abundant interferon-gamma production, which can be used to estimate the curative effect of ICIs. In this report, we present a case with a resected lung lesion, clinically suspected to be lung cancer, that consisted of a granulomatous lesion. A tumor was also found in the duodenum that was presumed to be derived from the pulmonary pleomorphic carcinoma. Duodenal tumor cells highly expressed PD-L1, suggesting PD-1/PD-L1 axis-mediated immune escape. As expected, pembrolizumab induced a complete response for the duodenal lesion. Interestingly, in histopathological analysis, the duodenal lesion was also replaced by an epithelial granuloma and multinucleated giant cells. We conclude that autoimmunity regressed the untreated primary lung lesion spontaneously, while the metastatic duodenal lesion responded to PD-1 blockade. Tumor-associated epithelioid granulomas, even before ICI administration, may be an important pathological finding indicating an immune response with interferon-gamma production by cytotoxic T cells to the tumor.

DOI： 10.1080/25785826.2023.2193283

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Evasion from immunity is a major obstacle to the achievement of successful cancer immunotherapy. Hybrids derived from cell-cell fusion are theoretically associated with tumor heterogeneity and progression by conferring novel properties on tumor cells, including drug resistance and metastatic capacity; however, their impact on immune evasion remains unknown. Here, we investigated the potency of tumor-macrophage hybrids in immune evasion. Hybrids were established by co-culture of a melanoma cell line (A375 cells) and type 2 macrophages. The hybrids showed greater migration ability and greater tumorigenicity than the parental melanoma cells. The hybrids showed heterogeneous sensitivity to New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T-cell receptor-transduced T (TCR-T) cells and two out of four hybrid clones showed less sensitivity to TCR-T compared with the parental cells. An in vitro tumor heterogeneity model revealed that the TCR-T cells preferentially killed the parental cells compared with the hybrids and the survival rate of the hybrids was higher than that of the parental cells, indicating that the hybrids evade killing by TCR-T cells efficiently. Analysis of a single-cell RNA sequencing dataset of patients with melanoma revealed that a few macrophages expressed RNA encoding melanoma differentiation antigens including melan A, tyrosinase, and premelanosome protein, which indicated the presence of hybrids in primary melanoma. In addition, the number of potential hybrids was correlated with a poorer response to immune checkpoint blockade. These results provide evidence that melanoma-macrophage fusion has a role in tumor heterogeneity and immune evasion. © 2023 The Pathological Society of Great Britain and Ireland.

DOI： 10.1002/path.6083

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BACKGROUND/AIM: Malignant melanoma is a fatal skin cancer and is among the most immunogenic malignancies expressing melanoma-differentiation antigens and neoantigens. SRY-related HMG-box 10 (SOX10) is a transcription factor and a neural-crest differentiation marker that is used as a diagnostic marker for melanoma whilst playing a role in melanoma initiation through activation of the SOX10-MITF axis. SOX10 was shown to play a role in melanoma initiation by inducing expression of immune checkpoint molecules (e.g., HVEM and CEACAM1). In this study, we aimed to investigate the relationship between SOX10 and the expression an immune checkpoint molecule, programmed death-1 ligand 1 (PD-L1). MATERIALS AND METHODS: SOX10 overexpression and knockdown was performed using SOX10 gene transfection and SOX10 siRNA transfection into A375 melanoma cells. PD-L1 expression was assessed by flow cytometry and western blotting. T cell response was evaluated using NY-ESO-1 specific TCR-transduced T (TCR-T) cells by IFNγ ELISPOT assay. RESULTS: SOX10 overexpression increased the expression of PD-L1, whereas SOX10 knockdown, using siRNA, decreased its expression. IFNγ ELISPOT assay revealed that overexpression of SOX10 decreased the susceptibility of cells to NY-ESO-1-specific TCR-T cells. CONCLUSION: SOX10 has a role in the intrinsic immune suppressive mechanisms of melanoma through expression of PD-L1.

DOI： 10.21873/anticanres.16296

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BACKGROUND: As therapy for solid tumours, various tumour antigens have been selected as targets, but CAR-T cells targeting these antigens have shown limited efficacy, in contrast to the effectiveness of CAR-T cells targeting haematological malignancies. In a previous report, we identified a cancer-testis antigen, DNAJB8. DNAJB8 plays a major role in tumorigenicity in cancer stem-like cells/cancer-initiating cells (CSCs/CICs). Here, we report a DNAJB8-reactive CAR yielding anti-tumour effects against renal cell carcinoma (RCC) and osteosarcoma. METHODS: We constructed a second-generation chimeric antigen receptor (CAR) against HLA-A*24:02/DNAJB8-derived peptide (DNAJB_143) complex (B10 CAR). The reactivity of B10-CAR T cells against T2-A24 cells pulsed with the cognate peptide and an RCC and osteosarcoma cell lines were quantified. The effects of adoptive cell transfer (ACT) therapy were assessed using in vivo xenografted mice models. RESULTS: B10 CAR-T cells recognised DNAJB8_143-pulsed T2-A24 cells and HLA-A*24:02(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. Moreover, ACT using B10 CAR-T cells showed anti-tumour effects against RCC and osteosarcoma cells. CONCLUSION: B10 CAR-T cells could show specific cytotoxicity against RCC and osteosarcoma cells in vitro and in vivo. B10 CAR-T cells targeting the CSC/CIC antigen DNAJB8 might be a candidate immunotherapy for carcinoma and sarcoma.

DOI： 10.1038/s41416-022-02100-1

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Bladder cancer is a major and fatal urological disease. Cisplatin is a key drug for the treatment of bladder cancer, especially in muscle-invasive cases. In most cases of bladder cancer, cisplatin is effective; however, resistance to cisplatin has a significant negative impact on prognosis. Thus, a treatment strategy for cisplatin-resistant bladder cancer is essential to improve the prognosis. In this study, we established a cisplatin-resistant (CR) bladder cancer cell line using an urothelial carcinoma cell lines (UM-UC-3 and J82). We screened for potential targets in CR cells and found that claspin (CLSPN) was overexpressed. CLSPN mRNA knockdown revealed that CLSPN had a role in cisplatin resistance in CR cells. In our previous study, we identified human leukocyte antigen (HLA)-A*02:01-restricted CLSPN peptide by HLA ligandome analysis. Thus, we generated a CLSPN peptide-specific cytotoxic T lymphocyte clone that recognized CR cells at a higher level than wild-type UM-UC-3 cells. These findings indicate that CLSPN is a driver of cisplatin resistance and CLSPN peptide-specific immunotherapy may be effective for cisplatin-resistant cases.

DOI： 10.1007/s00262-023-03388-5

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Arachidonate 5-lipoxygenase (ALOX5) is a cardinal enzyme in the synthesis of leukotrienes, which are powerful immune-regulating lipid mediators. We previously reported that ALOX5 is preferentially expressed in B lymphocytes in the mantle zone of human lymphoid tissue. In the context of physiological relevance, the loss of the Alox5 gene in mice significantly impairs the development of follicular B helper T cells and antibody production. However, ALOX5 expression in B-cell lymphomas has not been investigated in detail. In this study, we examined ALOX5 expression in representative B-cell lymphomas and non-neoplastic lymphoid tissues by immunohistochemistry with a commercially available anti-ALOX5 antibody that can be used on formalin-fixed paraffin-embedded specimens. Interestingly, 22/22 cases of mantle cell lymphoma, 7/7 cases of chronic lymphocytic leukemia/small cell lymphoma, and 20/20 cases of follicular lymphoma expressed ALOX5. A small proportion of extranodal marginal zone lymphoma/mucosa-associated lymphoid tissue lymphoma or nodal marginal zone lymphoma cases were positive for ALOX5 (2/13 or 1/3, respectively). In contrast, no cases with diffuse large B-cell lymphoma, regardless of germinal center B cell (GCB) or non-GCB type, expressed ALOX5 (0/25 cases). These findings suggest that ALOX5 may be a novel marker for identifying the cell of origin of B-cell lymphoma. Further investigation is required to clarify the biological significance of ALOX5 expression in lymphoma cells.

DOI： 10.1016/j.prp.2023.154328

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Immune checkpoint inhibitor-based cancer immunotherapy has provided an additional therapeutic option for oral squamous cell carcinoma (OSCC) with recurrence or distant metastases. However, further improvement of OSCC treatment is required to develop the optimal combination or order for chemoradiotherapy and immunotherapy. Along with the accumulation of clinical knowledge and evidence, it is also essential to clarify the biological impact of chemo-radiotherapeutic agents on the cancer immune microenvironment. In this study, we investigated the effects of cisplatin (CDDP), a key therapeutic agent for OSCC, on programmed death-ligand 1 (PD-L1) expression in OSCC lines. Although CDDP treatment increased the surface levels of PD-L1 on OSCC cell lines, the gene and total protein expression levels of PD-L1 were not altered. We also demonstrated that the phosphorylation of heat shock factor 1 and heat shock protein 90 was involved in this process. In addition, CDDP-induced PD-L1 attenuated the target-specific cytotoxic T lymphocyte reaction to OSCC. These results provide an immunobiological basis for the response of OSCC to CDDP and will contribute to our biological understanding of the action of novel combination therapy including immunotherapy together with platinum-based chemotherapy for OSCC.

DOI： 10.1002/cam4.5310

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Despite the considerable success of cancer immunotherapy with immune checkpoint inhibitors, their nonspecific release of the immunosuppressive mechanism is often associated with immune-related adverse events (irAEs). irAEs significantly disturb patients' quality of life and can even be life-threatening. Therefore, the appropriate management of irAEs is crucial for the development of further reliable cancer immunotherapies. irAEs have the appearance of ordinary autoimmune diseases in one aspect but often have distinct features. Although the detailed pathogenesis of irAEs remains unclear, increasing numbers of studies have provided numerous clues. Here, we review the current knowledge on irAEs, particularly from an immunopathological basis.

DOI： 10.1080/25785826.2021.1976942

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Recent studies have revealed that treatment-resistant cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be targeted by cytotoxic T lymphocytes (CTLs). CTLs recognize antigenic peptides derived from tumor-associated antigens; thus, the identification of tumor-associated antigens expressed by CSCs/CICs is essential. Human leucocyte antigen (HLA) ligandome analysis using mass spectrometry enables the analysis of naturally expressed antigenic peptides; however, HLA ligandome analysis requires a large number of cells and is challenging for CSCs/CICs. In this study, we established a novel bladder CSC/CIC model from a bladder cancer cell line (UM-UC-3 cells) using an ALDEFLUOR assay. CSCs/CICs were isolated as aldehyde dehydrogenase (ALDH)-high cells and several ALDHhigh clone cells were established. ALDHhigh clone cells were enriched with CSCs/CICs by sphere formation and tumorigenicity in immunodeficient mice. HLA ligandome analysis and cap analysis of gene expression using ALDHhigh clone cells revealed a distinctive antigenic peptide repertoire in bladder CSCs/CICs, and we found that a glutamate receptor, ionotropic, kainite 2 (GRIK2)-derived antigenic peptide (LMYDAVHVV) was specifically expressed by CSCs/CICs. A GRIK2 peptide-specific CTL clone recognized GRIK2-overexpressing UM-UC-3 cells and ALDHhigh clone cells, indicating that GRIK2 peptide can be a novel target for bladder CSC/CIC-targeting immunotherapy.

DOI： 10.1007/s00262-021-03025-z

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Immune checkpoint inhibitors (ICIs) are used in cancer immunotherapy to block programmed death-1 and cytotoxic T-lymphocyte antigen 4, but the response rate for ICIs is still low and tumor cell heterogeneity is considered to be responsible for resistance to immunotherapy. Tumor-infiltrating lymphocytes (TILs) have an essential role in the anti-tumor effect of cancer immunotherapy; however, the specificity of TILs in renal cell carcinoma (RCC) is elusive. In this study, we analyzed a 58-year-old case with clear cell RCC (ccRCC) with the tumor showing macroscopic and microscopic heterogeneity. The tumor was composed of low-grade and high-grade ccRCC. A tumor cell line (1226 RCC cells) and TILs were isolated from the high-grade ccRCC lesion, and a TIL clone recognized a novel neoantigen peptide (YVVPGSPCL) encoded by a missense mutation of the tensin 1 (TNS1) gene in a human leukocyte antigen-C*03:03-restricted fashion. The TNS1 gene mutation was not detected in the low-grade ccRCC lesion and the TIL clone did not recognized low-grade ccRCC cells. The missense mutation of TNS1 encoding the S1309Y mutation was found to be related to cell migration by gene over-expression. These findings suggest that macroscopically and microscopically heterogenous tumors might show heterogenous gene mutations and reactivity to TILs.

