研究者データベース

岡川 朋弘(オカガワ トモヒロ)
獣医学研究院
特任助教

基本情報

所属

  • 獣医学研究院

職名

  • 特任助教

学位

  • 博士(獣医学)(北海道大学)

J-Global ID

研究キーワード

  • 感染症学   獣医免疫学   抗体工学   家畜衛生   感染免疫   腫瘍免疫   ウイルス学   

研究分野

  • ライフサイエンス / 動物生命科学 / 抗体工学
  • ライフサイエンス / 獣医学 / 感染症学
  • ライフサイエンス / 獣医学 / 獣医免疫学

職歴

  • 2018年07月 - 現在 北海道大学 大学院獣医学研究院 特任助教
  • 2017年04月 - 2018年06月 北海道大学 大学院獣医学研究院 博士研究員
  • 2016年04月 - 2017年03月 北海道大学 大学院獣医学研究科 博士研究員
  • 2013年04月 - 2016年03月 日本学術振興会 特別研究員(DC1)

学歴

  • 2012年04月 - 2016年03月   北海道大学   大学院獣医学研究科
  • 2006年04月 - 2012年03月   北海道大学   獣医学部

所属学協会

  • 日本免疫学会   日本獣医学会   

研究活動情報

論文

  • Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Hayato Nakamura, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Developmental and Comparative Immunology 114 103847  2021年01月 [査読有り]
  • Naftaly Wang'ombe Githaka, Satoru Konnai, Masayoshi Isezaki, Shinya Goto, Marina Amaral Xavier, Sotaro Fujisawa, Shinji Yamada, Tomohiro Okagawa, Naoya Maekawa, Carlos Logullo, Itabajara da Silva Vaz, Shiro Murata, Kazuhiko Ohashi
    Ticks and Tick-borne Diseases 11 6 101547  2020年11月 [査読有り]
  • Sotaro Fujisawa, Shiro Murata, Masayoshi Isezaki, Eiji Oishi, Akira Taneno, Naoya Maekawa, Tomohiro Okagawa, Satoru Konnai, Kazuhiko Ohashi
    Parasitology International 78 102156  2020年06月 [査読有り]
  • Shinya Goto, Satoru Konnai, Yuki Hirano, Junko Kohara, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Kei Watari, Erina Minato, Atsuhi Kobayashi, Satoshi Gondaira, Hidetoshi Higuchi, Masateru Koiwa, Motoshi Tajima, Eiji Taguchi, Masaru Ishida, Ryoko Uemura, Shinji Yamada, Mika K. Kaneko, Yukinari Kato, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Japanese Journal of Veterinary Research 68 2 77 - 90 2020年05月 [査読有り][通常論文]
     
    Mycoplasma bovis (M. bovis) is a highly contagious pathogen and M. bovis-associated diseases, particularly pneumonia, occur predominantly as herd enzootics, causing considerable economic losses because of calf mortality, weight loss in surviving calves. In our previous studies, the programmed death-1 (PD-1)/ PD-ligand 1 (PD-L1) pathway and prostaglandin E-2 (PGE(2)) were shown to be involved in the immunosuppression during chronic infectious diseases in cattle. In this study, the efficacy of dual blockade of the PD-1/PD-L1 pathway and PGE(2) in M. bovis infection in vivo was investigated using anti-bovine PDLl rat-bovine chimeric antibody, Boch4G12, and cyclooxygenase 2 (COX-2) inhibitor, meloxicam. The calves treated with Boch4G12 and meloxicam significantly enhanced M. bovis-specific IFN--gamma response after the administration. On the other hand, IFN-gamma response was not activated in the controls and cattle treated with meloxicam alone throughout the experimental period. Interestingly, bacterial loads in nasal discharge and bronchoalveolar lavage fluid among calves treated with Boch4G12 with or without meloxicam were significantly decreased. These results suggest that the combination of anti-PD-L1 antibody with a COX-2 inhibitor is a candidate for therapeutic applications in calves infected with M. bovis.
  • Yamato Sajiki, Satoru Konnai, Akie Ochi, Tomohiro Okagawa, Naftaly Githaka, Masayoshi Isezaki, Shinji Yamada, Takuya Ito, Shuji Ando, Hiroki Kawabata, Carlos Logullo, Itabajara da Silva Vaz, Naoya Maekawa, Shiro Murata, Kazuhiko Ohashi
    Ticks and Tick-borne Diseases 11 2 101332 - 101332 2020年03月 [査読有り][通常論文]
  • Hiroto Takeuchi, Satoru Konnai, Naoya Maekawa, Erina Minato, Atsushi Kobayashi, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi
    Fronters in Veterinary Science 7 330  2020年 [査読有り][通常論文]
  • Shinya Goto, Satoru Konnai, Yuki Hirano, Junko Kohara, Tomohiro Okagawa, Naoya Maekawa, Yamato Sajiki, Kei Watari, Erina Minato, Atsuhi Kobayashi, Satoshi Gondaira, Hidetoshi Higuchi, Masateru Koiwa, Motoshi Tajima, Eiji Taguchi, Ryoko Uemura, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Frontiers in Veterinary Science 7 12  2020年 [査読有り][通常論文]
     
