Language：Japanese   Publisher：日本がん免疫学会  

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Language：Japanese   Publisher：(NPO)日本肺癌学会  

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Language：English   Publisher：(一社)日本癌学会  

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbagen.2022.130171

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

Epidermal growth factor receptor (EGFR) is the most frequently mutated driver oncogene in non-smoking-related, non-small-cell lung cancer (NSCLC). EGFR-mutant NSCLC has a non-inflamed tumor microenvironment (TME), with low infiltration by CD8+ T cells and, thus, immune checkpoint inhibitors, such as anti-programmed cell death-1 (anti-PD-1) have weak anti-tumor effects. Here, we showed that CD8+ T-cell responses were induced by an EGFR-tyrosine kinase inhibitor (TKI) in syngeneic Egfr-mutant NSCLC tumors, which was further pronounced by sequential dual blockade of PD-1 and vascular endothelial growth factor receptor 2 (VEGFR2). However, simultaneous triple blockade had no such effect. PD-1/VEGFR2 dual blockade did not exert tumor-inhibitory effects without pre-treatment with the EGFR-TKI, suggesting that treatment schedule is crucial for efficacy of the dual blockade therapy. Pre-treatment with EGFR-TKI increased the CD8+ T-cell/regulatory T-cell (Treg) ratio, while also increasing expression of immunosuppressive chemokines and chemokine receptors, as well as increasing the number of M2-like macrophages, in the TME. Discontinuing EGFR-TKI treatment reversed the transient increase of immunosuppressive factors in the TME. The subsequent PD-1/VEGFR2 inhibition maintained increased numbers of infiltrating CD8+ T cells and CD11c+ dendritic cells. Depletion of CD8+ T cells in vivo abolished tumor growth inhibition by EGFR-TKI alone and the sequential triple therapy, suggesting that EGFR inhibition is a prerequisite for the induction of CD8+ T-cell responses. Our findings could aid in developing an alternative immunotherapy strategy in patients with cancers that have driver mutations and a non-inflamed TME.

DOI： 10.1158/2326-6066.CIR-21-0751

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Language：Japanese   Publisher：日本がん免疫学会  

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

Prostaglandin E2 (PGE2), a product of the cyclooxygenase (COX) pathway, is produced by tumors and surrounding stromal cells. It stimulates tumor progression, promotes angiogenesis, and suppresses the antitumor response. Pharmacological inhibition of PGE2 synthesis has been shown to suppress tumor initiation and growth in vivo. In the current study, we demonstrated that the growth of the Ptgs2-deficient the 3LL lung adenocarcinoma cell line was downregulated in vivo through natural killer (NK) cell activation and a reduction in the population of polymorphonuclear leukocyte-myeloid-derived suppressor cells (PMN-MDSCs) and tumor associated macrophages (TAMs). Based on these results, the therapeutic effect of ONO-AE3-208 (EP4i), an inhibitor of EP4 (a PGE2 receptor), combined with anti-PD-1Ab was evaluated. EP4i, but not anti-PD-1 Ab, decreased tumor metabolism including glycolysis, fatty acid oxidation, and oxidative phosphorylation. EP4i induced IFNγ production from only NK cells (not from T cells) and a shift from M2- to M1-like macrophages in TAMs. These effects were further enhanced by anti-PD-1 Ab treatment. Although CD8T cell infiltration was increased, IFNγ production was not significantly altered, even with combination therapy. Tumor hypoxia was ameliorated by either EP4i or anti-PD-1 Ab treatment, which was further affected by the combination. Normalization of tumor vessels was significant only for the combination therapy. The results indicate a novel effect of EP4i for the metabolic reprogramming of tumors, revealed unique features of EP4i that can synergize with anti-PD-1Ab to promote IFNγ production of NK cells, polarize TAMs into the M1-phenotype, and reduce hypoxia through normalization of the tumor vasculature. (250 words).

DOI： 10.1093/intimm/dxac004

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

Metformin, a commonly prescribed drug for type 2 diabetes mellitus, has been shown to activate AMP-activated protein kinase (AMPK). Notably, AMPK activation has recently been observed to be associated with anti-inflammatory responses. Metformin is also reported to elicit anti-inflammatory responses in CD4+ T cells, resulting in improvement in experimental chronic inflammatory diseases, such as systemic lupus erythematosus. To investigate the effect of metformin on inflammatory bowel disease (IBD), we developed a T cell-transfer model of chronic colitis in which SCID mice were injected with CD4+ CD45RBhigh T cells to induce colitis. We examined the effects of metformin via in vitro and in vivo experiments on lamina propria (LP) CD4+ T cells. We observed that metformin suppresses the frequency of interferon (IFN) -γ-producing LP CD4+ T cells in vitro, which were regulated by AMPK activation, a process possibly induced by the inhibition of oxidative phosphorylation. Furthermore, we examined the effects of metformin on an in vivo IBD model. Metformin-treated mice showed AMPK activation in LP CD4+ T cells and ameliorated colitis. Our study demonstrates that metformin-induced AMPK activation in mucosal CD4+ T cells contributes to the improvement of IBD by suppressing IFN-γ production. Moreover, our results indicate that AMPK may be a target molecule for the regulation of mucosal immunity and inflammation. Thus, AMPK-activating drugs such as metformin may be potential therapeutic agents for the treatment of IBD.

DOI： 10.1096/fj.202100831RR

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Language：Japanese   Publisher：(有)科学評論社  

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

Metformin (Met), a first-line drug for type 2 diabetes, lowers blood glucose levels by suppressing gluconeogenesis in the liver, presumably through the liver kinase B1-dependent activation of AMP-activated protein kinase (AMPK) after inhibiting respiratory chain complex I. Met is also implicated as a drug to be repurposed for cancers; its mechanism is believed identical to that of gluconeogenesis inhibition. However, AMPK activation requires high Met concentrations at more than 1 mM, which are unachievable in vivo. The immune-mediated antitumor response might be the case in a low dose Met. Thus, we proposed activating or expanding tumor-infiltrating CD8+ T cells (CD8TILs) in a mouse model by orally administering Met in free drinking water. Here we showed that Met, at around 10 μM and a physiologically relevant concentration, enhanced production of IFNγ,TNFα and expression of CD25 of CD8+ T cells upon TCR stimulation. Under a glucose-rich condition, glycolysis was exclusively involved in enhancing IFNγ production. Under a low-glucose condition, fatty acid oxidation or autophagy-dependent glutaminolysis, or both, was also involved. Moreover, phosphoenolpyruvate carboxykinase 1 (PCK1), converting oxaloacetate to phosphoenolpyruvate, became essential. Importantly, the enhanced IFNγ production was blocked by a mitochondrial ROS scavenger and not by an inhibitor of AMPK. In addition, IFNγ production by CD8TILs relied on pyruvate translocation to the mitochondria and PCK1. Our results revealed a direct effect of Met on IFNγ production of CD8+ T cells that was dependent on differential metabolic pathways and determined by nutrient conditions in the microenvironment.

DOI： 10.3389/fimmu.2022.864225

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

<sec><title>Background</title>Metformin (Met) is the first-line treatment for type 2 diabetes mellitus and plays an effective role in treating various diseases, such as cardiovascular disease, neurodegenerative disease, cancer, and aging. However, the underlying mechanism of Met-dependent antitumor immunity remains to be elucidated.

</sec><sec><title>Methods</title>MitoTEMPO, a scavenger of mitochondrial superoxide, abolished the antitumor effect of Met, but not antiprogrammed cell death (PD-1) antibody (Ab) treatment. Consequently, we studied the mechanism of the Met-induced antitumor effect. Expressions of glucose transporter (Glut)-1, mitochondrial reactive oxygen species (mtROS), interferon (IFN)-γ, Ki67, autophagy markers, activation markers for NF-E2-related factor 2 (Nrf2), and mammalian target of rapamaycin complex 1 (mTORC1) in CD8⁺ tumor-infiltrating T lymphocytes (CD8TILs) were examined by flow cytometry analysis. In addition, conditional knockout mice for Nrf2 and p62 were used to detect these markers, together with the monitoring of in vivo tumor growth. RNA sequencing was performed for CD8TILs and tumor cells. Melanoma cells containing an IFN-γ receptor (IFNγR) cytoplasmic domain deletion mutant was overexpressed and used for characterization of the metabolic profile of those tumor cells using a Seahorse Flux Analyzer.

</sec><sec><title>Results</title>Met administration elevates mtROS and cell surface Glut-1, resulting in the production of IFN-γ in CD8TILs. mtROS activates Nrf2 in a glycolysis-dependent manner, inducing activation of autophagy, glutaminolysis, mTORC1, and p62/SQSTM1. mTORC1-dependent phosphorylation of p62 at serine 351 (p-p62(S351)) is also involved in activation of Nrf2. Conditional deletion of Nrf2 in CD8TILs abrogates mTORC1 activation and antitumor immunity by Met. In synergy with the effect of anti-PD-1 Ab, Met boosts CD8TIL proliferation and IFN-γ secretion, resulting in decreased glycolysis and oxidative phosphorylation in tumor cells. Consequently, Glut-1 is elevated in CD8TILs, together with the expansion of activated dendritic cells. Moreover, tumor cells lacking in IFNγR signaling abolish IFN-γ production and proliferation of CD8TILs.

</sec><sec><title>Conclusions</title>We found that Met stimulates production of mtROS, which triggers Glut-1 elevation and Nrf2 activation in CD8TILs. Nrf2 activates mTORC1, whereas mTORC1 activates Nrf2 in a p-p62(S351)-dependent manner, thus creating a feedback loop that ensures CD8TILs’ proliferation. In combination with anti-PD-1 Ab, Met stimulates robust proliferation of CD8TILs and IFN-γ secretion, resulting in an IFN-γ-dependent reprogramming of the tumor microenvironment.