DOI： 10.1007/s00262-021-03048-6

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BACKGROUND & AIMS: A detailed understanding of antitumor immunity is essential for optimal cancer immune therapy. Although defective mutations in the B2M and HLA-ABC genes, which encode molecules essential for antigen presentation, have been reported in several studies, the effects of these defects on tumor immunity have not been quantitatively evaluated. METHODS: Mutations in HLA-ABC genes were analyzed in 114 microsatellite instability-high colorectal cancers using a long-read sequencer. The data were further analyzed in combination with whole-exome sequencing, transcriptome sequencing, DNA methylation array, and immunohistochemistry data. RESULTS: We detected 101 truncating mutations in 57 tumors (50%) and loss of 61 alleles in 21 tumors (18%). Based on the integrated analysis that enabled the immunologic subclassification of microsatellite instability-high colorectal cancers, we identified a subtype of tumors in which lymphocyte infiltration was reduced, partly due to reduced expression of HLA-ABC genes in the absence of apparent genetic alterations. Survival time of patients with such tumors was shorter than in patients with other tumor types. Paradoxically, tumor mutation burden was highest in the subtype, suggesting that the immunogenic effect of accumulating mutations was counterbalanced by mutations that weakened immunoreactivity. Various genetic and epigenetic alterations, including frameshift mutations in RFX5 and promoter methylation of PSMB8 and HLA-A, converged on reduced expression of HLA-ABC genes. CONCLUSIONS: Our detailed immunogenomic analysis provides information that will facilitate the improvement and development of cancer immunotherapy.

DOI： 10.1053/j.gastro.2021.10.010

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The association between type 2 diabetes mellitus and prostate cancer is still under investigation, and the relationship between hyperinsulinemia and prostate cancer stem-like cells (CSCs) is elusive. Here, we investigated the function of insulin/AKT signaling in prostate CSCs. We isolated prostate CSCs as aldehyde dehydrogenase 1-high (ALDH1high) cells from the human prostate cancer 22Rv1 cell line using an ALDEFLUOR assay and established several ALDH1high and ALDH1low clones. ALDH1high clones showed high ALDH1 expression which is a putative CSC marker; however, they showed heterogeneity regarding tumorigenicity and resistance to radiation and chemotherapy. Interestingly, all ALDH1high clones showed lower phosphorylated AKT (Ser473) (pAKT) levels than the ALDH1low clones. PI3K/AKT signaling is a key cell survival pathway and we analyzed radiation resistance under AKT signaling activation by insulin. Insulin increased pAKT levels in ALDH1high and ALDH1low cells; the fold increase rate of pAKT was higher in ALDH1high cells than in ALDH1low cells. Insulin induced resistance to radiation and chemotherapy in ALDH1high cells, and the increased levels of pAKT induced by insulin were significantly related to radiation resistance. These results suggest that ALDH1 suppresses baseline pAKT levels, but AKT can be activated by insulin, leading to treatment resistance.

DOI： 10.1016/j.bbrc.2021.12.095

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To develop combination immunotherapies for gastric cancers, immunologically well-characterized preclinical models are crucial. Here, we leveraged two transplantable murine gastric cancer cell lines, YTN2 and YTN16, derived from the same parental line but differing in their susceptibility to immune rejection. We established their differential sensitivity to immune checkpoint inhibitors (ICI) and identified neoantigens. Although anti-CTLA-4 mAbs eradicated YTN16 tumors in 4 of 5 mice, anti-PD-1 and anti-PD-L1 mAbs failed to eradicate YTN16 tumors. Using whole-exome and RNA sequencing, we identified two and three neoantigens in YTN2 and YTN16, respectively. MHC class I ligandome analysis detected the expression of only one of these neoantigens, mutated Cdt1, but the exact length of MHC binding peptide was determined. Dendritic cell vaccine loaded with neoepitope peptides and adoptive transfer of neoantigen-specific CD8+ T cells successfully inhibited the YTN16 tumor growth. Targeting mutated Cdt1 had better efficacy for controlling the tumor. Therefore, mutated Cdt1 was the dominant neoantigen in these tumor cells. More mCdt1 peptides were bound to MHC class I and presented on YTN2 surface than YTN16. This might be one of the reasons why YTN2 was rejected while YTN16 grew in immune-competent mice.

DOI： 10.3390/cancers14010106

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BACKGROUND: Barrier disruption and an excessive immune response in keratinocytes are now considered to have important roles in the pathophysiology of atopic dermatitis (AD). Furthermore, disturbed keratinocyte differentiation is considered to underlie AD. ΔNp63, a p53-like transcription factor, is a major regulator of keratinocyte differentiation. However, the functional significance of ΔNp63 in AD has not been clarified. OBJECTIVE: In this study, we aimed to investigate the influence of the type 2 inflammatory environment on ΔNp63 expression and AD-associated molecules regulated by ΔNp63 in keratinocytes. METHODS: The immunohistochemical expression profiles of ΔNp63 and AD-related molecules were evaluated in human skin tissue. The function of ΔNp63 in the regulation of AD-related molecules and the influence of the type 2 inflammatory environment on ΔNp63 expression were investigated using human primary keratinocytes. Expression of ΔNp63 was manipulated using the RNA interfering method. RESULTS: In healthy skin tissue, we observed an inverse expression pattern between ∆Np63 and some barrier-related proteins including filaggrin, caspase-14, claudin-1, and claudin-4. ΔNp63 regulated expression of these genes and proteins. In addition, production of IL-1β and IL-33, pro-inflammatory cytokines, was modulated by ΔNp63. Furthermore, prolonged IL-13 exposure increased the thickness of the three-dimensional culture of keratinocytes. IL-13 interfered with ΔNp63 downregulation during calcium-induced keratinocyte differentiation. IL-13 modulated some barrier-related and inflammation-related molecules, which were regulated by ΔNp63. CONCLUSIONS: We have shown that ΔNp63 modulated AD-related barrier and inflammatory molecules. In addition, ΔNp63 expression was affected by IL-4/IL-13. IL-13-ΔNp63 axis would integrate two major factors of AD pathogenesis: dysregulated barrier and inflammation.

DOI： 10.1002/iid3.427

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The immune system is often called a double-edged sword, due to the inextricable link between cancer immunity and allergy/autoimmunity. Intriguingly, a growing number of cases have been reported in which PD-1 blockade triggers the exacerbation of tuberculosis (TB), an organ-invasive granulomatous disease caused by bacterial infection. As a result, the exacerbation of TB is now considered a severe adverse effect of nivolumab and pembrolizumab. In this letter, we report the strong expression of PD-L1 in epithelioid granulomatous lesions in tuberculosis, sarcoidosis, Crohn's disease, and foreign body granuloma. In addition, we discussed the exacerbation of tuberculosis after anti-PD-1 antibody-based cancer immunotherapy.

DOI： 10.1080/21645515.2020.1870364

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BACKGROUND/AIM: Immune checkpoint inhibitors (ICIs), including nivolumab and pembrolizumab, have recently been shown to have clinical benefits in patients with advanced non-small cell lung cancer (NSCLC). The novel tumour responses to these agents are changing the management of patients with cancer. Pseudo-progression of disease (pseudo-PD), that is, an initial flare followed by shrinkage of the tumour, has been described as a distinctive response to ICIs. However, pseudo-PD manifest initial progression and is difficult to segregate with hyper progressive disease (HPD). We, therefore, analysed a case with pseudo-PD histologically. PATIENTS AND METHODS: A 68-year-old Japanese man with stage IV non-small cell lung carcinoma (NSCLC) was treated by anti-PD-1 antibody (pembrolizumab). Four weeks later after second time treatment with pembrolizumab, the patient showed severe melena followed by Trousseau syndrome and died at day 174 after first treatment by pembrolizumab, suggesting HPD clinically. Primary lesion and metastatic lesions were analysed histologically. RESULTS: Histological analysis revealed that NSCLC cells expressed PD-L1, and CD8+ tumor-infiltrated lymphocytes (TILs) were observed. CD8+ TILs showed higher rates of PD-1 indicating that lesions were of the inflamed type and the case was pseudo-PD. Furthermore, it was found that cancer cells expressed MUC1. CONCLUSION: The clinical appearance of the case was aggressive after treatment by pembrolizumab, and the case seemed to be HPD; however, histological analysis revealed that the case was likely pseudo-PD. Therefore, careful histological evaluation is important when investigating the clinical response to an ICI and mucin expression might be a predictive marker for Trousseau syndrome.

DOI： 10.21873/anticanres.15161

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Effective management of immune-related adverse events in patients receiving immunotherapy for cancer is problematic. In this report, we present the case of a 58-year-old man with advanced clear cell renal cell carcinoma who responded well to a combination of ipilimumab and nivolumab. However, after two courses of treatment, he developed fulminant hepatitis and died. An autopsy confirmed that the primary lesion in the left kidney was more than 99% necrotic with only six small residual tumor lesions. These lesions were infiltrated by large numbers of CD8-positive/TIA-1-positive lymphocytes. However, a metastatic lesion in the right kidney harbored few lymphocytes. Furthermore, the tumor cells in the metastatic lesion and one of the residual lesions showed decreased expression of HLA class I molecules, which are a prerequisite for cytotoxic T-lymphocyte-mediated immunotherapy in tumor cells. In this patient, more than 80% of hepatocytes were destroyed and the parenchyma was infiltrated with CD8-positive/TIA-1-positive lymphocytes. The patient had polyuria, which was attributed to neurohypophysitis caused by the infiltration of CD8-positive/TIA-1-positive lymphocytes. We believe that this is an instructive case for immuno-oncologists.

DOI： 10.1080/25785826.2020.1788229

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Previously, we investigated gene expression in a high aldehyde dehydrogenase 1 expression (ALDH1high) population of urothelial carcinoma (UC) cells as UC cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) and found that NRG1 expression was upregulated in ALDH1high cells. NRG1 is a trophic factor that contains an epidermal growth factor (EGF)-like domain that signals by stimulating ERBB receptor tyrosine kinases and the cytoplasmic domain. NRG1 has been determined to be involved in frequent gene fusions with other partners in several malignancies and has a role in carcinogenesis through the NRG1 EGF-like domain and its cognitive receptor ERBBs. We thus aimed to elucidate the function of NRG1 in UC CSCs/CICs in this study. Both NRG1α and NRG1-β1 were preferentially expressed in ALDH1high cells compared with ALDH1low cells; however, siRNA experiments revealed that NRG1-β1 but not NRG1-α has a role in sphere formation. The EGF-like domain of NRG1 had a role in sphere formation of UC cells to some extent but was not essential. The intracellular domain of NRG1 did not have a role in sphere-formation. Inhibition of γ-secretase suppressed sphere formation. These findings indicate that cleavage of NRG1-β1 by γ-secretase plays an important role in UC CSC/CIC proliferation; however, the downstream targets of NRG1-β1 remain elusive.

DOI： 10.1016/j.bbrc.2021.03.038

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Immune checkpoint inhibitors (ICIs) have provided an additional treatment option for various types of human cancers. However, ICIs often induce various immune-related adverse events (irAEs). Enterocolitis is a major irAE with poorly understood histopathological characteristics. In this study, we retrospectively investigated the histopathology of colon tissue samples from 17 patients treated with ICIs. There were two major histological patterns of colitis: an ulcerative colitis-like pattern and a graft vs host disease-like pattern. Although these two patterns of colitis were mutually exclusive, both patterns often showed a characteristic that we call "subepithelial surface granulomatosis" (SSG), which has not been reported in other types of colitis. SSG was found even in colon tissue without symptoms or endoscopic findings of colitis. Given the increasing reports of sarcoid reaction or exacerbation of tuberculosis after treatment with ICIs, granuloma formation could be a histological hallmark of systemic immune activation by ICIs. Although statistical significance was not obtained, probably because of the small sample size, SSG may be a surrogate biomarker of systemic anticancer immune activation. We propose that a prospective study with larger sample size be performed.

