研究者基本情報

• 博士（医学）, 京都大学

• https://researchmap.jp/ksugata

• https://jglobal.jst.go.jp/detail?JGLOBAL_ID=202101001598990018

• 10650616

• 202101001598990018

• ライフサイエンス, ウイルス学, HTLV-1
• ライフサイエンス, 免疫学, 細胞性免疫

研究活動情報

• 2020年, 日本がん免疫学会総会 若手研究奨励賞
• 2015年, 上原記念生命科学財団リサーチフェローシップ

• Dynamics of lung-infiltrating virus-specific T cells associated with age-dependent SARS-CoV-2 pneumonia severity.
  Rise Kurokawa; Chatherine Silas Mtali; Innocent John Daniel; Thorbjorg Einarsdottir; Omnia Reda; Nobuko Irie; Wajihah Sakhor; Koki Niimura; Mitsuyoshi Takatori; Kenji Sugata; Chanidapa Adele Tye; Yorifumi Satou; Masahiro Ono; Takushi Nomura
  PLoS pathogens, 22, 1, e1013866, 2026年01月, ［国際誌］
  英語, 研究論文（学術雑誌）, Coronavirus disease 2019 (COVID-19) pneumonia is prevalent in the elderly infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, the mechanisms underlying its age-dependent pathogenesis remain unclear. In this study, we established a mouse-adapted SARS-CoV-2 strain infected Nr4a3-Tocky mouse model to examine T-cell dynamics associated with disease severity. Nr4a3-Tocky mice allow the analysis of the dynamics and induction of antigen-reactive T cells following antigen recognition in vivo using fluorescent Timer protein. SARS-CoV-2-infected adult mice exhibited transient body weight loss and recovery, whereas aged mice developed severe pneumonia. BALF viral RNA was comparable between 1-4 days post-inoculation (d.p.i.), but declined in adults at 5 d.p.i. Aged mice displayed stronger inflammation as indicated by scRNA-seq, and higher levels of inflammatory cytokines (TNF-α, CCL2, CXCL10 and IL-6) in BALF correlated with weight loss. Timer analysis revealed induction of antigen-reactive T cells in the adult lungs at 5 and 8 d.p.i., which inversely correlated with disease severity. Additionally, S-specific IFN-γ ⁺ CD8 ⁺ T cells were detected at 5 d.p.i. in adults, whereas detection of antigen-specific T cells was delayed in aged mice. These results suggest that the coexistence of age-related lung inflammation and delayed induction of antigen-specific T cells is linked to more severe pneumonia, while earlier T-cell responses are associated with improved viral control and milder disease. In this study, we utilized a novel mouse model enabling characterization of antigen-reactive T cells in the local tissue, and investigated inflammatory responses in the lung together with lung-infiltrating virus-specific T cells, finding the dynamics of these immunological parameters associated with the age of the mice. Our analysis provides new insights into understanding how age-related T-cell dysfunction is associated with the severity of SARS-CoV-2 pneumonia.
• Circulating Cell-Free DNA of Bovine Leukemia Virus: A Promising Biomarker for Enzootic Bovine Leukosis.
  M Ishrat Jahan; Toshiaki Inenaga; Sakurako Makimoto; Md Belal Hossain; Yuka Matsuoka; Sharmin Nahar Sithi; Samiul Alam Rajib; Arif Nur Muhammad Ansori; Kenji Sugata; Kazuhiko Imakawa; Tomoko Kobayashi; Yorifumi Satou
  Microbiology and immunology, 2025年07月07日, ［国際誌］
  英語, 研究論文（学術雑誌）, Bovine leukemia virus (BLV) causes Enzootic bovine leukosis (EBL) in approximately 1%-5% of infected cattle after a long latent period. Few biomarkers effectively distinguish non-EBL from EBL cattle. Given the rapid turnover of tumor cells, we hypothesized that cell-free DNA (cfDNA) in plasma could serve as a more effective biomarker for EBL diagnosis. We measured the proviral load (PVL) in whole blood and plasma by quantitative PCR targeting LTR and pol. Consistent with previous reports, PVL levels in whole blood in EBL cattle were generally higher than those in non-EBL with some overlap between these two groups. In contrast, PVL in plasma clearly distinguished non-EBL from EBL ones. The receiver operating characteristic analysis showed plasma PVL perfectly discriminated EBL from non-EBL (100% sensitivity and specificity), while whole-blood PVL achieved 70% sensitivity and 30% specificity. Additionally, length of PCR products played a role in PVL detection sensitivity in plasma. We compared the complete BLV sequence between genomic DNA from lymphoma tissue and cfDNA in plasma and found that the predominant BLV sequences were highly similar between them. By assessing the major tumor clone burden based on unique integration sites, we found that BLV cfDNA derived more from tumor clones in the tissues than from peripheral blood mononuclear cells (PBMCs). These data support the idea that BLV in cfDNA primarily originates from tumor cells in EBL cattle. These findings demonstrated that cfDNA could be a better indicator for EBL diagnosis, improving early detection and more timely intervention to reduce the economic loss in the meat and dairy industry.
• Intragenic viral silencer element regulates HTLV-1 latency via RUNX complex recruitment.
  Kenji Sugata; Akhinur Rahman; Koki Niimura; Kazuaki Monde; Takaharu Ueno; Samiul Alam Rajib; Mitsuyoshi Takatori; Wajihah Sakhor; Md Belal Hossain; Sharmin Nahar Sithi; M Ishrat Jahan; Kouki Matsuda; Mitsuharu Ueda; Yoshihisa Yamano; Terumasa Ikeda; Takamasa Ueno; Kiyoto Tsuchiya; Yuetsu Tanaka; Masahito Tokunaga; Kenji Maeda; Atae Utsunomiya; Kazu Okuma; Masahiro Ono; Yorifumi Satou
  Nature microbiology, 2025年05月13日, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Retroviruses integrate their genetic material into the host genome, enabling persistent infection. Human T cell leukaemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) share similarities in genome structure and target cells, yet their infection dynamics differ drastically. While HIV-1 leads to high viral replication and immune system collapse, HTLV-1 establishes latency, promoting the survival of infected cells and, in some cases, leading to leukaemia. The mechanisms underlying this latency preference remain unclear. Here we analyse blood samples from people with HTLV-1 and identify an open chromatin region within the HTLV-1 provirus that functions as a transcriptional silencer and regulates transcriptional burst. The host transcription factor RUNX1 binds to this open chromatin region, repressing viral expression. Mutation of this silencer enhances HTLV-1 replication and immunogenicity, while its insertion into HIV-1 suppresses viral production. These findings reveal a strategy by which HTLV-1 ensures long-term persistence, offering potential insights into retroviral evolution and therapeutic targets.
• Viral antigen mismatch affects antiviral T-cell response and may impair immunotherapeutic efficacy against adult T-cell leukemia/lymphoma.
  Kenji Sugata; Mitsuyoshi Takatori; Omnia Reda; Benjy Jek Yang Tan; Masahito Tokunaga; Tomoo Sato; Mitsuharu Ueda; Yoshihisa Yamano; Atae Utsunomiya; Yorifumi Satou
  The Journal of infectious diseases, 2024年09月20日, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, HTLV-1 transforms primary CD4+ T cells in vitro within a short time; however, majority of infected individuals maintain an asymptomatic condition, suggesting there is an equilibrium between the infected cells and the host immunity. In this study, we identified a variation in a major viral antigen epitope, HTLV-1 Tax301-309, in HLA-A24-positive individuals. Mismatch in A24/Tax301-309 multimers impaired detection of anti-Tax CTLs. Notably, over half of the TCRs of the anti-Tax CTLs did not recognize mismatched Tax301-309 peptides. These findings highlighted the importance of matching the viral antigen epitope type in T-cell-based immunotherapy against ATL by using viral antigen Tax.
• Clonal succession after prolonged antiretroviral therapy rejuvenates CD8+ T cell responses against HIV-1.