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DOI： 10.1136/jitc-2021-002954

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

Anti-programmed cell death protein-1 (PD-1) antibodies have become a standard treatment for advanced melanoma. However, a predictive biomarker for assessing the efficacy of anti-PD-1 antibodies has not been identified. In cancer, CD8+ T cells specific for tumor antigens undergo repeated T-cell receptor stimulation due to the persistence of cancer cells and gradually lose their ability to secrete interleukin 2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). We aimed to evaluate multi-cytokine production and immune exhaustion of peripheral CD8+ T cells in melanoma patients treated with anti-PD-1 antibodies. Twenty-four melanoma patients treated with nivolumab were included. Effector cytokine production (IL-2, TNF-α, and IFN-γ) and expression of an exhaustion marker (PD-1) in patients' CD8+ cells were analyzed with flow cytometry. The relationships between parameters such as the neutrophil-to-lymphocyte ratio (NLR) and clinical response to nivolumab were examined. Immunohistochemistry for programmed death-ligand 1 (PD-L1) expression in tumor cells and tumor-infiltrating lymphocytes (TILs) and analysis of their association with clinical response were performed. The clinical response rate to nivolumab was 29%. Regarding TILs, NLR, and several other parameters, no significant difference was found between responders and non-responders. The responder group showed an increase in the percentage of PD-1+ CD8+ /TNF-α+ IFN-γ+ or PD-1+ CD8+ /IFN-γ+ IL-2+ TNF-α+ T cells compared to non-responders. Positivity for PD-L1 expression was significantly higher in the responder group than the non-responder group. In advanced melanoma, the percentage of multifunctional CD8+ PD-1+ T cells and PD-L1 expression in the tumors may be a biomarker for a good response to anti-PD-1 antibody monotherapy.

DOI： 10.1111/1346-8138.15904

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>
The metabolic changes and dysfunction in CD8 + T cells may be involved in tumor progression and susceptibility to virus infection in type 2 diabetes (T2D). In C57BL/6JJcl mice fed with high fat-high sucrose chow (HFS), multifunctionality of CD8 + splenic and tumor-infiltrating lymphocytes (TILs) was impaired and associated with enhanced tumor growth, which were inhibited by metformin. In CD8 + splenic T cells from the HFS mice, glycolysis/basal respiration ratio was significantly reduced and reversed by metformin. In the patients with T2D (DM), multifunctionality of circulating CD8 + PD-1 + T cells stimulated with PMA/ionomycin as well as with HLA-A*24:02 CMV peptide was dampened, while metformin recovered multifunctionality. Both glycolysis and basal respiration were reduced in DM, and glycolysis was increased by metformin. The disturbance of the link between metabolism and immune function in CD8 + PD-1 + T cells in T2D was proved by recovery of antigen-specific and non-specific cytokine production via metformin-mediated increase in glycolytic activity.

DOI： 10.1038/s41598-020-71946-3

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Other Link： http://www.nature.com/articles/s41598-020-71946-3

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

The herbal medicine berberine (BBR) has been recently shown to be an AMP-activated protein kinase (AMPK) productive activator with various properties that induce anti-inflammatory responses. We investigated the effects of BBR on the mechanisms of mucosal CD4+T cell activation in vitro and on the inflammatory responses in T cell transfer mouse models of inflammatory bowel disease (IBD). We examined the favorable effects of BBR in vitro, using lamina propria (LP) CD4+ T cells in T cell transfer IBD models in which SCID mice had been injected with CD4+CD45RBhigh T cells. BBR suppressed the frequency of IFN-γ- and Il-17A-producing LP CD4+ T cells. This effect was found to be regulated by AMPK activation possibly induced by oxidative phosphorylation inhibition. We then examined the effects of BBR on the same IBD models in vivo. BBR-fed mice showed AMPK activation in the LPCD4+ T cells and an improvement of colitis. Our study newly showed that the BBR-induced AMPK activation of mucosal CD4+ T cells resulted in an improvement of IBD and underscored the importance of AMPK activity in colonic inflammation.

DOI： 10.1038/s41598-019-48331-w

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Other Link： http://www.nature.com/articles/s41598-019-48331-w

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Anticancer Research USA Inc.  

BACKGROUND/AIM: Metformin, a drug for type 2 diabetes, also exerts anticancer effects. This study addressed the immunological effects of metformin on peritoneal dissemination. MATERIALS AND METHODS: We developed a mouse model of peritoneal dissemination via intraperitoneal injection of RLmale1, an X-ray-induced leukemia cell line, into BALB/c mice. Cell-surface markers, cytokine production, and myeloid-derived suppressor cells (MDSCs) were examined in cells from spleen and peritoneal lavage fluid. RESULTS: Metformin-treated mice exhibited suppressed intraperitoneal tumor growth and extended survival, and these effects were lost in mice with severe combined immunodeficiency. MDSCs induction was inhibited in metformin-treated mice. Although MDSC mobilization into the peritoneal cavity was correlated with suppression of interferon-γ production by tumor-infiltrating lymphocytes, the T-helper 1 ability of these lymphocytes was preserved in metformin-treated mice. CONCLUSION: Our findings demonstrate the action of metformin on both intraperitoneal tumors and immune-suppressive cells and might contribute to the development of immunotherapy against peritoneal dissemination.

DOI： 10.21873/anticanres.13652

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

CD11b+ myeloid subpopulations, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), play crucial roles in the suppression of T-cell-mediated anti-tumor immunity. Regulation of these cell types is a primary goal for achieving efficient cancer immunotherapy. We found that metformin (Met) induces CD11b+-cell-mediated growth inhibition of a K7M2neo osteosarcoma independent of T cells, as growth inhibition of K7M2neo was still observed in wild-type (WT) mice depleted of T cells by antibodies and in SCID; this contrasted with the effect of Met on Meth A fibrosarcoma, which was entirely T-cell-dependent. Moreover, the inhibitory effect seen in SCID was abrogated by anti-CD11b antibody injection. PMN-MDSCs were significantly reduced in both spleens and tumors following Met treatment. In TAMs, production of IL-12 and TNF-α, but not IL-10, became apparent, and elevation of MHC class II with reduction of CD206 was observed, indicating a shift from an M2- to M1-like phenotype via Met administration. Metabolically, Met treatment decreased basal respiration and the oxygen consumption rate (OCR)/extracellular acidification rate (ECAR) ratio of CD11b+ cells in tumors, but not in the spleen. In addition, decreased reactive oxygen species (ROS) production and proton leakage in MDSCs and TAMs were consistently observed in tumors. Uptake of both 2-deoxy-2-d-glucose (2-NBDG) and BODIPY® decreased in MDSCs, but only BODIPY® incorporation was decreased in TAMs. Overall, our results suggest that Met redirects the metabolism of CD11b+ cells to lower oxidative phosphorylation (OXPHOS) while elevating glycolysis, thereby pushing the microenvironment to a state that inhibits the growth of certain tumors.

DOI： 10.1093/intimm/dxy079

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

Adaptive immune responses are critical for protection against infection with Plasmodium parasites. The metabolic state dramatically changes in T cells during activation and the memory phase. Recent findings suggest that metformin, a medication for treating type-II diabetes, enhances T-cell immune responses by modulating lymphocyte metabolism. In this study, we investigated whether metformin could enhance anti-malaria immunity. Mice were infected with Plasmodium yoelii and administered metformin. Levels of parasitemia were reduced in treated mice compared with those in untreated mice, starting at ~2 weeks post-infection. The number of γδ T cells dramatically increased in the spleens of treated mice compared with that in untreated mice during the later phase of infection, while that of αβ T cells did not. The proportions of Vγ1+ and Vγ2+ γδ T cells increased, suggesting that activated cells were selectively expanded. However, these γδ T cells expressed inhibitory receptors and had severe defects in cytokine production, suggesting that they were in a state of exhaustion. Metformin was unable to rescue the cells from exhaustion at this stage. Depletion of γδ T cells with antibody treatment did not affect the reduction of parasitemia in metformin-treated mice, suggesting that the effect of metformin on the reduction of parasitemia was independent of γδ T cells.

DOI： 10.3389/fimmu.2018.02942

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Although immune checkpoint inhibitors have shown significant survival benefits in the treatment of several cancers, optimal outcomes have been limited to certain subsets of patients. In a previous study, we found that the addition of metformin to nivolumab, an anti-programmed cell death protein 1 (PD-1) antibody, yielded substantial tumor regression in mouse models. Further analysis revealed that the number of tumor-infiltrating CD8 T cells had increased markedly. Based on this result, we have launched an investigator-initiated open-label phase-Ib clinical trial. The objectives of this trial are to investigate the safety, efficacy, and pharmacokinetics of a metformin-nivolumab combination treatment. This study consists of 2 parts. The recommended dose of metformin combined with nivolumab is determined in part 1. The safety and efficacy of the optimal dose of metformin to be delivered in conjunction with nivolumab are examined in part 2. Patient eligibility is based on the following criteria: pathologic diagnosis of refractory/recurrent solid tumor (part 1), and non-small-cell lung cancer or pancreatic cancer refractory to standard primary treatment (part 2); no prior use of immune checkpoint inhibitor; performance status 0 or 1; age ≥ 20 years; and adequate organ function. The primary endpoints are safety in part 1 and safety and pharmacokinetics in part 2. The maximum tolerated dose and recommended dose are determined in part 1 by the 3 + 3 cohort method, and the dose-limiting toxicity evaluation period for each patient is 4 weeks from the start of administration. In part 2, metformin is administered at the optimal dose determined in part 1. Total enrollment is 9 to 18 patients for part 1 and 30 patients for part 2. Enrollment began in 2017, and will be completed by 2019. The University Hospital Medical Information Network registration number for this study is 000028405.