DOI： 10.1111/cas.14773

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Mismatch repair protein deficiency (dMMR) is a favorable prognostic factor in colorectal cancer. It is also associated with aberrant expression of HLA class I molecules, which are required for cytotoxic T lymphocyte-mediated cancer immunotherapy. Because dMMR is frequently also found in endometrial cancers (ECs), we retrospectively investigated the expression of mismatch repair proteins and HLA class I molecules in 127 EC patients. In this study, EC patients being treated in our hospital were recruited from 2005 to 2009 and observed until December 2017. Lesion specimens were evaluated via immunohistochemistry for MSH6 and PMS2 (mismatch repair proteins) and HLA class I molecules. Expression of these molecules was statistically related to clinical and pathological factors and prognosis. dMMR was detected in 33 patients and did not correlate with the expression level of HLA class I molecules (P = 0.60). On the other hand, unexpectedly, multivariate analysis revealed that intact expression of HLA class I molecules was associated with p53 overexpression (P = 0.004). Neither dMMR nor decreased expression of HLA class I molecules were prognostic factors. These results are inconsistent with previous findings for colorectal cancer. A distinctive local tissue immune microenvironment would underlie the discrepancy in the results between EC and colorectal cancer.

DOI： 10.1007/s00795-020-00254-6

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Photodynamic therapy (PDT) using the photosensitizer talaporfin sodium (talaporfin) is a new mode of treatment for cancer. However, the metabolic mechanism of talaporfin has not been clarified. Thus, we investigated the uptake, transportation, and elimination mechanisms of talaporfin in carcinoma and sarcoma. The results showed that talaporfin co-localized in early endosomes and lysosomes. Talaporfin uptake was via clathrin- and caveolae-dependent endocytosis and a high amount of intracellular ATP was essential. Inhibition of lysosomal enzymes maintained intracellular talaporfin levels. Inhibition of K-Ras signaling reduced talaporfin uptake in carcinoma and sarcoma cell lines. Talaporfin was taken up by clathrin- and caveolae-dependent endocytosis, translocated from early endosomes to lysosomes, and finally degraded by lysosomes. We also demonstrated that ATP is essential for the uptake of talaporfin and that activation of K-Ras is involved as a regulatory mechanism. These results provide new insights into the metabolism of talaporfin in cancer cells for the enhancement of PDT for carcinoma and sarcoma.

DOI： 10.1111/cas.14735

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Palladium (Pd) is a widely used metal and extremely important biomaterial for the reconstruction of occlusions during dental restorations. However, metallic biomaterials can cause serious allergic reactions, such as Pd-related oral mucositis seen in dentistry. Metal allergy is categorized as a type IV allergy and we demonstrated that CD8 T cells play an important role in Pd allergy previously. As TCR of CD8 T cells recognizes MHC class I/peptide complex, the antigen specificity to this complex seems to be generated during Pd allergy. However, it remains unknown if Pd affects the MHC class I/peptide complex. In this study, we investigated the behavior of the MHC class I/peptide complex in response to Pd treatment. We found that PdCl2 treatment altered peptide presentation on MHC class I and that co-culture with Pd-treated DC2.4 cells induced activation of Pd-responsive TCR-expressing T cell line. Furthermore, PdCl2 treatment induced temporal MHC class I internalization and inhibition of membrane movement suppressed Pd-induced T cell-mediated antigenicity. These data suggest that Pd-induced MHC class I internalization is critical for generation of antigenicity through a mechanism including differential peptide loading on MHC class I, which results in Pd allergy.

DOI： 10.3389/fimmu.2021.736936

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Extensive research over 100 years has demonstrated that tumors can be eliminated by the autologous immune system. Without doubt, immunotherapy is now a standard treatment along with surgery, chemotherapy, and radiotherapy; however, the field of cancer immunotherapy is continuing to develop. The current challenges for the use of immunotherapy are to enhance its clinical efficacy, reduce side effects, and develop predictive biomarkers. Given that histopathological analysis provides molecular and morphological information on humans in vivo, its importance will continue to grow. This review article outlines the basic knowledge that is essential for the research and daily practice of immune checkpoint inhibitor-based cancer immunotherapy from the perspective of histopathology.

DOI： 10.3389/fonc.2021.679095

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INTRODUCTION: Immune checkpoint inhibitors are now a standard therapeutic option for lung adenocarcinoma. However, Immune checkpoint inhibitors often induce various immune-related adverse events. CASE PRESENTATION: The patient was a 78-year-old woman with lung adenocarcinoma who had a partial response to pembrolizumab. During treatment, she complained of pollakiuria and nocturia with painful micturition. Histological analysis revealed infiltration of CD8-positive and/or TIA-1 cytotoxic granule-associated RNA binding protein-positive lymphocytes and programmed death-ligand 1 expression in the urothelium. A diagnosis of immune-related adverse event cystitis was made based on these clinical and pathological findings. The patient's subjective symptoms and findings on cystoscopy improved dramatically after treatment with prednisolone. CONCLUSION: Immune checkpoint inhibitors-induced cystitis is extremely rare. This report is the first to include an immunohistochemical analysis of the urothelial epithelium in immune-related adverse event cystitis and describes an instructive case.

DOI： 10.1002/iju5.12211

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BACKGROUND: Peptide-vaccination therapy targeting tumour-associated antigens can elicit immune responses, but cannot be used to eliminate large tumour burden. In this study, we developed a therapeutic single-chain variable-fragment (scFv) antibody that recognises the cancer stem-like cell/cancer-initiating cell (CSC/CIC) antigen, DNAJB8. METHODS: We screened scFv clones reacting with HLA-A24:20/DNAJB8-derived peptide (DNAJB8_143) complex using naive scFv phage-display libraries. Reactivity and affinity of scFv clones against the cognate antigen were quantified using FACS and surface plasmon resonance. Candidate scFv clones were engineered to human IgG1 (hIgG1) and T-cell-engaging bispecific antibody (CD3xJB8). Complement-dependent cytotoxicity (CDC) and bispecific antibody-dependent cellular cytotoxicity (BADCC) were assessed. RESULTS: scFv clones A10 and B10 were isolated after bio-panning. Both A10-hIgG1 and B10-hIgG1 reacted with DNAJB8-143 peptide-pulsed antigen-presenting cells and HLA-A24(+)/DNAJB8(+) renal cell carcinoma and osteosarcoma cell lines. A10-hIgG1 and B10-hIgG1 showed strong affinity with the cognate HLA/peptide complex (KD = 2.96 × 10-9 M and 5.04 × 10-9 M, respectively). A10-hIgG1 and B10-hIgG1 showed CDC against HLA-A24(+)/DNAJB8(+) cell lines. B10-(CD3xJB8) showed superior BADCC to A10-(CD3xJB8). CONCLUSION: We isolated artificial scFv antibodies reactive to CSC/CIC antigen DNAJB8-derived peptide naturally present on renal cell carcinoma and sarcoma. Immunotherapy using these engineered antibodies could be promising.

DOI： 10.1038/s41416-020-1017-1

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BACKGROUND/AIM: The tumor microenvironment (TME) balances tumor growth and suppression through humoral factors and cell-cell interactions. In oral squamous cell carcinoma (OSCC), TMEs have been associated with prognosis of cancer patients and are evaluated by microscopy; however, these methods of evaluation vary among studies. MATERIALS AND METHODS: To evaluate the TME, borderline microenvironment fibrosis (bMF) was evaluated histologically in 236 OSCC cases and used to determine the clinicopathological status. RESULTS: bMF was observed in 47% (110 in 236 cases) of OSCC cases and associated with higher T category, N category, stage, histological grade and mode of invasion. bMF-positive was related to overall survival (OS) and progression-free survival (PFS). Multivariate analysis revealed that bMF-positive was an independent factor for OS in all cases [n=226; HR=1.683 (1.018-2.781); p=0.042], especially in T1+T2 cases [n=186; HR=1.926 (1.079-3.440); p=0.024], and PFS in all cases [n=226; HR=2.254 (1.397-3.637); p=0.001]. CONCLUSION: bMF may act as a novel biomarker for OSCC.

DOI： 10.21873/anticanres.14434

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Ewing sarcoma (EwS) is an aggressive cancer displaying an undifferentiated small-round-cell histomorphology that can be easily confused with a broad spectrum of differential diagnoses. Using comparative transcriptomics and immunohistochemistry (IHC), we previously identified BCL11B and GLG1 as potential specific auxiliary IHC markers for EWSR1-FLI1-positive EwS. Herein, we aimed at validating the specificity of both markers in a far larger and independent cohort of EwS (including EWSR1-ERG-positive cases) and differential diagnoses. Furthermore, we evaluated their intra-tumoral expression heterogeneity. Thus, we stained tissue microarrays from 133 molecularly confirmed EwS cases and 320 samples from morphological mimics, as well as a series of patient-derived xenograft (PDX) models for BCL11B, GLG1, and CD99, and systematically assessed the immunoreactivity and optimal cut-offs for each marker. These analyses demonstrated that high BCL11B and/or GLG1 immunoreactivity in CD99-positive cases had a specificity of 97.5% and an accuracy of 87.4% for diagnosing EwS solely by IHC, and that the markers were expressed by EWSR1-ERG-positive EwS. Only little intra-tumoral heterogeneity in immunoreactivity was observed for differential diagnoses. These results indicate that BCL11B and GLG1 may help as specific auxiliary IHC markers in diagnosing EwS in conjunction with CD99, especially if confirmatory molecular diagnostics are not available.

DOI： 10.3390/cancers12030644

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Peptide-based immunotherapy does not usually elicit strong immunological and clinical responses in patients with end-stage cancer, including sarcoma. Here we report a myxofibrosarcoma patient who showed a strong clinical response to peptide vaccinations and whose immune responses were reboosted by anti-PD1 therapy combined with peptide vaccinations. The 46-year-old man showed a strong response to the peptide vaccinations (papillomavirus binding factor peptide, survivin-2B peptide, incomplete Freund's adjuvant, and polyethylene glycol-conjugated interferon-alpha 2a) and subsequent wide necrosis and massive infiltration of CD8+ T cells in a recurrent tumor. The patient's immune responses weakened after surgical resection; however, they were reboosted following the administration of nivolumab combined with peptide vaccinations. Thus, anti-PD1 therapy combined with peptide vaccinations might be beneficial, as suggested by the observations in this sarcoma patient.

DOI： 10.1007/s00262-019-02455-0

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Osteosarcoma (OS) is a highly malignant bone tumor and the prognosis for non-responders to chemotherapy remains poor. Previous studies have shown that human sarcomas contain sarcoma-initiating cells (SIC), which have the characteristics of high tumorigenesis and resistance to chemotherapy. In the present study, we characterized SIC of a novel OS cell line, screened for SIC-related genes, and tried to regulate the proliferation of OS by metabolic interference. Initially, we established a new human OS cell line (OS13) and isolated clones showing higher tumorigenesis as SIC (OSHIGH ) and counterpart clones. OSHIGH cells showed chemoresistance and their metabolism highly depended on aerobic glycolysis and suppressed oxidative phosphorylation. Using RNA-sequencing, we identified LIN28B as a SIC-related gene highly expressed in OSHIGH cells. mRNA of LIN28B was expressed in sarcoma cell lines including OS13, but its expression was not detectable in normal organs other than the testis and placenta. LIN28B protein was also detected in various sarcoma tissues. Knockdown of LIN28B in OS13 cells reduced tumorigenesis, decreased chemoresistance, and reversed oxidative phosphorylation function. Combination therapy consisting of a glycolysis inhibitor and low-dose chemotherapy had antitumor effects. In conclusion, manipulation of glycolysis combined with chemotherapy might be a good adjuvant treatment for OS. Development of immunotherapy targeting LIN28B, a so-called cancer/testis antigen, might be a good approach.

DOI： 10.1111/cas.14229

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Bellwether Publishing, Ltd.  