    Bovine mycoplasmosis caused by Mycoplasma bovis results in pneumonia and mastitis in cattle. We previously demonstrated that the programmed death 1 (PD-1)/PD-ligand 1 (PD-L1) pathway is involved in immune dysfunction during M. bovis infection and that prostaglandin E2 (PGE2) suppressed immune responses and upregulated PD-L1 expression in Johne's disease, a bacterial infection in cattle. In this study, we investigated the role of PGE2 in immune dysfunction and the relationship between PGE2 and the PD-1/PD-L1 pathway in M. bovis infection. In vitro stimulation with M. bovis upregulated the expressions of PGE2 and PD-L1 presumably via Toll-like receptor 2 in bovine peripheral blood mononuclear cells (PBMCs). PGE2 levels of peripheral blood in infected cattle were significantly increased compared with those in uninfected cattle. Remarkably, plasma PGE2 levels were positively correlated with the proportions of PD-L1+ monocytes in M. bovis-infected cattle. Additionally, plasma PGE2 production in infected cattle was negatively correlated with M. bovis-specific interferon (IFN)-γ production from PBMCs. These results suggest that PGE2 could be one of the inducers of PD-L1 expression and could be involved in immunosuppression during M. bovis infection. In vitro blockade assays using anti-bovine PD-L1 antibody and a cyclooxygenase 2 inhibitor significantly upregulated the M. bovis-specific IFN-γ response. Our study findings might contribute to the development of novel therapeutic strategies for bovine mycoplasmosis that target PGE2 and the PD-1/PD-L1 pathway.
  • Immune inhibitory function of bovine CTLA-4 and the effects of its blockade in IFN-γ production
    Watari K, Konnai S, Maekawa N, Okagawa T, Suzuki Y, Murata S, Ohashi K
    BMC Veterinary Research 15 1 380  2019年09月 [査読有り][通常論文]
  • Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Kei Watari, Erina Minato, Atsushi Kobayashi, Junko Kohara, Shinji Yamada, Mika K Kaneko, Yukinari Kato, Hirofumi Takahashi, Nobuhiro Terasaki, Akira Takeda, Keiichi Yamamoto, Mikihiro Toda, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Journal of Immunology 203 5 1313 - 1324 2019年09月01日 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.
  • Vector transmission of bovine leukemia virus during summer season in Northern Hokkaido
    Inagaki H, Konnai S, Kaburagi H, Murota H, Takabatake N, Watari K, Okagawa T, Maekawa N, Murata S, Ohashi K
    Japanese Journal of Veterinary Research 2019年08月 [査読有り][通常論文]
  • Takehara M, Murata S, Katakura K, Hmoon M, Win S, Bawm S, Htun L, Aung Y, Win M, Isezaki M, Maekawa N, Okagawa T, Konnai S, Ohashi K
    Heliyon 5 4 e01544  2019年04月 [査読有り][通常論文]
  • Fujisawa S, Konnai S, Okagawa T, Maekawa N, Tanaka A, Suzuki Y, Murata S, Ohashi K
    BMC Veterinary Research 15 1 68  2019年02月 [査読有り][通常論文]
  • Ochirkhuu N, Konnai S, Odbileg R, Okagawa T, Murata S, Ohashi K
    Japanese Journal of Veterinary Research 66 3 177 - 192 2018年08月 [査読有り][通常論文]
  • Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Junko Kohara, Yasuhiko Suzuki, Shinji Yamada, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    Veterinary research 49 1 50 - 50 2018年06月19日 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) is a retrovirus that infects B cells in cattle and causes bovine leukosis after a long latent period. Progressive exhaustion of T cell functions is considered to facilitate disease progression of BLV infection. Programmed death-1 (PD-1) and lymphocyte activation gene-3 (LAG-3) are immunoinhibitory receptors that contribute to T-cell exhaustion caused by BLV infection in cattle. However, it is unclear whether the cooperation of PD-1 and LAG-3 accelerates disease progression of BLV infection. In this study, multi-color flow cytometric analyses of PD-1- and LAG-3-expressing T cells were performed in BLV-infected cattle at different stages of the disease. The frequencies of PD-1+LAG-3+ heavily exhausted T cells among CD4+ and CD8+ T cells was higher in the blood of cattle with B-cell lymphoma over that of BLV-uninfected and BLV-infected cattle without lymphoma. In addition, blockade assays of peripheral blood mononuclear cells were performed to examine whether inhibition of the interactions between PD-1 and LAG-3 and their ligands by blocking antibodies could restore T-cell function during BLV infection. Single or dual blockade of the PD-1 and LAG-3 pathways reactivated the production of Th1 cytokines, interferon-γ and tumor necrosis factor-α, from BLV-specific T cells of the infected cattle. Taken together, these results indicate that PD-1 and LAG-3 cooperatively mediate the functional exhaustion of CD4+ and CD8+ T cells and are associated with the development of B-cell lymphoma in BLV-infected cattle.
  • Yamato Sajiki, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Reiko Nagata, Satoko Kawaji, Yumiko Kagawa, Shinji Yamada, Yukinari Kato, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
    Infection and immunity 86 5 e00910-17  2018年05月 [査読有り][通常論文]
     
    Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, is a bovine chronic infection that is endemic in Japan and many other countries. The expression of immunoinhibitory molecules is upregulated in cattle with Johne's disease, but the mechanism of immunosuppression is poorly understood. Prostaglandin E2 (PGE2) is immunosuppressive in humans, but few veterinary data are available. In this study, functional and kinetic analyses of PGE2 were performed to investigate the immunosuppressive effect of PGE2 during Johne's disease. In vitro PGE2 treatment decreased T-cell proliferation and Th1 cytokine production and upregulated the expression of immunoinhibitory molecules such as interleukin-10 and programmed death ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) from healthy cattle. PGE2 was upregulated in sera and intestinal lesions of cattle with Johne's disease. In vitro stimulation with Johnin purified protein derivative (J-PPD) induced cyclooxygenase-2 (COX-2) transcription, PGE2 production, and upregulation of PD-L1 and immunoinhibitory receptors in PBMCs from cattle infected with M. avium subsp. paratuberculosis Therefore, Johnin-specific Th1 responses could be limited by the PGE2 pathway in cattle. In contrast, downregulation of PGE2 with a COX-2 inhibitor promoted J-PPD-stimulated CD8+ T-cell proliferation and Th1 cytokine production in PBMCs from the experimentally infected cattle. PD-L1 blockade induced J-PPD-stimulated CD8+ T-cell proliferation and interferon gamma production in vitro Combined treatment with a COX-2 inhibitor and anti-PD-L1 antibodies enhanced J-PPD-stimulated CD8+ T-cell proliferation in vitro, suggesting that the blockade of both pathways is a potential therapeutic strategy to control Johne's disease. The effects of COX-2 inhibition warrant further study as a novel treatment of Johne's disease.
  • Yamato Sajiki, Satoru Konnai, Asami Nishimori, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Masashi Nagano, Junko Kohara, Nana Kitano, Toshihiko Takahashi, Motoshi Tajima, Hirohisa Mekata, Yoichiro Horii, Shiro Murata, Kazuhiko Ohashi
    The Journal of veterinary medical science 79 12 2036 - 2039 2017年12月22日 [査読有り][通常論文]
     
