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

Brachyury is a transcription factor belonging to the T-box gene family and is involved in the posterior formation of the mesoderm and differentiation of chordates. As the overexpression of Brachyury is a poor prognostic factor in a variety of cancers, the establishment of Brachyury-targeted therapy would be beneficial for the treatment of aggressive tumors. Because transcription factors are difficult to treat with a therapeutic antibody, peptide vaccines are a feasible approach for targeting Brachyury. In this study, we identified Brachyury-derived epitopes that elicit antigen-specific and tumor-reactive CD4+ T cells that directly kill tumors. T cells recognizing Brachyury epitopes were present in patients with head and neck squamous cell carcinoma. Next, we focused on gemcitabine (GEM) as an immunoadjuvant to augment the efficacy of antitumor responses by T cells. Interestingly, GEM upregulated HLA class I and HLA-DR expression in tumor, followed by the upregulation of anti-tumor T cell responses. As tumoral PD-L1 expression was also augmented by GEM, PD-1/PD-L1 blockade and GEM synergistically enhanced the tumor-reactivity of Brachyury-reactive T cells. The synergy between the PD-1/PD-L1 blockade and GEM was also confirmed in a mouse model of head and neck squamous cell carcinoma. These results suggest that the combined treatment of Brachyury peptide with GEM and immune checkpoint blockade could be a promising immunotherapy against head and neck cancer.

DOI： 10.1007/s00262-023-03460-0

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The engagement of CD27 on lymphocytes with its ligand, CD70, on tumors is believed to mediate tumor immune evasion and the elevation of serum soluble CD27 (sCD27) levels in patients with CD70-positive malignancies. We previously showed that CD70 is expressed in extranodal natural killer/T-cell lymphoma, nasal type (ENKL), an Epstein-Barr virus (EBV)-related malignancy. However, little is known about serum sCD27 expression and its association with the clinical characteristics of, and the CD27/CD70 interaction in, ENKL. In the present study, we show that serum sCD27 is significantly elevated in the sera of patients with ENKL. The levels of serum sCD27 provided excellent diagnostic accuracy for discriminating patients with ENKL from healthy subjects, correlated positively with the levels of other diagnostic markers (lactate dehydrogenase, soluble interleukin-2 receptor, and EBV-DNA), and decreased significantly following treatment. Elevated serum sCD27 levels also correlated significantly with advanced clinical stage and tended to correspond with shorter survival, in patients with ENKL. Immunohistochemistry indicated that CD27-positive tumor-infiltrating immune cells exist adjacent to CD70-positive lymphoma cells. In addition, serum sCD27 levels in patients with CD70-positive ENKL were significantly higher than those in patients with CD70-negative ENKL, suggesting that the intra-tumoral CD27/CD70 interaction boosts the release of sCD27 in serum. Furthermore, the EBV-encoded oncoprotein latent membrane protein 1 upregulated CD70 expression in ENKL cells. Our results suggest that sCD27 may serve as a novel diagnostic biomarker and also may serve as a tool for evaluating the applicability of CD27/CD70-targeted therapies by predicting intra-tumoral CD70 expression and CD27/CD70 interaction in ENKL.

DOI： 10.1007/s00262-023-03394-7

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

Recent studies have shown that activation of the cGAS-STING pathway is a key process in antitumor immune responses and various kinds of STING agonists have been developed for cancer immunotherapy. Despite promising preclinical studies, preliminary clinical results have shown only a modest effect of STING agonists. There is therefore a need to develop more effective treatment strategies. Based on previous observations that COX-2 is frequently overexpressed not only in a variety of cancers but also in tumor myeloid cells and that it suppresses antitumor immunity and promotes tumor survival by producing PGE2, we investigated the antitumor effects of combination therapy with a STING agonist cGAMP and the selective COX-2 inhibitor celecoxib in mouse models. Combination treatment with cGAMP and celecoxib inhibited tumor growth compared with either monotherapy, and the combination therapy induced both local and systemic antitumor immunity. cGAMP treatment decreased PD-1 expression on tumor-infiltrating T-cells and enhanced T-cell activation in tumor-draining lymph nodes regardless of the presence of celecoxib. Meanwhile, although celecoxib treatment did not alter the frequency of CD4+ CD25+ Foxp3+ regulatory T-cells, it enhanced the expression of costimulatory molecules and glycolysis-associated genes in tumor-infiltrating CD11b+ Ly6G+ cells. Moreover, we also found that celecoxib decreased lactate efflux and increased the frequency of IFN-γ- and TNF-α-producing CD8+ T-cells in the tumor microenvironment. Taken together, our findings suggest that combined treatment with celecoxib may be an effective strategy to improve the antitumor efficacy of STING agonists.

DOI： 10.1002/ijc.34394

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Homeobox B7 (HOXB7) is a master regulatory gene that regulates cell proliferation and activates oncogenic pathways. Overexpression of HOXB7 correlates with aggressive behavior and poor prognosis in patients with cancer. However, the expression and role of HOXB7 in head and neck squamous cell carcinoma (HNSCC) remain unclear. In this study, we observed that most samples from patients with oropharyngeal cancer and HNSCC expressed HOXB7. As no direct inhibitor has been reported, we identified a potent peptide epitope to target HOXB7-expressing tumors through immune cells. A novel HOXB7-derived peptide epitope (HOXB78-25 ) elicited antigen-specific and tumor-reactive promiscuous CD4+ T cell responses. These CD4+ T cells produced γ-interferon (IFN-γ) and had the direct ability to kill tumors through granzyme B. Notably, downregulation of HOXB7 using siRNA enhanced human leukocyte antigen class II expression on tumor cells by decreasing the phosphorylation of MAPK. Mitogen-activated protein kinase inhibition augmented IFN-γ production by HOXB7-reactive CD4+ T cell responses without decreasing the expression of HOXB7. These results suggest that combining HOXB7 peptide-based vaccine with MAPK inhibitors could be an effective immunological strategy for cancer treatment.

DOI： 10.1111/cas.15619

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

Although neoantigens are one of the most favorable targets in cancer immunotherapy, it is less versatile and costly to apply neoantigen-derived cancer vaccines to patients due to individual variation. It is, therefore, important to find highly immunogenic antigens between tumor-specific or associated antigens that are shared among patients. Considering the cancer immunoediting theory, immunogenic tumor cells cannot survive in the early phase of tumor progression including two processes: elimination and equilibrium. We hypothesized that highly immunogenic molecules are allowed to be expressed in tumor cells after an immune suppressive tumor microenvironment was established, if these molecules contribute to tumor survival. In the current study, we focused on TWIST1 as a candidate for highly immunogenic antigens because it is upregulated in tumor cells under hypoxia and promotes tumor metastasis, which is observed in the late phase of tumor progression. We demonstrated that TWIST1 had an immunogenic peptide sequence TWIST1140-162 , which effectively activated TWIST1-specific CD4+ T-cells. In a short-term culture system, we detected more TWIST1-specific responses in breast cancer patients compared with in healthy donors. Vaccination with the TWIST1 peptide also showed efficient expansion of TWIST1-reactive HTLs in humanized mice. These findings indicate that TWIST1 is a highly immunogenic shared antigen and a favorable target for cancer immunotherapy.

DOI： 10.1111/cas.15429

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/cas.15429

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Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis, FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However, the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here, we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway, which is a crucial pathway for cancer development through FGFR signaling. Moreover, we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably, FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy, such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor, could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells.

DOI： 10.1080/2162402X.2021.2021619

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

Activation of STING signaling plays an important role in anti-tumor immunity, and we previously reported the anti-tumor effects of STING through accumulation of M1-like macrophages in tumor tissue treated with a STING agonist. However, myeloid cells express SIRPα, an inhibitory receptor for phagocytosis, and its receptor, CD47, is overexpressed in various cancer types. Based on our findings that breast cancer patients with highly expressed CD47 have poor survival, we evaluated the therapeutic efficacy and underlying mechanisms of combination therapy with the STING ligand cGAMP and an antagonistic anti-CD47 mAb using E0771 mouse breast cancer cells. Anti-CD47 mAb monotherapy did not suppress tumor growth in our setting, whereas cGAMP and anti-CD47 mAb combination therapy inhibited tumor growth. The combination therapy enhanced phagocytosis of tumor cells and induced systemic anti-tumor immune responses, which rely on STING and type I IFN signaling. Taken together, our findings indicate that coadministration of cGAMP and an antagonistic anti-CD47 mAb may be promising for effective cancer immunotherapy.