DOI： 10.1080/15421406.2020.1743458

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The prognosis of advanced pancreatic adenocarcinoma is still extremely poor. This study sought to determine the efficacy of, and immunological response to, peptide vaccination therapy in patients with this disease. In this multicenter randomized phase II study, patients with advanced pancreatic adenocarcinoma after gemcitabine and/or tegafur/gimeracil/oteracil were randomly assigned to 3 groups that each received a 2-step treatment course. In Step 1, the groups received treatments of: (i) survivin 2B peptide (SVN-2B) plus interferon-β (IFNβ); (ii) SVN-2B only; or (iii) placebo until the patients show progression. In Step 2, all patients who consented to participate received 4 treatments with SVN-2B plus IFNβ. The primary endpoint was progression-free survival (PFS) after initiation of Step 1 treatment. Secondary endpoints included immunological effects assessed by analysis of PBMCs after Step 1. Eighty-three patients were randomly assigned to receive SVN-2B plus IFNβ (n = 30), SVN-2B (n = 34), or placebo (n = 19). No significant improvement in PFS was observed. Survivin 2B-specific CTLs were found to be increased in the SVN-2B plus IFNβ group by tetramer assay. Among patients who participated in Step 2, those who had received SVN-2B plus IFNβ in Step 1 showed better overall survival compared with those who had received placebo in Step 1. Patients vaccinated with SVN-2B plus IFNβ did not have improved PFS, but showed significant immunological reaction after vaccination. Subgroup analysis suggested that a longer SVN-2B plus IFNβ vaccination protocol might confer survival benefit. (Clinical trial registration number: UMIN 000012146).

DOI： 10.1111/cas.14106

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Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer by providing new options in addition to existing therapies. However, peptide vaccination therapies still represent an attractive approach, because of the antigen specificity. We identified survivin 2B peptide (SVN-2B), a 9-mer antigenic peptide encoded by survivin, and an SVN-2B peptide vaccine-based phase II randomized clinical trial targeting unresectable and refractory pancreatic carcinoma was undertaken. The SVN-2B peptide vaccine did not have any statistically significant clinical benefits in that study. Therefore, we undertook an autopsy study to analyze the immune status of the pancreatic cancer lesions at the histological level. Autopsies were carried out in 13 patients who had died of pancreatic cancer, including 7 who had received SVN-2B peptide vaccination and 6 who had not, as negative controls. The expression of immune-related molecules was analyzed by immunohistochemical staining. Cytotoxic T lymphocytes were analyzed by tetramer staining and enzyme-linked immunospot assay. Histological analysis revealed dense infiltration of CD8+ T cells in some lesions in patients who had received the SVN-2B peptide vaccine. A high rate of programmed cell death ligand 1 expression in cancer cells was observed in these cases, indicating that CTLs were induced by SVN-2B peptide vaccination and had infiltrated the lesions. The lack of a significant antitumor effect was most likely attributable to the expression of immune checkpoint molecules. These findings suggest that the combination of a tumor-specific peptide vaccine and an ICI might be a promising approach to the treatment of pancreatic carcinoma in the future.

DOI： 10.1111/cas.14099

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Uterine endometrial carcinoma is one of the common cancers in females. Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are tumorigenic and are resistant to treatments, thus they are focused as treatment targets. However, the heterogeneity of CSCs/CICs is still elusive, and we therefore analyzed CSCs/CICs at the clonal level. We previously established sphere-cultured CSCs/CICs from primary human uterine endometrial carcinoma, and we isolated several clones from CSCs/CICs in this study. Interestingly, we established two types of clones based on the growth pattern. The clones were termed sphere clones (S clones) and leukemia-like clones (LL clones). Functional analysis revealed that S clones are resistant to chemotherapy, whereas LL clones are sensitive to chemotherapy. On the other hand, S clones are less tumorigenic, while LL clones are highly tumorigenic. Transcriptome analysis using serial analysis of gene expression sequencing (SAGE-Seq) revealed distinctive gene expression profiles in S clone cells and LL clone cells. The results indicate that CSCs/CICs are composed of functionally heterogenic subpopulations including highly tumorigenic clones and treatment-resistant clones and that the characteristics of CSCs/CICs might be determined by the characteristics of different clones that compose CSCs/CICs.

DOI： 10.1016/j.yexmp.2018.11.013

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Bronchial epithelial cells serve as a physical barrier at the forefront of the immune system. Barrier disruption and an excessive immune response of the bronchial epithelium contribute to the pathophysiology of asthma, a chronic bronchial inflammatory disease. The purpose of this study was to investigate the functional significance of ΔNp63, a p53-like transcription factor expressed by the basal bronchial epithelium. The immunohistochemical expression profile of ΔNp63 was evaluated in human bronchial tissue derived from asthma patients. The role of ΔNp63 in apoptosis inhibition and production of soluble mediators was investigated in vitro with cultured BEAS-2B bronchial epithelial cells using molecular biological analysis. In healthy bronchial tissue, ΔNp63-positive basal epithelial cells were covered with differentiated ΔNp63-negative cells but in the asthmatic airway, ΔNp63-positive cells were directly exposed to the bronchial lumen due to severe epithelial shedding. ΔNp63 regulated bronchial apoptosis in response to Toll-like receptor 3 stimulation. On the other hand, expression of ΔNp63 was modulated by stimulation with trypsin and SLIGKV, protease-activated receptor 2 ligands. Further phenotypic analysis revealed that ΔNp63 controlled the transcriptional expression and protein release of some epithelium-derived proinflammatory cytokines and endogenous protease inhibitors. We conclude that ΔNp63 modulates the bronchial epithelial response to viral infection. At the same time, ΔNp63 expression is influenced by proteases, which are abundant in house dust mites. Therefore, the ΔNp63 axis would be intimately involved in these two major triggers of asthma exacerbations, viral infection and protease overload.

DOI： 10.1038/s41374-018-0132-6

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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.

DOI： 10.1111/cas.13854

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Photodynamic diagnosis/therapy (PDD/PDT) are novel modalities for the diagnosis and treatment of cancer. The photosensitizer protoporphyrin IX is metabolized from 5-aminolevulinic acid (5-ALA) intracellularly, and PDD/PDT using 5-ALA have been approved in dermatologic malignancies and gliomas. However, the molecular mechanism that defines the efficacy of PDD/PDT is unknown. In this study, we analyzed the functions of ATP-binding cassette (ABC) transporters in PDD using 5-ALA. Most of the human gastrointestinal cancer line cells examined showed a homogenous staining pattern with 5-ALA, except for the pancreatic cancer line PANC-1, which showed heterogeneous staining. To analyze this heterogeneous staining pattern, single cell clones were established from PANC-1 cells and the expression of ABC transporters was assessed. Among the ABC transporter genes examined, ABCG2 showed an inverse correlation with the rate of 5-ALA-positive staining. PANC-1 clone #2 cells showed the highest level of ABCG2 expression and the lowest level of 5-ALA staining, with only a 0.6% positive rate. Knockdown of the ABCG2 gene by small interfering RNAs increased the positive rate of 5-ALA staining in PANC-1 wild-type and clone cells. Interestingly, PANC-1 clone #2 cells showed the high sphere-forming ability and tumor-formation ability, indicating that the cells contained high numbers of cancer stem cells (CSCs). Knockdown or inhibition of ABCG2 increased the rate of 5-ALA staining, but did not decrease sphere-forming ability. These results indicate that gastrointestinal cancer cell lines expressing high levels of ABCG2 are enriched with CSCs and show low rates of 5-ALA staining, but 5-ALA staining rates can be improved by inhibition of ABCG2.

DOI： 10.1371/journal.pone.0216503

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A 62-year-old woman who underwent surgery to treat pancreatic cancer provided written, informed consent to undergo adjuvant therapy with gemcitabine, tegafur, and uracil. However, this was stopped after only 14 days due to Grade 4 neutropenia. She was then started on vaccine therapy with Survivin 2B peptide (SVN-2B) including IFA and INF-α. Although metastatic lung tumors were identified and resected at 82 months after surgery, the patient has remained free of new or relapsed disease for 12 years thereafter. Tetramer and ELISPOT assays revealed the continuous circulation of SVN-2B-restricted cytotoxic T-lymphocytes (CTLs) in her peripheral blood, and CTL clones had specific activity for SVN-2B at 12 years after surgery. The adverse effects of the peptide vaccination were tolerable and comprised low-grade headache, nausea, and fatigue. A prognosis beyond 10 years in the face of pancreatic cancer with distant metastasis is extremely rare. This experience might indicate the value of cancer vaccination therapy.

DOI： 10.1007/s00262-018-2217-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

Immunotherapy can revolutionize anti-cancer therapy if specific targets are available. Immunogenic peptides encoded by cancer-specific genes (CSGs) may enable targeted immunotherapy, even of oligo-mutated cancers, which lack neo-antigens generated by protein-coding missense mutations. Here, we describe an algorithm and user-friendly software named RAVEN (Rich Analysis of Variable gene Expressions in Numerous tissues) that automatizes the systematic and fast identification of CSG-encoded peptides highly affine to Major Histocompatibility Complexes (MHC) starting from transcriptome data. We applied RAVEN to a dataset assembled from 2,678 simultaneously normalized gene expression microarrays comprising 50 tumor entities, with a focus on oligo-mutated pediatric cancers, and 71 normal tissue types. RAVEN performed a transcriptome-wide scan in each cancer entity for gender-specific CSGs, and identified several established CSGs, but also many novel candidates potentially suitable for targeting multiple cancer types. The specific expression of the most promising CSGs was validated in cancer cell lines and in a comprehensive tissue-microarray. Subsequently, RAVEN identified likely immunogenic CSG-encoded peptides by predicting their affinity to MHCs and excluded sequence identity to abundantly expressed proteins by interrogating the UniProt protein-database. The predicted affinity of selected peptides was validated in T2-cell peptide-binding assays in which many showed binding-kinetics like a very immunogenic influenza control peptide. Collectively, we provide an exquisitely curated catalogue of cancer-specific and highly MHC-affine peptides across 50 cancer types, and a freely available software (https://github.com/JSGerke/RAVENsoftware) to easily apply our algorithm to any gene expression dataset. We anticipate that our peptide libraries and software constitute a rich resource to advance anti-cancer immunotherapy.

DOI： 10.1080/2162402x.2018.1481558

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In a previous study, we found that DNAJB8, a heat shock protein (HSP) 40 family member is expressed in kidney cancer stem-like cells (CSC)/cancer-initiating cells (CIC) and that it has a role in the maintenance of kidney CSC/CIC. Heat shock factor (HSF) 1 is a key transcription factor for responses to stress including heat shock, and it induces HSP family expression through activation by phosphorylation. In the present study, we therefore examined whether heat shock (HS) induces CSC/CIC. We treated the human kidney cancer cell line ACHN with HS, and found that HS increased side population (SP) cells. Western blot analysis and qRT-PCR showed that HS increased the expression of DNAJB8 and SOX2. Gene knockdown experiments using siRNAs showed that the increase in SOX2 expression and SP cell ratio depends on DNAJB8 and that the increase in DNAJB8 and SOX2 depend on HSF1. Furthermore, treatment with a mammalian target of rapamycin (mTOR) inhibitor, temsirolimus, decreased the expression of DNAJB8 and SOX2 and the ratio of SP cells. Taken together, the results indicate that heat shock induces DNAJB8 by activation of HSF1 and induces cancer stem-like cells.