  Eoghann White; Laura Papagno; Assia Samri; Kenji Sugata; Boris Hejblum; Amy R Henry; Daniel C Rogan; Samuel Darko; Patricia Recordon-Pinson; Yasmine Dudoit; Sian Llewellyn-Lacey; Lisa A Chakrabarti; Florence Buseyne; Stephen A Migueles; David A Price; Marie-Aline Andreola; Yorifumi Satou; Rodolphe Thiebaut; Christine Katlama; Brigitte Autran; Daniel C Douek; Victor Appay
  Nature immunology, 25, 9, 1555, 1564, 2024年08月23日, ［国際誌］
  英語, 研究論文（学術雑誌）, Human immunodeficiency virus 1 (HIV-1) infection is characterized by a dynamic and persistent state of viral replication that overwhelms the host immune system in the absence of antiretroviral therapy (ART). The impact of prolonged treatment on the antiviral efficacy of HIV-1-specific CD8+ T cells has nonetheless remained unknown. Here, we used single-cell technologies to address this issue in a cohort of aging individuals infected early during the pandemic and subsequently treated with continuous ART. Our data showed that long-term ART was associated with a process of clonal succession, which effectively rejuvenated HIV-1-specific CD8+ T cell populations in the face of immune senescence. Tracking individual transcriptomes further revealed that initially dominant CD8+ T cell clonotypes displayed signatures of exhaustion and terminal differentiation, whereas newly dominant CD8+ T cell clonotypes displayed signatures of early differentiation and stemness associated with natural control of viral replication. These findings reveal a degree of immune resilience that could inform adjunctive treatments for HIV-1.
• A micro-disc-based multiplex method for monitoring emerging SARS-CoV-2 variants using the molecular diagnostic tool Intelli-OVI.
  Md Belal Hossain; Yoshikazu Uchiyama; Samiul Alam Rajib; Akhinur Rahman; Mitsuyoshi Takatori; Benjy Jek Yang Tan; Kenji Sugata; Mami Nagashima; Mamiyo Kawakami; Hitoshi Ito; Ryota Kumagai; Kenji Sadamasu; Yasuhiro Ogi; Tatsuya Kawaguchi; Tomokazu Tamura; Takasuke Fukuhara; Masahiro Ono; Kazuhisa Yoshimura; Yorifumi Satou
  Communications medicine, 4, 1, 161, 161, 2024年08月09日, ［国際誌］
  英語, 研究論文（学術雑誌）, BACKGROUND: Highly transmissible viruses including SARS-CoV-2 frequently accumulate novel mutations that are detected via high-throughput sequencing. However, there is a need to develop an alternative rapid and non-expensive approach. Here we developed a novel multiplex DNA detection method Intelli-OVI for analysing existing and novel mutations of SARS-CoV-2. METHODS: We have developed Intelli-OVI that includes the micro-disc-based method IntelliPlex and computational algorithms of objective variant identification (OVI). More than 250 SARS-CoV-2 positive samples including wastewater ones were analysed to verify the efficiency of the method. RESULTS: IntelliPlex uses micro-discs printed with a unique pictorial pattern as a labelling conjugate for DNA probes, and OVI allows simultaneous identification of several variants using multidimensional data obtained by the IntelliPlex method. Importantly, de novo mutations can be identified by decreased signals, which indicates that there is an emergence of de novo variant virus as well as prompts the need to design additional primers and probes. We have upgraded probe panel according to the emergence of new variants and demonstrated that Intelli-OVI efficiently identified more than 20 different SARS-CoV-2 variants by using 35 different probes simultaneously. CONCLUSIONS: Intelli-OVI can be upgraded to keep up with rapidly evolving viruses as we showed in this study using SARS-CoV-2 as an example and may be suitable for other viruses but would need to be validated.
• HIV-Tocky system to visualize proviral expression dynamics
  Omnia Reda; Kazuaki Monde; Kenji Sugata; Akhinur Rahman; Wajihah Sakhor; Samiul Alam Rajib; Sharmin Nahar Sithi; Benjy Jek Yang Tan; Koki Niimura; Chihiro Motozono; Kenji Maeda; Masahiro Ono; Hiroaki Takeuchi; Yorifumi Satou
  Communications Biology, 7, 1, 344, 344, Springer Science and Business Media LLC, 2024年03月20日, ［国際誌］
  英語, 研究論文（学術雑誌）, Abstract
  
  Determinants of HIV-1 latency establishment are yet to be elucidated. HIV reservoir comprises a rare fraction of infected cells that can survive host and virus-mediated killing. In vitro reporter models so far offered a feasible means to inspect this population, but with limited capabilities to dissect provirus silencing dynamics. Here, we describe a new HIV reporter model, HIV-Timer of cell kinetics and activity (HIV-Tocky) with dual fluorescence spontaneous shifting to reveal provirus silencing and reactivation dynamics. This unique feature allows, for the first time, identifying two latent populations: a directly latent, and a recently silenced subset, with the latter having integration features suggestive of stable latency. Our proposed model can help address the heterogeneous nature of HIV reservoirs and offers new possibilities for evaluating eradication strategies.
• Antiviral immune response against HTLV-1 invalidates T-SPOT.TB® results in patients with HTLV-1-positive rheumatic diseases.
  Masatoshi Kimura; Kunihiko Umekita; Chihiro Iwao; Katsumi Kawano; Yuki Hashikura; Yayoi Hashiba; Toshihiko Hidaka; Kenji Sugata; Yorifumi Satou; Taiga Miyazaki
  Frontiers in immunology, 15, 1480506, 1480506, 2024年, ［国際誌］
  英語, 研究論文（学術雑誌）, BACKGROUND: T-SPOT.TB®, one of the screening tests for latent tuberculosis infection (LTBI), yields invalid results in human T-cell leukemia virus type 1 (HTLV-1)-positive patients with rheumatoid arthritis. However, the detailed mechanisms behind this invalidation are unclear. Additionally, it remains unclear whether T-SPOT.TB® or QuantiFERON-TB (QFT) is more useful in HTLV-1-positive patients with rheumatic disease (RD). METHOD: Among all of the HTLV-1-positive RD patients who visited our department between August 2012 and December 2022, 44 patients who were screened using T-SPOT.TB® were included in the analysis. QFT testing was performed in 33 of the 44 patients, and the results were compared with that of T-SPOT.TB®. Furthermore, we performed a culture experiment mimicking T-SPOT.TB® using peripheral blood mononuclear cells (PBMCs) obtained from HTLV-1-positive patients with RD. Additionally, T-cell subsets with autonomous product IFN-γ were analyzed using a flow cytometer. RESULTS: Of the included patients, 13 (29.5%) were invalid for T-SPOT.TB® because of the increased number of negative control spots. The median HTLV-1 proviral load in the invalid group was higher than that in the valid group (2.45 vs. 0.49 copies/100 PBMCs, respectively, p = 0.002). QFT was performed in all 33 patients, including 13 patients who were invalid in T-SPOT.TB®. The main source of IFN-γ production was CD8+ T-cells in the T-SPOT.TB® mimic experiment. Furthermore, Tax-expressing CD4+ T-cells and Tax-specific cytotoxic CD8+ T-cells were more frequently observed in patients with invalid results than in patients with valid results. CD4+ T-cell depletion in the T-SPOT.TB® mimic experiment reduced the population of IFN-γ producing CD8+ T cells. CONCLUSION: T-SPOT.TB® may be invalidated by the interaction between Tax-expressing CD4+ T-cells and cytotoxic CD8+ T-cells. Moreover, HTLV-1-associated immune reactions due to contact between these cells may be unlikely to occur in QFT using whole blood. Therefore, our results reveal the superiority of QFT over T-SPOT.TB® as a screening test for LTBI in HTLV-1-positive patients with RD.
• Association of Clostridium butyricum Therapy Using the Live Bacterial Product CBM588 with the Survival of Patients with Lung Cancer Receiving Chemoimmunotherapy Combinations.