DOI： 10.1084/jem.20200792

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

OX40 (CD134) is a co-stimulatory molecule mostly expressed on activated T lymphocytes. Previous reports have shown that OX40 can be an immuno-oncology target and a clinical biomarker for cancers of various organs. In this study, we collected formalin-fixed paraffin-embedded tumor samples from 124 patients with small-cell lung cancer (SCLC) who had undergone surgery. We analyzed the expression profiles of OX40 and other relevant molecules, such as CD4, CD8, and Foxp3, in tumor stroma and cancer nest using immunohistochemistry and investigated their association with survival. High infiltration of OX40+ lymphocytes (OX40high) in tumor stroma was positively associated with relapse-free survival (RFS) and overall survival (OS) compared with low infiltration of OX40+ lymphocytes (OX40low) (RFS, median, 26.0 months [95% confidence interval (CI), not reached (NR)-NR] vs 13.2 months [9.1-17.2], p = .024; OS, NR [95% CI, NR-NR] vs 29.8 months [21.3-38.2], p = .049). Multivariate analysis revealed that OX40high in tumor stroma was an independent indicator of prolonged RFS. Moreover, RFS of patients with OX40high/CD4high in tumor stroma was significantly longer than that of patients with OX40low/CD4low. The RFS of patients with tumor stroma with OX40high/CD8high was significantly longer than that of patients with tumor stroma with OX40low/CD8high, OX40high/CD8low, or OX40low/CD8low. These findings suggest that OX40+ lymphocytes in tumor stroma play a complementary role in regulating the relapse of early-stage SCLC. Reinforcing immunity by coordinating the recruitment of OX40+ lymphocytes with CD4+ and CD8+ T cells in tumor stroma may constitute a potential immunotherapeutic strategy for patients with SCLC.

DOI： 10.1080/2162402x.2021.1971430

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

Stimulator of interferon genes (STING) contributes to anti-tumor immunity by activating antigen-presenting cells and inducing mobilization of tumor-specific T cells. A role for tumor-migrating neutrophils in the anti-tumor effect of STING-activating therapy has not been defined. We used mouse tumor transplantation models for assessing neutrophil migration into the tumor triggered by intratumoral treatment with STING agonist, 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumoral STING activation with cGAMP enhanced neutrophil migration into the tumor in an NF-κB/CXCL1/2-dependent manner. Blocking the neutrophil migration by anti-CXCR2 monoclonal antibody impaired T cell activation in tumor-draining lymph nodes (dLNs) and efficacy of intratumoral cGAMP treatment. Moreover, the intratumoral cGAMP treatment did not show any anti-tumor effect in type I interferon (IFN) signal-impaired mice in spite of enhanced neutrophil accumulation in the tumor. These results suggest that both neutrophil migration and type I interferon (IFN) induction by intratumoral cGAMP treatment were critical for T-cell activation of dLNs and the anti-tumor effect. In addition, we also performed in vitro analysis showing enhanced cytotoxicity of neutrophils by IFN-β1. Extrinsic STING activation triggers anti-tumor immune responses by recruiting and activating neutrophils in the tumor via two signaling pathways, CXCL1/2 and type I IFNs.

DOI： 10.1007/s00262-021-02864-0

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

Recent studies have revealed that tumor cells decrease their immunogenicity by epigenetically repressing the expression of highly immunogenic antigens to survive in immunocompetent hosts. We hypothesized that these epigenetically hidden "stealth" antigens should be favorable targets for cancer immunotherapy due to their high immunogenicity. To identify these stealth antigens, we treated human lung cell line A549 with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza) and its prodrug guadecitabine for 3 d in vitro and screened it using cDNA microarray analysis. We found that the gene encoding sperm equatorial segment protein 1 (SPESP1) was re-expressed in cell lines including solid tumors and leukemias treated with 5Aza, although SPESP1 was not detected in untreated tumor cell lines. Using normal human tissue cDNA panels, we demonstrated that SPESP1 was not detected in normal human tissue except for testis and placenta. Moreover, we found using immunohistochemistry SPESP1 re-expression in xenografts in BALB/c-nu/nu mice that received 5Aza treatment. To assess the antigenicity of SPESP1, we stimulated human CD4+ T-cells with a SPESP1-derived peptide designed using a computer algorithm. After repetitive stimulation, SPESP1-specific helper T-cells were obtained; these cells produced interferon-γ against HLA-matched tumor cell lines treated with 5Aza. We also detected SPESP1 expression in freshly collected tumor cells derived from patients with acute myeloid leukemia or lung cancer. In conclusion, SPESP1 can be classified as a stealth antigen, a molecule encoded by a gene that is epigenetically silenced in tumor cells but serves as a highly immunogenic antigen suitable for cancer immunotherapy.

DOI： 10.1111/cas.14973

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BMJ  

<sec><title>Background</title>While adoptive transfer of T-cells has been a major medical breakthrough for patients with B cell malignancies, the development of safe and effective T-cell-based immunotherapy for central nervous system (CNS) tumors, such as glioblastoma (GBM), still needs to overcome multiple challenges, including effective homing and persistence of T-cells. Based on previous observations that interleukin (IL)-17-producing T-cells can traffic to the CNS in autoimmune conditions, we evaluated CD8⁺ T-cells that produce IL-17 and interferon-γ (IFN-γ) (Tc17-1) cells in a preclinical GBM model.

</sec><sec><title>Methods</title>We differentiated Pmel-1 CD8⁺ T-cells into Tc17-1 cells and compared their phenotypic and functional characteristics with those of IFN-γ-producing CD8⁺ T (Tc1) and IL-17-producing CD8⁺ T (Tc17) cells. We also evaluated the therapeutic efficacy, persistence, and tumor-homing of Tc17-1 cells in comparison to Tc1 cells using a mouse GL261 glioma model.

</sec><sec><title>Results</title>In vitro, Tc17-1 cells demonstrated profiles of both Tc1 and Tc17 cells, including production of both IFN-γ and IL-17, although Tc17-1 cells demonstrated lesser degrees of antigen-specific cytotoxic activity compared with Tc1 cells. In mice-bearing intracranial GL261-Quad tumor and treated with temozolomide, Tc1 cells, but not Tc17-1, showed a significant prolongation of survival. However, when the T-cell transfer was combined with poly-ICLC and Pmel-1 peptide vaccine, both Tc1 and Tc17-1 cells exhibited significantly prolonged survival associated with upregulation of very late activation antigen−4 on Tc17-1 cells in vivo. Glioma cells that recurred following the therapy lost the susceptibility to Pmel-1-derived cytotoxic T-cells, indicating that immuno-editing was a mechanism of the acquired resistance.

</sec><sec><title>Conclusions</title>Tc17-1 cells were equally effective as Tc1 cells when combined with poly-ICLC and peptide vaccine treatment.

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DOI： 10.1136/jitc-2021-002426

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Identification of immunogenic tumor antigens, their corresponding T cell epitopes and the selection of effective adjuvants are prerequisites for developing effective cancer immunotherapies such as therapeutic vaccines. Murine double minute 2 (MDM2) is an E3 ubiquitin-protein ligase that negatively regulates tumor suppressor p53. Because MDM2 overexpression serves as a poor prognosis factor in various types of tumors, it would be beneficial to develop MDM2-targeted cancer vaccines. In this report, we identified an MDM2-derived peptide epitope (MDM232-46) that elicited antigen-specific and tumor-reactive CD4+ T cell responses. These CD4+ T cells directly killed tumor cells via granzyme B. MDM2 is expressed in head and neck cancer patients with poor prognosis, and the T cells that recognize this MDM2 peptide were present in these patients. Notably, Nutlin-3 (MDM2-p53 blocker), inhibited tumor cell proliferation, was shown to augment antitumor T cell responses by increasing MDM2 expression, HLA-class I and HLA-DR through class II transactivator (CIITA). These results suggest that the use of this MDM2 peptide as a therapeutic vaccine combined with MDM2 inhibitors could represent an effective immunologic strategy to treat cancer.