DOI： 10.1111/cas.13501

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Colorectal cancer (CRC) is a mortal disease due to treatment resistance, recurrence and distant metastasis. Emerging evidence has revealed that a small sub-population of cancer cells termed cancer stem cells (CSCs)/ cancer-initiating cells (CICs) is endowed with high levels of tumor-initiating ability, self-renewal ability and differentiation ability and is responsible for treatment resistance, recurrence and distant metastasis. Eradication of CSCs/CICs is essential to improve current treatments. However, the molecular mechanisms by which CSCs/CICs are maintained are still elusive. In this study, we aimed to determine the molecular mechanisms by which colorectal (CR)-CSCs/CICs in are maintained human primary CRC cells. CR-CSCs/CICs were isolated by sphere-culture and the ALDEFLUOR assay, and transcriptome analysis revealed that the gene ST6 N-Acetylgalactosaminide Alpha-2,6-Sialyltransferase 1 (ST6GALNAC1) was expressed at high levels in CR-CSCs/CICs. Overexpression of ST6GALNAC1 enhanced the expression of sialyl-Tn (STn) antigen, which is carried by the CSC marker CD44, and increased the sphere-forming ability and resistance to a chemotherapeutic reagent. The opposite phenomena were observed by gene knockdown using siRNA. Furthermore, the Akt pathway was activated in ST6GANAC1-overexpressed cells, and activation of the pathway was cancelled by gene knockdown of galectin-3. The results indicate that ST6GALNAC1 has a role in the maintenance of CR-CSCs/CICs by activating the Akt pathway in cooperation with galectin-3 and that ST6GalNAC1 (or STn antigen) might be a reasonable molecule for CSC/CIC-targeting therapy.

DOI： 10.18632/oncotarget.22545

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BACKGROUND: It is essential that cytotoxic T-lymphocytes (CTLs) recognize tumor-associated antigens (TAAs) and exhibit antitumor effects in immunological responses for tumor rejection. However, only a few cases with oral squamous cell carcinoma (OSCC) have been studied for these antitumor mechanisms. We established a cancer cell line and autologous CTL pair for identifying TAAs in OSCC, despite difficulties in establishing such a pair. MATERIALS AND METHODS: A cell line (OTM) from a primary lesion and a CTL line (TcOTM) were derived from OSCC in a 64-year-old female patient. CTL clones were generated by repetitive limiting dilutions. Accurate characterization was performed by in vitro analysis. RESULTS: The TcOTM clone showed specific cytotoxic activity against OTM cells in an human leukocyte antigen (HLA)-A24-restricted manner. Furthermore, it exerted cytotoxicity against the allogenic HLA-A24 cell lines. CONCLUSION: These data indicated that the TcOTM clone recognized a TAA presented by HLA-A24 cells. This unique method will allow for identification of unknown TAAs for OSCC in the future.

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Chemotherapy has improved the prognosis of patients with sarcomas. However, it may suppress anti-tumor immunity. Recently, we reported a novel CD8+ memory T cell population with a chemo-resistance property, "young memory" T (TYM ) cells. In this study, we investigated the proportion and function of TYM cells in peripheral blood of healthy donors and sarcoma patients who received chemotherapy and those who did not. The proportion of TYM cells was significantly decreased in patients compared with that in healthy donors. In healthy donors, anti-EBV CTLs were induced using mixed lymphocyte peptide culture, from not only TYM cells but also TCM and TEM cells. No CTLs directed to tumor-associated antigens were induced. In sarcoma patients who did not receive chemotherapy, in addition to anti-EBV CTLs, CTLs directed to the tumor-associated antigen PBF were induced from TYM , TCM and TEM cells. In sarcoma patients who received chemotherapy, EBV-specific CTLs were induced from TYM cells but were hardly induced from TEM cells. Interestingly, CTLs directed to the anti-tumor-associated antigen PBF were induced from TYM cells but not from the TCM and TEM cells in sarcoma patients who received chemotherapy. The findings suggest that TYM cells are resistant to chemotherapy and can firstly recover from the nadir. TYM cells might be important for immunological memory, especially in sarcoma patients receiving chemotherapy.

DOI： 10.1111/cas.13319

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Cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) are defined by their higher tumor-initiating ability, self-renewal capacity and differentiation capacity. CSCs/CICs are resistant to several therapies including chemotherapy and radiotherapy. CSCs/CICs thus are thought to be responsible for recurrence and distant metastasis, and elucidation of the molecular mechanisms of CSCs/CICs are essential to design CSC/CIC-targeting therapy. In this study, we analyzed the molecular aspects of gynecological CSCs/CICs. Gynecological CSCs/CICs were isolated as ALDH1high cell by Aldefluor assay. The gene expression profile of CSCs/CICs revealed that several genes related to stress responses are preferentially expressed in gynecological CSCs/CICs. Among the stress response genes, a small heat shock protein HSP27 has a role in the maintenance of gynecological CSCs/CICs. The upstream transcription factor of HSP27, heat shock factior-1 (HSF1) was activated by phosphorylation at serine 326 residue (pSer326) in CSCs/CICs, and phosphorylation at serine 326 residue is essential for induction of HSP27. Immunohistochemical staining using clinical ovarian cancer samples revealed that higher expressions of HSF1 pSer326 was related to poorer prognosis. These findings indicate that activation of HSF1 at Ser326 residue and transcription of HSP27 is related to the maintenance of gynecological CSCs/CICs.

DOI： 10.18632/oncotarget.16361

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Colorectal carcinoma (CRC) is one of the most frequently diagnosed cancers and the leading cause of cancer-related death for both men and women. Recent studies have revealed that a small sub-population of cancer cells, termed cancer stem-like cells (CSCs)/cancer-initiating cells (CICs), are endowed with tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs are resistant to current therapies including chemotherapy and radiotherapy. Thus, CSCs/CICs are responsible for recurrence and metastasis, and eradication of CSCs/CICs is essential to cure cancer. In this study, we isolated CR-CSCs/CICs as sphere-cultured cells and found that a product derived from LY6/PLAUR domain containing 3 (LYPD3) is preferentially expressed in CSCs/CICs. Gene overexpression and gene knockdown experiments revealed that LYPD3 has a role in the maintenance of CR-CSCs/CICs. The findings provide a novel molecular insight into CR-CSCs/CICs.

DOI： 10.1016/j.bbrc.2017.02.112

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Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are small sub-population of cancer cells that are endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability. CSCs/CICs could be isolated as high aldehyde dehydrogenase 1 activity cells (ALDH1high) from various cancer samples. In this study, we isolated urothelial carcinoma CSCs/CICs as ALDHhigh cells and investigated the molecular aspects. ALDH1high cells showed greater sphere-forming ability and higher tumor-initiating ability in immune-deficient mice than those of ALDH1low cells, indicating that CSCs/CICs were enriched in ALDH1high cells. cDNA microarray analysis revealed that an ionotropic glutamate receptor glutamate receptor, ionotropic, kainate 2 (GRIK2) was expressed in ALDH1high cells at a higher level than that in ALDH1low cells. GRIK2 gene knockdown by siRNAs decreased the sphere-forming ability and invasion ability, whereas GRIK2 overexpression increased the sphere-forming ability, invasion ability and tumorigenicity, indicating that GRIK2 has a role in the maintenance of CSCs/CICs. Immunohistochemical staining revealed that higher levels of GRIK2 and ALDH1 expression were related to poorer prognosis in urinary tract carcinoma cases. The findings indicate that GRIK2 has a role in the maintenance of urothelial CSCs/CICs and that GRIK2 and ALDH1 can be prognosis prediction markers for urinary tract carcinomas.

DOI： 10.18632/oncotarget.16259

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Many human cancers have been reported to have enhanced expression of the immune checkpoint molecule programmed death-ligand 1 (PD-L1), which binds to programmed cell death-1 (PD-1) expressed on immune cells. PD-L1/PD-1 plays a role in inhibition of antitumor immunity by inducing T cell apoptosis and tolerance. Thus, it is crucial to elucidate mechanisms of PD-L1 expression on cancer cells. ERO1-α is an oxidase located in the endoplasmic reticulum. It is overexpressed in a variety of tumor types and it plays a role in disulfide bond formation in collaboration with PDI. Here, we investigated the influence of ERO1-α on expression of PD-L1 and immune escape. We demonstrated that ERO1-α augmented the expression of PD-L1 via facilitation of oxidative protein folding within PD-L1. In addition, we showed that overexpression of ERO1-α increased HIF-1α protein expression, resulting in an increase of PD-L1 mRNA as well as protein. In clinical cases, we observed that the expression of ERO1-α in triple negative breast cancer was related to the expression of PD-L1. Moreover, apoptosis of Jurkat leukemia T cells, which express PD-1, induced by tumor PD-L1 was inhibited when ERO1-α was depleted. The results suggest that targeting ERO1-α in tumor cells can be a novel approach for cancer immunotherapy. Therefore, the role of ERO1-α in tumor-mediated immunosuppression should be further explored.

DOI： 10.18632/oncotarget.14960

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Colorectal cancer (CRC) is one of the most common malignancy, and the prognosis is not still satisfactory due to treatment resistance, recurrence and distant metastasis. Cancer stem cells (CSCs)/cancer-initiating cells (CICs) is endowed with higher tumor-initiating ability, self-renewal ability and differentiation ability, and CSCs/CICs are resistant to treatments. Thus, CSCs/CICs are thought to be responsible for recurrence and distant metastasis, and eradication of CSCs/CICs is essential to cure CRCs. However, the molecular mechanisms of CSCs/CICs are remain unknown, and we aimed to elucidate molecular aspects of CR-CSCs/CICs in this study. We screened the transcriptome data of primary human CR-CSCs/CICs that we previously established, and found that LEM domain containing 1 (LEMD1) is preferentially expressed in CR-CSCs/CICs. LEMD1 belongs to cancer-testis (CT) antigen, and has five transcript variants (variant 1 [V1] - variant 5 [V5]). We found that LEMD1 V1, V2 and V3 is expressed in testis and CR-CSCs/CICs, whereas LEMD1 V4 and V5 is ubiquitously expressed. LEMD1 gene knockdown experiments using siRNAs and gene overexpression experiments revealed that LEMD1 has a role in the maintenance of CR-CSCs/CICs. These observations indicate that CR-CSC/CIC-specific LEMD1 variants are reasonable target of CR-CSC/CIC-targeted therapy.

DOI： 10.1016/j.bbrc.2017.02.081

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Accumulating evidence indicates that immune checkpoint inhibition-mediated cancer immunotherapies greatly improve the prognosis of certain types of cancer. This approach is now becoming a standard therapy, joining surgery, radiotherapy, and chemotherapy. Because the costs of antibody drugs are now a socioeconomic burden in many countries, an urgent need in cancer immunotherapy is the identification of relevant biomarkers that can predict therapy efficacy. Recent studies have reported that colorectal adenocarcinoma with hereditary or sporadic deficiency in mismatch repair (MMR) proteins has high antigenicity and that detection of these proteins could be a promising way to estimate clinical response. In this study of 135 patients with colorectal cancer, we used immunohistochemistry to investigate the correlation between deficiency in MMR proteins and expression of human leukocyte antigen (HLA) class I molecules, a prerequisite of cytotoxic T-cell-based immunotherapy. Interestingly, MMR protein deficiency was an independent risk factor for the impaired expression of HLA class I molecules (odds ratio (OR)=10.44, 95% confidence interval (CI)=3.15-34.62, p<0.001), suggesting the existence of a putative entity that we have named "adaptive immune escape". Moreover, our results might provide a potential novel biomarker for the selection of patients who would respond to cancer immunotherapies. At the same time, the results suggest that we have to overcome the impaired expression of HLA class I molecules to further improve the cure rate of cancer immunotherapies.

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Lung cancer is one of the most common malignancies with a high rate of mortality. Lung cancer stem-like cells (CSCs)/ cancer-initiating cells (CICs) play major role in resistance to treatments, recurrence and distant metastasis and eradication of CSCs/CICs is crucial to improve recent therapy. Cytotoxic T lymphocytes (CTLs) are major effectors of cancer immunotherapy, and CTLs recognize antigenic peptides derived from antigens that are presented by major histocompatibility complex (MHC) class I molecules. In this study, we analyzed the potency of a cancer-testis (CT) antigen, brother of the regulator of the imprinted site variant subfamily 6 (BORIS sf6), in lung CSC/CIC immunotherapy. BORIS sf6 mRNA was expressed in lung carcinoma cells (9/19), especially in sphere-cultured lung cancer stem-like cells, and in primary lung carcinoma tissues (4/9) by RT-PCR. Immunohistochemical staining using BORIS sf6-specific antibody revealed that high expression of BORIS sf6 is related to poorer prognosis. CTLs could be induced by using a human leukocyte antigen, (HLA)-A2 restricted antigenic peptide (BORIS C34_24(9)), from all of 3 HLA-A2-positive individuals, and CTL clone cells specific for BORIS C34_24(9) peptide could recognize BORIS sf6-positive, HLA-A2-positive lung carcinoma cells. These results indicate that BORIS sf6 is a novel target of lung cancer immunotherapy that might be useful for targeting treatment-resistant lung cancer stem-like cells.