  Yusuke Tomita; Shinya Sakata; Kosuke Imamura; Shinji Iyama; Takayuki Jodai; Koichi Saruwatari; Shohei Hamada; Kimitaka Akaike; Moriyasu Anai; Kazuaki Fukusima; Akira Takaki; Hirotake Tsukamoto; Yoshihiko Goto; Chihiro Motozono; Kenji Sugata; Yorifumi Satou; Takamasa Ueno; Tokunori Ikeda; Takuro Sakagami
  Cancers, 16, 1, 2023年12月21日, ［国際誌］
  英語, 研究論文（学術雑誌）, The gut microbiota has emerged as a key regulator of immune checkpoint inhibitor (ICI) efficacy. Therapeutic approaches aimed at manipulating the microbiota through targeted reconstitution to enhance cancer treatment outcomes have garnered considerable attention. A single live microbial biotherapeutic bacterium, Clostridium butyricum MIYAIRI 588 strain (CBM588), has been shown to enhance the effects of ICI monotherapy in patients with advanced lung cancer. However, whether CBM588 affects the outcomes of chemoimmunotherapy combinations in lung cancer remains unknown. We hypothesized that CBM588 augments the effect of chemoimmunotherapy combinations and restores diminished effectiveness in patients with non-small cell lung cancer (NSCLC) receiving dysbiosis-inducing drugs. To validate this hypothesis, we retrospectively analyzed 106 patients with stage IV or recurrent metastatic NSCLC consecutively treated with chemoimmunotherapy combinations. A survival analysis was performed employing univariate and multivariate Cox proportional hazard models with inverse probability of treatment weighting (IPTW) using propensity scores. Forty-five percent of patients received Clostridium butyricum therapy. CBM588 significantly extended overall survival in patients with NSCLC receiving chemoimmunotherapy. The favorable impact of CBM588 on the efficacy of chemoimmunotherapy combinations varied based on tumor-programmed cell death ligand 1 (PD-L1) expression. The survival benefit of CBM588 in the PD-L1 <1% cohort was higher than that in the PD-L1 1-49% and PD-L1 ≥ 50% cohorts. Furthermore, CBM588 was associated with improved overall survival in patients receiving proton pump inhibitors and/or antibiotics. CBM588-induced manipulation of the commensal microbiota holds the potential to enhance the efficacy of chemoimmunotherapy combinations, warranting further exploration of the synergy between CBM588 and immunotherapy.
• Understanding the Immunopathology of HTLV-1-Associated Adult T-Cell Leukemia/Lymphoma: A Comprehensive Review.
  Shingo Nakahata; Daniel Enriquez-Vera; M Ishrat Jahan; Kenji Sugata; Yorifumi Satou
  Biomolecules, 13, 10, 2023年10月19日, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type-1 (HTLV-1) causes adult T-cell leukemia/lymphoma (ATL). HTLV-1 carriers have a lifelong asymptomatic balance between infected cells and host antiviral immunity; however, 5-10% of carriers lose this balance and develop ATL. Coinfection with Strongyloides promotes ATL development, suggesting that the immunological status of infected individuals is a determinant of HTLV-1 pathogenicity. As CD4+ T cells play a central role in host immunity, the deregulation of their function and differentiation via HTLV-1 promotes the immune evasion of infected T cells. During ATL development, the accumulation of genetic and epigenetic alterations in key host immunity-related genes further disturbs the immunological balance. Various approaches are available for treating these abnormalities; however, hematopoietic stem cell transplantation is currently the only treatment with the potential to cure ATL. The patient's immune state may contribute to the treatment outcome. Additionally, the activity of the anti-CC chemokine receptor 4 antibody, mogamulizumab, depends on immune function, including antibody-dependent cytotoxicity. In this comprehensive review, we summarize the immunopathogenesis of HTLV-1 infection in ATL and discuss the clinical findings that should be considered when developing treatment strategies for ATL.
• Clone Dynamics and Its Application for the Diagnosis of Enzootic Bovine Leukosis.
  Md Belal Hossain; Tomoko Kobayashi; Sakurako Makimoto; Misaki Matsuo; Kohei Nishikaku; Benjy Jek Yang Tan; Akhinur Rahman; Samiul Alam Rajib; Kenji Sugata; Nagaki Ohnuki; Masumichi Saito; Toshiaki Inenaga; Kazuhiko Imakawa; Yorifumi Satou
  Journal of virology, 97, 1, e0154222, 2023年01月31日, ［国際誌］
  英語, 研究論文（学術雑誌）, Bovine leukemia virus (BLV) infection results in polyclonal expansion of infected B lymphocytes, and ~5% of infected cattle develop enzootic bovine leukosis (EBL). Since BLV is a retrovirus, each individual clone can be identified by using viral integration sites. To investigate the distribution of tumor cells in EBL cattle, we performed viral integration site analysis by using a viral DNA capture-sequencing method. We found that the same tumor clones existed in peripheral blood, with a dominance similar to that in lymphoma tissue. Additionally, we observed that multiple tumor tissues from different sites harbored the identical clones, indicating that tumor cells can circulate and distribute systematically in EBL cattle. To investigate clonal expansion of BLV-infected cells during a long latent period, we collected peripheral blood samples from asymptomatic cattle every 2 years, among which several cattle developed EBL. We found that no detectable EBL clone existed before the diagnosis of EBL in some cases; in the other cases, clones that were later detected as malignant clones at the EBL stage were present several months or even years before the disease onset. To establish a feasible clonality-based method for the diagnosis of EBL, we simplified a quick and cost-effective method, namely, rapid amplification of integration sites for BLV infection (BLV-RAIS). We found that the clonality values (Cvs) were well correlated between the BLV-RAIS and viral DNA capture-sequencing methods. Furthermore, receiver operating characteristic (ROC) curve analysis identified an optimal Cv cutoff value of 0.4 for EBL diagnosis, with excellent diagnostic sensitivity (94%) and specificity (100%). These results indicated that the RAIS method efficiently and reliably detected expanded clones not only in lymphoma tissue but also in peripheral blood. Overall, our findings elucidated the clonal dynamics of BLV- infected cells during EBL development. In addition, Cvs of BLV-infected cells in blood can be used to establish a valid and noninvasive diagnostic test for potential EBL onset. IMPORTANCE Although BLV has been eradicated in some European countries, BLV is still endemic in other countries, including Japan and the United States. EBL causes huge economic damage to the cattle industry. However, there are no effective drugs or vaccines to control BLV infection and related diseases. The strategy of eradication of infected cattle is not practical due to the high endemicity of BLV. Furthermore, how BLV-infected B cell clones proliferate during oncogenesis and their distribution in EBL cattle have yet to be elucidated. Here, we provided evidence that tumor cells are circulating in the blood of diseased cattle. Thus, the Cv of virus-infected cells in blood is useful information for the evaluation of the disease status. The BLV-RAIS method provides quantitative and accurate clonality information and therefore is a promising method for the diagnosis of EBL.
• Modification of the HLA-A*24:02 Peptide Binding Pocket Enhances Cognate Peptide-Binding Capacity and Antigen-Specific T Cell Activation.
  Kenji Murata; Dalam Ly; Hiroshi Saijo; Yukiko Matsunaga; Kenji Sugata; Fumie Ihara; Daisuke Oryoji; Yota Ohashi; Kayoko Saso; Chung-Hsi Wang; Evey Y F Zheng; Brian D Burt; Marcus O Butler; Naoto Hirano
  Journal of immunology (Baltimore, Md. : 1950), 209, 8, 1481, 1491, 2022年09月09日, ［国際誌］
  英語, 研究論文（学術雑誌）, The immunogenicity of a T cell Ag is correlated with the ability of its antigenic epitope to bind HLA and be stably presented to T cells. This presents a challenge for the development of effective cancer immunotherapies, as many self-derived tumor-associated epitopes elicit weak T cell responses, in part due to weak binding affinity to HLA. Traditional methods to increase peptide-HLA binding affinity involve modifying the peptide to reflect HLA allele binding preferences. Using a different approach, we sought to analyze whether the immunogenicity of wild-type peptides could be altered through modification of the HLA binding pocket. After analyzing HLA class I peptide binding pocket alignments, we identified an alanine 81 to leucine (A81L) modification within the F binding pocket of HLA-A*24:02 that was found to heighten the ability of artificial APCs to retain and present HLA-A*24:02-restricted peptides, resulting in increased T cell responses while retaining Ag specificity. This modification led to increased peptide exchange efficiencies for enhanced detection of low-avidity T cells and, when expressed on artificial APCs, resulted in greater expansion of Ag-specific T cells from melanoma-derived tumor-infiltrating lymphocytes. Our study provides an example of how modifications to the HLA binding pocket can enhance wild-type cognate peptide presentation to heighten T cell activation.
• Identification and characterization of a novel enhancer in the HTLV-1 proviral genome.