DOI： 10.1007/s00262-021-02940-5

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Placenta-specific 1 (PLAC1) is expressed primarily in placental trophoblasts but not in normal tissues and is a targetable candidate for cancer immunotherapy because it is a cancer testis antigen known to be up-regulated in various tumors. Although peptide epitopes capable of stimulating CD8 T cells have been previously described, there have been no reports of PLAC1 CD4 helper T lymphocyte (HTL) epitopes and the expression of this antigen in head and neck squamous cell carcinoma (HNSCC). Here, we show that PLAC1 is highly expressed in 74.5% of oropharyngeal and 51.9% of oral cavity tumors from HNSCC patients and in several HNSCC established cell lines. We also identified an HTL peptide epitope (PLAC131-50) capable of eliciting effective antigen-specific and tumor-reactive T cell responses. Notably, this peptide behaves as a promiscuous epitope capable of stimulating T cells in the context of more than one human leukocyte antigen (HLA)-DR allele and induces PLAC1-specific CD4 T cells that kill PLAC1-positive HNSCC cell lines in an HLA-DR-restricted manner. Furthermore, T-cells reactive to PLAC131-50 peptide were detected in the peripheral blood of HNSCC patients. These findings suggest that PLAC1 represents a potential target antigen for HTL based immunotherapy in HNSCC.

DOI： 10.1080/2162402X.2020.1856545

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

Colorectal cancer (CRC) patients with metastatic lesions have low 5-year survival rates. During metastasis, cancer cells often obtain unique characteristics such as epithelial-mesenchymal transition (EMT). Vimentin a biomarker contributes to EMT by changing cell shape and motility. Since abnormal phosphorylation is a hallmark of malignancy, targeting phosphorylated vimentin is a feasible approach for the treatment of metastatic tumors while sparing non-tumor cells. Recent evidence has revealed that both CD8 cytotoxic T lymphocytes (CTLs) and also CD4 helper T lymphocytes (HTLs) can distinguish post-translationally modified antigens from normal antigens. Here, we showed that the expression of phosphorylated vimentin was upregulated in metastatic sites of CRC. We also showed that a chemotherapeutic reagent augmented the expression of phosphorylated vimentin. The novel phosphorylated helper peptide epitopes from vimentin could elicit a sufficient T cell response. Notably, precursor lymphocytes that specifically reacted to these phosphorylated vimentin-derived peptides were detected in CRC patients. These results suggest that immunotherapy targeting phosphorylated vimentin could be promising for metastatic CRC patients.

DOI： 10.1007/s00262-020-02524-9

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

Although cisplatin (CDDP) has been used as a major chemotherapeutic drug for head and neck squamous cell carcinoma (HNSCC), its impact on T-cell functions is controversial. Therefore, we investigated the immunologic effects of CDDP and antitumor effects by combination therapy of CDDP with a ligand for stimulator of interferon genes, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Direct impacts of CDDP on T-cell functions were addressed by comparing T-cell functions between human subjects treated and untreated with CDDP. The immune responses and the efficacy of combination therapy using CDDP and cGAMP were assessed using BALB/c mice inoculated with mouse squamous cell carcinoma (SCC) cell lines. CDDP inhibited T-cell proliferation in a dose-dependent manner. T-cell functions of CDDP-treated HNSCC patients were comparable to those of healthy donors and CDDP-untreated HNSCC patients. In the mice bearing SCC cell lines, combination therapy using CDDP and cGAMP enhanced the gene expressions of CXCL9 and CXCL10 in the tumor tissues and inhibited tumor growth. The antitumor effect was cancelled by anti-CXCR3 monoclonal antibody. These findings suggest that the combination therapy using CDDP and an immunomodulating drug like cGAMP would be a rational cancer immunotherapy for patients with HNSCC.

DOI： 10.1016/j.bbrc.2019.11.107

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

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) originates from squamous epithelium of the upper aerodigestive tract and is the most common malignancy in the head and neck region. Among HNSCCs, oropharynx squamous cell carcinoma (OSCC) has a unique profile and is associated with human papillomavirus infection. Recently, anti-programmed cell death-1 monoclonal antibody has yielded good clinical responses in recurrent and/or metastatic HNSCC patients. Therefore, programmed death-ligand 1 (PD-L1) may be a favorable target molecule for cancer immunotherapy. Although PD-L1-expressing malignant cells could be targeted by PD-L1-specific CD8+ cytotoxic T lymphocytes, it remains unclear whether CD4+ helper T lymphocytes (HTLs) recognize and kill tumor cells in a PD-L1-specific manner. METHODS: The expression levels of PD-L1 and HLA-DR were evaluated using immunohistochemical analyses. MHC class II-binding peptides for PD-L1 were designed based on computer algorithm analyses and added into in vitro culture of HTLs with antigen-presenting cells to evaluate their stimulatory activity. RESULTS: We found that seven of 24 cases of OSCC showed positive for both PD-L1 and HLA-DR and that PD-L1241-265 peptide efficiently activates HTLs, which showed not only cytokine production but also cytotoxicity against tumor cells in a PD-L1-dependent manner. Also, an adoptive transfer of the PD-L1-specific HTLs significantly inhibited growth of PD-L1-expressing human tumor cell lines in an immunodeficient mouse model. Importantly, T cell responses specific for the PD-L1241-265 peptide were detected in the HNSCC patients. CONCLUSIONS: The cancer immunotherapy targeting PD-L1 as a helper T-cell antigen would be a rational strategy for HNSCC patients.

DOI： 10.1186/s12967-019-1957-5

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Nasal natural killer/T-cell lymphoma (NNKTL) is closely associated with Epstein-Barr virus (EBV) and is characterized by poor prognosis, resulting from rapid progression of lesions in the affected organs. Recent data have shown that NNKTL is associated with the aberrant expression of cyclin-dependent kinase 1 (CDK1) and its downstream target survivin, but little is known about the functional roles of CDK1 and survivin in NNKTL. In the current study, we show that knockdown of the EBV-encoded oncoprotein latent membrane protein 1 (LMP1) induces downregulation of CDK1 and survivin in NNKTL cells. Immunohistochemistry detected CDK1 and survivin expression in LMP1-positive cells of NNKTL biopsy specimens. Inhibition of CDK1 and survivin in NNKTL cells with several inhibitors led to a dose-dependent decrease in cell proliferation. In addition, the Sp1 inhibitor mithramycin, which can downregulate both CDK1 and survivin, significantly suppressed the growth of established NNKTL in a murine xenograft model. Our results suggest that LMP1 upregulation of CDK1 and survivin may be essential for NNKTL progression. Furthermore, targeting CDK1 and survivin with Sp1 inhibitors such as mithramycin may be an effective approach to treat NNKTL, which is considered to be a treatment-refractory lymphoma.

DOI： 10.1038/s41374-018-0182-9

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Informa UK Limited  

DOI： 10.1080/2162402x.2018.1466771

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

Stimulator of interferon genes (STING) was defined as an important molecule for promoting antitumor immunity through mediating type I interferon (IFN) production by sensing its ligands such as cyclic GMP-AMP (cGAMP). Our recent study indeed demonstrated that intratumoral injection of cGAMP showed effective antitumor responses via accumulating activated macrophages in the tumor microenvironment in a STING-dependent manner. Because the antitumor effect of cGAMP was abrogated when macrophages were depleted, the existence of the activated macrophages in the tumor site would be important for effective antitumor immune responses. Macrophages show phenotypic diversity and plasticity and are categorized into several groups by stimulation factors, e.g. IFN-γ and IL-4 for M1 and M2 macrophages, respectively. However, the impact of STING stimulation on the macrophage activation status remains to be evaluated. Here we summarize the complex polarized status of macrophages and the signaling cascade triggered by STING stimulation and also discuss the impact of STING signaling on the macrophage activation status for future directions.

DOI： 10.1080/21645515.2017.1395995

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

Despite recent advances in treatment for melanoma patients through using immune checkpoint inhibitors, these monotherapies have limitations and additional treatments have been explored. Type I IFNs have been used to treat melanoma and possess immunomodulatory effects including enhancement of T-cell infiltration. T-cell plays a critical role in immune checkpoint therapies via restoration of effector functions and tumor infiltration by T-cells predicts longer survival in a variety of cancer types. Moreover, tumor-infiltrating T-cells are associated with the expression of chemokines such as CCL5 and CXCR3 ligands in tumor tissues. We therefore investigated whether intratumoral injection of IFN-β induces the expression of CCL5 and CXCR3 ligands in melanoma cells and has additional antitumor effects when combined with anti-PD-L1 mAb treatment. IFN-β treatment enhanced CD8+ T-cell infiltration into tumors and CCL5 and CXCR3 ligand expression. In vivo studies using a mouse model showed that monotherapy with IFN-β, but not with anti-PD-L1 mAb, inhibited tumor growth in comparison to control. However, the therapeutic efficacy of IFN-β was significantly enhanced by the addition of anti-PD-L1 mAb. This antitumor response of combination therapy was abrogated by anti-CD8 mAb and IFN-β augmented the neoantigen-specific T-cell response of anti-PD-L1 mAb. Our findings suggest that IFN-β induces the expression of CCL5 and CXCR3 ligands in melanoma, which could play a role in T-cell recruitment, and enhances the efficacy of anti-PD-L1 mAb treatment in a CD8-dependent manner.