DOI： 10.1016/j.cllc.2018.07.010

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

Despite years of effort and investment, there are few topical or systemic medications for skin wounds. Identifying natural drivers of wound healing could facilitate the development of new and effective treatments. When skin is injured, there is a massive increase of heat shock protein (Hsp) 90α inside the wound bed. The precise role for these Hsp90α proteins, however, was unclear. The availability of a unique mouse model that lacked the intracellular ATPase-driven chaperoning, but spared the extracellular fragment-5-supported pro-motility function of Hsp90α allowed us to test specifically the role of the non-chaperone function of Hsp90α in normal wound closure. We found that the chaperone-defective Hsp90α-Δ mutant mice showed similar wound closure rate as the wild-type Hsp90α mice. We generated recombinant proteins from the mouse cDNAs encoding the Hsp90α-Δ and wild-type Hsp90α. Topical application of Hsp90α-Δ mutant protein promoted wound closure as effectively as the full-length wild-type Hsp90α protein. More importantly, selective inhibition of the extracellular Hsp90α-Δ protein function by a monoclonal antibody targeting the fragment-5 region disrupted normal wound closure in both wild-type Hsp90α and Hsp90α-Δ mice. Thus, this study provides direct support for non-chaperone, extracellular Hsp90α as a potential driver for normal wound closure.

DOI： 10.1016/j.jid.2017.08.043

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Association of Immunologists  

In Legionella pneumophila infection, macrophages play a critical role in the host defense response. Metformin, an oral drug for type 2 diabetes, is attracting attention as a new supportive therapy against a variety of diseases, such as cancer and infectious diseases. The novel mechanisms for metformin actions include modulation of the effector functions of macrophages and other host immune cells. In this study, we have examined the effects of metformin on L. pneumophila infection in vitro and in vivo. Metformin treatment suppressed growth of L. pneumophila in a time- and concentration-dependent fashion in bone marrow-derived mac-rophages, RAW cells (mouse), and U937 cells (human). Metformin induced phosphorylation of AMP-activated protein kinase (AMPK) in L. pneumophila-infected bone marrow-derived macrophages, and the AMPK inhibitor Compound C negated metformin-mediated growth suppression. Also, metformin induced mitochondrial reactive oxygen species but not phagosomal NADPH oxidase-derived reactive oxygen species. Metformin-mediated growth suppression was mitigated in the presence of the reactive oxygen species scavenger glutathione. In a murine L. pneumophila pneumonia model, metformin treatment improved survival of mice, which was associated with a significant reduction in bacterial number in the lung. Similar to in vitro observations, induction of AMPK phosphorylation and mitochondrial ROS was demonstrated in the infected lungs of mice treated with metformin. Finally, glutathione treatment abolished metformin effects on lung bacterial clearance. Collectively, these data suggest that metformin promotes mitochondrial ROS production and AMPK signaling and enhances the bactericidal activity of macro-phages, which may contribute to improved survival in L. pneumophila pneumonia.

DOI： 10.4049/jimmunol.1700474

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

CD4+ CD25+ regulatory T cells (Treg), an essential subset for preventing autoimmune diseases, is implicated as a negative regulator in anti-tumor immunity. We found that metformin (Met) reduced tumor-infiltrating Treg (Ti-Treg), particularly the terminally-differentiated CD103+ KLRG1+ population, and also decreased effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naïve CD4+ T cells into inducible Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein, caused by mTORC1 activation that was determined by the elevation of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C, an inhibitor of AMP-activated protein kinase (AMPK) restored the iTreg generation, further indicating the involvement of mTORC1 and AMPK. The metabolic profile of iTreg, increased Glut1-expression, and reduced mitochondrial membrane-potential and ROS production of Ti-Treg aided in identifying enhanced glycolysis upon Met-treatment. The negative impact of Met on Ti-Treg may help generation of the sustained antitumor immunity.

DOI： 10.1016/j.ebiom.2017.10.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pone.0169260

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Language：English   Publisher：Okayama University Medical School  

DOI： 10.18926/AMO/54514

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC CANCER RESEARCH  

Purpose: FoxP3(+) Tregs inhibit immune responses against tumors. KW-0761 is a humanized anti-human CCR4 monoclonal antibody (mAb) that has antibody-dependent cellular cytotoxicity activity. Depletion of CCR4-expressing FoxP3(+) CD4 Tregs by KW-0761 infusion was investigated in solid cancer patients.
Experimental Design: We conducted a phase Ia clinical trial of KW-0761 infusion in 7 lung and 3 esophageal cancer patients. Toxicity, clinical efficacy, changes in lymphocyte subpopulations, including Tregs, and induction of immune responses were analyzed.
Results: The results showed that KW-0761 infusion in a dose range between0.1mg/kg and 1.0mg/kgwas safe andwell tolerated. Nodose-limiting toxicitywas observed. Four of 10 patients showed stable disease during treatment and were long survivors. The monitoring of FoxP3(+) Tregs in the peripheral blood mononuclear cells during treatment indicated efficient depletion of those cells, even at the lowest dose of 0.1 mg/kg used. The reduction in Th 1 CD4 T cells and CD8 T cells was limited, whereas a significant reduction was observed with Th 2 and Th 17 CD4 T cells. Immune responses to cancer/testis (CT) antigens and an autoantibody response to thyroid peroxidase were observed in some patients.
Conclusions: The findings showed Tregs depletion and the possible occurrence of an immune response following KW-0761 infusion. Combined use of KW-0761 to deplete FoxP3(+) Tregs with other immunotherapies, such as cancer vaccines or checkpoint inhibitors, is a promising approach to augment immune responses. (C) 2015 AACR.

DOI： 10.1158/1078-0432.CCR-15-0357

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Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS can potentially better estimate the actual frequency of antigen-specific T cells and thus provide more accurate patient monitoring.

DOI： 10.1371/journal.pone.0136086

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

Metformin, a prescribed drug for type 2 diabetes, has been reported to have anti-cancer effects; however, the underlying mechanism is poorly understood. Here we show that this mechanism may be immune-mediated. Metformin enabled normal but not T-cell-deficient SCID mice to reject solid tumors. In addition, it increased the number of CD8+ tumor-infiltrating lymphocytes (TILs) and protected them from apoptosis and exhaustion characterized by decreased production of IL-2, TNFα, and IFNγ. CD8+ TILs capable of producing multiple cytokines were mainly PD-1-Tim-3+, an effector memory subset responsible for tumor rejection. Combined use of metformin and cancer vaccine improved CD8+ TIL multifunctionality. The adoptive transfer of antigen-specific CD8+ T cells treated with metformin concentrations as low as 10 μM showed efficient migration into tumors while maintaining multifunctionality in a manner sensitive to the AMP-activated protein kinase (AMPK) inhibitor compound C. Therefore, a direct effect of metformin on CD8+ T cells is critical for protection against the inevitable functional exhaustion in the tumor microenvironment.

DOI： 10.1073/pnas.1417636112

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Authorship：Last author,　Corresponding author   Language：English   Publisher：HINDAWI LTD  

Although multimodal therapies including surgery, chemotherapy, and radiotherapy have improved clinical outcomes of patients with bone and soft tissue sarcomas, the prognosis of patients has plateaued over these 20 years. Immunotherapies have shown the effectiveness for several types of advanced tumors. Immunotherapies, such as cytokine therapies, vaccinations, and adoptive cell transfers, have also been investigated for bone and soft tissue sarcomas. Cytokine therapies with interleukin-2 or interferons have limited efficacy because of their cytotoxicities. Liposomal muramyl tripeptide phosphatidylethanolamine (L-MTP-PE), an activator of the innate immune system, has been approved as adjuvant therapeutics in combination with conventional chemotherapy in Europe, which has improved the 5-year overall survival of patients. Vaccinations and transfer of T cells transduced to express chimeric antigen receptors have shown some efficacy for sarcomas. Ipilimumab and nivolumab are monoclonal antibodies designed to inhibit immune checkpoint mechanisms. These antibodies have recently been shown to be effective for patients with melanoma and also investigated for patients with sarcomas. In this review, we provide an overview of various trials of immunotherapies for bone and soft tissue sarcomas, and discuss their potential as adjuvant therapies in combination with conventional therapies.

DOI： 10.1155/2015/820813

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

HSP90, found in all kingdoms of life, is a major chaperone protein regulating many client proteins. We demonstrated that HSP90α, one of two paralogs duplicated in vertebrates, plays an important role in the biogenesis of fetal PIWI-interacting RNAs (piRNA), which act against the transposon activities, in mouse male germ cells. The knockout mutation of Hsp90α resulted in a large reduction in the expression of primary and secondary piRNAs and mislocalization of MIWI2, a PIWI homolog. Whereas the mutation in Fkbp6 encoding a co-chaperone reduced piRNAs of 28-32 nucleotides in length, the Hsp90α mutation reduced piRNAs of 24-32 nucleotides, suggesting the presence of both FKBP6-dependent and -independent actions of HSP90α. Although DNA methylation and mRNA levels of L1 retrotransposon were largely unchanged in the Hsp90α mutant testes, the L1-encoded protein was increased, suggesting the presence of post-transcriptional regulation. This study revealed the specialized function of the HSP90α isofom in the piRNA biogenesis and repression of retrotransposons during the development of male germ cells in mammals.

DOI： 10.1093/nar/gku881

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：LIPPINCOTT WILLIAMS & WILKINS  

We conducted a clinical trial of an NY-ESO-1 cancer vaccine using 4 synthetic overlapping long peptides (OLP; peptides #1, 79-108; #2, 100-129; #3, 121-150; and #4, 142-173) that include a highly immunogenic region of the NY-ESO-1 molecule. Nine patients were immunized with 0.25 mg each of three 30-mer and a 32-mer long NY-ESO-1 OLP mixed with 0.2 KE Picibanil OK-432 and 1.25 mL Montanide ISA-51. The primary endpoints of this study were safety and NY-ESO-1 immune responses. Five to 18 injections of the NY-ESO-1 OLP vaccine were well tolerated. Vaccine-related adverse events observed were fever and injection site reaction (grade 1 and 2). Two patients showed stable disease after vaccination. An NY-ESO-1-specific humoral immune response was observed in all patients and an antibody against peptide #3 (121-150) was detected firstly and strongly after vaccination. NY-ESO-1 CD4 and CD8 T-cell responses were elicited in these patients and their epitopes were identified. Using a multifunctional cytokine assay, the number of single or double cytokine-producing cells was increased in NY-ESO-1-specific CD4 and CD8 T cells after vaccination. Multiple cytokine-producing cells were observed in PD-1 (-) and PD-1 (+) CD4 T cells. In conclusion, our study indicated that the NY-ESO-1 OLP vaccine mixed with Picibanil OK-432 and Montanide ISA-51 was well tolerated and elicited NY-ESO-1-specific humoral and CD4 and CD8 T-cell responses in immunized patients.