  Misaki Matsuo; Takaharu Ueno; Kazuaki Monde; Kenji Sugata; Benjy Jek Yang Tan; Akhinur Rahman; Paola Miyazato; Kyosuke Uchiyama; Saiful Islam; Hiroo Katsuya; Shinsuke Nakajima; Masahito Tokunaga; Kisato Nosaka; Hiroyuki Hata; Atae Utsunomiya; Jun-Ichi Fujisawa; Yorifumi Satou
  Nature communications, 13, 1, 2405, 2405, 2022年05月03日, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes adult T-cell leukemia/lymphoma (ATL), a cancer of infected CD4+ T-cells. There is both sense and antisense transcription from the integrated provirus. Sense transcription tends to be suppressed, but antisense transcription is constitutively active. Various efforts have been made to elucidate the regulatory mechanism of HTLV-1 provirus for several decades; however, it remains unknown how HTLV-1 antisense transcription is maintained. Here, using proviral DNA-capture sequencing, we found a previously unidentified viral enhancer in the middle of the HTLV-1 provirus. The transcription factors, SRF and ELK-1, play a pivotal role in the activity of this enhancer. Aberrant transcription of genes in the proximity of integration sites was observed in freshly isolated ATL cells. This finding resolves certain long-standing questions concerning HTLV-1 persistence and pathogenesis. We anticipate that the DNA-capture-seq approach can be applied to analyze the regulatory mechanisms of other oncogenic viruses integrated into the host cellular genome.
• HTLV-1 persistence and leukemogenesis: A game of hide-and-seek with the host immune system.
  Benjy J Y Tan; Kenji Sugata; Masahiro Ono; Yorifumi Satou
  Frontiers in immunology, 13, 991928, 991928, 2022年, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus which mainly infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATL), is primarily transmitted via direct cell-to-cell transmission. This feature generates a wide variety of infected clones in hosts, which are maintained via clonal proliferation, resulting in the persistence and survival of the virus. The maintenance of the pool of infected cells is achieved by sculpting the immunophenotype of infected cells and modulating host immune responses to avoid immune surveillance. Here, we review the processes undertaken by HTLV-1 to modulate and subvert host immune responses which contributes to viral persistence and development of ATL.
• A widely distributed HIV-1 provirus elimination assay to evaluate latency-reversing agents in vitro.
  Kouki Matsuda; Saiful Islam; Toru Takada; Kiyoto Tsuchiya; Benjy Jek Yang Tan; Shin-Ichiro Hattori; Hiroo Katsuya; Kosaku Kitagawa; Kwang Su Kim; Misaki Matsuo; Kenji Sugata; Nicole S Delino; Hiroyuki Gatanaga; Kazuhisa Yoshimura; Shuzo Matsushita; Hiroaki Mitsuya; Shingo Iwami; Yorifumi Satou; Kenji Maeda
  Cell reports methods, 1, 8, 100122, 100122, 2021年12月20日, ［国際誌］
  英語, 研究論文（学術雑誌）, Persistence of HIV-1 latent reservoir cells during antiretroviral therapy (ART) is a major obstacle for curing HIV-1. Even though latency-reversing agents (LRAs) are under development to reactivate and eradicate latently infected cells, there are few useful models for evaluating LRA activity in vitro. Here, we establish a long-term cell culture system called the "widely distributed intact provirus elimination" (WIPE) assay. It harbors thousands of different HIV-1-infected cell clones with a wide distribution of HIV-1 provirus similar to that observed in vivo. Mathematical modeling and experimental results from this in vitro infection model demonstrates that the addition of an LRA to ART shows a latency-reversing effect and contributes to the eradication of replication-competent HIV-1. The WIPE assay can be used to optimize therapeutics against HIV-1 latency and investigate mechanistic insights into the clonal selection of heterogeneous HIV-1-infected cells.
• HTLV-1 infection promotes excessive T cell activation and transformation into adult T cell leukemia/lymphoma.
  Benjy Jy Tan; Kenji Sugata; Omnia Reda; Misaki Matsuo; Kyosuke Uchiyama; Paola Miyazato; Vincent Hahaut; Makoto Yamagishi; Kaoru Uchimaru; Yutaka Suzuki; Takamasa Ueno; Hitoshi Suzushima; Hiroo Katsuya; Masahito Tokunaga; Yoshikazu Uchiyama; Hideaki Nakamura; Eisaburo Sueoka; Atae Utsunomiya; Masahiro Ono; Yorifumi Satou
  The Journal of clinical investigation, 131, 24, 2021年12月15日, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T cell leukemia virus type 1 (HTLV-1) mainly infects CD4+ T cells and induces chronic, persistent infection in infected individuals, with some developing adult T cell leukemia/lymphoma (ATL). HTLV-1 alters cellular differentiation, activation, and survival; however, it is unknown whether and how these changes contribute to the malignant transformation of infected cells. In this study, we used single-cell RNA-sequencing and T cell receptor-sequencing to investigate the differentiation and HTLV-1-mediated transformation of T cells. We analyzed 87,742 PBMCs from 12 infected and 3 uninfected individuals. Using multiple independent bioinformatics methods, we demonstrated the seamless transition of naive T cells into activated T cells, whereby HTLV-1-infected cells in an activated state further transformed into ATL cells, which are characterized as clonally expanded, highly activated T cells. Notably, the greater the activation state of ATL cells, the more they acquire Treg signatures. Intriguingly, the expression of HLA class II genes in HTLV-1-infected cells was uniquely induced by the viral protein Tax and further upregulated in ATL cells. Functional assays revealed that HTLV-1-infected cells upregulated HLA class II molecules and acted as tolerogenic antigen-presenting cells to induce anergy of antigen-specific T cells. In conclusion, our study revealed the in vivo mechanisms of HTLV-1-mediated transformation and immune escape at the single-cell level.
• Affinity-matured HLA class II dimers for robust staining of antigen-specific CD4+ T cells.
  Kenji Sugata; Yukiko Matsunaga; Yuki Yamashita; Munehide Nakatsugawa; Tingxi Guo; Levon Halabelian; Yota Ohashi; Kayoko Saso; Muhammed A Rahman; Mark Anczurowski; Chung-Hsi Wang; Kenji Murata; Hiroshi Saijo; Yuki Kagoya; Dalam Ly; Brian D Burt; Marcus O Butler; Tak W Mak; Naoto Hirano
  Nature biotechnology, 39, 8, 958, 967, 2021年08月, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Peptide-major histocompatibility complex (pMHC) multimers enable the detection of antigen-specific T cells in studies ranging from vaccine efficacy to cancer immunotherapy. However, this technology is unreliable when applied to pMHC class II for the detection of CD4+ T cells. Here, using a combination of molecular biological and immunological techniques, we cloned sequences encoding human leukocyte antigen (HLA)-DP, HLA-DQ and HLA-DR molecules with enhanced CD4 binding affinity (with a Kd of 8.9 ± 1.1 µM between CD4 and affinity-matured HLA-DP4) and produced affinity-matured class II dimers that stain antigen-specific T cells better than conventional multimers in both in vitro and ex vivo analyses. Using a comprehensive library of dimers for HLA-DP4, which is the most frequent HLA allele in many ancestry groups, we mapped 103 HLA-DP4-restricted epitopes derived from diverse tumor-associated antigens and cloned the cognate T-cell antigen receptor (TCR) genes from in vitro-stimulated CD4+ T cells. The availability of affinity-matured class II dimers across HLA-DP, HLA-DQ and HLA-DR alleles will aid in the investigation of human CD4+ T-cell responses.
• In vivo dynamics and adaptation of HTLV-1-infected clones under different clinical conditions.
  Mikiko Izaki; Jun-Ichirou Yasunaga; Kisato Nosaka; Kenji Sugata; Hayato Utsunomiya; Youko Suehiro; Takafumi Shichijo; Asami Yamada; Yasuhiko Sugawara; Taizo Hibi; Yukihiro Inomata; Hirofumi Akari; Anat Melamed; Charles Bangham; Masao Matsuoka
  PLoS pathogens, 17, 2, e1009271, 2021年02月, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) spreads through cell contact. Therefore, this virus persists and propagates within the host by two routes: clonal proliferation of infected cells and de novo infection. The proliferation is influenced by the host immune responses and expression of viral genes. However, the detailed mechanisms that control clonal expansion of infected cells remain to be elucidated. In this study, we show that newly infected clones were strongly suppressed, and then stable clones were selected, in a patient who was infected by live liver transplantation from a seropositive donor. Conversely, most HTLV-1+ clones persisted in patients who received hematopoietic stem cell transplantation from seropositive donors. To clarify the role of cell-mediated immunity in this clonal selection, we suppressed CD8+ or CD16+ cells in simian T-cell leukemia virus type 1 (STLV-1)-infected Japanese macaques. Decreasing CD8+ T cells had marginal effects on proviral load (PVL). However, the clonality of infected cells changed after depletion of CD8+ T cells. Consistent with this, PVL at 24 hours in vitro culture increased, suggesting that infected cells with higher proliferative ability increased. Analyses of provirus in a patient who received Tax-peptide pulsed dendritic cells indicate that enhanced anti-Tax immunity did not result in a decreased PVL although it inhibited recurrence of ATL. We postulate that in vivo selection, due to the immune response, cytopathic effects of HTLV-1 and intrinsic attributes of infected cells, results in the emergence of clones of HTLV-1-infected T cells that proliferate with minimized HTLV-1 antigen expression.
• Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy.
  Yuki Kagoya; Tingxi Guo; Brian Yeung; Kayoko Saso; Mark Anczurowski; Chung-Hsi Wang; Kenji Murata; Kenji Sugata; Hiroshi Saijo; Yukiko Matsunaga; Yota Ohashi; Marcus O Butler; Naoto Hirano
  Cancer immunology research, 8, 7, 926, 936, 2020年07月, ［国際誌］
  英語, 研究論文（学術雑誌）, Adoptive immunotherapy can induce sustained therapeutic effects in some cancers. Antitumor T-cell grafts are often individually prepared in vitro from autologous T cells, which requires an intensive workload and increased costs. The quality of the generated T cells can also be variable, which affects the therapy's antitumor efficacy and toxicity. Standardized production of antitumor T-cell grafts from third-party donors will enable widespread use of this modality if allogeneic T-cell responses are effectively controlled. Here, we generated HLA class I, HLA class II, and T-cell receptor (TCR) triple-knockout (tKO) T cells by simultaneous knockout of the B2M, CIITA, and TRAC genes through Cas9/sgRNA ribonucleoprotein electroporation. Although HLA-deficient T cells were targeted by natural killer cells, they persisted better than HLA-sufficient T cells in the presence of allogeneic peripheral blood mononuclear cells (PBMC) in immunodeficient mice. When transduced with a CD19 chimeric antigen receptor (CAR) and stimulated by tumor cells, tKO CAR-T cells persisted better when cultured with allogeneic PBMCs compared with TRAC and B2M double-knockout T cells. The CD19 tKO CAR-T cells did not induce graft-versus-host disease but retained antitumor responses. These results demonstrated the benefit of HLA class I, HLA class II, and TCR deletion in enabling allogeneic-sourced T cells to be used for off-the-shelf adoptive immunotherapy.
• Landscape mapping of shared antigenic epitopes and their cognate TCRs of tumor-infiltrating T lymphocytes in melanoma.
  Kenji Murata; Munehide Nakatsugawa; Muhammed A Rahman; Linh T Nguyen; Douglas G Millar; David T Mulder; Kenji Sugata; Hiroshi Saijo; Yukiko Matsunaga; Yuki Kagoya; Tingxi Guo; Mark Anczurowski; Chung-Hsi Wang; Brian D Burt; Dalam Ly; Kayoko Saso; Alexandra Easson; David P Goldstein; Michael Reedijk; Danny Ghazarian; Trevor J Pugh; Marcus O Butler; Tak W Mak; Pamela S Ohashi; Naoto Hirano
  eLife, 9, 2020年04月21日, ［国際誌］
  英語, 研究論文（学術雑誌）, HLA-restricted T cell responses can induce antitumor effects in cancer patients. Previous human T cell research has largely focused on the few HLA alleles prevalent in a subset of ethnic groups. Here, using a panel of newly developed peptide-exchangeable peptide/HLA multimers and artificial antigen-presenting cells for 25 different class I alleles and greater than 800 peptides, we systematically and comprehensively mapped shared antigenic epitopes recognized by tumor-infiltrating T lymphocytes (TILs) from eight melanoma patients for all their class I alleles. We were able to determine the specificity, on average, of 12.2% of the TILs recognizing a mean of 3.1 shared antigen-derived epitopes across HLA-A, B, and C. Furthermore, we isolated a number of cognate T cell receptor genes with tumor reactivity. Our novel strategy allows for a more complete examination of the immune response and development of novel cancer immunotherapy not limited by HLA allele prevalence or tumor mutation burden.
• Chaperones of the class I peptide-loading complex facilitate the constitutive presentation of endogenous antigens on HLA-DP84GGPM87.
  Mark Anczurowski; Kenji Sugata; Yukiko Matsunaga; Yuki Yamashita; Chung-Hsi Wang; Tingxi Guo; Kenji Murata; Hiroshi Saijo; Yuki Kagoya; Kayoko Saso; Marcus O Butler; Naoto Hirano
  Journal of autoimmunity, 102, 114, 125, 2019年08月, ［国際誌］
  英語, 研究論文（学術雑誌）, Recent work has delineated key differences in the antigen processing and presentation mechanisms underlying HLA-DP alleles encoding glycine at position 84 of the DPβ chain (DP84GGPM87). These DPs are unable to associate with the class II-associated Ii peptide (CLIP) region of the invariant chain (Ii) chaperone early in the endocytic pathway, leading to continuous presentation of endogenous antigens. However, little is known about the chaperone support involved in the loading of these endogenous antigens onto DP molecules. Here, we demonstrate the proteasome and TAP dependency of this pathway and reveal the ability of HLA class I to compete with DP84GGPM87 for the presentation of endogenous antigens, suggesting that shared subcellular machinery may exist between the two classes of HLA. We identify physical interactions of prototypical class I-associated chaperones with numerous DP alleles, including TAP2, tapasin, ERp57, calnexin, and calreticulin, using a conventional immunoprecipitation and immunoblot approach and confirm the existence of these interactions in vivo through the use of the BioID2 proximal biotinylation system in human cells. Based on immunological assays, we then demonstrate the ability of each of these chaperones to facilitate the presentation of endogenously derived, but not exogenously derived, antigens on DP molecules. Considering previous genetic and clinical studies linking DP84GGPM87 to disease frequency and severity in autoimmune disease, viral infections, and cancer, we suggest that the above chaperones may form the molecular basis of these observable clinical differences through facilitating the presentation of endogenously derived antigens to CD4+ T cells.
• Arginine methylation of FOXP3 is crucial for the suppressive function of regulatory T cells.
  Yuki Kagoya; Hiroshi Saijo; Yukiko Matsunaga; Tingxi Guo; Kayoko Saso; Mark Anczurowski; Chung-Hsi Wang; Kenji Sugata; Kenji Murata; Marcus O Butler; Cheryl H Arrowsmith; Naoto Hirano
  Journal of autoimmunity, 97, 10, 21, 2019年02月, ［国際誌］
  英語, 研究論文（学術雑誌）, Forkhead box transcription factor 3 (FOXP3) plays a pivotal role in the suppressive function of regulatory T cells. In addition to mRNA levels, FOXP3 activity can also be controlled by posttranslational mechanisms, which have not been studied in a comprehensive manner. Through extensive screening using selective inhibitors, we demonstrate that the inhibition of type I protein arginine methytransferases (PRMTs) attenuates the suppressive functions of regulatory T cells. FOXP3 undergoes methylation on arginine residues at positions 48 and 51 by interacting with protein arginine methyltransferase 1 (PRMT1). The inhibition of arginine methylation confers gene expression profiles representing type I helper T cells to FOXP3+ T cells, which results in attenuated suppressive activity. A methylation-defective mutant of FOXP3 displays less potent activity to suppress xenogeneic graft-versus-host disease in vivo. These results elucidate an important role of arginine methylation to enhance FOXP3 functions and are potentially applicable to modulate regulatory T cell functions.
• Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo.
  Rie Furuta; Jun-Ichirou Yasunaga; Michi Miura; Kenji Sugata; Akatsuki Saito; Hirofumi Akari; Takaharu Ueno; Norihiro Takenouchi; Jun-Ichi Fujisawa; Ki-Ryang Koh; Yusuke Higuchi; Mohamed Mahgoub; Masakazu Shimizu; Fumihiko Matsuda; Anat Melamed; Charles R Bangham; Masao Matsuoka
  PLoS pathogens, 13, 11, e1006722, 2017年11月, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.
• HTLV-1 Viral Factor HBZ Induces CCR4 to Promote T-cell Migration and Proliferation.