DOI： 10.1016/j.bbrc.2017.06.072

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Nasal natural killer/T-cell lymphoma (NNKTL) is an aggressive neoplasm with poor therapeutic responses and prognosis. The programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) pathway plays an important role in immune evasion of tumor cells through T-cell exhaustion. The aim of the present study was to examine the expression of PD-L1 and PD-1 molecules in NNKTL. We detected the expression of PD-L1 in biopsy samples from all of the NNKTL patients studied. PD-L1 was found on both malignant cells and tumor-infiltrating macrophages, while PD-1-positive mononuclear cells infiltrated the tumor tissues in 36% of patients. Most significantly, soluble PD-L1 (sPD-L1) was present in sera of NNKTL patients at higher levels as compared to healthy individuals and the levels of serum sPD-L1 in patients positively correlated with the expression of PD-L1 in lymphoma cells of tumor tissues. In addition, the high-sPD-L1 group of patients showed significantly worse prognosis than the low-sPD-L1 group. Furthermore, we confirmed that membrane and soluble PD-L1 was expressed on the surface and in the culture supernatant, respectively, of NNKTL cell lines. The expression of PD-L1 was observed in tumor tissues and sera from a murine xenograft model inoculated with an NNKTL cell line. Our results suggest that sPD-L1 could be a prognostic predictor for NNKTL and open up the possibility of immunotherapy of this lymphoma using PD-1/PD-L1 axis inhibitors.

DOI： 10.1007/s00262-017-1987-x

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

Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2',5')pA(3',5')p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45+ CD11bmid Ly6C+ cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8+ T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11, IFN-induced molecules, MX dynamin-like GTPase 1 (Mx1) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1), nitric oxide synthase 2 (Nos2), and interferon beta 1 (Ifnb1). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds.

DOI： 10.1007/s00262-017-1975-1

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

Tumor immune escape has been a major problem for developing effective immunotherapy. The human leukocyte antigen G (HLA-G) is a non-classical MHC class I molecule whose primary function is to protect the fetus from the mother's immune system. While HLA-G is hardly found in normal adult tissues, various tumor cells are known to express it, aiding their escape from the immune system. Thus, HLA-G is an attractive immunotherapy target. CD4(+) helper T lymphocytes (HTLs) play an important role in the immune reaction against tumors by assisting in the generation and persistence of CD8(+) cytotoxic T lymphocytes (CTLs) or by displaying direct antitumor effects. We report here that HLA-G expression in breast cancer significantly correlates with a poor prognosis. Also, we describe that the MHC class II-binding peptide HLA-G26-40 was effective in eliciting tumor-reactive CD4(+) T cell responses. Furthermore, treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased HLA-G expression in tumors and subsequently enhanced recognition by HLA-G26-40-specific HTLs. These findings predict that a combination immunotherapy targeting HLA-G together with a DNA methyltransferase inhibitor could be useful against some cancers.

DOI： 10.1080/2162402X.2016.1169356

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HER-3 expression has been reported to act as an important oncoprotein in head and neck squamous cell carcinoma. This protein is known to control tumor proliferation and acquisition of resistance by tumor cells towards EGFR inhibitors, therefore, development of a HER-3-targeted therapy is desirable. In this study, we found that HER-3 expression on tumor cells was increased after EGFR inhibition. To establish a novel therapeutic approach for HER-3-positive head and neck carcinoma, we identified a HER-3 helper epitope that could elicit effective helper T cell responses to the naturally processed HER-3-derived epitope presented in a HER-3 expressing tumors. This epitope induced potent cytolytic activity of CD4 T cells against such tumor cells. Moreover, pan HER-family tyrosine kinase inhibitor augmented the responses of HER-3-reactive CD4 T cells via upregulation of HLA-DR protein on the surface of tumor cells. Our results supports the validity of CD4 T cell-dependent HER-3-targeted therapy combined with a broad inhibitor of HER-family.

DOI： 10.1038/srep16280

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To elucidate mechanisms underlying epidemiological findings of decreased risk of glioma development in patients with allergies and asthma, gliomas were induced in mice deficient for histidine decarboxylase (HDC), the enzyme responsible for histamine production. These mice exhibited shortened survival and enhanced tumor growth compared to wild-type (WT) mice. Previous studies have shown a pivotal role of HDC in maturation of bone marrow (BM)-derived myeloid cells. In our glioma models, brain-infiltrating leukocytes (BIL) demonstrated an increased frequency of CD11b+Gr1+ immature myeloid cells (IMC; both CD11b+Ly6G+ and CD11b+Ly6C+ subpopulations) as well as diminished CD8+ T cell infiltration and their effector functions in HDC-/- mice compared with WT mice. Furthermore, HDC-/- IMC demonstrated a more profound immune suppression of CD8+ T cell proliferation and functions associated with increased prostaglandin E2 (PGE2) expression levels. Celecoxib, a cyclooxygenase-2 inhibitor, which is vital for PGE2 production, abrogated suppressive capabilities of HDC-/- IMC. In addition, glioma-bearing HDC-eGFP mice, in which HDC promoter drives green fluorescence protein (GFP) expression, exhibited decreased HDC promoter activities in CD11b+Gr1+ cells in the BM, spleen, and intracranial tumor site compared with non-tumor bearing HDC-eGFP mice. Additionally, in vitro culture with glioma supernatants decreased GFP expression in CD11b+Gr1+, CD11b+Ly6G+, and CD11b+Ly6C+ IMC. HDC expression levels inversely correlated with suppressive functions of CD11b+Gr1+ IMC, as GFP-CD11b+Gr1+ more profoundly inhibited CD8+ T cell proliferation compared with CD11b+Gr1+GFP+ cells. Taken together, these data show a significant role of HDC in the glioma microenvironment via maturation of myeloid cells and resulting activation of CD8+ T cells.

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Nasal natural killer/T-cell lymphoma (NNKTL) is associated with Epstein-Barr virus and has a poor prognosis because of local invasion and/or multiple dissemination. Various chemokines play a role in tumor proliferation and invasion, and chemokine receptors including the C-C chemokine receptor 4 (CCR4) are recognized as potential targets for treating hematologic malignancies. The aim of the present study was to determine whether specific chemokines are produced by NNKTL. We compared chemokine expression patterns in culture supernatants of NNKTL cell lines with those of other lymphoma or leukemia cell lines using chemokine protein array and ELISA. Chemokine (C-C motif) ligand (CCL) 17 and CCL22 were highly produced by NNKTL cell lines as compared to the other cell lines. In addition, CCL17 and CCL22 were readily observed in the sera of NNKTL patients. The levels of these chemokines were significantly higher in patients than in healthy controls. Furthermore, we detected the expression of CCR4 (the receptor for CCL17 and CCL22) on the surface of NNKTL cell lines and in tissues of NNKTL patients. Anti-CCR4 monoclonal antibody (mAb) efficiently induced antibody-dependent cellular cytotoxicity mediated by natural killer cells against NNKTL cell lines. Our results suggest that CCL17 and CCL22 may be important factors in the development of NNKTL and open up the possibility of immunotherapy of this lymphoma using anti-CCR4 mAb.

DOI： 10.1007/s00262-015-1675-7

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We recently reported that STING contributes to antiglioma immunity by triggering type I IFN induction in glioma microenvironment. Moreover, intratumoral administration of STING agonist improved the efficacy of peptide vaccination in a mouse glioma model, suggesting the rational use of STING agonists in the immunotherapy of brain tumor.

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MicroRNAs (miRs) play important roles in regulation of a variety of cell functions, including immune responses. We have previously demonstrated that miR-17-92 expression in T-cells enhances Th1 phenotype and provides a long-term protection against glioblastoma when co-expressed as a transgene in T-cells along with a chimeric antigen receptor. To further elucidate the function of miR-17-92 in tumor antigen-specific CD8(+) T-cells, we generated transgenic (Tg) mice in which CD8(+) T-cells overexpress transgene-derived miR-17-92 under the lck promoter as well as T-cell receptor specific for human gp10025-33 (Pmel-1) (miR-17-92/Pmel-Tg). CD8(+) T-cells from miR-17-92/Pmel-Tg mice demonstrated enhanced interferon (IFN)-γ production and cytotoxicity in response to the cognate antigen compared with those from control Pmel-Tg mice without the transgene for miR-17-92. In addition, miR-17-92/Pmel-Tg mouse-derived CD8(+)CD44(+) T-cells demonstrated increased frequencies of cells with memory phenotypes and IFN-γ production. We also found that miR-17-92/Pmel-Tg-derived CD8(+) T-cells expressed decreased levels of transforming growth factor (TGF)-β type II receptor (TGFBR2) on their surface, thereby resisting against suppressive effects of TGF-β1. Our findings suggest that engineering of tumor antigen-specific CD8(+) T-cells to express miR-17-92 may improve the potency of cancer immunotherapy.