    Enzootic bovine leukemia is caused by the bovine leukemia virus (BLV). BLV is transmitted vertically or horizontally through the transfer of infected cells via direct contact, through milk, insect bites and contaminated iatrogenic procedures. However, we lacked direct evidence of intrauterine infection. The purpose of this study was to confirm intrauterine BLV infection in two pregnant dams with high viral load by cesarean delivery. BLV was detected in cord and placental blood, and the BLV in the newborns showed 100% nucleotide identity with the BLV-env sequence from the dams. Notably, a newborn was seropositive for BLV but had no colostral antibodies. In this study, we presented a direct evidence of intrauterine BLV transmission in pregnant dam with a high proviral load. These results could aid the development of BLV control measures targeting viral load.
  • Goto S, Konnai S, Okagawa T, Nishimori A, Maekawa N, Gondaira S, Higuchi H, Koiwa M, Tajima M, Kohara J, Ogasawara S, Kato Y, Suzuki Y, Murata S, Ohashi K
    Immunity, inflammation and disease 5 3 355 - 363 2017年09月 [査読有り][通常論文]
     
    INTRODUCTION: Bovine mycoplasma, chiefly Mycoplasma bovis, is a pathogen that causes pneumonia, mastitis, arthritis, and otitis media in cattle. This pathogen exerts immunosuppressive effects, such as the inhibition of interferon production. However, the mechanisms involved in bovine mycoplasmosis have not been fully elucidated. In this study, we investigated the role of the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway in immunosuppression in bovine mycoplasmosis. METHODS: In the initial experiments, we used enzyme-linked immunosorbent assay to measure interferon-γ (IFN-γ) from peripheral blood mononuclear cells (PBMCs) isolated from cattle with mycoplasmosis. RESULTS: Expectedly, IFN-γ production significantly decreased in cattle with mycoplasmosis compared with that in clinically healthy cattle. Concomitantly, flow cytometric analysis revealed that the proportions of PD-1+ CD4+ and PD-L1+ CD14+ cells significantly increased in peripheral blood of the infected cattle. Interestingly, the number of PD-1+ CD4+ and PD-1+ CD8+ T cells were negatively correlated with IFN-γ production from PBMCs in bovine mycoplasmosis. Additionally, blockade of the PD-1/PD-L1 pathway in vitro by anti-bovine PD-1- and anti-bovine PD-L1 antibodies significantly upregulated the production of IFN-γ from anti-mycoplasma-specific cells. CONCLUSIONS: These results suggest that the PD-1/PD-L1 pathway could be involved in immune exhaustion of bovine mycoplasma-specific T cells. In conclusion, our study opens up a new perspective in the therapeutic strategy for bovine mycoplasmosis by targeting the immunoinhibitory receptor pathways.
  • Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Shinya Goto, Ryoyo Ikebuchi, Ayako Nakahara, Yuzumi Chiba, Masaho Ikeda, Shiro Murata, Kazuhiko Ohashi
    CLINICAL AND VACCINE IMMUNOLOGY 24 9 2017年09月 [査読有り][通常論文]
     
    Bovine leukemia is classified into two types: enzootic bovine leukosis (EBL) and sporadic bovine leukosis (SBL). EBL is caused by infection with bovine leukemia virus (BLV), which induces persistent lymphocytosis and B-cell lymphoma in cattle after a long latent period. Although it has been demonstrated that BLVassociated lymphoma occurs predominantly in adult cattle of > 3 to 5 years, suspicious cases of EBL onset in juvenile cattle were recently reported in Japan. To investigate the current status of bovine leukemia in Japan, we performed immunophenotypic analysis of samples from 50 cattle that were clinically diagnosed as having bovine leukemia. We classified the samples into five groups on the basis of the analysis and found two different types of EBL: classic EBL (cEBL), which has the familiar phenotype commonly known as EBL, and polyclonal EBL (pEBL), which exhibited neoplastic proliferation of polyclonal B cells. Moreover, there were several atypical EBL cases even in cEBL, including an early onset of EBL in juvenile cattle. A comparison of the cell marker expressions among cEBL, pEBL, and B-cell-type SBL (B-SBL) revealed characteristic patterns in B-cell leukemia, and these patterns could be clearly differentiated from those of healthy phenotypes, whereas it was difficult to discriminate between cEBL, pEBL, and B-SBL only by the expression patterns of cell markers. This study identified novel characteristics of bovine leukemia that should contribute to a better understanding of the mechanism underlying tumor development in BLV infection.
  • Naoya Maekawa, Satoru Konnai, Satoshi Takagi, Yumiko Kagawa, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Tatsuya Deguchi, Chie Nakajima, Yukinari Kato, Keiichi Yamamoto, Hidetoshi Uemura, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    SCIENTIFIC REPORTS 7 1 8951  2017年08月 [査読有り][通常論文]
     
    Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated in vitro, demonstrating significantly enhanced cytokine production and proliferation of dog peripheral blood mononuclear cells. A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM (14.3%, 1/7) and one with undifferentiated sarcoma (50.0%, 1/2) when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.
  • Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Chie Nakajima, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    FRONTIERS IN IMMUNOLOGY 8 650  2017年06月 [査読有り][通常論文]
     
    Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1)/PD-ligand 1 (PD-L1), is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat-bovine chimeric monoclonal antibody 5D2 (Boch5D2) was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV). Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4(+) T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.
  • Asami Nishimori, Satoru Konnai, Tomohiro Okagawa, Naoya Maekawa, Ryoyo Ikebuchi, Shinya Goto, Yamato Sajiki, Yasuhiko Suzuki, Junko Kohara, Satoshi Ogasawara, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    PLOS ONE 12 4 e0174916  2017年04月 [査読有り][通常論文]
     
    Programmed death-1 (PD-1), an immunoinhibitory receptor on T cells, is known to be involved in immune evasion through its binding to PD-ligand 1 (PD-L1) in many chronic diseases. We previously found that PD-L1 expression was upregulated in cattle infected with bovine leukemia virus (BLV) and that an antibody that blocked the PD-1/PD-L1 interaction reactivated T-cell function in vitro. Therefore, this study assessed its antivirus activities in vivo. First, we inoculated the anti-bovine PD-L1 rat monoclonal antibody 4G12 into a BLV-infected cow. However, this did not induce T-cell proliferation or reduction of BLV provirus loads during the test period, and only bound to circulating IgM(+) B cells until one week post-inoculation. We hypothesized that this lack of in vivo effects was due to its lower stability in cattle and so established an anti-PD-L1 rat-bovine chimeric antibody (Boch4G12). Boch4G12 was able to bind specifically with bovine PD-L1, interrupt the PD-1/PD-L1 interaction, and activate the immune response in both healthy and BLV-infected cattle in vitro. Therefore, we experimentally infected a healthy calf with BLV and inoculated it intravenously with 1 mg/kg of Boch4G12 once it reached the aleukemic (AL) stage. Cultivation of peripheral blood mononuclear cells (PBMCs) isolated from the tested calf indicated that the proliferation of CD4(+) T cells was increased by Boch4G12 inoculation, while BLV provirus loads were significantly reduced, clearly demonstrating that this treatment induced antivirus activities. Therefore, further studies using a large number of animals are required to support its efficacy for clinical application.
  • Increase of cells expressing PD-1 and PD-L1 and enhancement of IFN-γ production via PD-1/PD-L1 blockade in bovine mycoplasmosis.
    Goto, S, Konnai, S, Okagawa, T, Nishimori, A, Maekawa, N, Gondaira, S, Higuchi, H, Koiwa, M, Tajima, M, Kohara, J, Ogasawara, S, Kato, Y, Suzuki, Y, Murata, S, Ohashi, K
    Immunity, Inflammation and Disease 10 1 - 9 2017年 [査読有り][通常論文]
  • Tomohiro Okagawa, Satoru Konnai, James R. Deringer, Massaro W. Ueti, Glen A. Scoles, Shiro Murata, Kazuhiko Ohashi, Wendy C. Brown
    Infection and immunity 84 10 2779 - 2790 2016年10月 [査読有り][通常論文]
     
    The CD4(+) T-cell response is central for the control of Anaplasma marginale infection in cattle. However, the infection induces a functional exhaustion of antigen-specific CD4(+) T cells in cattle immunized with A. marginale outer membrane proteins or purified outer membranes (OMs), which presumably facilitates the persistence of this rickettsia. In the present study, we hypothesize that T-cell exhaustion following infection is induced by the upregulation of immunoinhibitory receptors on T cells, such as programmed death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3). OM-specific T-cell responses and the kinetics of PD-1-positive (PD-1(+)) LAG-3(+) exhausted T cells were monitored in A. marginale-challenged cattle previously immunized with OMs. Consistent with data from previous studies, OM-specific proliferation of peripheral blood mononuclear cells (PBMCs) and interferon gamma (IFN-gamma) production were significantly suppressed in challenged animals by 5 weeks postinfection (wpi). In addition, bacteremia and anemia also peaked in these animals at 5 wpi. Flow cytometric analysis revealed that the percentage of PD-1(+) LAG-3(+) T cells in the CD4(+), CD8(+), and delta gamma T-cell populations gradually increased and also peaked at 5 wpi. A large increase in the percentage of LAG-3(+) gamma delta T cells was also observed. Importantly, in vitro, the combined blockade of the PD-1 and LAG-3 pathways partially restored OM-specific PBMC proliferation and IFN-gamma production at 5 wpi. Taken together, these results indicate that coexpression of PD-1 and LAG-3 on T cells contributes to the rapid exhaustion of A. marginale-specific T cells following infection and that these immunoinhibitory receptors regulate T-cell responses during bovine anaplasmosis.
  • Naoya Maekawa, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ryoyo Ikebuchi, Yusuke Izumi, Satoshi Takagi, Yumiko Kagawa, Chie Nakajima, Yasuhiko Suzuki, Yukinari Kato, Shiro Murata, Kazuhiko Ohashi
    PLOS ONE 11 6 e0157176  2016年06月 [査読有り][通常論文]
     