  Kenji Sugata; Jun-Ichirou Yasunaga; Haruka Kinosada; Yuichi Mitobe; Rie Furuta; Mohamed Mahgoub; Chiho Onishi; Kazutaka Nakashima; Koichi Ohshima; Masao Matsuoka
  Cancer research, 76, 17, 5068, 79, 2016年09月01日, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and other inflammatory diseases in infected individuals. However, a complete understanding of how HTLV-1 transforms T cells is lacking. Expression of the chemokine receptor CCR4 on ATL cells and HTLV-1-infected cells suggested the hypothesis that CCR4 may mediate features of ATL and inflammatory diseases caused by HTLV-1. In this study, we show that the constitutively expressed HTLV-1 bZIP factor (HBZ) encoded by HTLV-1 is responsible for inducing CCR4 and its ability to promote T-cell proliferation and migration. Ectopic expression of HBZ was sufficient to stimulate expression of CCR4 in human and mouse T cells. Conversely, HBZ silencing in ATL cell lines was sufficient to inhibit CCR4 expression. Mechanistic investigations showed that HBZ induced GATA3 expression in CD4(+) T cells, thereby activating transcription from the CCR4 promoter. In an established air pouch model of ATL, we observed that CD4(+) T cells of HBZ transgenic mice (HBZ-Tg mice) migrated preferentially to the pouch, as compared with those in nontransgenic mice. Migration of CD4(+) T cells in HBZ-Tg mice was inhibited by treatment with a CCR4 antagonist. Proliferating (Ki67(+)) CD4(+) T cells were found to express high levels of CCR4 and CD103. Further, CD4(+) T-cell proliferation in HBZ-Tg mice was enhanced by coordinate treatment with the CCR4 ligands CCL17 and 22 and with the CD103 ligand E-cadherin. Consistent with this finding, we found that ATL cells in clinical skin lesions were frequently positive for CCR4, CD103, and Ki67. Taken together, our results show how HBZ activates CCR4 expression on T cells to augment their migration and proliferation, two phenomena linked to HTLV-1 pathogenesis. Cancer Res; 76(17); 5068-79. ©2016 AACR.
• Impact of the SCF signaling pathway on leukemia stem cell-mediated ATL initiation and progression in an HBZ transgenic mouse model.
  Wakako Kuribayashi; Kazuya Takizawa; Kenji Sugata; Madoka Kuramitsu; Haruka Momose; Eita Sasaki; Yuki Hiradate; Keiko Furuhata; Yoshihisa Asada; Atsushi Iwama; Masao Matsuoka; Takuo Mizukami; Isao Hamaguchi
  Oncotarget, 7, 32, 51027, 51043, 2016年08月09日, ［国際誌］
  英語, 研究論文（学術雑誌）, Adult T-cell leukemia (ATL) is a malignant disease caused by human T-lymphotropic virus type 1. In aggressive ATL, the response to chemotherapy is extremely poor. We hypothesized that this poor response is due to the existence of chemotherapy-resistant cells, such as leukemic stem cells. Previously, we successfully identified an ATL stem cell (ATLSC) candidate as the c-kit+/CD38-/CD71- cells in an ATL mouse model using Tax transgenic mice. Here, with a new ATL mouse model using HBZ-transgenic mice, we further discovered that the functional ATLSC candidate, which commonly expresses c-kit, is drug-resistant and has the ability to initiate tumors and reconstitute lymphomatous cells. We characterized the ATLSCs as c-kit+/CD4-/CD8- cells and found that they have a similar gene expression profile as T cell progenitors. Additionally, we found that AP-1 gene family members, including Junb, Jund, and Fosb, were up-regulated in the ATLSC fraction. The results of an in vitro assay showed that ATLSCs cultured with cytokines known to promote stem cell expansion, such as stem cell factor (SCF), showed highly proliferative activity and maintained their stem cell fraction. Inhibition of c-kit-SCF signaling with the neutralizing antibody ACK2 affected ATLSC self-renewal and proliferation. Experiments in Sl/Sld mice, which have a mutation in the membrane-bound c-kit ligand, found that ATL development was completely blocked in these mice. These results clearly suggest that the c-kit-SCF signal plays a key role in ATLSC self-renewal and in ATL initiation and disease progression.
• Enhancement of anti-STLV-1/HTLV-1 immune responses through multimodal effects of anti-CCR4 antibody.
  Kenji Sugata; Jun-Ichirou Yasunaga; Michi Miura; Hirofumi Akari; Atae Utsunomiya; Kisato Nosaka; Yuko Watanabe; Hitoshi Suzushima; Ki-Ryang Koh; Masanori Nakagawa; Michinori Kohara; Masao Matsuoka
  Scientific reports, 6, 27150, 27150, 2016年06月02日, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia and inflammatory diseases. Because anti-HTLV-1 immune responses are critical for suppressing infected cells, enhancing cellular immunity is beneficial for the treatment of HTLV-1-associated diseases. Using simian T-cell leukemia virus type 1 (STLV-1) infected Japanese macaques, we analyzed the immune responses to viral antigens and the dynamics of virus-infected cells. The chemokine receptor CCR4 is expressed on STLV-1 infected cells, and administration of humanized monoclonal antibody to CCR4, mogamulizumab, dramatically decreased the number of STLV-1-infected cells in vivo. Concurrently, mogamulizumab treatment enhanced STLV-1 specific CD4(+) and CD8(+) T cell responses by simultaneously targeting CCR4(+) effector regulatory T (Treg) cells and infected cells. Mogamulizumab promoted the phagocytosis of CCR4(+) infected cells by macrophages, which likely enhanced antigen presentation. Vaccination with recombinant vaccinia virus (rVV) expressing viral antigens suppressed the proviral load and the number of Tax-expressing cells. Enhanced T-cell responses were also observed in some ATL patients who were treated with mogamulizumab. This study shows that mogamulizumab works not only by killing CCR4(+) infected cells directly, but also by enhancing T cell responses by increasing the phagocytosis of infected cells by antigen-presenting cells and suppressing CCR4(+) effector Treg cells.
• HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule, T Cell Immunoglobulin and ITIM Domain (TIGIT).
  Keiko Yasuma; Jun-ichirou Yasunaga; Keiko Takemoto; Kenji Sugata; Yuichi Mitobe; Norihiro Takenouchi; Masanori Nakagawa; Yutaka Suzuki; Masao Matsuoka
  PLoS pathogens, 12, 1, e1005372, 2016年01月, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) infects CD4+ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4+ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4+ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT+CD4+ cells of HBZ-Tg mice compared with TIGIT-CD4+ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4+ T cells from HBZ-Tg mice were stimulated with TIGIT's ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.
• Protective effect of cytotoxic T lymphocytes targeting HTLV-1 bZIP factor.
  Kenji Sugata; Jun-Ichirou Yasunaga; Yuichi Mitobe; Michi Miura; Paola Miyazato; Michinori Kohara; Masao Matsuoka
  Blood, 126, 9, 1095, 105, 2015年08月27日, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia-lymphoma (ATL) and inflammatory diseases in a small percentage of infected individuals. Host immune responses, in particular cytotoxic T lymphocytes (CTLs), influence the proliferation and survival of ATL cells and HTLV-1-infected cells. We generated recombinant vaccinia viruses (rVVs) expressing HTLV-1 basic leucine zipper (bZIP) factor (HBZ) or Tax to study the immunogenic potential of these viral proteins. Vaccination with rVV expressing Tax or HBZ induced specific T-cell responses, although multiple boosters were needed for HBZ. HBZ-stimulated T cells killed HBZ peptide-pulsed T cells and CD4(+) T cells from HBZ transgenic (HBZ-Tg) mice. The anti-lymphoma effect of the CTLs targeting HBZ was tested in mice inoculated with a lymphoma cell line derived from an HBZ-Tg mouse. Transfer of splenocytes from HBZ-immunized mice increased the survival of the lymphoma cell-inoculated mice, suggesting that the anti-HBZ CTLs have a protective effect. The rVV could also induce specific T-cell responses to HBZ and Tax in HTLV-1-infected rhesus monkeys. On the basis of the results of rVV-vaccinated mice and macaques, we identified a candidate peptide (HBZ157-176) for vaccine development. Dendritic cells pulsed with this peptide could generate HBZ-specific CTLs from human CD8(+) T cells. This study demonstrates that HBZ could be a target for immunotherapy of patients with ATL.
• Characterization of simian T-cell leukemia virus type 1 in naturally infected Japanese macaques as a model of HTLV-1 infection.