DOI： 10.1016/j.bbrc.2015.02.003

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Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).

DOI： 10.1126/scitranslmed.aaa4963

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Background: The expression of c-Met and its ligand HGF plays a critical role in cell proliferation and is involved in numerous malignancies. Because c-Met expression and its role in NK/T-cell lymphoma remain unclear, we studied the expression and function of c-Met in NK/T-cell lymphoma cells. In addition, we investigated the possibility that c-Met could function as a tumor-associated antigen for helper T lymphocytes (HTLs). Methods: We evaluated whether HGF and c-Met were expressed in NK/T-cell lymphoma and the capacity of predicted c-Met HTL epitopes to induce antitumor responses in vitro. In addition, c-Met inhibitor was evaluated for the ability to inhibit TGF-β production in tumor and subsequently increase HTL recognition. Results: c-Met and HGF were expressed in NK/T-cell lymphoma cell lines, nasal NK/T-cell lymphoma specimens and patient serum samples. Moreover, HGF was shown to promote NK/T cell lymphoma (NKTCL) proliferation in an autocrine manner. Furthermore, we have identified three novel c-Met HTL epitopes that were restricted by several HLA-DR molecules. Notably, peptide-induced HTL lines directly recognized and killed c-Met expressing NK/T-cell lymphomas and various epithelial solid tumors. The c-Met specific HTLs could also recognize dendritic cells (DCs) pulsed with c-Met expressing tumor cell lysates. In addition, we observed that c-Met inhibition augmented HTL recognition by decreasing TGF-β production by tumor cells. Lastly, autophagy partly regulated the HTL responses against tumors. Conclusions: We identified novel c-Met HTL epitopes that can elicit effective antitumor responses against tumors expressing c-Met. Our results provide the rationale of combining c-Met targeting therapy and immunotherapy for NKTCLs and epithelial tumors.

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Although type I IFNs play critical roles in antiviral and antitumor activity, it remains to be elucidated how type I IFNs are produced in sterile conditions of the tumor microenvironment and directly affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b(+) brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing Sting(Gt) (/Gt) mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of Sting(Gt) (/Gt) mice showed increased CD11b(+) Gr-1(+) immature myeloid suppressor and CD25(+) Foxp3(+) regulatory T cells (Treg) and decreased IFNγ-producing CD8(+) T cells. CD4(+) and CD8(+) T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations.

DOI： 10.1158/2326-6066.CIR-14-0099

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We identified novel helper epitope peptides of Survivin cancer antigen, which are presented to both HLA-DRB1*01:01 and DQB1*06:01. The helper epitope also contained three distinct Survivin-killer epitopes presented to HLA-A*02:01 and A*24:02. This 19 amino-acids epitope peptide (SU18) induced weak responses of Survivin-specific CD4(+) and CD8(+) T cells though it contained both helper and killer epitopes. To enhance the vaccine efficacy, we synthesized a long peptide by conjugating SU18 peptide and another DR53-restricted helper epitope peptide (SU22; 12 amino-acids) using glycine-linker. We designated this artificial 40 amino-acids long peptide containing two helper and three killer epitopes as Survivin-helper/killer-hybrid epitope long peptide (Survivin-H/K-HELP). Survivin-H/K-HELP allowed superior activation of IFN-γ-producing CD4(+) Th1 cells and CD8(+) Tc1 cells compared with the mixture of its component peptides (SU18 and SU22) in the presence of OK-432-treated monocyte-derived DC (Mo-DC). Survivin-H/K-HELP-pulsed Mo-DC pretreated with OK-432 also exhibited sustained antigen-presentation capability of stimulating Survivin-specific Th1 cells compared with Mo-DC pulsed with a mixture of SU18 and SU22 short peptides. Moreover, we demonstrated that Survivin-H/K-HELP induced a complete response in a breast cancer patient with the induction of cellular and humoral immune responses. Thus, we believe that an artificially synthesized Survivin-H/K-HELP will become an innovative cancer vaccine.

DOI： 10.1016/j.imlet.2014.04.010

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Malignant gliomas are heavily infiltrated by immature myeloid cells that mediate immunosuppression. Agonistic CD40 monoclonal antibody (mAb) has been shown to activate myeloid cells and promote antitumor immunity. Our previous study has also demonstrated blockade of cyclooxygenase-2 (COX-2) reduces immunosuppressive myeloid cells, thereby suppressing glioma development in mice. We therefore hypothesized that a combinatory strategy to modulate myeloid cells via two distinct pathways, i.e., CD40/CD40L stimulation and COX-2 blockade, would enhance anti-glioma immunity. We used three different mouse glioma models to evaluate therapeutic effects and underlying mechanisms of a combination regimen with an agonist CD40 mAb and the COX-2 inhibitor celecoxib. Treatment of glioma-bearing mice with the combination therapy significantly prolonged survival compared with either anti-CD40 mAb or celecoxib alone. The combination regimen promoted maturation of CD11b(+) cells in both spleen and brain, and enhanced Cxcl10 while suppressing Arg1 in CD11b(+)Gr-1(+) cells in the brain. Anti-glioma activity of the combination regimen was T-cell dependent because depletion of CD4(+) and CD8(+) cells in vivo abrogated the anti-glioma effects. Furthermore, the combination therapy significantly increased the frequency of CD8(+) T-cells, enhanced IFN-γ-production and reduced CD4(+)CD25(+)Foxp3(+) T regulatory cells in the brain, and induced tumor-antigen-specific T-cell responses in lymph nodes. Our findings suggest that the combination therapy of anti-CD40 mAb with celecoxib enhances anti-glioma activities via promotion of type-1 immunity both in myeloid cells and T-cells.

DOI： 10.1007/s00262-014-1561-8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Cancer/testis (CT) antigens, which are expressed in various cancer cells but not in normal cells except germline cells of the testis, have been used as targets for cancer vaccine therapy. 5-Aza-2'-deoxycytidine (DAC), a potent inhibitor of genomic and promoter-specific DNA methylation, inhibits DNA methyltransferase activity and is reported to induce the expression of certain CT antigens by the demethylation of promoter CpG islands of the treated cells. Here, using DAC-treated cancer cells, we searched for novel attractive target molecules that would be useful for cancer immunotherapy and found a meiosis-specific protein, meiosis specific with OB domains (MEIOB), to be a novel CT antigen. Indeed, the MEIOB gene is expressed only in the testis and not in other normal tissues. The mRNA expression of MEIOB was greatly enhanced in several lung cancer cell lines after the treatment with DAC. Furthermore, we identified a variety of helper epitopes of the MEIOB antigen, which were recognized by MEIOB antigen-specific T cells in a HLA-restriction manner. Finally, we demonstrated that IFN-gamma production of MEIOB peptide-specific helper T cells in response to HLA-matched cancer cells was greatly augmented by treatment with DAC and IFN-gamma. Taken together, these findings show DAC to be a promising tool for finding novel CT antigens and for developing a future novel combination cancer vaccine chemotherapy. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.imlet.2014.01.004

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BACKGROUND: Expression of miR-17-92 enhances T-cell survival and interferon (IFN)-γ production. We previously reported that miR-17-92 is down-regulated in T-cells derived from glioblastoma (GBM) patients. We hypothesized that transgene-derived co-expression of miR17-92 and chimeric antigen receptor (CAR) in T-cells would improve the efficacy of adoptive transfer therapy against GBM. METHODS: We constructed novel lentiviral vectors for miR-17-92 (FG12-EF1a-miR-17/92) and a CAR consisting of an epidermal growth factor receptor variant III (EGFRvIII)-specific, single-chain variable fragment (scFv) coupled to the T-cell receptor CD3ζ chain signaling module and co-stimulatory motifs of CD137 (4-1BB) and CD28 in tandem (pELNS-3C10-CAR). Human T-cells were transduced with these lentiviral vectors, and their anti-tumor effects were evaluated both in vitro and in vivo. RESULTS: CAR-transduced T-cells (CAR-T-cells) exhibited potent, antigen-specific, cytotoxic activity against U87 GBM cells that stably express EGFRvIII (U87-EGFRvIII) and, when co-transduced with miR-17-92, exhibited improved survival in the presence of temozolomide (TMZ) compared with CAR-T-cells without miR-17-92 co-transduction. In mice bearing intracranial U87-EGFRvIII xenografts, CAR-T-cells with or without transgene-derived miR-17-92 expression demonstrated similar levels of therapeutic effect without demonstrating any uncontrolled growth of CAR-T-cells. However, when these mice were re-challenged with U87-EGFRvIII cells in their brains, mice receiving co-transduced CAR-T-cells exhibited improved protection compared with mice treated with CAR-T-cells without miR-17-92 co-transduction. CONCLUSION: These results warrant the development of novel CAR-T-cell strategies that incorporate miR-17-92 to improve therapeutic potency, especially in patients with GBM.