DOI： 10.1097/CJI.0000000000000017

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

CD8 T cells play a critical role in the host defense against cancers and infectious diseases. However, the presence of antigen-specific CD8 T cells does not always imply that cancers and/or pathogens are efficiently eliminated in the body. Concerning this point, the recent studies suggest the concept of immune exhaustion of CD8 T cells, characterized by their decreased production of IL-2, TNFα, and IFNγ even after antigen stimulation. Thus, continuous stimulation of CD8 T cells by the persistent antigens results in immune exhaustion, which eventually causes immune tolerance against cancers and chronic infections. The identification of immune effector and/or exhausted CD8 T cells by monitoring multiple parameters including T cell exhaustion markers such as PD-1 and Tim-3 and intracellular cytokines is, therefore, crucial to understand the real-time, ongoing immune status. For this purpose, polychromatic flow cytometry is the most common and reliable tool to monitor T cell functions. We describe here the method for detection of immune-exhaustion status of CD8 T cells from human peripheral blood mononuclear cells (PBMCs). By stimulation of PBMCs with PMA/ionomycin for 6 h, more than 1-2 % of total CD8 T cells are identified as positive in terms of multifunctionality, thus producing multiple cytokines--IL-2, TNFα, and IFNγ--at single-cell level in case of all healthy donors. By contrast, CD8 T cells from certain populations of cancer patients are significantly less effective; less than 0.5 % of CD8 T cells are positive in producing such multiple cytokines. The cutoff value around 0.5 % of CD8 T cells might distinguish patients who would receive beneficial effect by cancer vaccine from those who would not respond to the vaccine. Thus, the remaining capacity to produce multiple cytokines of CD8 T cells might be a critical parameter determining the outcome of cancer patients who receive various kinds of cancer vaccines. The method to monitor the state of multifunctionality of CD8 T cells, as described here, would become more important to understand the immune statues in cancers and chronic infectious diseases such as AIDS and malaria infections.

DOI： 10.1007/978-1-4939-0404-4_2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

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

DOI： 10.1002/ijc.27682

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Heat shock protein 90 (HSP90) is a molecular chaperone required for efficient antigen presentation and cross-presentation. In addition, HSP90 was recently reported to interact with and stabilize the activation-induced cytidine deaminase (AID) and plays a critical role in immunoglobulin gene hypermutation and class switch recombination. In mice and humans, there are two HSP90 isoforms, HSP90 and HSP90, but the in vivo role of each isoform remains largely unknown. Here we have analyzed humoral immune responses in HSP90-deficient mice. We found that HSP90 deficiency did not affect AID protein expression. B cell development and maturation, as well as immunoglobulin gene hypermuation and class switch, occurred normally in HSP90-deficient mice. However, antibody production to a T-dependent antigen was elevated in the mutant mice and this was associated with enhanced MHC class II antigen presentation to T helper cells by dendritic cells. Our results reveal a previously unidentified inhibitory role for HSP90 isoform in MHC class II antigen presentation and the humoral immune response. Along with our recent finding that HSP90 is required for antigen cross-presentation, these results suggest that HSP90 controls the balance of humoral and cellular immunity by dictating the fate of presentation of exogenous antigen.

DOI： 10.1093/intimm/dxs076

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Background/Aim: The cross-presentation system of tumor antigen by monocyte-derived dendritic cells (mo-DCs) has been observed under appropriate conditions. Both CD14- negative and CD1a-positive phenotypes were critical in our previous study. This study compared the phenotype of mo-DCs and identified the conditions that favored T helper-1 (Th1) cytokine production after stimulation with the hsc70 and NY-ESO- 1 p157-165 epitope fusion protein (hsc70/ESO p157- 165). Materials and Methods: The mo-DCs were induced from healthy donors. Their surface markers and cytokine production were examined after stimulation with hsc70/ESO p157-165. Results: CD1a+ and CD1a- mo-DCs were generated in half of the healthy donors. The concentration of fetal calf serum in the culture medium was critical for the induction of CD1a+ DCs, which were able to produce interleukin-12 (IL-12), but not IL-10. Neutralizing IL-6 and IL-6R antibodies affected the expression of CD1a. Conclusion: Anti IL-6 analogs may be effective adjuvants for the development of mo-DC-based cancer vaccine.

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

<title>Summary</title>
It is controversial whether a functional androgen receptor (AR) on germ cells, including spermatogonia, is essential for their development into sperm and, thus, initiation and maintenance of spermatogenesis. It was recently shown that many spermatocytes underwent apoptosis in the testes of Hsp90α KO mice. We had generated Hsp90α KO mice independently and confirmed this phenotype. However, the important question of whether Hsp90α is required to maintain spermatogenesis in adult mice in which testicular maturation is already completed could not be addressed using these conventional KO mice. To answer this question, we generated a tamoxifen-inducible deletion mutant of Hsp90α and found that conditional deletion of Hsp90α in adult mice caused even more severe apoptosis in germ cells beyond the pachytene stage, leading to complete arrest of spermatogenesis and testicular atrophy. Importantly, immunohistochemical analysis revealed that AR expression in WT testis was more evident in spermatogonia than in spermatocytes, whereas its expression was aberrant and ectopic in Hsp90α KO testis, raising the possibility that an AR abnormality in primordial germ cells is involved in spermatogenesis arrest in the Hsp90α KO mice. Our results suggest that the AR, specifically chaperoned by Hsp90α in spermatogonia, is critical for maintenance of established spermatogenesis and for survival of spermatocytes in adult testis, in addition to setting the first wave of spermatogenesis before puberty.

DOI： 10.1242/bio.2012646

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

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Priming of CD8(+) T cells requires two signals, one produced by T-cell receptor recognition of antigen, and a second that is often provided by the innate immune response. In this context, antigens non-covalently or covalently associated with heat shock proteins (HSP) are internalized and processed in antigen-presenting cells (APC) to be presented by MHC I molecules to CD8(+) T cells, thus, signal 1 has been well characterized in this pathway of cross-presentation. Signal 2 is not fully understood, although there are reports that Toll-like receptors (TLRs) interact with HSP and activate APC. The ability of HSP to activate APC through TLRs is, however, controversial because of the possibility of endotoxin contamination. Using a variety of TLR KO mice, we present evidence that TLRs (TLR2, 3, 4, 7, and 9) and their adaptor molecules MyD88 and IRAK4 are dispensable in cross-priming by a mycobacterial HSP70-antigen (ovalbumin as a model antigen) fusion protein; in contrast, MyD88/IRAK4, but not TLRs, are required for tumor rejection induced by the same reagent. Our results indicate that HSP-mediated cross-priming uses a second signal produced by mechanisms other than TLR cascades. We hypothesize that efficient cross-priming by HSP70 alone is insufficient for tumor rejection and that MyD88/IRAK4-dependent inflammatory stimulation, which might contribute to maintenance of the initially primed effector cells, is required to eradicate tumor burden.

DOI： 10.1111/j.1349-7006.2012.02233.x

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Authorship：Lead author,　Corresponding author   Language：English   Publisher：Okayama University Medical School  

DOI： 10.18926/AMO/48075

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

Antigens (Ag) from cancer or virus-infected cells must be internalized by dendritic cells (DCs) to be presented to CD8(+) T cells, which eventually differentiate into Ag-specific cytotoxic T lymphocytes (CTLs) that destroy cancer cells and infected cells. This pathway is termed cross-presentation and is also implicated as an essential step in triggering autoimmune diseases such as Type I diabetes. Internalized Ag locates within endosomes, followed by translocation through a putative pore structure spanning endosomal membranes into the cytosol, where it is degraded by the proteasome to generate antigen peptides. During translocation, Ag is believed to be unfolded since the pore size is too narrow to accept native Ag structure. Here, we show that paraformaldehyde-fixed, structurally inflexible Ag is less efficient in cross-presentation because of diminished translocation into the cytosol, supporting the "unfolded Ag" theory. We also show that HSP70 inhibitors block both endogenous and cross-presentation. ImageStream analysis revealed that the inhibition in cross-presentation is not due to blocking of Ag translocation because a HSP70 inhibitor rather facilitates the translocation, which is in marked contrast to the effect of an HSP90 inhibitor that blocks Ag translocation. Our results indicate that Ag translocation to the cytosol in cross-presentation is differentially regulated by HSP70 and HSP90.

DOI： 10.1155/2012/745962

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Other Link： http://downloads.hindawi.com/journals/ad/2012/745962.xml

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

In antigen (Ag) cross-presentation, dendritic cells (DCs) take up extracellular Ag and translocate them from the endosome to the cytosol for proteasomal degradation. The processed peptides can enter the conventional MHC I pathway. The molecules responsible for the translocation of Ag across the endosomal membrane into the cytosol are unknown. Here we demonstrate that heat shock protein 90 (HSP90) is critical for this step. Cross-presentation and -priming were decreased in both HSP90α-null DCs and mice. CD8α(+) DC apoptosis mediated by translocation of exogenous cytochrome c to the cytosol was also eliminated in HSP90α-null mice. Ag translocation into the cytosol was diminished in HSP90α-null DCs and in DCs treated with an HSP90 inhibitor. Internalized Ag was associated with HSP90 and translocated to the cytosol, a process abrogated by the HSP90 inhibitor. Ag within purified phagosomes was released in an HSP90-dependent manner. These results demonstrate the important role of HSP90 in cross-presentation by pulling endosomal Ag out into the cytosol.