  Michi Miura; Jun-ichiro Yasunaga; Junko Tanabe; Kenji Sugata; Tiejun Zhao; Guangyong Ma; Paola Miyazato; Koichi Ohshima; Akihisa Kaneko; Akino Watanabe; Akatsuki Saito; Hirofumi Akari; Masao Matsuoka
  Retrovirology, 10, 118, 118, 2013年10月24日, ［国際誌］
  英語, 研究論文（学術雑誌）, BACKGROUND: Human T-cell leukemia virus type 1 (HTLV-1) causes chronic infection leading to development of adult T-cell leukemia (ATL) and inflammatory diseases. Non-human primates infected with simian T-cell leukemia virus type 1 (STLV-1) are considered to constitute a suitable animal model for HTLV-1 research. However, the function of the regulatory and accessory genes of STLV-1 has not been analyzed in detail. In this study, STLV-1 in naturally infected Japanese macaques was analyzed. RESULTS: We identified spliced transcripts of STLV-1 corresponding to HTLV-1 tax and HTLV-1 bZIP factor (HBZ). STLV-1 Tax activated the NFAT, AP-1 and NF-κB signaling pathways, whereas STLV-1 bZIP factor (SBZ) suppressed them. Conversely, SBZ enhanced TGF-β signaling and induced Foxp3 expression. Furthermore, STLV-1 Tax activated the canonical Wnt pathway while SBZ suppressed it. STLV-1 Tax enhanced the viral promoter activity while SBZ suppressed its activation. Then we addressed the clonal proliferation of STLV-1⁺ cells by massively sequencing the provirus integration sites. Some clones proliferated distinctively in monkeys with higher STLV-1 proviral loads. Notably, one of the monkeys surveyed in this study developed T-cell lymphoma in the brain; STLV-1 provirus was integrated in the lymphoma cell genome. When anti-CCR4 antibody, mogamulizumab, was administered into STLV-1-infected monkeys, the proviral load decreased dramatically within 2 weeks. We observed that some abundant clones recovered after discontinuation of mogamulizumab administration. CONCLUSIONS: STLV-1 Tax and SBZ have functions similar to those of their counterparts in HTLV-1. This study demonstrates that Japanese macaques naturally infected with STLV-1 resemble HTLV-1 carriers and are a suitable model for the investigation of persistent HTLV-1 infection and asymptomatic HTLV-1 carrier state. Using these animals, we verified that mogamulizumab, which is currently used as a drug for relapsed ATL, is also effective in reducing the proviral load in asymptomatic individuals.
• HTLV-1 bZIP factor impairs cell-mediated immunity by suppressing production of Th1 cytokines.
  Kenji Sugata; Yorifumi Satou; Jun-ichirou Yasunaga; Hideki Hara; Kouichi Ohshima; Atae Utsunomiya; Masao Mitsuyama; Masao Matsuoka
  Blood, 119, 2, 434, 44, 2012年01月12日, ［筆頭著者］, ［国際誌］
  英語, 研究論文（学術雑誌）, Adult T-cell leukemia (ATL) patients and human T-cell leukemia virus-1 (HTLV-1) infected individuals succumb to opportunistic infections. Cell mediated immunity is impaired, yet the mechanism of this impairment has remained elusive. The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the viral DNA and is constitutively expressed in infected cells and ATL cells. To test the hypothesis that HBZ contributes to HTLV-1-associated immunodeficiency, we challenged transgenic mice that express the HBZ gene in CD4 T cells (HBZ-Tg mice) with herpes simplex virus type 2 or Listeria monocytogenes, and evaluated cellular immunity to these pathogens. HBZ-Tg mice were more vulnerable to both infections than non-Tg mice. The acquired immune response phase was specifically suppressed, indicating that cellular immunity was impaired in HBZ-Tg mice. In particular, production of IFN-γ by CD4 T cells was suppressed in HBZ-Tg mice. HBZ suppressed transcription from the IFN-γ gene promoter in a CD4 T cell-intrinsic manner by inhibiting nuclear factor of activated T cells and the activator protein 1 signaling pathway. This study shows that HBZ inhibits CD4 T-cell responses by directly interfering with the host cell-signaling pathway, resulting in impaired cell-mediated immunity in vivo.
• HTLV-1 bZIP factor enhances TGF-β signaling through p300 coactivator.
  Tiejun Zhao; Yorifumi Satou; Kenji Sugata; Paola Miyazato; Patrick L Green; Takeshi Imamura; Masao Matsuoka
  Blood, 118, 7, 1865, 76, 2011年08月18日, ［国際誌］
  英語, 研究論文（学術雑誌）, Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus that is etiologically associated with adult T-cell leukemia. The HTLV-1 bZIP factor (HBZ), which is encoded by the minus strand of the provirus, is involved in both regulation of viral gene transcription and T-cell proliferation. We showed in this report that HBZ interacted with Smad2/3, and enhanced transforming growth factor-β (TGF-β)/Smad transcriptional responses in a p300-dependent manner. The N-terminal LXXLL motif of HBZ was responsible for HBZ-mediated TGF-β signaling activation. In a serial immunoprecipitation assay, HBZ, Smad3, and p300 formed a ternary complex, and the association between Smad3 and p300 was markedly enhanced in the presence of HBZ. In addition, HBZ could overcome the repression of the TGF-β response by Tax. Finally, HBZ expression resulted in enhanced transcription of Pdgfb, Sox4, Ctgf, Foxp3, Runx1, and Tsc22d1 genes and suppression of the Id2 gene; such effects were similar to those by TGF-β. In particular, HBZ induced Foxp3 expression in naive T cells through Smad3-dependent TGF-β signaling. Our results suggest that HBZ, by enhancing TGF-β signaling and Foxp3 expression, enables HTLV-1 to convert infected T cells into regulatory T cells, which is thought to be a critical strategy for virus persistence.
• The vIL-10 gene of the Epstein-Barr virus (EBV) is conserved in a stable manner except for a few point mutations in various EBV isolates.
  Kyosuke Kanai; Yukio Satoh; Hiroyuki Yamanaka; Asako Kawaguchi; Kazutaka Horie; Kenji Sugata; Yoshiko Hoshikawa; Tetsutaro Sata; Takeshi Sairenji
  Virus genes, 35, 3, 563, 9, 2007年12月, ［国際誌］
  英語, 研究論文（学術雑誌）, A gene of the Epstein-Barr virus (EBV), BamHI-C fragment rightward reading frame 1 (BCRF1), codes viral interleukin-10 (vIL-10), which is a close homolog to human IL-10. EBV strain variations are known at EBV latent membrane protein 1 (LMP1), and the distinct forms of LMP1 have been identified. In order to further elucidate the variations of EBV strains, the BCRF1 (vIL-10) gene was analyzed using PCR-direct sequencing in African Burkitt's lymphoma (BL) cell lines Raji, P3HR-1, EB1 and Daudi, Japanese BL cell line Akata, lymphoblastoid cell line OB and 22 wild EBV isolates from eight gastric carcinoma tissues and 14 throat washes. We found only five variations of the vIL-10 gene in them with one silent mutation and three non-silent mutations. Raji had no mutation to the prototype gene of B95-8. EB1 and P3HR-1 had non-silent mutations in the sequences leading to the arginine/serine and threonine/proline interchanges at residues 4 and 166, respectively. The silent mutation was detected at valine 102 in Daudi and also in the Japanese cell lines Akata, OB and 20 (90.9%) of the wild EBV isolates. The type of variations in the vIL-10 gene had a common relationship with those in the LMP1 gene. All of the variants of valine 102 had China1-type LMP1 sequences except for Daudi with Med-type LMP1 and other minorities with B95-8 type LMP1. The conservativeness of vIL-10 with a few variations suggests the indispensability of the vIL-10 gene in EBV and that the variations of the vIL-10 gene may depend upon the geographical prevalence of the EBV strains. This is the first report regarding the variations of the vIL-10 gene in cell lines and other wild isolates.