    Spontaneous cancers are common diseases in dogs. Among these, some malignant cancers such as oral melanoma, osteosarcoma, hemangiosarcoma, and mast cell tumor are often recognized as clinical problems because, despite their high frequencies, current treatments for these cancers may not always achieve satisfying outcomes. The absence of effective systemic therapies against these cancers leads researchers to investigate novel therapeutic modalities, including immunotherapy. Programmed death 1 (PD-1) is a costimulatory receptor with immunosuppressive function. When it binds its ligands, PD-ligand 1 (PD-L1) or PD-L2, PD-1 on T cells negatively regulates activating signals from the T cell receptor, resulting in the inhibition of the effector function of cytotoxic T lymphocytes. Aberrant PD-L1 expression has been reported in many human cancers and is considered an immune escape mechanism for cancers. In clinical trials, anti-PD-1 or anti-PD-L1 antibodies induced tumor regression for several malignancies, including advanced melanoma, non-small cell lung carcinoma, and renal cell carcinoma. In this study, to assess the potential of the PD-1/PD-L1 axis as a novel therapeutic target for canine cancer immunotherapy, immunohistochemical analysis of PD-L1 expression in various malignant cancers of dogs was performed. Here, we show that dog oral melanoma, osteosarcoma, hemangiosarcoma, mast cell tumor, mammary adenocarcinoma, and prostate adenocarcinoma expressed PD-L1, whereas some other types of cancer did not. In addition, PD-1 was highly expressed on tumor-infiltrating lymphocytes obtained from oral melanoma, showing that lymphocytes in this cancer type might have been functionally exhausted. These results strongly encourage the clinical application of PD-1/PD-L1 inhibitors as novel therapeutic agents against these cancers in dogs.
  • Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Ayako Nakahara, Shiro Murata, Kazuhiko Ohashi
    Journal of veterinary medical science 78 5 791 - 796 2016年05月 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.
  • Nyamsuren Ochirkhuu, Satoru Konnai, Raadan Odbileg, Asami Nishimori, Tomohiro Okagawa, Shiro Murata, Kazuhiko Ohashi
    Archives of virology 161 4 985 - 991 2016年04月 [査読有り][通常論文]
     
    Epidemiological studies have indicated that bovine leukemia virus (BLV) infection is globally distributed. However, no information regarding the disease and genetic diversity of the virus in the cattle of Mongolia is currently available. In this study, the prevalence of BLV was assessed using PCR, and the genetic diversity was analyzed through DNA sequencing. Of the 517 samples tested, 20 positives were identified. Phylogenetic analysis showed that six, one, and four isolates were classified into genotype 4, 7, and 1, respectively. Most isolates were clustered with isolates from Eastern Europe and Russia. This study is the first to investigate the BLV genotype in Mongolia.
  • Ohira K, Nakahara A, Konnai S, Okagawa T, Nishimori A, Maekawa N, Ikebuchi R, Kohara J, Murata S, Ohashi K
    Immunity, inflammation and disease 4 1 52 - 63 2016年03月 [査読有り][通常論文]
  • Tomohiro Okagawa, Satoru Konnai, Asami Nishimori, Ryoyo Ikebuchi, Seiko Mizorogi, Reiko Nagata, Satoko Kawaji, Shogo Tanaka, Yumiko Kagawa, Shiro Murata, Yasuyuki Mori, Kazuhiko Ohashi
    Infection and immunity 84 1 77 - 89 2016年01月 [査読有り][通常論文]
     
    Johne's disease (paratuberculosis) is a chronic enteritis in cattle that is caused by intracellular infection with Mycobacterium avium subsp. paratuberculosis. This infection is characterized by the functional exhaustion of T-cell responses to M. avium subsp. paratuberculosis antigens during late subclinical and clinical stages, presumably facilitating the persistence of this bacterium and the formation of clinical lesions. However, the mechanisms underlying T-cell exhaustion in Johne's disease are poorly understood. Thus, we performed expression and functional analyses of the immunoinhibitory molecules programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) and lymphocyte activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC-II) in M. avium subsp. paratuberculosis-infected cattle during the late subclinical stage. Flow cytometric analyses revealed the upregulation of PD-1 and LAG-3 in T cells in infected animals, which suffered progressive suppression of interferon gamma (IFN-gamma) responses to the M. avium subsp. paratuberculosis antigen. In addition, PD-L1 and MHC-II were expressed on macrophages from infected animals, consistent with PD-1 and LAG-3 pathways contributing to the suppression of IFN-gamma responses during the subclinical stages of M. avium subsp. paratuberculosis infection. Furthermore, dual blockade of PD-L1 and LAG-3 enhanced M. avium subsp. paratuberculosis-specific IFN-gamma responses in blood from infected animals, and in vitro LAG-3 blockade enhanced IFN-gamma production from M. avium subsp. paratuberculosis-specific CD4(+) and CD8(+) T cells. Taken together, the present data indicate that M. avium subsp. paratuberculosis-specific T-cell exhaustion is in part mediated by PD-1/PD-L1 and LAG-3/MHC-II interactions and that LAG-3 is a molecular target for the control of M. avium subsp. paratuberculosis-specific T-cell responses.
  • Nyamsuren Ochirkhuu, Satoru Konnai, Claro N. Mingala, Tomohiro Okagawa, Marvin Villanueva, Flor Marie Immanuelle R. Pilapil, Shiro Murata, Kazuhiko Ohashi
    VETERINARY PARASITOLOGY 212 3-4 161 - 167 2015年09月 [査読有り][通常論文]
     
    In the Philippines, vector-borne disease is one of the important problems in the livestock industry. To elucidate the epidemiology of vector-borne diseases in cattle on Luzon Island, the Philippines, the prevalence of five protozoan agents was assessed by polymerase chain reaction. Out of the 339 samples, 324 (95.5%), 154 (45.4%), 209 (61.6%), 140 (41.3%), and 2 (0.6%) were positive for Anaplasma marginale, Babesia bigemina, Babesia bovis, Theileria spp., and Trypanosoma evansi infections, respectively. Mixed infections were detected in 290 (85.5%) samples, of which 115 (33.9%) had two pathogens, 144 (42.5%) had three pathogens, and 31 (9.1%) had four kinds of pathogens. 16S rRNA gene was 100% identical in A. marginale compared with the same lineage across the world. B. bovis RAP-1 and B. bigemina AMA-1 genes were identical with 92.27%-100% and 97.07%-100% sequences, respectively, in the database (Asian isolates). MPSP genes of Theileria spp. were 83.51%-100% identical with the one another. Phylogenetic analysis showed that they belong to the groups of T. sergenti and T. buffeli. Positive rates of the tick-borne pathogens were extremely high in this area. These findings provide vital information that can be used for the planning and execution of effective control measures for vector-borne diseases in the Philippine cattle industry. (C) 2015 Elsevier B.V. All rights reserved.
  • Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Asami Nishimori, Junko Kohara, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 163 3-4 115 - 124 2015年02月 [査読有り][通常論文]
     
    Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-gamma expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CfLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4+ T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-beta mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-gamma mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection. (C) 2014 Elsevier B.V. All rights reserved.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Immunology 142 4 551 - 561 2014年08月 [査読有り][通常論文]
     
    Programmed death-ligand 1 (PD-L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death-1 (PD-1). However, the mechanism of PD-L1-mediated inhibitory signalling after PD-L1 cross-linking by anti-PD-L1 monoclonal antibody (mAb) or PD-1-immunogloblin fusion protein (PD-1-Ig) is still unknown, although it may induce cell death of PD-L1(+) cells required for regular immune reactions. In this study, PD-1-Ig or anti-PD-L1 mAb treatment was tested in cell lines that expressed PD-L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD-L1-mediated cell death. PD-L1 cross-linking by PD-1-Ig or anti-PD-L1 mAb primarily increased the number of dead cells in PD-L1(high) cells, but not in PD-L1(low) cells; these cells were prepared from Cos-7 cells in which bovine PD-L1 expression was induced by transfection. The PD-L1-mediated cell death also occurred in Cos-7 and HeLa cells transfected with vectors only encoding the extracellular region of PD-L1. In bovine lymphocytes, the anti-PD-L1 mAb treatment up-regulated interferon-gamma (IFN-gamma) production, whereas PD-1-Ig treatment decreased this cytokine production and cell proliferation. The IFN-gamma production in B-cell-depleted peripheral blood mononuclear cells was not reduced by PD-1-Ig treatment and the percentages of dead cells in PD-L1(+) B cells were increased by PD-1-Ig treatment, indicating that PD-1-Ig-induced immunosuppression in bovine lymphocytes could be caused by PD-L1-mediated B-cell death. This study provides novel information for the understanding of signalling through PD-L1.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Asami Nishimori, Ayako Nakahara, Shiro Murata, Kazuhiko Ohashi
    Journal of general virology 95 Pt 8 1832 - 1842 2014年08月 [査読有り][通常論文]
     
    Bovine leukemia virus (BLV) induces abnormal B-cell proliferation and B-cell lymphoma in cattle, where the BLV provirus is integrated into the host genome. BLV-infected B-cells rarely express viral proteins in vivo, but short-term cultivation augments BLV expression in some, but not all, BLV-infected B-cells. This observation suggests that two subsets, i.e. BLV-silencing cells and BLV-expressing cells, are present among BLV-infected B-cells, although the mechanisms of viral expression have not been determined. In this study, we examined B-cell markers and viral antigen expression in B-cells from BLV-infected cattle to identify markers that may discriminate BLV, expressing cells from BLV-silencing cells. The proportions of IgM(high) B-cells were increased in blood lymphocytes from BLV-infected cattle. IgM(high) B-cells mainly expressed BLV antigens, whereas IgM(low) B-cells did not, although the provirus load was equivalent in both subsets. Several parameters were investigated in these two subsets to characterize their cellular behaviour. Real-time PCR and microarray analyses detected higher expression levels of some proto-oncogenes (e.g. Maf, Jun and Fos) in IgM(low) B-cells than those in IgM(high) B-cells. Moreover, lymphoma cells obtained from the lymph nodes of 14 BLV-infected cattle contained IgM(low) or IgM(-) B-cells but no IgM(high) B-cells. To our knowledge, this is the first study to demonstrate that IgM(high) B-cells mainly comprise BLV-expressing cells, whereas IgM(low) B-cells comprise a high proportion of BLV-silencing B-cells in BLV-infected cattle.
  • Asami Nishimori, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Chie Nakajima, Yasuhiko Suzuki, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
    Microbiology and immunology 58 7 388 - 397 2014年07月 [査読有り][通常論文]
     
    Previous reports from this group have indicated that the immunoinhibitory programmed death (PD)-1 receptor and its ligand, PD-L1, are involved in the mechanism of immune evasion of bovine chronic infection. However, no functional analysis of bovine PD-L2 in cattle has been reported. Thus, in this study, the molecular function of bovine PD-L2 was analyzed in vitro. Recombinant PD-L2 (PD-L2-Ig), which comprises an extracellular domain of bovine PD-L2 fused to the Fc portion of rabbit IgG1, was prepared based on the cloned cDNA sequence for bovine PD-L2. Bovine PD-L2-Ig bound to bovine PD-1-expressing cells and addition of soluble bovine PD-1-Ig clearly inhibited the binding of PD-L2-Ig to membrane PD-1 in a dose-dependent manner. Cell proliferation and IFN-gamma production were significantly enhanced in the presence of PD-L2-Ig in peripheral blood mononuclear cells (PBMCs) from cattle. Moreover, PD-L2-Ig significantly enhanced IFN-gamma production from virus envelope peptides-stimulated PBMCs derived from bovine leukemia virus-infected cattle. Interestingly, PD-L2-Ig-induced IFN-gamma production was further enhanced by treatment with anti-bovine PD-1 antibody. These data suggest potential applications of bovine PD-L2-Ig as a therapy for bovine diseases.
  • Maekawa N, Konnai S, Ikebuchi R, Okagawa T, Adachi M, Takagi S, Kagawa Y, Nakajima C, Suzuki Y, Murata S, Ohashi K
    PLOS ONE 9 6 e98415  2014年06月 [査読有り][通常論文]
     