DOI： 10.1371/journal.pone.0171460

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To establish an effective cancer immunotherapy, it is crucial that cancer cells present a cancer-specific antigen in a hypoxic area, a hallmark of the tumor microenvironment. Here, we show the impact of hypoxia on MHC class I antigen presentation in vitro and in vivo in murine tumors. Activation of antigen-specific CTLs by tumor cells that had been pre-incubated under a condition of hypoxia was enhanced compared with that by tumor cells pre-incubated under a condition of normoxia. Cell surface expression of MHC class I-peptide complex on the tumor cells was increased under a condition of hypoxia, thereby leading to higher susceptibility to specific CTLs. We show that the hypoxia-inducible ER-resident oxidase ERO1-α plays an important role in the hypoxia-induced augmentation of MHC class I-peptide complex expression. ERO1-α facilitated oxidative folding of MHC class I heavy chains, thereby resulting in the augmentation of cell surface expression of MHC class I-peptide complex under hypoxic conditions. These results suggest that since the expression of MHC class I-peptide complex is augmented in a hypoxic tumor microenvironment, strategies for inhibiting the function of regulatory T cells and myeloid-derived suppressor cells and/or immunotherapy with immune checkpoint inhibitors are promising for improving cancer immunotherapy.

DOI： 10.1002/eji.201646525

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The aim of peptide vaccination therapy is to stimulate and activate peptide-specific T cells to reject tumor cells. This strategy has been promoted by the discovery of tumor-associated antigens recognized by T cells. Peptide vaccination therapy can induce immune responses in some cancer patients but the objective clinical response rates are still low. To improve of the efficacy of peptide vaccination therapy: (i) cancer stem cell antigens specifically expressed in carcinoma/sarcoma stem-like cells but not in normal cells are needed; (ii) peptide vaccination therapy should be performed in the earlier stages; and (iii) memory T stem cells should be regulated to maintain long-lasting immune responses to the peptide vaccination.

DOI： 10.1111/pin.12438

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Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are reasonable targets for cancer therapy. However, recent studies have revealed that some non-CSCs/CICs have plastic ability and can dedifferentiate into CSCs/CICs. Therefore, an understanding of the molecular mechanisms that control the plasticity is essential to achieve CSC/CIC-targeting therapy. In this study, we analyzed the plasticity of lung cancer cells and found that lung non-CSCs/CICs can dedifferentiate into CSCs/CICs in accordance with the expression of stem cell transcription factor SOX2. SOX2 expression was induced by the transcription factor HOXA5. Oxidative stress repressed the expression of HDAC8 and then induced histone 3 acetylation and increased the expression of HOXA5 and SOX2. These findings indicate that lung cancer cells have plasticity under a condition of oxidative stress and that HOAX5 has a critical role in dedifferentiation.

DOI： 10.18632/oncotarget.10571

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INTRODUCTION: The use of immunotherapeutic challenges for sarcoma has a long history. Despite the existence of objective responses, immunotherapy has been overshadowed by the results of chemotherapy, especially for osteosarcoma. However, the prognosis for non-responders to chemotherapy is still poor and immunotherapy is now focused on again. AREAS COVERED: We reviewed the following types of clinical trials of immunotherapy for sarcoma: (i) vaccination with autologous tumor cells, (ii) vaccination with peptides derived from tumor-associated antigens, (iii) adoptive cell transfer using engineered T cells expressing T cell receptor directed at NY-ESO-1 and (iv) immune checkpoint inhibitors targeting CTLA-4 and PD1/PDL1. EXPERT OPINION: The immunogenicity of sarcoma might be lower than that of melanoma. Patients with small lesions who have not received any chemotherapy are good candidates for peptide-based immunotherapy. Combining peptide vaccination and immune checkpoint inhibitors is an attractive option, and long-lived memory T cells are attracting attention. Memory T stem cells defined by CD95+ are long-lived and have the capacity for self-renewal and multidifferentiation. We also identified a novel memory T cell population, young memory T cells defined by CD73+CXCR3+. Regulation of such memory T stem cells will be useful for peptide vaccination and adoptive cell transfer.

DOI： 10.1080/14712598.2016.1188075

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PURPOSE: Cancer-initiating cells (CICs) are thought to be essential for tumor maintenance, recurrence, and distant metastasis, and they are therefore reasonable targets for cancer therapy. Cancer immunotherapy is a novel approach to target cancer. In this study, we aimed to establish novel CIC-targeting immunotherapy. EXPERIMENTAL DESIGN: Colorectal cancer (CRC) CICs were isolated as side population (SP) cells. The gene expression profile of CRC CICs was analyzed by cDNA microarray and RT-PCR. Protein expression of olfactory receptor family 7 subfamily C member 1 (OR7C1) were analyzed by Western blot and immunohistochemical staining. The functions of OR7C1 were analyzed by gene overexpression and gene knockdown using siRNAs. OR7C1-positive cells were isolated by a flow cytometer and analyzed. CTLs specific for OR7C1 peptide were generated, and the antitumor effect was addressed by mice adoptive transfer model. RESULTS: OR7C1 has essential roles in the maintenance of colon CICs, and the OR7C1-positive population showed higher tumorigenicity than that of the OR7C1-negative population, indicating that OR7C1 is a novel functional marker for colon CIC. Immunohistochemical staining revealed that OR7C1 high expression was correlated with poorer prognosis in CRC patients. OR7C1-derived antigenic peptide-specific CTLs showed specific cytotoxicity for CICs, and an OR7C1-specific CTL clone showed a greater antitumor effect than did a CTL clone targeting all cancer cells in a CTL adoptive transfer mouse model. CONCLUSIONS: OR7C1 is a novel marker for colon CICs and can be a target of potent CIC-targeting immunotherapy. Clin Cancer Res; 22(13); 3298-309. ©2016 AACR.

DOI： 10.1158/1078-0432.CCR-15-1709

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Polyglutamine (polyQ) diseases comprise neurodegenerative disorders caused by expression of expanded polyQ-containing proteins. The cytotoxicity of the expanded polyQ-containing proteins is closely associated with aggregate formation. In this study, we report that a novel J-protein, DNAJ (HSP40) Homolog, Subfamily C, Member 8 (DNAJC8), suppresses the aggregation of polyQ-containing protein in a cellular model of spinocerebellar ataxia type 3 (SCA3), which is also known as Machado-Joseph disease. Overexpression of DNAJC8 in SH-SY5Y neuroblastoma cells significantly reduced the polyQ aggregation and apoptosis, and DNAJC8 was co-localized with the polyQ aggregation in the cell nucleus. Deletion mutants of DNAJC8 revealed that the C-terminal domain of DNAJC8 was essential for the suppression of polyQ aggregation, whereas the J-domain was dispensable. Furthermore, 22-mer oligopeptide derived from C-termilal domain could suppress the polyQ aggregation. These results indicate that DNAJC8 can suppress the polyQ aggregation via a distinct mechanism independent of HSP70-based chaperone machinery and have a unique protective role against the aggregation of expanded polyQ-containing proteins such as pathogenic ataxin-3 proteins.

DOI： 10.1016/j.bbrc.2016.03.152

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High-dose chemotherapy may kill not only tumor cells but also immunocytes, and frequently induces severe lymphocytopenia. On the other hand, patients who recover from the nadir maintain immunity against infection, suggesting the existence of an unknown memory T-cell population with stress resistance, long-living capacity, proliferation and differentiation. Recently, the differentiation system of T-cell memory has been clarified using mouse models. However, the human T-cell memory system has great diversity induced by natural antigens derived from many pathogens and tumor cells throughout life, and profoundly differs from the mouse memory system constructed using artificial antigens and transgenic T cells. Here we report a novel human T-cell memory population, "young memory" T (TYM) cells. TYM cells are defined by positive expression of CD73, which represents high aldehyde dehydrogenase 1 (ALDH1) activity and CXCR3 among CD8(+)CD45RA(+)CD62L(+) T cells. TYM proliferate upon TCR stimulation, with differentiation capacity into TCM and TEM and drug resistance. Moreover, TYM are involved in memory function for viral and tumor-associated antigens in healthy donors and cancer patients, respectively. Regulation of TYM might be very attractive for peptide vaccination, adoptive cell-transfer therapy and hematopoietic stem cell transplantation.

DOI： 10.1080/2162402X.2016.1165376

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BACKGROUND: Endoplasmic reticulum disulfide oxidase 1-α (ERO1-α) is an oxidase that exists in the endoplasmic reticulum and has a role in the formation of disulfide bonds of secreted proteins and cell-surface proteins. Recently, we reported that ERO1-α is present in high levels in various types of tumours, and that ERO1-α is a novel factor of poor prognosis. However, how ERO1-α affects a tumour in vivo and why patients who have a tumour with a high expression level of ERO1-α have a poor prognosis are still unknown. Therefore, to clarify the mechanism, we investigated the effect of ERO1-α on a tumour from the point of view of angiogenesis. METHODS: The effect of ERO1-α on tumour growth and angiogenesis was analysed by using non-obese diabetic-severe combined immunodeficient mice. The production of vascular endothelial growth factor (VEGF) in MDA-MB-231 cells with ERO1-α- overexpression or with ERO1-α knockdown was measured. The role of ERO1-α on VEGF expression was investigated. In triple-negative breast cancer cases, the relationship between expression of ERO1-α and angiogenesis was analysed. RESULTS: We found that the expression of ERO1-α promoted tumour growth in a mouse study and angiogenesis. The effects of ERO1-α on angiogenesis were mediated via oxidative protein folding of VEGF and enhancement of VEGF mRNA expression by using MDA-MB-231. In triple-negative breast cancer cases, the expression of ERO1-α related to the number of the blood vessel. Furthermore, we found that ERO1-α was a poor prognosis factor in triple-negative breast cancer. CONCLUSIONS: Our study has established a novel link between expression of ERO1-α and secretion of VEGF, providing new evidence for the effectiveness of ERO1-α-targeted therapy in patients with ERO1-α-expressed cancer.

DOI： 10.1038/bjc.2016.105

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Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as small subpopulation of cancer cells that are endowed with higher tumor-initiating ability. CSCs/CICs are resistant to standard cancer therapies including chemotherapy and radiotherapy, and they are thus thought to be responsible for cancer recurrence and metastasis. Therefore, elucidation of molecular mechanisms of CSCs/CICs is essential to cure cancer. In this study, we analyzed the gene expression profiles of gynecological CSCs/CICs isolated as aldehyde dehydrogenase high (ALDH(high)) cells, and found that MAPK13, PTTG1IP, CAPN1 and UBQLN2 were preferentially expressed in CSCs/CICs. MAPK13 is expressed in uterine, ovary, stomach, colon, liver and kidney cancer tissues at higher levels compared with adjacent normal tissues. MAPK13 gene knockdown using siRNA reduced the ALDH(high) population and abrogated the tumor-initiating ability. These results indicate that MAPK13 is expressed in gynecological CSCs/CICs and has roles in the maintenance of CSCs/CICs and tumor-initiating ability, and MAPK13 might be a novel molecular target for treatment-resistant CSCs/CICs.

DOI： 10.1016/j.bbrc.2016.03.004

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Cancer stem-like cells (CSC)/cancer-initiating cells (CIC) are defined as minor subpopulations of cancer cells that are endowed with properties of higher tumor-initiating ability, self-renewal ability and differentiation ability. Accumulating results of recent studies have revealed that CSC/CIC are resistant to standard cancer therapies, including chemotherapy, radiotherapy and molecular targeting therapy, and eradiation of CSC/CIC is, thus, critical to cure cancer. Cancer immunotherapy is expected to become the "fourth" cancer therapy. Cytotoxic T lymphocytes (CTL) play an essential role in immune responses to cancers, and CTL can recognize CSC/CIC in an antigen-specific manner. CSC/CIC express several tumor-associated antigens (TAA), and cancer testis (CT) antigens are reasonable sources for CSC/CIC-targeting immunotherapy. In this review article, we discuss CSC/CIC recognition by CTL, regulation of immune systems by CSC/CIC, TAA expression in CSC/CIC, and the advantages of CSC/CIC-targeting immunotherapy.