DOI： 10.1073/pnas.1108372108

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Other Link： http://orcid.org/0000-0001-9049-8102

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

Extracellular HSP90 associated with Ag peptides have been demonstrated to efficiently cross-prime T cells, following internalization by dendritic cells (DCs). In addition, the nature of cell-associated Ags required for cross-priming is implicated as peptides and proteins chaperoned by heat shock protein (HSP). However, the role of endogenous HSP in DCs during cross-presentation remains elusive. In this paper, we show that endogenous HSP90 is essential for cross-presentation of both soluble and cell-associated Ags in DCs. Cross-presentation of soluble OVA and OVA-loaded transporter associated with Ag processing-1-deficient cells by bone marrow-derived DCs and DC-like cell line DC2.4 was profoundly blocked by HSP90 inhibitors, whereas presentation of endogenously expressed OVA was only partially suppressed. Assays using small interfering RNA and heat shock factor-1-deficient DCs (with defective expression of HSP90alpha) revealed the pivotal role of HSP90alpha in cross-presentation. The results suggest that in addition to HSP90 in Ag donor cells, endogenous HSP90 in DCs plays an essential role during Ag cross-presentation and, moreover, points to a link between heat shock factor-1-dependent induction of HSP90alpha within DC and cytotoxic T cell immunity.

DOI： 10.4049/jimmunol.1000821

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Cancer-induced immunotolerance mediated by inducible Treg (iTreg) is a major obstacle to cancer immunotherapy. In a basic study of immunotolerance, injection of an endogenous superantigen, i.e. the minor lymphocyte stimulatory (Mls)-1(a), into specific TCR Vbeta8.1-Tg mice enabled generation of anergic CD25(-) iTreg, the immunosuppressive function of which was maintained by IL-10 production via p38-MAPK activation. Interestingly, although p38-chemical inhibitor (p38-inhibitor) is capable of breaking CD25(-) iTreg-induced immunotolerance, the p38-inhibitor had hardly any immunotolerance breaking effect when CD25(+) Treg were present, suggesting that depletion of CD25(+) Treg is necessary for p38-inhibitor to be effective. Peptide OVA(323-339) iv.-injection into its specific TCR-Tg (OT-II) mice also induced adaptive tolerance by iTreg. Peptide immunotherapy with p38-inhibitor after CD25(+) Treg-depletion was performed in an OVA-expressing lymphoma E.G7-bearing tolerant model established by adoptive transfer of OT-II CD25(-) iTreg, which resulted in suppression of tumor growth. Similarly, the antitumor immunity induced by peptide immunotherapy in colon carcinoma CT26-bearing mice, in which the number of IL-10-secreting iTreg is increased, was augmented by treatment with p38-inhibitor after CD25(+) Treg-depletion and resulted in inhibition of tumor progression. These results suggest that simultaneous inhibition of two distinct Treg-functions may be important to the success of cancer immunotherapy.

DOI： 10.1002/eji.200939513

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

Heat shock proteins (HSP) are molecular chaperones implicated in facilitation of protein folding and translocation between distinct compartments, and hence in preventing protein from aggregation. In terms of proteolysis, HSP act as a double-edged sword, stimulating proteasome-dependent proteolysis while preventing the degradation of the same proteins, even though in both cases association of unfolded proteins with HSP is the initial step. The proteasomal degradation products are utilised as ligands of major histocompatibility complex (MHC) class I molecules to be recognised by CD8(+) T cells, leading to activation of cytotoxic T cell immunity indispensable in fighting virus infections and cancers. In this context, HSP-mediated antigen traffic towards proteasomal degradation is coupled with acquired T cell immunity. In addition, exogenous antigens internalised by dendritic cells (DC) are also forwarded to the proteasome, possibly through the ER-associated degradation (ERAD) system, based on the fusion of the ER-membrane to the endosome containing the antigens. Thus, antigens within endosomes might be translocated to the cytosol, possibly through the Sec61 complex recruited from ER and degraded by the proteasome, rendering their peptides presentable by MHC class I molecules, a process known as cross-presentation. Since binding protein (Bip) facilitates degradation of most ER luminal soluble proteins in yeast, it is possible that endosomal HSP in DC, mimicking the action of Bip, facilitate the degradation of internalised soluble antigens. This may explain why the HSP-peptide/protein complex is extremely efficient in terms of cross-presentation ability. In this review, we discuss how HSP are linked to the ubiquitin-dependent proteasome system to generate peptides presentable by MHC molecules.

DOI： 10.3109/02656730902902183

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Heat shock protein 90 (hsp90) and the proteasome activator PA28 stimulate major histocompatibility complex (MHC) class I antigen processing. It is unknown whether hsp90 influences the proteasome activity to produce T cell epitopes, although association of PA28 with the 20 S proteasome stimulates the enzyme activity. Here, we show that hsp90 is essential in assembly of the 26 S proteasome and as a result, is involved in epitope production. Addition of recombinant hsp90alpha to cell lysate enhanced chymotrypsin-like activity of the 26 S proteasome in an ATP-dependent manner as determined by an in-gel hydrolysis assay. We successfully pulled down histidine-tagged hsp90alpha- and PA28alpha-induced, newly assembled 26 S proteasomes from the cell extracts for in vitro epitope production assay, and we found these structures to be sensitive to geldanamycin, an hsp90 inhibitor. We found a cleaved epitope unique to the proteasome pulled down by both hsp90alpha and PA28alpha, whereas two different epitopes were identified in the hsp90alpha- and PA28alpha-pulldowns, respectively. Processing of these respective peptides in vivo was enhanced faithfully by the protein combinations used for the proteasome pulldowns. Inhibition of hsp90 in vivo by geldanamycin partly disrupted the 26 S proteasome structure, consistent with down-regulated MHC class I expression. Our results indicate that hsp90 facilitates MHC class I antigen processing through epitope production in a complex of the 26 S proteasome.

DOI： 10.1074/jbc.M803077200

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PA28 is an IFN-γ-inducible proteasome activator and its genetic ablation causes complete loss of processing of certain Ags, but not all of them. The reason why this occurs and how PA28 influences the formation of peptide repertoires for MHC class I molecules remains unknown. In this study, we show the allele-specific role of PA28 in Ag processing. Retrovirus-transduced overexpression of PA28α decreased expression of Kd (D d) while it increased Kb and Ld on the cell surface. By contrast, overexpression of PA28αΔC5, a mutant carrying a deletion of its five C-terminal residues and capable of attenuating the activity of endogenous PA28, produced the opposite effect on expression of those MHC class I molecules. Moreover, knockdown of both PA28α and β by small-interfering RNA profoundly augmented expression of Kd and Dd, but not of Ld, on the cell surface. Finally, we found that PA28-associated proteasome preferentially digested within epitopic sequences of Kd, although correct C-terminal flankings were removed, which in turn hampered production of Kd ligands. Our results indicate that whereas PA28 negatively influences processing of Kd (Dd) ligands, thereby, down-regulating Ag presentation by those MHC class I molecules, it also efficiently produces Kb (Ld) epitopes, leading to up-regulation of the MHC molecules. Copyright © 2008 by The American Association of Immunologists, Inc.

DOI： 10.4049/jimmunol.181.3.1655

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

Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8(+) T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8(+) T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4(+) T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)-derived 8 mer peptide, OVA(257-264), and 16mer peptide, OVA(265-280), were used as CD8(+) and CD4(+) T cell epitopes, respectively. Vaccination with hsc70-OVA(257-264) generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA(265-280) to the amino-terminus of hsc70-OVA(257-264) (OVA(265-280)-hsc70-OVA(257-264)) enhanced the generation of the OVA(257-264)-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA(257-264). Our results suggest that fusion of both CD4(+) and CD8(+) T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8(+) T cell epitope alone.

DOI： 10.1111/j.1349-7006.2008.00788.x

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DOI： 10.1111/j.1349-7006.2007.00654.x

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A member of the IFN regulatory factor (IRF) family of transcription factors, IRF-4 is expressed in lymphocytes and macrophage/dendritic cells. Studies using IRF-4-deficient mice have revealed the critical roles of IRF-4 in lymphocyte responses. However, the role of IRF-4 in innate immune responses is not clearly understood. Here, we demonstrate that IRF-4 negatively regulates the production of proinflammatory cytokines by macrophages in response to Toll-like receptor (TLR) stimulation. Mice lacking IRF-4 are sensitive to LPS-induced shock, and their macrophages produce high levels of proinflammatory cytokines, including TNF-alpha and IL-6, in response to TLR ligands. The inhibitory role of IRF-4 in response to TLR stimulation was confirmed by the down-regulation of IRF-4 expression in normal macrophages by using the small interfering RNA technique and by the overexpression of IRF-4 in macrophage line RAW264.7. Activation of the important signaling pathways for cytokine production, NF-kappaB and JNK (c-Jun N-terminal kinase), was enhanced after LPS stimulation in IRF-4(-/-) macrophages. These results imply that IRF-4 negatively regulates TLR signaling and is inhibitory to the production of proinflammatory cytokines in response to TLR stimulation.

DOI： 10.1073/pnas.0504226102

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DOI： 10.1128/MCB.25.19.8763.2005

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In anergic T cells, T-cell receptor (TCR)-mediated responses are functionally inactivated by negative regulatory signals whose mechanisms are poorly understood. Here, we show that CD4+ T cells anergized in vivo by superantigen Mls-1a express a scaffolding protein, transforming growth factor β-activated protein kinase 1-binding protein 1 (TAB1), that negatively regulates TCR signaling through the activation of mitogen-activated protein kinase p38α. TAB1 was not expressed in naive and activated CD4+ T cells. Inhibition of p38 activity in anergic T cells by a chemical inhibitor resulted in the recovery of interleukin 2 (IL-2) and the inhibition of IL-10 secretion. T-cell hybridoma 2B4 cells transduced with TAB1-containing retrovirus (TAB1-2B4 cells) showed activated p38α, inhibited extracellular signal-regulated kinase (ERK) activity, culminating in reduced IL-2 levels and increased IL-10 production. The use of a p38 inhibitor or cotransfection of a dominant-negative form of p38 in TAB1-2B4 cells resulted in the recovery of ERK activity and IL-2 production. These results imply that TAB1-mediated activation of p38α in anergic T cells regulates the maintenance of T-cell unresponsiveness both by inhibiting IL-2 production and by promoting IL-10 production.