    Programmed death 1 (PD-1), an immunoinhibitory receptor, and programmed death ligand 1 (PD-L1), its ligand, together induce the "exhausted'' status in antigen-specific lymphocytes and are thus involved in the immune evasion of tumor cells. In this study, canine PD-1 and PD-L1 were molecularly characterized, and their potential as therapeutic targets for canine tumors was discussed. The canine PD-1 and PD-L1 genes were conserved among canine breeds. Based on the sequence information obtained, the recombinant canine PD-1 and PD-L1 proteins were constructed; they were confirmed to bind each other. Antibovine PD-L1 monoclonal antibody effectively blocked the binding of recombinant PD-1 with PD-L1-expressing cells in a dose-dependent manner. Canine melanoma, mastocytoma, renal cell carcinoma, and other types of tumors examined expressed PD-L1, whereas some did not. Interestingly, anti-PD-L1 antibody treatment enhanced IFN-gamma production from tumor-infiltrating cells. These results showed that the canine PD-1/PD-L1 pathway is also associated with T-cell exhaustion in canine tumors and that its blockade with antibody could be a new therapeutic strategy for canine tumors. Further investigations are needed to confirm the ability of anti-PD-L1 antibody to reactivate canine antitumor immunity in vivo, and its therapeutic potential has to be further discussed.
  • Saori Suzuki, Satoru Konnai, Tomohiro Okagawa, Ryoyo Ikebuchi, Tatsuya Shirai, Yuji Sunden, Claro N. Mingala, Shiro Murata, Kazuhiko Ohashi
    Microbiology and Immunology 57 8 600 - 604 2013年08月 [査読有り][通常論文]
     
    In the present study, we monitored Foxp3+ T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3+CD4+ cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3+CD4+ cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3+CD4+ T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection. © 2013 The Societies and Wiley Publishing Asia Pty Ltd.
  • Suzuki S, Konnai S, Okagawa T, Ikebuchi R, Shirai T, Sunden Y, Mingala CN, Murata S, Ohashi K
    Microbiology and immunology 2013年06月 [査読有り][通常論文]
  • 免疫抑制受容体PD-1のリガンドPD-L2の機能的特徴と臨床応用研究
    西森朝美, 今内 覚, 池渕良洋, 岡川朋弘, 村田史郎, 大橋和彦
    動物用ワクチンーバイオ医薬品研究会ニュースレター 7 19 - 20 2013年06月 [査読無し][招待有り]
  • Satoru Konnai, Saori Suzuki, Tatsuya Shirai, Ryoyo Ikebuchi, Tomohiro Okagawa, Yuji Sunden, Claro N. Mingala, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    COMPARATIVE IMMUNOLOGY MICROBIOLOGY AND INFECTIOUS DISEASES 36 1 63 - 69 2013年01月 [査読有り][通常論文]
     
    An immunoinhibitory receptor, lymphocyte activation gene-3 (LAG-3), which is mainly expressed in T-cells, is involved in the immune evasion of several pathogens causing chronic infections and tumors. However, unlike human or mouse LAG-3, no functional analysis of LAG-3 has been reported in domestic animals. Thus, in this study, bovine LAG-3 expression was analyzed in bovine leukemia virus (BLV)-infected cattle. In persistent lymphocytotic (PL) cattle, the numbers of LAG-3(+)CD4(+) cells and LAG-3(+)CD8(+) cells were conserved whilst the number of MHC class II+ cells was remarkably higher than in the control animals. In contrast, the mean fluorescence intensity (MFI) for LAG-3 on PBMCs from PL cattle was significantly increased compared to control and asymptomatic (AL) cattle. Specifically, the LAG-3 expression level was significantly increased in both CD4(+) and CD8(+) T cells from PL cattle. LAG-3 expression correlated positively with increased numbers of lymphocytes and MHC class II+ cells in infected animals. Preliminary results from PD-L1 and LAG-3 blockade assay revealed that IFN-gamma and IL-2 expressions were significantly up-regulated by addition of anti- PD-L1 and LAG-3 antibodies in PBMCs from PL cattle. These findings suggest that LAG-3 might be involved in the inhibition of T-cell function through its binding and signaling on MHC class II molecule during BLV infection. (c) 2012 Elsevier Ltd. All rights reserved.
  • Ryoyo Ikebuchi, Satoru Konnai, Tomohiro Okagawa, Kazumasa Yokoyama, Chie Nakajima, Yasuhiko Suzuki, Shiro Murata, Kazuhiko Ohashi
    Veterinary Research 44 1 59  2013年 [査読有り][通常論文]
     
    Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1+ CD4+ T cells in blood and lymph node and PD-1 + CD8+ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.© 2013 Ikebuchi et al. licensee BioMed Central Ltd.
  • Tomohiro Okagawa, Satoru Konnai, Hirohisa Mekata, Naftaly Githaka, Saori Suzuki, Edward Kariuki, Francis Gakuya, Esther Kanduma, Tatsuya Shirai, Ryoyo Ikebuchi, Yoshinori Ikenaka, Mayumi Ishizuka, Shiro Murata, Kazuhiko Ohashi
    VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY 148 3-4 373 - 379 2012年08月 [査読有り][通常論文]
     
    Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus coffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo. (c) 2012 Elsevier B.V. All rights reserved.
  • Tomohiro Okagawa, Satoru Konnai, Ryoyo Ikebuchi, Saori Suzuki, Tatsuya Shirai, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    VETERINARY RESEARCH 43 45  2012年05月 [査読有り][通常論文]
     
    The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4(+) and CD8(+) cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4(+) and CD8(+) cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-gamma and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-gamma mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.
  • S. Suzuki, S. Konnai, T. Okagawa, N. W. Githaka, E. Kariuki, F. Gakuya, E. Kanduma, T. Shirai, R. Ikebuchi, Y. Ikenaka, M. Ishizuka, S. Murata, K. Ohashi
    INTERNATIONAL JOURNAL OF IMMUNOGENETICS 39 2 170 - 182 2012年04月 [査読有り][通常論文]
     