DOI： 10.1111/cas.12830

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Human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) can be isolated as side population (SP) cells, aldehyde dehydrogenase high (ALDHhigh) cells or cell surface marker-positive cells including CD44+ cells and CD133+ cells. CSCs/CICs and non-CSCs/CICs are unstable in in vitro culture, and CSCs/CICs can differentiate into non-CSCs/CICs and some non-CSCs/CICs can dedifferentiate into CSCs/CICs. Therefore, experiments using a large amount of CSCs/CICs are technically very difficult. In this study, we isolated single cell clones from SP cells and main population (MP) cells derived from the human colon cancer cell line SW480. SP analysis revealed that SP clone cells had relatively high percentages of SP cells, whereas MP clone cells showed very few SP cells, and the phenotypes were sustainable for more than 2 months of in vitro culture. Xenograft transplantation revealed that SP clone cells have higher tumor-initiating ability than that of MP clone cells and SP clone cell showed higher chemo-resistance compared with MP clone cells. These results indicate that SP clone cells derived from SW480 cells are enriched with CSCs/CICs, whereas MP clone cells are pure non-CSCs/CICs. SP clone cells and MP clone cells are a very stable in vitro CSC/CIC-enriched and non-CSC/CIC model for further analysis.

DOI： 10.1371/journal.pone.0158903

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We report the case of a 76-year-old man diagnosed with angioimmunoblastic T-cell lymphoma (AITL) with high serum vascular endothelial growth factor (VEGF) preceded by Remitting seronegative symmetrical synovitis with pitting edema syndrome. He suffered respiratory discomfort caused by large amounts of pleural effusion. Interestingly, changes in serum VEGF measured over time were similar to changes in pleural effusion. Whether VEGF is related to the pathological condition of AITL is a very important question.

DOI： 10.3109/14397595.2013.857836

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Plasmacytoid dendritic cells (pDCs) are the main producers of IFN-α in response to unmethylated DNA molecules, including cytosine guanine dinucleotide (CpG)-DNA in vivo. pDCs specifically express toll-like receptor (TLR) 9 and are therefore able to recognize the unmethylated DNAs. It has recently been shown that not only conventional DCs (cDCs) but also pDCs efficiently cross-present exogenous antigens after TLR9 activation. However, the precise molecular mechanism has remained unclear. Here, we show that pDCs secreted heat shock protein 72 (Hsp72) in response to CpG-A administration in a TLR9-dependent manner. Extracellular Hsp72 bound to an Hsp90-peptide complex and enhanced binding of Hsp90-peptide complex to pDC, resulting in efficient cross-presentation. Our experiments therefore suggest a mechanism for orchestration of immune responses by stimulation of pDCs with CpG-A.

DOI： 10.1016/j.imlet.2015.06.014

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ERO1-α is an oxidizing enzyme that exists in the endoplasmic reticulum and is induced under hypoxia. It reoxidizes the reduced form of protein disulfide isomerase that has oxidized target proteins. We found that ERO1-α is overexpressed in a variety of tumor types. MHC class I H chain (HC) has two disulfide bonds in the α2 and α3 domains. MHC class I HC folding is linked to the assembly of MHC class I molecules because only fully disulfide-bonded class I HCs efficiently assemble with β2-microglobulin. In this study, we show that ERO1-α associates with protein disulfide isomerase, calnexin, and immature MHC class I before being incorporated into the TAP-1-associated peptide-loading complex. Importantly, ERO1-α regulates the redox state as well as cell surface expression of MHC class I, leading to alteration of susceptibility by CD8(+) T cells. Similarly, the ERO1-α expression within cancer cells was associated with the expression level of MHC class I in colon cancer tissues. Thus, the cancer-associated ERO1-α regulates the expression of the MHC class I molecule via oxidative folding.

DOI： 10.4049/jimmunol.1303228

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The presentation of an exogenous antigen in a major histocompatibility complex class-I- restricted fashion to CD8(+) T cells is called cross-presentation. Heat shock proteins (HSPs) such as Hsp70, gp96, and Hsp90 have been shown to elicit efficient CTL responses by cross-presentation through an as-yet entirely unknown mechanism. Hsp90 is the most abundant cytosolic HSP and is known to act as a molecular chaperone. We have shown that a tumor antigen peptide complexed with Hsp90 could be cross-presented by dendritic cells (DCs) through an endosomal pathway in a murine system. However, it has not been determined whether human DCs also cross-present an Hsp90-peptide complex and induce peptide-specific CTLs. In this study, we found that an Hsp90-cancer antigen peptide complex was efficiently cross-presented by human monocyte-derived DCs and induced peptide-specific CTLs. Furthermore, we observed that the internalized Hsp90-peptide complex was strictly sorted to the Rab5(+), EEA1(+) static early endosome and the Hsp90-chaperoned peptide was processed and bound to MHC class I molecules through an endosome-recycling pathway. Our data indicate that targeting of the antigen to a "static" early endosome by Hsp90 is essential for efficient cross-presentation.

DOI： 10.1111/cas.12570

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The aim of the present study was to establish cancer stem-like cell/cancer-initiating cell (CSC/CIC)-targeting immunotherapy. The CSC/CIC are thought to be essential for tumor maintenance, recurrence and distant metastasis. Therefore they are reasonable targets for cancer therapy. In the present study, we found that a heat shock protein (HSP) 40 family member, DnaJ (Hsp40) homolog, subfamily B, member 8 (DNAJB8), is preferentially expressed in CSC/CIC derived from colorectal cancer (CRC) cells rather than in non-CSC/CIC. Overexpression of DNAJB8 enhanced the expression of stem cell markers and tumorigenicity, indicating that DNAJB8 has a role in CRC CSC/CIC. A DNAJB8-specific cytotoxic T lymphocyte (CTL) response could be induced by a DNAJB8-derived antigenic peptide. A CTL clone specific for DNAJB8 peptide showed higher killing activity to CRC CSC/CIC compared with non-CSC/CIC, and CTL adoptive transfer into CRC CSC/CIC showed an antitumor effect in vivo. Taken together, the results indicate that DNAJB8 is expressed and has role in CRC CSC/CIC and that DNAJB8 is a novel target of CRC CSC/CIC-targeting immunotherapy.

DOI： 10.1111/cas.12362

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Identification of antigenic peptides derived from tumor-associated antigens (TAA) enables cancer vaccine therapy using antigenic peptides. Here, we summarize the design of antigenic peptides and induction of cytotoxic T lymphocytes (CTL) using antigenic peptides and validation of CTL.

DOI： 10.1007/978-1-4939-0345-0_28

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Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are considered to be essential for tumor maintenance, recurrence and metastasis. Therefore, eradication of CSCs/CICs is essential to cure cancers. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we investigated the molecular mechanism of the cell growth of oral CSCs/CICs. Oral CSCs/CICs were isolated as aldehyde dehydrogenase 1 bright (ALDH1(br)) cells by the ALDEFLUOR assay. Small proline-rich protein-1B (SPRR1B) gene was shown to be overexpressed in ALDH1(br) cells by a cDNA microarray and RT-PCR. SPRR1B was shown to have a role in cell growth and maintenance of ALDH1(br) cells by SPRR1B overexpression and knockdown experiments. To elucidate the molecular mechanism by which SPRR1B regulates cell growth, further cDNA microarray analysis was performed using SPRR1B-overexpressed cells and cells with SPRR1B knocked down by siRNA. Expression of the tumor suppressor gene Ras association domain family member 4 (RASSF4) was found to be suppressed in SPRR1B-overexpressed cells. On the other hand, the expression of RASSF4 was enhanced in cells in which SPRR1B expression was knocked down by SPRR1B-specific siRNA. RASSF4 has an RA (Ras association) domain, and we thus hypothesized that RASSF4 modulates the MAP kinase signal downstream of the Ras signal. MAP kinase signal was activated in SPRR1B-overexpressed cells, whereas the signal was suppressed in SPRR1B knocked down cells. Taken together, the results indicate that the expression of SPRR1B is upregulated in oral CSCs/CICs and that SPRR1B has a role in cell growth by suppression of RASSF4.

DOI： 10.1016/j.bbrc.2013.08.021

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DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.

DOI： 10.1016/j.yexmp.2012.10.004

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Invasion into the matrix is one of hallmarks of malignant diseases and is the first step for tumor metastasis. Thus, analysis of the molecular mechanisms of invasion is essential to overcome tumor cell invasion. In the present study, we screened for colon carcinoma-specific genes using a cDNA microarray database of colon carcinoma tissues and normal colon tissues, and we found that fermitin family member-1 (FERMT1) is overexpressed in colon carcinoma cells. FRRMT1, FERMT2 and FERMT3 expression was investigated in colon carcinoma cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that only FERMT1 had cancer cell-specific expression. Protein expression of FERMT1 was confirmed by western blotting and immunohistochemical staining. To address the molecular functions of FERMT genes in colon carcinoma cells, we established FERMT1-, FERMT2- and FERMT3-overexpressing colon carcinoma cells. FERMT1-overexpressing cells exhibited greater invasive ability than did FERMT2- and FERMT3-overexpressing cells. On the other hand, FERMT1-, FERMT2- and FERMT3-overexpressing cells exhibited enhancement of cell growth. Taken together, the results of this study indicate that FERMT1 is expressed specifically in colon carcinoma cells, and has roles in matrix invasion and cell growth. These findings indicate that FERMT1 is a potential molecular target for cancer therapy.

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Language：English   Publishing type：Research paper (scientific journal)  

Survivin, a member of the inhibitor of apoptosis protein (IAP) family containing a single baculovirus IAP repeat domain, is highly expressed in cancerous tissues but not in normal counterparts. Our group identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), that is recognized by CD8 + CTLs and functions as an immunogenic molecule in patients with cancers of various histological origins such as colon, breast, lung, oral, and urogenital malignancies. Subsequent clinical trials with this epitope peptide alone resulted in clinical and immunological responses. However, these were not strong enough for routine clinical use as a therapeutic cancer vaccine, and our previous study of colon cancer patients indicated that treatment with a vaccination protocol of survivin-2B80-88 plus incomplete Freund's adjuvant (IFA) and α-interferon (IFNα) conferred overt clinical improvement and enhanced the immunological responses of patients. In the current study, we further investigated whether this vaccination protocol could efficiently provide not only improved immune responses but also better clinical outcomes for advanced pancreatic cancers. Tetramer and enzyme-linked immunosorbent spot analysis data indicated that more than 50% of the patients had positive clinical and immunological responses. In contrast, assessment of treatment with IFNα only to another group of cancer patients resulted in no obvious increase in the frequency of survivin-2B80-88 peptide-specific CTLs. Taken together, our data clearly indicate that a vaccination protocol of survivin-2B80-88 plus IFA and IFNα is very effective and useful in immunotherapy for this type of poor-prognosis neoplasm. This trial was registered with the UMIN Clinical Trials Registry, no. UMIN000000905.

DOI： 10.1111/cas.12046

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1111/tan.12113

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Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have self-renewal ability, differentiation ability and high tumor-initiating ability. CSCs/CICs are resistant to cancer therapies including chemotherapy and radiotherapy. Therefore, CSCs/CICs are thought to be responsible for cancer recurrence and distant metastasis after treatment. However, the molecular mechanisms of CSCs/CICs are still elusive. In this study, we isolated CSCs/CICs as side population (SP) cells from lung carcinoma, colon carcinoma and breast carcinoma cells and analyzed the molecular mechanisms of CSCs/CICs. cDNA micro-array screening and RT-PCR analysis revealed that sperm mitochondria-associated cysteine-rich protein (SMCP) is ectopically expressed in SP cells. 5'-Rapid amplification of cDNA end (RACE) analysis revealed that the SMCP transcript in SP cells was a variant form (termed vt2) which is composed from only one exon. SMCP vt2 was detected in only cancer cells, whereas the wild-type (vt1) form of SMCP was expressed in the testis. SMCP was shown to have a role in tumor initiation by SMCP overexpression and SMCP knockdown using siRNAs in lung cancer cells. Taken together, the initiation results indicate that an ectopically expressed variant form of SMCP has a role in tumor initiation of CSCs/CICs and that the variant form of SMCP might be a novel CSC/CIC marker and a potential and promising target of CSC/CIC-targeting therapy.