DOI： 10.1128/MCB.24.16.6957-6966.2004

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Because a definite diagnosis of prion diseases relies on the detection of the abnormal isoform of prion protein (PrPSc), it has been urgently necessary to establish a non-invasive diagnostic test to detect PrP Sc in human prion diseases. To evaluate diagnostic usefulness and reliability of the detection of protease-resistant prion protein in urine, we extensively analyzed proteinase K (PK)-resistant proteins in patients affected with prion diseases and control subjects by Western blot, a coupled liquid chromatography and mass spectrometry analysis, and N-terminal sequence analysis. The PK-resistant signal migrating around 32 kDa previously reported by Shaked et al. (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482) was not observed in this study. Instead, discrete protein bands with an apparent molecular mass of ∼37 kDa were detected in the urine of many patients affected with prion diseases and two diseased controls. Although these proteins also gave strong signals in the Western blot using a variety of anti-PrP antibodies as a primary antibody, we found that the signals were still detectable by incubation of secondary antibodies alone, i.e. in the absence of the primary anti-PrP antibodies. Mass spectrometry and N-terminal protein sequencing analysis revealed that the majority of the PK-resistant 37-kDa proteins in the urine of patients were outer membrane proteins (OMPs) of the Enterobacterial species. OMPs isolated from these bacteria were resistant to PK and the PK-resistant OMPs from the Enterobacterial species migrated around 37 kDa on SDS-PAGE. Furthermore, nonspecific binding of OMPs to antibodies could be mistaken for PrPSc. These findings caution that bacterial contamination can affect the immunological detection of prion protein. Therefore, the presence of Enterobacterial species should be excluded in the immunological tests for PrPSc in clinical samples, in particular, urine.

DOI： 10.1074/jbc.M400187200

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Immunization with antigenic peptide non-covalently associated with HSP elicits the peptide specific CD8(+)T cell response. The evidence encourages us to test the vaccination effect of recombinant HSP to which antigenic peptides are genetically fused. In the fusion protein, there should be no empty HSP molecules that failed to associate with the peptide of interest, like in vitro reconstitution method, therefore, promising effect may be easily obtained. Recombinant proteins expressed in Escherichia coli often form inclusion bodies and are thereby obtained as insoluble proteins or as proteins lacking their original functions. We describe here a simple and rapid refolding method of histidine-tagged recombinant hsp70/hsp70-peptide complex using a Ni2+-agarose column chromatography, without taking a process of dialysis to remove denaturants. The hsp70(hsp70-peptide complex) expressed in E coli as a form of inclusion body was solubilized in 8 M urea containing buffer and applied to a Ni2+,agarose column. The bound hsp70 was refolded on the column by quick removal of urea with urea-free buffer and eluted with a denaturant-free and imidazole-containing buffer. The purified hsp70 was homogeneous and soluble. In addition, it had a very high ATPase activity and strong CTL inducing activity, whereas hsp70 prepared by conventional dialysis method had a negligible ATPase activity. This simple and rapid refolding method may provide a general method for a restoration of function (and/or immunization effect) and solubility of histidine-tagged recombinant HSP. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S1046-2023(03)00182-8

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Heat shock proteins (Hsp) 70 and Hsp 40 are stress proteins that cooperate as chaperones in mammalian cells. We determined the expression of Hsp 70 and Hsp 40 in 81 gastric cancers. Immunoreactivities to Hsp 70 and Hsp 40 were detected in 67.9 and 22.2% of tumors, respectively. Immunohistochemical analysis showed enhanced Hsp 70 and Hsp 40 expression in gastric tumor tissue, relative to the surrounding normal tissue. Overexpression of Hsp 70 and Hsp 40 was also confirmed by immunoblotting. Among various clinicopathological parameters, low histopathological differentiation was associated with reduced expression of both proteins.

DOI： 10.1016/S0304-3835(03)00305-7

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IFN-regulatory factor (IRF)-4 is a member of the IRF family of transcription factors expressed in lymphocytes and macrophages. The previous studies using mice deficient in the IRF-4 gene showed profound defects in function of both B and T cells. To further investigate the role of IRF-4 in CD4+ T cell function, IRF-4-/- mice were challenged with the intracellular pathogen Leishmania major. The mice were protected against L. major during the early phase of the infection and CD4+ T cells of the infected mice produced IFN-γ in response to L. major antigen. However, during the late phase of infection, lymphocyte numbers were dramatically reduced in the draining lymph nodes, resulting in the deterioration of the lesion, indicating that IRF-4 was required for sustained immune responses against L. major infection. The function of CD4+ T cells was further investigated using TCR transgenic mice lacking the IRF-4 gene. CD4+ T cells from IRF-4-/- mice produced IFN-γ and expressed T-bet after culture under Th1-skewing conditions in vitro. However, Th2 cell development was not observed after culture under Th2-polarizing conditions. Proliferation of CD4+ T cells to IL-4 was reduced in IRF-4-/- mice, suggesting the defects in the responsiveness to IL-4. Furthermore, stimulation of the IRF-4-/- CD4+ T cells with IL-4-induced activation of signal transducer and activator of transcription 6, but not expression of growth factor independent-1. Thus, development of CD4+ T cell subsets differentially depends on IRF-4; induction of Th1 response does not depend on IRF-4, while Th2 response depends entirely on IRF-4.

DOI： 10.1093/intimm/dxg001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

One of the difficulties in developing an effective malaria vaccine is the antigenic change of the parasite during the life cycle. It is desirable that vaccine-induced protective immunity be effective at different stages of parasite development. Merozoite surface protein 1 (MSP1) is a candidate vaccine antigen against blood-stage malaria, but it is also expressed in the exoerythrocytic forms. It was not known, however, whether the anti-MSP1 immune response is effective against the liver-stage malaria parasite. We generated a recombinant protein of MSP1 fused to heat-shock cognate protein 70 (hsc70) and studied its vaccination effect. When C57BL/6 mice were immunized with the fusion protein prior to challenge infection with Plasmodium yoelii sporozoites, the onset of parasitemia was delayed or no parasitemia was observed. To determine whether this was due to the protective immunity against liver-stage parasites, P. yoelii-specific rRNA in the infected liver was quantitated by real-time reverse transcription-PCR analysis. The level of parasite-specific rRNA was reduced in mice immunized with the fusion protein of MSP1 and hsc70 but not with hsc70 alone, indicating that MSP1-specific immunity can be protective against the exoerythrocytic form of the parasite. Furthermore, the adoptive transfer experiments of immune lymphocytes and serum into naive mice suggested that the protective immunity was dependent on cellular and not Immoral immunity. Finally, the vaccine-induced protection was also observed in A/J, C3H, and BALB/c mice, suggesting that MSP1-specific protective immunity at the exoerythrocytic stage can be induced in animals over a wide range of genetic backgrounds.

DOI： 10.1128/IAI.70.11.6075-6082.2002

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Major histocompatibility complex (MHC) class I ligands are mainly produced by the proteasome. Herein, we show that the processing of antigens is regulated by two distinct pathways. one requiring PA28 and the other hsp90. Both hsp90 and PA28 enhanced the antigen processing of ovalbumin (OVA). Geldanamycin, an inhibitor of hsp90, almost completely suppressed OVA antigen presentation in PA28alpha(-/-)/beta(-/-) lipopolysaccharide blasts, but not in wild-type cells, indicating that hsp90 compensates for the loss of PA28 and is essential in the PA28-independent pathway. In contrast, treatment of cells with interferon (IFN)-gamma, which induces PA28 expression, abrogated the requirement of hsp90, suggesting that IFN-gamma enhances the PA28-dependent path Nay, whereas it diminishes hsp90-dependent pathway. Importantly, IFN-gamma did not induce MHC class I expressions in PA28-deficient cells, indicating a prominent role for PA28 in IFN-gamma-stiniulated peptide supply. Thus, these two pathways operate either redundantly or specifically, depending on antigen species and cell type.

DOI： 10.1084/jem.20011922

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

Two members of the proteasome activator, PA28alpha and PA28beta, form a heteropolymer that binds to both ends of the 20S proteasome. Evidence in vitro indicates that this interferon-gamma (IFN-gamma)-inducible heteropolymer is involved in the processing of intracellular antigens, but its functions in vivo remain elusive. To investigate the role of PA28alpha/beta in vivo, we generated mice deficient in both PA28alpha and PA28beta genes. The ATP-dependent proteolytic activities were decreased in PA28alpha(-/-)/beta(-/-) cells, suggesting that 'hybrid proteasomes' are involved in protein degradation. Treatment of PA28alpha(-/-)/beta(-/-) cells with IFN-gamma resulted in sufficient induction of the 'immunoproteasome'. Moreover, splenocytes from PA28alpha(-/-)/beta(-/-) mice displayed no apparent defects in processing of ovalbumin. These results are in marked contrast to the previous finding that immunoproteasome assembly and immune responses were impaired in PA28beta(-/-) mice. PA28alpha(-/-)/beta(-/-) mice also showed apparently normal immune responses against infection with influenza A virus. However, they almost completely lost the ability to process a melanoma antigen TRP2-derived peptide. Hence, PA28alpha/beta is not a prerequisite for antigen presentation in general, but plays an essential role for the processing of certain antigens.

