Elucidation of Signaling Mechanisms Involved in Human Regulatory T Cell Differentiation by Multi-omics Analysis

• Project/Area Number: 22K15493
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 49070:Immunology-related
• Research Institution: Osaka University
• Principal Investigator: Takeshima Yusuke 大阪大学, 免疫学フロンティア研究センター, 特任助教 (70893288)
• Project Period (FY): 2022-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2023: ¥390,000 (Direct Cost: ¥300,000、Indirect Cost: ¥90,000); Fiscal Year 2022: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
• Keywords: マルチオミクス解析 / シングルセル解析 / 制御性T細胞 / 免疫学

Outline of Research at the Start

近年, 次世代シークエンサーの進歩により単一細胞レベルでの多彩な表現型観測法が開発されているものの, 同一細胞に関する多層的な解析は未だin silicoでの推測に依存しているため, 転写-翻訳に関わる機構の連関については未だ完全に解明されていない. そこで、 本研究では申請者らが開発した単一細胞からepigenome-transcriptome-proteomeの3層にわたる情報を同時に取得できるDOGMA-seqに, CRISPR-Cas9を組み合わせることで制御性T細胞(Treg)への分化に関与する生物学的応答シグナル伝達ネットワークの解明を目指す.

Outline of Final Research Achievements

Regulatory T cells (Tregs) are a population of T cells that specialize in immunosuppressive functions. Although human CD4-positive T cells transiently express FOXP3 upon strong T cell receptor stimulation, FOXP3 expression is not stable. Therefore, it remains difficult to efficiently induce functionally stable Tregs. In this study, we developed a large-scale screen for the effects of target gene knockout using CRISPR-Cas9 by combining genome-wide open chromatin analysis, transcriptome analysis and quantification of cell surface and intracellular proteins at the single cell level. We revealed the biological response signalling network that occurs during Treg differentiation induction and successfully identified candidate regulators of differentiation.

Academic Significance and Societal Importance of the Research Achievements

本研究により、多岐にわたるCRISPR screenを事前に選択肢を狭めることなく一括で行うことができるとともに、既存の手法で解析可能な影響因子の同定だけにとどまらず、アンバイアスにゲノムワイドな探索が可能となることが期待され、影響因子の探索過程ならびにネットワークの詳述において大幅な技術革新がもたらされうると考える。また、誘導に対して正の制御を行うことで安定して効率的な誘導性Tregを分化させることができ、自己由来の末梢性T細胞から誘導Tregを産生することで自己免疫疾患に臨床応用することが可能となる。逆に、負の制御を行うことで自己免疫寛容を抑制し悪性腫瘍に対する治療応用が可能となる。

Research Products

• [Journal Article] Single-cell transcriptome landscape of circulating CD4+ T cell populations in autoimmune diseases (2024)
  • Author(s): Yasumizu Yoshiaki、Takeuchi Daiki、Morimoto Reo、Takeshima Yusuke、Okuno Tatsusada、Kinoshita Makoto、Morita Takayoshi、Kato Yasuhiro、Wang Min、Motooka Daisuke、Okuzaki Daisuke、Nakamura Yamami、Mikami Norihisa、Arai Masaya、Zhang Xuan、Kumanogoh Atsushi、Mochizuki Hideki、Ohkura Naganari、Sakaguchi Shimon
  • Journal Title: Cell Genomics Volume: 4 Issue: 2 Pages: 100473-100473
  • DOI: 10.1016/j.xgen.2023.100473
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Transcriptome Profiling of Immune Cell Types in Peripheral Blood Reveals Common and Specific Pathways Involved in the Pathogenesis of <scp>Myositis‐Specific Antibody‐Positive</scp> Inflammatory Myopathies (2023)
  • Author(s): Sugimori Yusuke、Iwasaki Yukiko、Takeshima Yusuke、Okubo Mai、Kobayashi Satomi、Hatano Hiroaki、Yamada Saeko、Nakano Masahiro、Yoshida Ryochi、Ota Mineto、Tsuchida Yumi、Nagafuchi Yasuo、Shimane Kenichi、Yoshida Ken、Kurosaka Daitaro、Sumitomo Shuji、Shoda Hirofumi、Yamamoto Kazuhiko、Okamura Tomohisa、Fujio Keishi
  • Journal Title: ACR Open Rheumatology Volume: 5 Issue: 2 Pages: 93-102
  • DOI: 10.1002/acr2.11521
  • Peer Reviewed / Open Access
• [Journal Article] Single-cell analyses and host genetics highlight the role of innate immune cells in COVID-19 severity (2023)
  • Author(s): Japan COVID-19 Task Force
  • Journal Title: Nature Genetics Volume: 55 Issue: 5 Pages: 753-767
  • DOI: 10.1038/s41588-023-01375-1
  • Peer Reviewed / Open Access
• [Journal Article] Immunomics analysis of rheumatoid arthritis identified precursor dendritic cells as a key cell subset of treatment resistance. (2023)
  • Author(s): Yamada S, Nagafuchi Y, Wang M, Ota M, Hatano H, Takeshima Y, Okubo M, Kobayashi S, Sugimori Y, Masahiro N, Yoshida R, Hanata N, Suwa Y, Tsuchida Y, Iwasaki Y, Sumitomo S, Kubo K, Shimane K, Setoguchi K, Azuma T, Kanda H, Shoda H, Zhang X, Yamamoto K, Ishigaki K, Okamura T, Fujio K
  • Journal Title: Ann Rheum Dis Volume: Epub ahead of print Issue: 6 Pages: 1-11
  • DOI: 10.1136/ard-2022-223645
  • Peer Reviewed / Open Access / Int'l Joint Research