DOI： 10.1371/journal.pone.0069095

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Focal inflammation causes systemic fever. Cancer hyperthermia therapy results in shrinkage of tumors by various mechanisms, including induction of adaptive immune response. However, the physiological meaning of systemic fever and mechanisms of tumor shrinkage by hyperthermia have not been completely understood. In this study, we investigated how heat shock influences the adaptive immune system. We established a cytotoxic T lymphocyte (CTL) clone (#IM29) specific for survivin, one of the tumor-associated antigens (TAAs), from survivin peptide-immunized cancer patients' peripheral blood, and the CTL activities were investigated in several temperature conditions (37-41 °C). Cytotoxicity and IFN-γ secretion of CTL were greatest under 39 °C condition, whereas they were minimum under 41 °C. To address the molecular mechanisms of this phenomenon, we investigated the apoptosis status of CTLs, expression of CD3, CD8, and TCRαβ by flow cytometry, and expression of perforin, granzyme B, and Fas ligand by western blot analysis. The expression of perforin and granzyme B were upregulated under temperature conditions of 39 and 41 °C. On the other hand, CTL cell death was induced under 41 °C condition with highest Caspase-3 activity. Therefore, the greatest cytotoxicity activity at 39 °C might depend on upregulation of cytotoxic granule proteins including perforin and granzyme B. These results suggest that heat shock enhances effector phase of the adaptive immune system and promotes eradication of microbe and tumor cells.

DOI： 10.1007/s12192-012-0348-0

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Cancer stem-like cells (CSC) are a small population of cancer cells with superior tumor initiating, self-renewal, and differentiation properties. In this study, we show that the cancer-testis antigen and HSP40 family member DNAJB8 contributes to the CSC phenotype in renal cell carcinoma (RCC). DNAJB8 overexpression increased the percentage of side population (SP) cells representing CSCs in RCC cells, enhancing their tumor-initiating ability. Conversely, attenuation of DNAJB8 decreased SP cells and reduced tumor-initiating ability. The utility of DNAJB8 as an immunologic target was established in DNA vaccination experiments. Compared with immunization with the tumor-associated antigen survivin, which was expressed in both CSCs and non-CSCs in RCC, immunization with Dnajb8 expression plasmids yielded stronger antitumor effects. Together, our findings suggest that DNAJB8 plays a role in CSC maintenance and that it offers a candidate for CSC-targeting immunotherapy in RCC.

DOI： 10.1158/0008-5472.CAN-11-3062

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A novel monoclonal anti-pan human leukocyte antigen (HLA) class I heavy chain antibody, EMR8-5, was established. It could detect HLA-A, -B, and -C antigens in formalin-fixed paraffin embedded tissues. By immunohistochemical staining using the EMR8-5 antibody, various cancer tissues from 246 cases were examined for HLA class I expression. It was found that HLA class I expression was decreased in 20% to 42% of the cases of lung cancer, hepatocellular carcinoma, colon cancer, renal cell carcinoma, and urothelial carcinoma. In contrast, 85% of breast cancer cases had loss of or decreased HLA class I expression. Of the 35 breast cancer cases that had decreased HLA class I heavy chain expression, 33 (94%) also had decreased beta2-microglobulin expression detected by immunohistochemical staining. It was suggested that HLA class I down-regulation might be a common characteristic of breast cancer mostly caused by the down-regulation of beta2-microglobulin expression.

DOI： 10.1111/j.1440-1827.2012.02789.x

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We previously identified a human leukocyte antigen (HLA)-A24-restricted antigenic peptide, survivin-2B80-88, recognized by CD8+ cytotoxic T lymphocytes (CTL). Subsequently, we attempted clinical trials with this epitope peptide alone for some malignancies, resulting in clinical and immunological responses, although their potential was not strong enough for routine clinical use as a cancer vaccine. In the current study, to assess whether immunogenicity of the survivin-2B80-88 peptide could be enhanced with other vaccination protocols, we performed clinical trials in advanced colon cancer patients with two vaccination protocols: (i) survivin-2B80-88 plus incomplete Freund's adjuvant (IFA); and (ii) survivin-2B80-88 plus IFA and a type-I interferon (IFN), IFNα. Our data clearly indicated that, although the effect of survivin-2B80-88 plus IFA was not significantly different from that with survivin-2B80-88 alone, treatment with the vaccination protocol of survivin-2B80-88 plus IFA and IFNα resulted in clinical improvement and enhanced immunological responses of patients. Tetramer analysis of survivin-2B80-88 peptide-specific CTL demonstrated that such CTL were increased at least twofold after vaccination with this protocol in four of eight patients. In these patients, enzyme-linked immunosorbent spot (ELISPOT) results were also enhanced. Subsequent study of single-cell clone separation by cell sorting of peptide-specific CTL showed that each CTL clone was indeed not only peptide-specific but also cytotoxic against human cancer cells in the context of the expression of both HLA-A24 and survivin molecules. Taken together, these results indicate that vaccination of colon cancer patients with survivin-2B80-88 plus IFA and IFNα can be considered to be a very potent immunotherapeutic regimen, and that this protocol might work for other cancers.

DOI： 10.1111/j.1349-7006.2011.01918.x

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Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs.

DOI： 10.1016/j.yexmp.2009.09.021

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We reported previously a HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL), recognized by CD8(+) CTL. This peptide was derived from survivin protein, an inhibitor of apoptosis proteins, expressed in a variety of tumors, such as adenocarcinoma, squamous cell carcinoma, and malignant melanoma. In this report, we provide further evidence that survivin-2B80-88 peptide might serve as a potent immunogenic cancer vaccine for various cancer patients. Overexpression of survivin was detected in surgically resected primary tumor specimens of most breast and colorectal cancers and some gastric cancers as assessed by immunohistochemical study. HLA-A24/survivin-2B80-88 tetramer analysis revealed that there existed an increased number of CTL precursors in peripheral blood mononuclear cells (PBMC) of HLA-A24(+) cancer patients, and in vitro stimulation of PBMCs from six breast cancer patients with survivin-2B80-88 peptide could lead to increases of the CTL precursor frequency. Furthermore, CTLs specific for this peptide were successfully induced from PBMCs in all 7 (100%) patients with breast cancers, 6 of 7 (83%) patients with colorectal cancers, and 4 of 7 (57%) patients with gastric cancers. These data indicate that survivin expressed in tumor tissues is antigenic in cancer patients, and survivin-2B80-88-specific CTLs are present in PBMCs of various cancer patients. Our study raises the possibility that this peptide may be applicable as a general cancer vaccine to a large proportion of HLA-A24(+) cancer patients.

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Tightly regulated at the level of transcription, expression of MHC class II molecules varies significantly among gastrointestinal cancers. High levels of MHC class II expression are often associated with a better prognosis, which is indicative of the involvement of CD4+ lymphocytes in tumor suppression, but the molecular mechanism by which MHC class II expression is regulated remains unclear. In the present study, we investigated the expression of one inducible MHC class II molecule, HLA-DR, and its coactivators in a panel of colorectal and gastric cancer cell lines. Interferon-gamma induced expression of HLA-DR in 14 of 20 cell lines tested; the remaining six cell lines did not express HLA-DR. Analysis of the expression of transcription factors and coactivators associated with HLA-DR revealed that the loss of CIITA expression was closely associated with the absence of HLA-DR induction. Moreover, DNA methylation of the 5' CpG island of CIITA-PIV was detected in all cancer cells that lacked CIITA. The methylation and resultant silencing of CIITA-PIV depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3B, and their genetic inactivation restored CIITA-PIV expression. It thus appears that CIITA methylation is a key mechanism that enables some gastrointestinal cancer cells to escape immune surveillance.

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We first classified 12 malignant glioma cell lines into three different groups (types 1-3) with respect to major histocompatibility complex (MHC) class II expression and analyzed each group based on the different expression status of the class II transactivator (CIITA) gene. Glioma type 1 (2 of 12) showed constitutive expression of all class II molecules that might be mediated by activation of B cell-specific CIITA promoter III. Glioma type 2 represented the major phenotype (66.7 %) of malignant glioma cell lines, and MHC class II expression was induced by interferon-gamma (IFN-gamma) in this phenotype. Analysis of glioma tissue samples revealed that CIITA promoter IV was detected in 9 of 11 patients (81.8%); however, promoter III was only in two (18.2%). Moreover, cultured glioma cells obtained from a fresh tumor sample upregulated expression of CIITA and class II molecules in the presence of IFN-gamma, strongly suggesting that glioma type 2 might be predominant in glioma tissues. Glioma type 3 (2 of 12) showed CIITA transcripts but loss of MHC class II expression even in the presence of IFN-gamma. In addition, we determined that the constitutive MHC class II expression in the glioma cell lines (type 1) was the result of transcriptional activation of the CIITA gene. This phenomenon was mediated by global histone acetylation over 6 kb upstream from the transcriptional start site of CIITA promoter III. Moreover, stable transfection of CIITA promoter IV as well as promoter III into MHC class II inducible cell lines restored the constitutive expression of all class II molecules. These studies lay the foundation to understand the molecular basis for the expression of class II molecules in gliomas.

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A large number of human tumor antigens recognized by CD8+ cytotoxic T lymphocytes (CTL) have been identified. Some of them have been employed in clinical trials and have achieved some objective responses. However, little is known about those that are recognized by CD4+ T cells, except for a very few that were identified from melanomas. Previously, we reported that an oral squamous cell carcinoma (SCC) cell line, OSC-20, was effectively lysed by HLA-DRB1*08032 (HLA-DR8)-restricted autologous CD4+ T cell line, TcOSC-20. In this study, we performed two steps of chromatographic purification of the tumor cell lysate in combination with mass spectrometry. We found one reverse-phase high-performance liquid chromatography (RP-HPLC) fraction that was effectively recognized by the T cells. We analyzed the fraction by nano-liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/MS/MS) and found six representative ions. We could determine the primary amino acid sequence of each of the six ions. Three of them contained a potential HLA-DR8 binding motif, and TcOSC-20 showed a rather strong cytotoxic response to one of the synthetic peptides, namely, amino acid residues 321-336 of human alpha-enolase. Thus, several gene products of squamous cancer cells are endogenously processed and may be presented on HLA class II molecules, so that they could constitute target molecules for autologous CD4+ T cells.

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Mutations of the tumor suppressor gene PTEN have been reported in patients with Cowden disease (CD) and in several malignant tumors. We analyzed a germline mutation of the PTEN gene in a patient with CD and identified a 4-bp deletion in exon 8 of the PTEN gene. The same germline mutation was detected in 3 members of her family. The mutated gene was predicted to encode a C-terminal truncated PTEN protein. Immunoblotting analysis revealed that the expression level of the wild-type PTEN protein in the patient's lymphocytes was reduced to almost half the level of the control lymphocytes, and the predicted truncated mutant PTEN could not be detected. Since PTEN is known to function as a negative regulator of the phosphatidylinositol-3-kinase signal pathway that promotes cell survival, the patient's lymphocytes were tested for the resistance against the apoptotic stimulus. It was shown that the patient's lymphocytes were more resistant to apoptosis induced by calcium ionophore than the healthy control lymphocytes. These results indicate that the germline mutation of the PTEN gene and the consequent loss of heterozygous expression may lead to an increase in the survival potential of cells, thereby elucidating a role of PTEN in the pathogenesis of tumor generation and hyperplasia of lymphoid tissue in CD.

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Language：Japanese  

CiNii Article

CiNii Books

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Other Link： https://search.jamas.or.jp/link/ui/2021218154

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Language：Japanese  

CiNii Article

CiNii Books

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Other Link： https://search.jamas.or.jp/link/ui/2018307809

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Event date： 2021

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Event date： 2019

Presentation type：Symposium, workshop panel (nominated)  

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Event date： 2018

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Event date： 2018

Language：English  

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Event date： 2017

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