DOI： 10.1093/emboj/20.21.5898

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

Immunization with gp96 and heat shock cognate protein 70 (hsc70) purified with in vivo bound naturally occurring peptides or bound to synthetic peptides by in vitro reconstitution has been shown to induce peptide-specific cytotoxic T lymphocytes (CTL). In addition, mycobacterial heat shock protein 70 covalently fused to ovalbumin (OVA)-derived fragments has been shown to generate MHC class I-restricted CTL responses. Here, we genetically fused five different CTL epitopes, including peptides derived from Plasmodium yoelli circumsporozoite protein, tumor antigens, HY antigen and OVA, to either the N- or C-terminus of murine hsc70 and expressed the resulting proteins in Escherichia coli. Vaccination with all five fusion proteins induced peptide-specific CTL, indicating that no cognate flanking regions of CTL epitopes are necessary for the immune response. The point of injection was crucial for CTL induction. CD4(+) T cells were not required for the priming of CD8(+) T cells and vaccination with bone marrow-derived dendritic cells pulsed with hsc70 fusion proteins also elicited CTL responses. Furthermore, by using deletion mutants of hsc70, we identified amino acid residues 280-385 of hsc70 as the region most critical for inducing the CTL response.

DOI： 10.1093/intimm/13.10.1233

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academy of Cancer Immunology  

Tumors elicit an immune response in hosts and yet, paradoxically, often grow progressively with fatal consequences. This phenomenon has been attributed to the possible expression by tumor cells of immunomodulatory factors that overcome the anti-tumor effector functions of both specific and non-specific immune cells. This study reports on the ability of the methylcholanthrene- induced fibrosarcoma, Meth A, as well as other tumors of varied histological origins to downregulate the lytic activity of CD8+ T cells. The suppressive activity is contact-dependent and reversible. As tumor-bearing hosts are rarely immunosuppressed systemically, these findings may explain how local events within the tumor bed subvert the specific anti-tumor immune response.

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

Background: Proinflammatory cytokines, such as tumor necrosis factor (TNF-α) and interleukin-1 (IL-1), play important roles in acute allograft rejection. FR167653 is an inhibitor of these cytokines that acts through inhibition of the mitogen-activated protein kinase p38 pathway. We examined the effect of FR167653 on allograft rejection. Methods: We used Brown-Norway and Lewis rats as donors and recipients, respectively. We performed heterotopic cardiac transplantation. The control group consisted of untreated rats. In the experimental groups, recipients were intraperitoneally injected with FR167653 just after operation, followed by daily injection of the drug from Day 1 to 10. We divided 20 rats into 5 groups, which received varying doses of FR167653, ranging from 75 to 300 mg/kg/day. Results: In the control group, the mean graft survival was 6.8 ± 0.3 days. FR167653 at 150 mg/kg/day significantly prolonged the survival period (up to 12.1 ± 1.5 days, p = 0.002). Histologically, FR167653 markedly suppressed cellular infiltration on Day 5 post-transplantation. The serum level of TNF-α in the control group was persistently elevated from 9.3 ± 3.9 pg/ml to 11.3 ± 3.8 pg/ml, whereas FR167653 significantly suppressed the level to &lt
1.4 ± 1.4 pg/ml.ConclusionsFR167653 prolonged rat cardiac allograft survival by suppressing the action of proinflammatory cytokines. Copyright © 2001 International Society for Heart and Lung Transplantation.

DOI： 10.1016/S1053-2498(00)00324-7

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE LTD  

Cryptococcus neoformans causes infection in individuals with defective T cell function, such as AIDS, as well as without underlying disease. It has been suggested that humoral as well as cellular immunity might play an important role in the immune response to C. neoformans infection. We have recently shown, using immunoblotting, that the 70-kD hsp family of C. neoformans was the major target molecule of the humoral response in murine pulmonary cryptococcosis. In this study we also used immunoblotting to define the antibody responses in the sera of 24 patients with pulmonary cryptococcosis: 21 proven and three suspected diagnoses. Anti-C. neoformans hsp70 antibody was detected in 16 of 24 (66.7%) patients with pulmonary cryptococcosis. Fourteen of 17 (82.3%) patients with high antigen titres (greater than or equal to 1:8) and two of seven (28.6%) patients with low titres (less than or equal to 1:4) had detectable levels of anti-hsp70 antibody. Sera from patients positive for anti-hsp70 antibody showed high titres in the Eiken latex agglutination test for the detection of serum cryptococcal antigen. Our results indicate that the 70-kD hsp family from C. neoformans appears to be a major target molecule of the humoral response, not only in murine pulmonary cryptococcosis, but also in human patients with pulmonary cryptococcosis.

DOI： 10.1046/j.1365-2249.1999.00821.x

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER ASSOC IMMUNOLOGISTS  

We have previously demonstrated that vaccination with heat shock proteins hsp70, hsp90, and gp96 elicits specific immunity against the tumor from which the hsps were purified. Although the association of tumor Ag peptides with these hsps have been suggested, the identification of the peptides or their precursors stripped from the hsps remained to be resolved. We show in this report that an L-d-restricted cytotoxic T lymphocyte epitope of a mouse leukemia RL male 1 and its precursors are associated with the chaperones hsp90 and hsp70 in the cytosol and gp96 in the lumen of the endoplasmic reticulum, Hsp70 was associated,vith only final sized octamer, while hsp90 was found to associate with the octamer and two distinct precursor peptides, The gp96,vas associated with the octamer and one of the two precursors. Thus, each of the hsps bound a distinct set of peptides, Our results have demonstrated for the first time that the hsps associate not only with final sized tumor Ag peptide but also with its precursors. The implication of this evidence is also discussed in terms of the roles of hsps in MHC class I Ag processing/presentation.

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Language：Japanese   Publishing type：Research paper (international conference proceedings)  

Adjuvant-free vaccination with tumor-derived heat shock protein (HSP)- peptide complex has been previously shown to elicit tumor-specific protective and therapeutic immunity in murine systems. We investigated the in vitro effect of hsp70 preparations purified from surgically resected human colon cancer on autologous and allogeneic peripheral blood mononuclear cells (PBMC). The secretion of a considerable amount of TNF-α and INF-γ was observed both in autologous and allogeneic PBMC, while the proliferative response and tumor-specific cytotoxic activity were induced only in autologous PBMC. These results suggest that autologous tumor-derived HSP may not only serve as a nonspecific adjuvant but also act as a specific tumor vaccine for cancer immunotherapy.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PROFESSOR D A SPANDIDOS  

Cytotoxic T lymphocytes (CTL) specific for HLA-A2-binding MAGE-3 peptide (FLWGPRALV) were generated by repetitive stimulation of PBMC with the peptide in the presence of EBV-transformed B blasts and IL-2. Using these CTL, we investigated the expression of the HLA-A2-binding MAGE-3 peptide on lung cancer cell lines. Of 14 cell lines investigated, 1-87, PC-9, OU-LC-KI, 11-18 and LK87 were derived from HLA-A2 positive patients. But cytofluorometry analysis showed that 1-87, PC-9 and OU-LC-KI, but not 11-18 or LK87 expressed the HLA-A2 antigen. All five cell lines expressed MAGE-3 gene mRNA. Twelve of thirteen CTL lines from two HLA-A2 positive donors showed no cytotoxicity against any of the 14 lung cancer cell lines. CTL line TI-1 showed cytotoxicity against 1-87 but not against any of the other cell lines. Treatment of 1-87 with IFN-gamma greatly augmented the cytotoxicity of TI-1 and induced it in the other 12 CTL lines, confirming the expression of the peptide on 1-87. No cytotoxicity was induced by IFN- gamma treatment of PC-9 or OU-LC-KI. However, PC-9 and OU-LC-KI pulsed with the peptide were killed efficiently by all of the CTL lines, suggesting no expression of the peptide on those cells. A low level of cytotoxicity was induced on 11-18 but not LK87 by IFN-gamma treatment, although expression of the HLA-A2 antigen was not observed by cytofluorometry. These findings showed that expression of the HLA-A2-binding MAGE-3 peptide recognized by CTL was variable on lung cancer cell lines.

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Language：Japanese   Publishing type：Research paper (international conference proceedings)  

Heat shock proteins (HSPs) facilitate intracellular translocation of antigenic peptides as chaperones and play an essential role in antigen processing and presentation. Tumor-derived HSPs function as tumor rejection antigens to induce tumor-specific transplantation resistance. To determine if tumor-derived HSPs chaperone tumor-antigenic peptides, we purified hsp70, gp96 and hsp90 from mouse radiation-induced leukemia RL ♂ 1 and dissociated peptides from the HSPs. Tandem mass spectrometry of the dissociated peptide fractions with which RL ♂ 1-specificity CTL, Y-15 exhibited cytotoxicity against peptide-pulsed P815 revealed that hsp70 was associated with a known tumor-rejection-antigenic peptide, pRL1a, gp96 was associated with pRL1 and its precursor peptide, pRL1b, and hsp90 was associated with pRL1, pRL1b and other unknown antigenic peptide. Hsp70 and gp96 purified from surgically resected human colon cancer strongly stimulated autologous peripheral blood mononuclear cells for the production of interferon γ, tumor necrosis factor α and proliferative responses. The cytotoxicity of tumor-specific cytotoxic T lymphocytes was also induced by the culture with the hsp70. These results suggest that the autologous cancer-derived HSP, which is associated with both known and unknown tumor-antigenic peptides, is an effective tumor vaccine for the immunotherapy of cancers without the HLA haplotype restriction.

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Publishing type：Research paper (scientific journal)   Publisher：Rockefeller University Press  

Heat shock protein (HSP) preparations derived from cancer cells and virus-infected cells have been shown previously to elicit cancer-specific or virus-specific immunity. The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs. The studies reported here demonstrate that immunogenic HSP–peptide complexes can also be reconstituted in vitro. The studies show that (a) complexes of hsp70 or gp96 HSP molecules with a variety of synthetic peptides can be generated in vitro; (b) the binding of HSPs with peptides is specific in that a number of other proteins tested do not bind synthetic peptides under the conditions in which gp96 molecules do; (c) HSP–peptide complexes reconstituted in vitro are immunologically active, as tested by their ability to elicit antitumor immunity and specific CD8+ cytolytic T lymphocyte response; and (d) synthetic peptides reconstituted in vitro with gp96 are capable of being taken up and re-presented by macrophage in the same manner as gp96– peptides complexes generated in vivo. These observations demonstrate that HSPs are CD8+ T cell response–eliciting adjuvants.