• HAM患者脳脊髄液での特異的CD8T細胞の遺伝子発現とTCR avidityの解析
  菅田謙治; TAN Jek Yan; 新村光輝; 高鳥光徳; 徳永雅仁; 中島誠; 佐藤知雄; 佐藤知雄; 植田光晴; 宇都宮與; 山野嘉久; 山野嘉久; 佐藤賢文, 日本HTLV-1学会学術集会, 10th, 2024年
• HTLV-1による成人T細胞白血病・リンパ腫の発症機構
  菅田謙治; 佐藤賢文, 日本臨牀 81 (増刊号3) 7-12, 2023., 2023年
• HAM患者末梢血および脳脊髄液のシングルセル解析によるHAM病態解明と疾患サロゲートマーカーの探索研究
  菅田謙治; TAN Benjy Jek Yang; HOSSAIN Md Belal; RAJIB Samiul Alam; REDA Omnia; 徳永雅仁; 野村隼也; 増田曜章; 佐藤知雄; 佐藤知雄; 植田光晴; 宇都宮與; 山野嘉久; 山野嘉久; 佐藤賢文, 日本HTLV-1学会学術集会, 8th, 2022年
• HTLV-1プロウイルスにおける新規エンハンサーの特定と分子メカニズム解析
  松尾美沙希; 松尾美沙希; 上野孝治; 門出和精; TAN Benjy Jek Yang; TAN Benjy Jek Yang; 菅田謙治; 宮里パオラ; 宮里パオラ; 内山喬介; 内山喬介; ISLAM Saiful; ISLAM Saiful; 勝屋弘雄; 勝屋弘雄; 中嶋伸介; 徳永雅仁; 野坂生郷; 畑裕之; 宇都宮與; 藤澤順一; 佐藤賢文; 佐藤賢文, 日本エイズ学会誌, 23, 4, 2021年
• 【リンパ腫学-最新の研究動向-】リンパ腫の疫学と危険因子 HTLV-1感染の疫学と病原性発現機構
  菅田 謙治; 松岡 雅雄, 日本臨床, 73, 増刊8 リンパ腫学, 49, 54, 2015年10月
  (株)日本臨床社, 日本語
• HBZはCCR4発現を誘導する
  菅田謙治; 安永純一朗; 古田梨愛; 松岡雅雄, 日本HTLV-1学会学術集会, 2nd, 2015年
• HTLV-1感染動物モデルとしてのサルT細胞白血病ウイルス1型感染ニホンザルの解析
  三浦未知; 田邉順子; 菅田謙治; 趙鉄軍; 安永純一朗; 松岡雅雄, ATLシンポジウム/HTLV-1国際シンポジウム/HTLV-1研究会プログラム・抄録集, 1st-2nd-5th, 2012年

• 2021年 - 現在
  日本HTLV-1学会
• 2020年 - 現在
  日本がん免疫学会
• 2008年 - 現在
  日本ウイルス学会

• HBZ発現マウスを用いたTCRシグナルを介した腫瘍化機構の解明
  科学研究費助成事業
  2025年04月01日 - 2028年03月31日
  菅田 謙治
  日本学術振興会, 基盤研究(C), 熊本大学, 25K10381
• HTLV-1特異的CTLが発現するHAM関連遺伝子の探索
  科学研究費助成事業 基盤研究(C)
  2021年04月01日 - 2024年03月31日
  菅田 謙治
  ヒトT細胞白血病ウイルス1型（HTLV-1）はCD4T細胞に感染することで難治性の腫瘍性疾患と炎症性疾患を感染者に惹起する。主に後者はHTLV-1関連脊髄症(HAM: HTLV-1-associated myelopathy)がよく知られており、難治性の慢性神経疾患である。多くのHTLV-1キャリアは無症状で経過するものの感染者の一部で発症することから、その発症には宿主免疫が強く関与している可能性が示唆されている。HAM患者ではHTLV-1抗原に対する強いT細胞応答が観察される。また、病巣である脊髄ではHTLV-1感染CD4T細胞とHTLV-1特異的細胞傷害性T細胞（CTL）の浸潤とそれに伴う炎症像が確認される。近年ではHAMに対する新規治療法としてHTLV-1感染細胞で発現が上昇しているCCR4（C-C chemokine receptor type 4）に対するヒト化モノクローナル抗体（モガムリズマブ）を投与することで生体内の感染細胞を排除する試みがなされている。HAMが難治性の疾患であることから、末梢血を用いた病態解析に基づいた発症予測マーカーの発見が重要な意味を持つことは明らかであるものの、これまで有用なHAM特異的マーカーは未だに見つかっていない。HAM患者の末梢血と脊髄では病態が異なり、末梢血での感染CD4T細胞やCTLの機能解析は必ずしも病巣のT細胞の病態を反映していない可能性が考えられた。本研究ではHAM患者の脊髄病変に浸潤しているHTLV-1抗原特異的CTLの遺伝子発現プロファイルに基づき、末梢血での同CTLが発現するHAMの発症リスク遺伝子を発見することを目的としている。
  日本学術振興会, 基盤研究(C), 熊本大学, 21K08494
• 高親和性HTLV1特異的TCRの網羅的探索
  科学研究費助成事業 研究活動スタート支援
  2020年09月11日 - 2022年03月31日
  菅田 謙治
  ATLの新規治療法の開発を目的とし、本研究では近年がん患者で治療成果をあげているT細胞移入療法に適した高親和性HTLV1特異的TCRの探索をシングルセル解析より得られたTCR配列を元に行った。ATL患者やHAM患者由来の検体を用いて行ったシングルセル解析結果からTCRレパトアが優位に増加しているCD8T細胞クローン由来のHLA-I拘束性TCRの配列情報を抽出した。得られたTCRをPrimary T細胞で発現させてHTLV-1抗原の探索を行ったところ、特にHAM患者由来のTCRがHLA-A*24:02拘束性Tax301-309に特異的なTCRであった。
  日本学術振興会, 研究活動スタート支援, 熊本大学, 20K22783
• HBZ特異的CTLの作製とその機能評価
  科学研究費助成事業 若手研究(B)
  2014年04月01日 - 2016年03月31日
  菅田 謙治
  新たな抗HTLV-1療法の開発の為にHBZ抗原を発現するrecombinant vaccine virus (rVV)を用いて特異的CTLを誘導し、機能評価を行った。そのrVVを接種したマウスではHBZ特異的CTLの誘導が確認され、そのCTLはHBZ発現細胞に対して細胞障害機能を有していた。それらの結果はRhesus macaqueにrVVを接種した際も観察された。それらのHBZ特異的CTLはHBZ157-176ペプチドに反応した。HBZ157-176はin vitroでCD8T細胞からHBZ特異的CTLを誘導した。この研究でHBZがHTLV-1療法の標的になりうることを示した。
  日本学術振興会, 若手研究(B), 京都大学, 26860301
• 組換えワクシニアウイルスを用いた抗ＨＴＬＶ－１ワクチンの開発
  科学研究費助成事業 特別研究員奨励費
  2013年04月01日 - 2016年03月31日
  菅田 謙治
  HTLV-1(Human T cell leukemia virus type-1)はCD4陽性Tリンパ球に感染し、腫瘍性疾患である成人T細胞白血病(ATL)を引き起こす。これまでの研究で様々な抗HTLV-1治療法の開発が行われてきた。抗HTLV-1免疫を誘導する樹状細胞療法などが行われており、抗ウイルス免疫を賦活する免疫療法はHTLV-1関連疾患に対して有望な治療法になることが期待されている。本研究では新たなツールとして HTLV-1抗原を発現する遺伝子組換えワクシニアウイルス(rVV)を用いた抗HTLV-1ワクチンの作製と機能評価を行い、HTLV-1霊長類モデルを用いて体内でのウイルス感染細胞の動態を解析した。
  これまでの研究で私は点変異HBZ抗原(HBZ LL/AA:L27A/L28A)を発現する組換えワクシニアウイルスがマウスでのHBZ-induced ATLモデルの生存率を延長させることを見出した。生体内でのrVVによるワクチン効果を調べる為にSTLV-1感染ニホンザルをHTLV-1感染の霊長類モデルとして実験に使用した。STLV-1のウイルス抗原であるSTLV-1 Tax(sTax)とSTLV-1 bZIP factor(SBZ)を発現するワクシニアウイルスを接種し、生体内に感染したSTLV-1の体内動態を解析した。その結果、STLV-1抗原を発現するワクシニアウイルスはSTLV-1感染CD4 T細胞を減少させることが示された。
  本研究ではSTLV-1感染ニホンザルをHTLV-1感染霊長類モデルとして用いて、組換えワクシニアウイルスを組み合わせてより効果的な抗HTLV-1療法の開発を行った。
  日本学術振興会, 特別研究員奨励費, 京都大学, 13J05302