    The African buffalo (Syncerus caffer) has been implicated as the reservoir of several bovine infectious agents. However, there is insufficient information on the protective immune responses in the African buffalo, particularly in infected animals. In this study, we analysed Th1 cytokines IL-2 and IFN-?, and Th2 cytokines IL-4 and IL-10. The cloned cDNA of IL-2, IL-4, IL-10 and IFN-? contained an open reading frame of 468, 501, 408 and 540 nucleotides, encoding polypeptides of 155, 166, 135 and 179 amino acids, respectively. Nucleotide sequence homology of IL-2, IFN-? and IL-4 was more than 98% between the African buffalo and cattle, which resulted in identical polypeptides. Meanwhile, IL-10 gene of African buffalo and cattle had 95% homology in nucleotide sequence, corresponding to thirteen amino acid residues substitution. Cysteine residues and potential glycosylation sites were conserved within the family Bovinae. Phylogenetic analyses including cytokines of the African buffalo placed them within a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle, water buffalo, sheep, goat, pig and artiodactyl wildlife. A deeper understanding of the structure of these cytokines will shed light on their protective role in the disease-resistant African buffalo in comparison with other closely related species.
  • S. Suzuki, S. Konnai, T. Okagawa, N. W. Githaka, E. Kariuki, F. Gakuya, E. Kanduma, T. Shirai, R. Ikebuchi, Y. Ikenaka, M. Ishizuka, S. Murata, K. Ohashi
    International Journal of Immunogenetics 39 2 170 - 182 2012年04月 [査読有り][通常論文]
     
    The African buffalo (Syncerus caffer) has been implicated as the reservoir of several bovine infectious agents. However, there is insufficient information on the protective immune responses in the African buffalo, particularly in infected animals. In this study, we analysed Th1 cytokines IL-2 and IFN-γ, and Th2 cytokines IL-4 and IL-10. The cloned cDNA of IL-2, IL-4, IL-10 and IFN-γ contained an open reading frame of 468, 501, 408 and 540 nucleotides, encoding polypeptides of 155, 166, 135 and 179 amino acids, respectively. Nucleotide sequence homology of IL-2, IFN-γ and IL-4 was more than 98% between the African buffalo and cattle, which resulted in identical polypeptides. Meanwhile, IL-10 gene of African buffalo and cattle had 95% homology in nucleotide sequence, corresponding to thirteen amino acid residues substitution. Cysteine residues and potential glycosylation sites were conserved within the family Bovinae. Phylogenetic analyses including cytokines of the African buffalo placed them within a cluster comprised mainly of species belonging to the order Artiodactyla, including cattle, water buffalo, sheep, goat, pig and artiodactyl wildlife. A deeper understanding of the structure of these cytokines will shed light on their protective role in the disease-resistant African buffalo in comparison with other closely related species. © 2011 Blackwell Publishing Ltd.
  • Tatsuya Shirai, Satoru Konnai, Ryoyo Ikebuchi, Tomohiro Okagawa, Saori Suzuki, Yuji Sunden, Misao Onuma, Shiro Murata, Kazuhiko Ohashi
    Veterinary immunology and immunopathology 144 3-4 462 - 467 2011年12月 [査読有り][通常論文]
     
    Lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II binding CD4 homologue has recently been shown as one of the mechanisms for down-regulating immune responses during chronic disease progression. For the first time, we cloned LAG-3 from two breeds of cattle (Holstein and Japanese Black), and analyzed its expression levels in cattle infected with bovine leukemia virus (BLV), a chronic viral infection that leads to immuno-suppression. The cloned cDNA of bovine LAG-3 have an open reading frame of 1551 nucleotides, encoding a polypeptide of 515 amino acids in length. Similar to the swine LAG-3, the bovine LAG-3 protein sequence consisted of four extracellular domains, a transmembrane domain and an inhibitory motif, KTGELE. We found that the bovine LAG-3 mRNA transcripts were expressed predominantly on T-cells such as CD4(+) and CD8(+) cells, among peripheral blood mononuclear cells. In subsequent expression analysis, LAG-3 mRNA expression on CD4(+) T-cells from BLV-infected cattle was upregulated compared to that in normal cattle. Comparable results were obtained with CD8(+) T-cells from cattle infected with BLV. We further observed strong upregualtion of MHC class II molecule, the ligand for LAG-3 in BLV-infected cattle. These findings indicate an important role for inhibitory receptor molecules such as LAG-3 in chronic bovine infections and future studies will elucidate the specific role of LAG-3 in bovine diseases. (C) 2011 Elsevier B.V. All rights reserved.

書籍

  • 実験医学別冊 ラボ必携 フローサイトメトリーQ&A〜正しいデータを出すための100箇条
    岡川 朋弘 (担当:分担執筆範囲:第八章 Q97)
    2017年11月
  • 動物用ワクチンとバイオ医薬品―新たな潮流―
    岡川 朋弘, 今内 覚 (担当:分担執筆範囲:第一章 4. 抗体医薬:免疫チェックポイント阻害薬等)
    文永堂出版 2017年07月

その他活動・業績

受賞

  • 2016年09月 日本獣医学会 獣医学奨励賞
     
    受賞者: 岡川 朋弘
  • 2015年09月 日本獣医学会微生物分科会 第6回微生物学分科会若手奨励賞
     
    受賞者: 岡川 朋弘
  • 2014年10月 Veterinary Immunology Committee of the International Union of Immunological Societies IUIS-VIC PhD/DVM Student Travel Award
     
    受賞者: Tomohiro Okagawa
  • 2012年09月 The Global COE Program “Establishment of International Collaboration Center for Zoonosis Control” Best Poster Presentation Award at the 4th international Young Researcher Seminar for Zoonosis Control
     
    受賞者: Tomohiro Okagawa

共同研究・競争的資金等の研究課題

  • 家畜悪性感染症における免疫破綻の分子機序の解明および新規制御法の開発
    日本学術振興会:科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))
    研究期間 : 2019年10月 -2023年03月 
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