DOI： 10.1084/jem.186.8.1315

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Heat shock proteins have been implicated as tumor specific transplantation antigens of which the precise cellular mechanisms have been unknown. For the past several years animal model experiments have revealed some answers to this mystery which will be discussed in this review as follows. Firstly, hsps as carrier proteins for antigenic peptides
secondly, novel priming of CD8+ T cells by hsp peptide complex
and thirdly, involvement of hsps in antigen processing.

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

DOI： 10.1073/pnas.91.8.3077

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Vaccination of mice with heat shock protein 70 (hsp70) preparations derived from the Meth A sarcoma, but not from normal tissues, renders the mice immune to a substantial challenge with Meth A sarcoma. The immunogenicity is dose dependent and tumor specific. Treatment of an antigenically active hsp70 preparation with ATP followed by removal of low-molecular weight material leaves hsp70 intact, as judged by SDS-PAGE but results in loss of antigenicity, as judged by tumor rejection assays. Separation of this low-molecular weight material on a C18 reverse-phase column shows a diverse array of peptides with molecular mass between 1,000 and 5,000 daltons. Our data indicate that antigenicity of hsp70 preparations derives, not from hsp70 per se, but from associated peptides. These observations may suggest a novel method of using the peptide-binding property of hsp70 for specific vaccination against cancer and infectious diseases.

DOI： 10.1084/jem.178.4.1391

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：VCH PUBLISHERS INC  

DOI： 10.1002/eji.1830200931

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

DOI： 10.1084/jem.171.4.1141

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Authorship：Lead author   Language：English   Publisher：JAPANESE CANCER ASSOCIATION  

DOI： 10.1111/j.1349-7006.1989.tb01692.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：GUSTAV FISCHER VERLAG  

DOI： 10.1016/s0171-2985(89)80061-0

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER SOC CLINICAL ONCOLOGY  

DOI： 10.1200/JCO.2019.37.15_suppl.e14182

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Language：Japanese   Publisher：日本医学会  

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Language：Japanese   Publisher：中部大学生命健康科学研究所  

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

＜文献概要＞糖尿病により,がん罹患リスクが上昇することはよく知られている。だが,現在のところ血糖コントロールを厳密にすることでがん罹患率が減少するといった良質なエビデンスは存在しない。しかし,メトホルミン内服によりがん罹患率,がん死亡率が低下することは,メタアナリシスから示唆されている。In vivo,in vitroの実験からメトホルミンの抗腫瘍効果のメカニズムは徐々に解明されつつあるが,近年免疫細胞を介して抗腫瘍効果を発現しているとの報告があり,注目を浴びている。メトホルミンはUKPDS(United Kingdom Prospective Diabetes Study)の報告以降「古くて新しい薬」と言われているが,現在でもさまざまな薬理作用が研究されていることから,今でも「古くて新しい薬」である。

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近年、T細胞のエフェクター機能について、細胞内代謝の視点からアプローチした研究報告が散見されている。特にT細胞受容体(TCR)シグナルの下流では、解糖系の亢進が種々の機序によってT細胞のエフェクター機能につながっていることが明らかになってきた。さらには、アミノ酸代謝やエネルギーセンサー分子であるAMPKの活性化がそれらに深くかかわっていることも報告されている。CD8陽性T細胞の代謝を調節し、そのエフェクター機能を高めることで抗腫瘍効果をもたらすがん免疫療法のさらなる進歩に期待したい。(著者抄録)

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY  

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代謝はT細胞応答に密接に関与しており、感染やがんなどの生体防御においても重要である。特に解糖系(糖代謝)はエフェクターT細胞機能に不可欠な代謝経路である。近年、がん微小環境下は低酸素/低栄養状態であり、このような環境下ではエフェクターT細胞は本来の機能が発揮し難いことが明らかにされている。このような環境におけるT細胞の機能を制御するために、様々な代謝薬を用いたがん免疫療法に関する研究が展開されつつある。本稿では、がん微小環境における"代謝競合"とそれに伴う腫瘍浸潤T細胞の機能低下について言及する。(著者抄録)

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2017-5592

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T細胞は、その分化・成熟および抗原刺激に続くclonal expansion、さらには記憶T細胞への移行において、刻々と代謝システムを変化させている。これは、T細胞抗原受容体シグナルにはしまる各種転写因子の活性化と制御による。一方、薬剤等で強制的に代謝を改変した場合、逆にT細分の分化・増殖と機能を変化させうることが徐々に明らかになってきた。すなわち、代謝改変は免疫細胞のあり方を変化させることのできる新たな免疫調節法を提示する。とりわけ、がんに対する免疫応答を改善できる可能性がある。(著者抄録)

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：AMER ASSOC CANCER RESEARCH  

DOI： 10.1158/1538-7445.AM2016-4865

Web of Science

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Language：Japanese   Publisher：(公社)日本整形外科学会  

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Language：Japanese   Publisher：日本がん免疫学会  

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Language：Japanese   Publisher：(一社)日本細胞生物学会  

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Language：Japanese   Publisher：(株)医薬ジャーナル社  

近年、2型糖尿病薬メトホルミンは抗腫瘍作用があるとの報告があり、岡山大学ではマウス腫瘍移植モデルにおいて、その抗腫瘍作用は免疫系、特にCD8 T細胞の機能を介した作用であることを報告している。腫瘍内でCD8 T細胞の多くはアポトーシスを起こし、機能も低下しているが、メトホルミン投与によりアポトーシスが抑制され、高機能なエフェクターメモリー型のCD8 T細胞が誘導される。その作用機序としては、AMPK(AMP活性化プロテインキナーゼ)の活性化を介した解糖系(糖代謝)の制御が重要ではないかと考えられる。現在、代謝と免疫応答の関係は非常に注目されたトピックスであり、効果的ながん免疫治療を考える上でも代謝の観点からのアプローチは必要であろう。本邦では、糖尿病薬メトホルミンについて、またその抗腫瘍作用の機序、糖代謝とT細胞応答、T細胞疲弊の関係について言及したい。(著者抄録)

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Language：Japanese   Publisher：(有)科学評論社  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GENETICS SOC JAPAN  

Web of Science

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Language：Japanese   Publisher：(株)癌と化学療法社  

感染症やがんでは、CD8T細胞は繰り返す抗原刺激により疲弊(exhaustion)する。疲弊の過程で抗原特異的なCD8T細胞はしだいにサイトカイン産生能を消失し、最終的にはアポトーシスにより死滅する。また、CD8T細胞は持続的な抗原刺激により、疲弊分子PD-1、CTLA-4、TIM-3、LAG-3を発現する。これら疲弊分子とそのリガンドとの結合により発生する負のシグナルによりCD8T細胞はより機能を消失する。2013年、抗PD-1、抗CTLA-4抗体により悪性黒色腫患者で劇的な腫瘍縮小効果が報告された。このようなCD8T細胞疲弊の制御は、今後のがん治療に新たな展開をもたらすと考えられる。(著者抄録)

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Language：Japanese   Publisher：日本がん免疫学会  

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Language：Japanese   Publisher：岡山医学会  

がんワクチンによる免疫治療では,如何にCD8T細胞を感作(プライミング)しその数を増やすか(免疫増強)という点に多大の努力が払われて来た.樹状細胞への抗原デリバリーと抗原プロセシング/提示,Toll様受容体などの刺激,即ち自然免疫系の活性化の併用などはそれに該当する.しかし十分に活性化されたT細胞をもってしても癌の拒絶は容易ではない.それには癌組織という特殊な環境が禍している.T細胞は癌塊内に入り込み莫大な数の癌細胞と遭遇する.癌組織内での繰り返す抗原認識の過程でT細胞は疲弊し,次第に本来あるべき機能を喪失していく.この疲弊(exhaustion)と呼ばれる現象は,T細胞に発現する複数の免疫抑制性分子-免疫チェックポイント分子-と腫瘍に発現するそのリガンドの結合によってもたらされる.代表的なチェックポイント分子の機能を抑制し,エフェクターT細胞が疲弊することなくその機能を長く維持できれば,これからのがん免疫治療に飛躍的な進展がみられるかもしれない.(著者抄録)

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Language：Japanese   Publisher：(公社)日本生化学会  

これまでの熱ショックタンパク質(HSP)研究の主流は生化学的手法によるものあるいは特定の細胞株を用いた実験系が大多数であったが、遺伝子改変マウスの作製が進むにつれその意義が生体レベルでも明らかにされるようになってきた。HSPは細胞をストレスから防御する、ということがその第一義的な役割だが、免疫系においてはがん抗原やウイルス抗原の抗原プロセシングひいては主要組織適合抗原(MHC)による抗原提示に重要な役割をもつことが示唆されている。凝集性を帯び始めたタンパク質はHSPに認識され、抗原プロセシングの過程に組み込まれる。Hsp90αKOマウス作製とそれを用いた研究により、これまでの「HSPと免疫研究」に確信と前進がもたらされた。遺伝子改変マウスを用いた実験結果と生化学的解析さらには分子イメージングから得られるHSPの免疫系、特に交差抗原提示機構における役割について最新の知見を論述・考察したい。(著者抄録)

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細胞は内在性抗原と外来性抗原を分解するために,各々ユビキチン-プロテアソーム系とライソゾーム系を具備している.しかし,抗原側からみるとこの両者は隔絶されたものではなく,互いに行きかうことが可能であり,独特の連続した分解システムを構築している.de novo合成されたMHCクラスI分子だけではなく,細胞膜に存在するMHCクラスI分子でもリサイクルシステムにより内在性及び外来性抗原とアクセスできるようである.シャペロン分子(HSPs)は,この2つの分解系にタンパク質を輸送する役割を担い,MHCクラスI,II抗原提示経路に予想を上回るダイナミズムを与えている

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DOI： 10.7601/mez.52.123_3

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