DOI： 10.1186/2051-1426-1-21

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A patient with pulmonary metastasis of colon cancer was treated with artificially synthesized helper/killer-hybrid epitope long peptide (H/K-HELP) of MAGE-A4 cancer antigen. The patient was vaccinated with MAGE-A4-H/K-HELP combined with OK432 and Montanide ISA-51. There were no severe side-effects except for a skin reaction at the injection site. MAGE-A4-H/K-HELP induced MAGE-A4-specific Th1 and Tc1 immune responses and the production of MAGE-A4-specific complement-fixing IgG antibodies. Tumor growth and carcinoembryonic antigen tumor marker were significantly decreased in the final diagnosis. This is the first report that artificially synthesized MAGE-A4-H/K-HELP induces Th1-dependent cellular and humoral immune responses in a human cancer patient.

DOI： 10.1111/j.1349-7006.2011.02106.x

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Graft-versus-host reaction (GVHR) is considered as a problem in hematopoietic cell transplantation. We found that CD45RB(high) CD62L(+) naïve CD4(+) T cells from wild-type B10D2 (H-2d MMTV6(-)) mice immediately differentiated into effector T cells producing high-levels of various cytokines after the transfer into BALB/c RAG2(-/-) (H-2d MMTV6(+)) mice. The expanded CD4(+) T cells, which have almost TCR Vβ3 chain, recognized the minor antigen of recipient mice and brought typical severe GVHR symptoms such as eyelid irritation, diarrhea, and liver failure. Eventually, all of the recipient mice transferred CD4(+) T cells was dead within 10 days. We demonstrated here that blockade of IL-6 signaling by administration of anti-IL-6 receptor (IL-6R) monoclonal antibody (mAb) remarkably inhibited the CD4(+) T cell-mediated lethal GVHR. In addition, we confirmed that the in vivo injection of anti-IL-6R mAb prevented the generation of effector CD4(+) T cells which produce the inflammatory cytokines such as IFN-γ, TNF-α, and IL-17. These findings indicated that IL-6 was a critical factor in the CD4(+) T cell-dependent acute GVHR induced by a minor-antigen, suggesting that IL-6-mediated signaling pathway would be a strong therapeutic target in T cell-mediated GVHR as well as other diseases including autoimmune and inflammation.

DOI： 10.1016/j.imlet.2011.01.004

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Type-1 immunity has an essential role for our host defenses against cancer and outer pathogens such as bacteria and virus. We demonstrated here that the edible plant extract of Chrysanthemum coronarium L. (C. coronarium) remarkably activates Type-1 immunity in a Toll-like receptor (TLR)2-, TLR4-, and TLR9-dependent manner. In the present experiments, the extract of C. coronarium significantly induces interferon (IFN)-γ production by mouse spleen cells. In addition, the IFN-γ production by spleen cells was completely blocked by the addition of anti-Interleukin (IL)-12 monoclonal antibodies. We confirmed that NK1.1(+) natural killer (NK) cells, NKT cells, and CD11c(+) dendritic cells (DC) were immediately activated after the stimulation with the extract of C. coronarium and the IFN-γ production was abolished in NK1.1(+) cell-depleted spleen cells. The stimulation with the extract of C. coronarium caused DC maturation involving with up-regulations of surface expression levels of MHC class I, MHC class II, CD40, and CD86 as well as induction of IL-12 production. The IFN-γ production induced by the extract was significantly reduced in the spleen cells depleted CD11c(+) cells. Furthermore, the IFN-γ production after the stimulation was strongly reduced in TLR4- and partially in TLR2- and TLR9-deficient spleen cells. Thus, we demonstrated the cellular mechanism for the activation of Type-1 immunity via NK cells, NKT cells, and DC by the extract of C. coronarium. These findings strongly suggest that C. coronarium would be a promising immuno-improving adjuvant, which might be useful for prevention of infectious, cancer, and allergic diseases through the activation of Type-1 immunity.

DOI： 10.1016/j.intimp.2010.11.026

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1α,25-Dihydroxyvitamin D(3) (1,25D3), the active form of vitamin D(3), is an immunoregulatory hormone with beneficial effects on Th1 cell-mediated inflammatory diseases. Although IL-17-producing CD4(+) T helper (Th17) cells have been recently identified as novel effector cells, the immunomodulating effects of 1,25D3 on Th17 cells have not been well defined. We confirmed here that 1,25D3 inhibited the generation of Th17 cells in vitro. Interestingly, 1,25D3 synergistically suppressed the generation of Th17 cells by the combination with all-trans retinoic acid (ATRA). 1,25D3 and ATRA suppressed the development of allergen-induced contact hypersensitivity (CHS) in a mouse ear swelling model. In addition, we found that 1,25D3 and ATRA significantly inhibited the development of human Th17 cells from both naïve and memory human CD4(+) T cells. 1,25D3 and ATRA effectively suppressed mRNA expressions of IL-1R1, IL-21R, IL-23R, RORC, and AHR in human T cells. ATRA further suppressed IL-6R, whereas 1,25D3 did not. Finally, we found that 1,25D3 and ATRA remarkably blocked IL-22 as well as IL-17 mRNA expression in human memory CD4(+) T cells. Thus, we initially reveal that 1,25D3 and ATRA have synergistic effects on the generation of Th17 cells, suggesting that the combination with ATRA would provide a promising novel therapy for Th17 cell-related immune diseases including skin inflammation.

DOI： 10.1016/j.imlet.2010.07.002

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Based on the evidence that IL-17 is a key cytokine involved in various inflammatory diseases, we explored the critical role of IL-17-producing gammadelta T cells for tumor development in tumor-bearing mouse model. IL-17(-/-) mice exhibited a significant reduction of tumor growth, concomitantly with the decrease of vascular density at lesion area, indicating a pro-tumor property of IL-17. Among tumor-infiltrating lymphocytes (TIL), gammadelta T cells were the major cellular source of IL-17. Analysis of TCR repertoires in TIL-gammadelta T cells showed that circulating gammadelta T cells, but not skin resident Vgamma5(+)gammadelta T cells, produced IL-17. Neutralizing antibodies against IL-23, IL-6, and TGF-beta, which were produced within the tumor microenvironment, inhibited the induction of IL-17-producing gammadelta T cells. IL-17 production by tumor-infiltrating gammadelta T cells was blocked by anti-gammadeltaTCR or anti-NKG2D antibodies, indicating that these ligands, expressed within the tumor microenvironment, are involved in gammadelta T-cell activation. The IL-17-producing TIL-gammadelta T cells exhibited reduced levels of perforin mRNA expression, but increased levels of COX-2 mRNA expression. Together, our findings support the novel concept that IL-17-producing gammadelta T cells, generated in response to tumor microenvironment, act as tumor-promoting cells by inducing angiogenesis.

DOI： 10.1002/eji.200940157

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Adoptive cell transfer of an ovalbumin (OVA)-specific T(h)17-polarized cell population from transgenic DO11.10 mice into BALB/c mice followed by OVA inhalation caused airway hyperresponsiveness (AHR) with severe neutrophilia. The transferred T(h)17 cell population-previously polarized in vitro with IL-6, transforming growth factor-beta and IL-23-contained negligible numbers of IFN-gamma-producing cells; however, during T(h)17-cell-dependent airway inflammation, significant numbers of IFN-gamma-producing cells-including cells producing both IL-17 and IFN-gamma and cells producing only IFN-gamma-were detected in the lung in addition to cells producing only IL-17. Using T(h)17-polarized cell populations derived from IL-17(-/-) or IFN-gamma(-/-) mice, it was demonstrated that IL-17 is essential for inducing neutrophilic airway inflammation and that IFN-gamma is required for the AHR elevation. IFN-gamma appeared to be derived from cells producing both IL-17 and IFN-gamma and/or from cells producing only IFN-gamma, which were converted from the transferred T(h)17-polarized cell population. We also found that mAbs that neutralize IL-12 significantly suppressed the conversion of the T(h)17-polarized cell population toward IFN-gamma producers in the lung; concomitantly, with this decreased conversion, IL-12 neutralization also attenuated the AHR elevation in the lung. IL-12-dependent conversion of the transferred T(h)17-polarized cell population into IFN-gamma producers in the lung thus appeared to be a crucial process for inducing AHR elevation in T(h)17-cell-dependent airway inflammation.

DOI： 10.1093/intimm/dxq034

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Radiation therapy is one of the primary treatment modalities for cancer along with chemotherapy and surgical therapy. The main mechanism of the tumor reduction after irradiation has been considered to be damage to the tumor DNA. However, we found that tumor-specific CTL, which were induced in the draining lymph nodes (DLN) and tumor tissue of tumor-bearing mice, play a crucial role in the inhibition of tumor growth by radiation. Indeed, the therapeutic effect of irradiation was almost completely abolished in tumor-bearing mice by depleting CD8(+) T cells through anti-CD8 monoclonal antibody administration. In mice whose DLN were surgically ablated or genetically defective (Aly/Aly mice), the generation of tetramer(+) tumor-specific CTL at the tumor site was greatly reduced in parallel with the attenuation of the radiation-induced therapeutic effect against the tumor. This indicates that DLN are essential for the activation and accumulation of radiation-induced CTL, which are essential for inhibition of the tumor. A combined therapy of local radiation with Th1 cell therapy augmented the generation of tumor-specific CTL at the tumor site and induced a complete regression of the tumor, although radiation therapy alone did not exhibit such a pronounced therapeutic effect. Thus, we conclude that the combination treatment of local radiation therapy and Th1 cell therapy is a rational strategy to augment antitumor activity mediated by tumor-specific CTL.

DOI： 10.1158/0008-5472.CAN-09-2982

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Regulatory T cells (Tregs) are major immunosuppressors in tumor-bearing hosts. Although Treg-depletion therapy has been shown to induce a complete cure in tumor-bearing mice, this treatment is not always successful. Using 3-methylcholanthrene-induced primary mouse tumors, we examined the distinct regulation of Treg-mediated immunosuppression between carcinomas and sarcomas. We showed that the number of Tregs was greatly increased in squamous cell carcinoma (SCC)-bearing mice compared with sarcoma-bearing mice. This appeared to be because SCC produced higher levels of active transforming growth factor (TGF)-beta, which is essential for inducing Tregs, compared with sarcoma. Moreover, SCC, but not sarcomas, were refractory to Treg-depletion therapy by treatment with anti-CD25 mAb. The refractoriness of SCC against Treg-depletion therapy was due to the rapid recovery of Tregs in SCC-bearing mice compared with sarcoma-bearing mice. However, combination treatment of anti-TGF-beta mAb with anti-CD25 mAb caused a significant reduction in Treg recovery and induced a complete cure in SCC-bearing mice. Thus, we showed the refractoriness of mouse carcinoma against Treg-depletion therapy using anti-CD25 mAb treatment. We also proposed a novel Treg-blocking combination therapy using anti-CD25 mAb and anti-TGF-beta mAb to induce a complete cure of tumor-bearing hosts.

DOI： 10.1111/j.1349-7006.2009.01469.x

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3-Methylcholanthrene (MCA)-induced sarcomas have been used as conventional tools for investigating immunosurveillance against tumor development. However, MCA-induced sarcoma is not always an ideal model for the study of the human cancer system because carcinomas and not sarcomas are the dominant types of human cancers. To resolve this problem, we established a novel and simple method to induce mouse squamous cell carcinomas (SCCs). As well known, the subcutaneous injection of MCA caused the formation of sarcomas at 100% incidence. However, we here first succeeded at inducing SCC at 60% of incidence within 2 months by a single intra-dermal injection of MCA. Using this primary SCC model, we demonstrated the critical role of interferon (IFN)-gamma-dependent type 1 immunity in immunosurveillance against SCC from the following results: (i) The incidence of SCC was accelerated in IFN-gamma-deficient mice compared with that in wild-type mice; (ii) In vivo injection of CpG-oligodeoxynucleotides (CpG-ODN) caused a marked reduction in the incidence of SCC in parallel with the activation of type 1-dependent antitumor immunity and (iii) The antitumor activity of CpG-ODN was significantly decreased in IFN-gamma-deficient mice. Thus, our established MCA-induced mouse SCC model could be a powerful tool for evaluating immunosurveillance mechanisms during the development of SCC and might result in a novel strategy to address immunosurveillance mechanisms of human cancer.

DOI： 10.1093/carcin/bgp144

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Language：English   Publisher：日本癌学会  

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Unmethylated cytosine-phosphorothioate-guanine containing oligodeoxynucleotides (CpG-ODN) is known as a ligand of toll-like receptor 9 (TLR9), which selectively activates type-1 immunity. We have already reported that the vaccination of tumor-bearing mice with liposome-CpG coencapsulated with model-tumor antigen, ovalbumin (OVA) (CpG + OVA-liposome) caused complete cure of the mice bearing OVA-expressing EG-7 lymphoma cells. However, the same therapy was not effective to eradicate Lewis lung carcinoma (LLC)-OVA-carcinoma. To overcome the refractoriness of LLC-OVA, we tried the combination therapy of radiation with CpG-based tumor vaccination. When LLC-OVA-carcinoma intradermally (i.d.) injected into C57BL/6 became palpable (7-8 mm), the mice were irradiated twice with a dose of 14 Gy at intervals of 24 h. After the second radiation, CpG + OVA-liposome was i.d. administered near the draining lymph node (DLN) of the tumor mass. The tumor growth of mice treated with radiation plus CpG + OVA-liposome was greatly inhibited and approximately 60% of mice treated were completely cured. Moreover, the combined therapy with radiation and CpG + OVA-liposome allowed the augmented induction of OVA-tetramer(+) LLC-OVA-specific cytotoxic T lymphocyte (CTL) in DLN of tumor-bearing mice. These results indicate that the combined therapy of radiation with CpG-based tumor vaccine is a useful strategy to eradicate intractable carcinoma.

DOI： 10.1111/j.1349-7006.2009.01114.x

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

MAGE-A4 has been considered as an attractive cancer-testis (CT) antigen for tumour immunotherapy. It has been well accepted that T-helper type 1 (Th1) cell-dominant immunity is critical for the successful induction of antitumour immunity in a tumour-bearing host. The adoptive Th1 cell therapy has been shown to be an attractive strategy for inducing tumour eradication in mouse systems. However, Th1-cell therapy using human tumour-specific Th1 cells, which were expanded from peripheral blood mononuclear cells (PBMCs) in a clinically useful protocol, has never been performed. Here, we first identified MAGE-A4-derived promiscuous helper epitope, peptide (MAGE-A4 280-299), bound to both HLA-DPB1(*)0501 and DRB1(*)1403. Using the peptide, we established a suitable protocol for the propagation of MAGE-A4-specific Th1 cells in vitro. Culture of CD4(+) T cells with IFN-gamma-treated PBMC-derived adherent cells in the presence of helper epitope peptide resulted in a great expansion of MAGE-A4-reactive Th cells producing IFN-gamma , but not IL-4. Moreover, it was shown that ligation of MAGE-A4-reactive Th1 cells with the cognate peptide caused the production of IFN-gamma and IL-2. Thus, our identified MAGE-A4 helper epitope peptide will become a good tool for the propagation of tumour-specific Th1 cells applicable to adoptive immunotherapy of human cancer.

DOI： 10.1038/sj.bjc.6604966

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

Evaluation of immunosuppressive tumor-escape mechanisms in tumor-bearing hosts is of great importance for the development of an efficient tumor immunotherapy. We document here the functional characteristics of CD11b+Gr-1+ immature myeloid cells (ImC), which increase abnormally in tumor-bearing mice. Although it has been reported that ImC exhibit a strong immunosuppressive activity against T cell responses, we demonstrate that ImC derived from tumor-bearing mouse spleens (TB-SPL) did not exhibit a strong inhibitory activity against CTL generation in MLR. However, ImC isolated from TB-SPL and induced to differentiate into CD11b+Gr-1+F4/80+ suppressor macrophages (MΦ) under the influence of tumor-derived factors were immunosuppressive. Furthermore, we also demonstrate that ImC isolated from TB-SPL had a capability of differentiating into immunostimulatory dendritic cells (DC1) supportive of the generation of IFN-γ producing CTL if the ImC were cultured with Th1 cytokines plus GM-CSF and IL-3. Thus, our findings indicate that tumor bearing mouse-derived CD11b+Gr-1+ ImC are not committed to development into immunosuppressor cells but have dual differentiation ability into both immunosuppressive myeloid cells and immunostimulatory DC1.

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Language：English   Publisher：日本癌学会  

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

Cytokine production by memory T cells in secondary immune responses has a critical role in host defenses. Previously, we had demonstrated that a unique antigen composed of sialyl lewis(x) (sLe(x)) was expressed on CD45RO(+) memory-phenotype subsets of human T cells. Here, we found that the sLe(x) antigen was up-regulated on CD45RA(+) naïve human CD4(+) T and CD8(+) T cells by TCR stimulation. In addition, sLe(x) antigen-expressing CD4(+) T and CD8(+) T cells in human PBMCs were activated immediately by cytokine stimulations composed of IL-2 plus IL-12 or IL-15 in an antigen-independent manner. Moreover, the sLe(x)-positive human CD8(+) T cells significantly enhanced reverse antibody-dependent cellular cytotoxicity compared with a sLe(x)-negative population. These findings clearly indicate that sLe(x) antigen-expressing memory phenotype CD4(+) T and CD8(+) T cells contribute to early-stage immunity by providing a source of IFN-gamma and cytotoxicity, suggesting that they would be a key immunomodulator in host defenses.

DOI： 10.1189/jlb.0907599

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

NY-ESO-1 is a cancer-testis antigen that elicits strong cellular and humoral immune responses against NY-ESO-1-expressing tumors. Although CD4(+) T cells play a critical role in inducing antitumor immunity, little is known about MHC class II-restricted helper epitopes of the NY-ESO-1 antigen compared with MHC class I-restricted epitopes. Here, we searched for new NY-ESO-1 helper epitopes presented by MHC class II molecules, especially those found frequently in the Japanese population. We established five NY-ESO-1-specific helper T-cell lines from healthy Japanese donors using NY-ESO-1 recombinant protein and peptide. Using MHC class II-specific antibodies and a panel of Epstein-Barr virus-transformed B-cell lines, it was demonstrated that four out of the five T-cell lines recognized a region within NY-ESO-1(119-143) in the context of HLA-DRB1*0802, DRB1*0901, DRB1*1502 or DRB1*0405/*0410. In addition, using a set of overlapping 15-mer synthetic peptides, we found that NY-ESO-1(122-138) was a promiscuous region that bound to four distinct HLA-DR molecules found in the Japanese population. These findings expand the usefulness of NY-ESO-1 as a tool for tumor vaccine therapy in eliciting NY-ESO-1-specific helper T-cell responses, especially in Japanese cancer patients.

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

Since the prognosis of human osteosarcoma in advanced stage remains poor, the development of new and effective therapies including immunotherapy is required. To identify tumor-associated antigens of osteosarcoma applicable to the immunotherapy of this malignancy, we employed the serological analysis of recombinant cDNA expression library (SEREX) technique that defines tumor antigens recognized by the humoral immune system. Screening a cDNA library derived from an osteosarcoma cell line MG63 with sera from osteosarcoma patients identified 43 positive clones, representing 14 distinct antigens. Among them, CLUAP1 (clusterin-associated protein 1) was highly expressed in osteosarcoma tissue samples and cell lines. Overexpression of CLUAP1 was observed in other malignancies including ovarian, colon, and lung cancers. Our results suggest that CLUAP1 may be useful as a prognostic/diagnostic marker and/or for a target of immunotherapy of osteosarcoma.

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

To generate tumor-specific and interferon (IFN)-gamma-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3zeta. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2-/- mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-gamma in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer.

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

Cancer-testis (CT) antigens were identified as a group of highly attractive targets for cancer immunotherapy because of their expression in a variety of malignant tumors but solely in the testis among the normal adult tissues. To evaluate the potential of two members of this family, MAGE-A4 and NY-ESO-1 antigens, for cancer vaccine in non-small cell lung carcinoma (NSCLC), we examined the expression of these antigens and T cell infiltration in tumor tissue, and evaluated their prognostic significance. One hundred fifty-seven patients with NSCLC were studied. Reverse transcription-PCR was performed to evaluate MAGE-A4 and NY-ESO-1 expression. Immunohistochemistry was performed for NY-ESO-1 expression and T cell infiltration into the tumor site. Survival analysis was also performed. MAGE-A4 and NY-ESO-1 were expressed in 40 of 141 (28.4%) and 13 of 157 (8.3%) NSCLC respectively. Both CT antigens were more frequently expressed in squamous cell carcinoma (SCC) than in adenocarcinoma. An inverse correlation was found between MAGE-A4 expression and patient survival in advanced stage cancers. Combined infiltration of both CD4+ and CD8+ T cells into tumor nest predicted better survival. There was no correlation, however, between lymphocyte infiltration and antigen expression in the tumor. MAGE-A4 expression in advanced group and T cell infiltration may provide prognostic information. Lastly, these CT antigens, especially MAGE-A4, may represent potential targets for cancer immunotherapy in patients with NSCLC.

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

Tumor antigen-specific CD4+ and CD8+ T lymphocytes, especially interferon-gamma (IFN-gamma)-producing type-1 helper T (Th1) and type-1 cytotoxic T (Tc1) cells, play a crucial role in tumor eradication. Adoptive transfer using tumor-specific Th1 and Tc1 cells is a promising therapeutic strategy for tumor immunotherapy. However, its clinical application has been hampered because of difficulties in generating tumor-specific Th1 cells from patients with tumors. To overcome this problem, we have developed an efficient method to prepare tumor-specific Th1 and Tc1 cells. T-cell receptor (TCR) alpha and beta genes obtained from an HLA-A24-restricted, Wilms tumor 1 (WT1) peptide-specific Tc clone were lentivirally transduced to polyclonally activated Th1 and Tc1 cells. As expected, TCR gene-modified Tc1 cells showed cytotoxicity and IFN-gamma production in response to peptide-loaded lymphoblastoid cell lines, WT1 gene-transduced cells, and freshly isolated leukemia cells expressing both WT1 and HLA-A24. Surprisingly, we further demonstrated that Th1 cells transduced with HLA-class I-restricted TCR genes also showed both cytotoxicity and cytokine production in an HLA-A24-restricted manner. In contrast to gene-modified Tc1 cells, Th1 cells produced high amounts of interleukin-2 (IL-2) in addition to IFN-gamma, which is beneficial for induction of antitumor cellular immunity. Thus, TCR gene-modified HLA-class I-restricted Th1 and Tc1 cells are a powerful strategy for the application to adoptive immunotherapy of human cancer.

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

CD4+ Th cells, in particular IFN-gamma-producing Th1 cells, play a critical role in the activation and maintenance of Tc1 cells that are essential for tumor eradication. Here, we report the generation of artificial tumor-specific Th1 and Tc1 cells from nonspecifically activated T cells using a lentiviral transduction system. Anti-CD3-activated T cells from healthy human donors were transduced with a lentivirus containing a chimeric immunoglobulin T-cell receptor gene composed of single-chain variable fragments derived from an anticarcinoembryonic antigen (CEA)-specific monoclonal antibody fused to an intracellular signaling domain derived from the cytoplasmic portions of membrane-bound CD28 and CD3zeta. These artificial tumor-specific Tc1 and Th1 cells, termed Tc1- and Th1-T bodies, respectively, could be targeted to CEA+ tumor cells independently of MHC restriction. Specifically, Tc1-T bodies demonstrated high cytotoxicity and produced IFN-gamma in response to CEA+ tumor cell lines but not CEA- tumors. Although Th1-T bodies exhibited low cytotoxicity, they secreted high levels of IFN-gamma and interleukin-2 in response to CEA+ tumor cells. Such CEA+ tumor-specific activation was not observed in mock gene-transduced nonspecific Tc1 and Th1 cells. Moreover, Tc1- and Th1-T bodies exhibited strong antitumor activities against CEA+ human lung cancer cells implanted into RAG2(-/-) mice. Furthermore, combined therapy with Tc1- and Th1-T bodies resulted in enhanced antitumor activities in vivo. Taken together, our findings demonstrate that Tc1- and Th1-T bodies represent a promising alternative to current methods for the development of effective adoptive immunotherapies.

PubMed

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Symposium, workshop panel (nominated)  

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Presentation type：Oral presentation (invited, special)  

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Presentation type：Oral presentation (invited, special)  

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