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2019 年度 実施状況報告書

The role of the transcription factor Tox2 in Treg and Tfh biology

研究課題

研究課題/領域番号 18K07175
研究機関大阪大学

研究代表者

ウィング ジェイムス  大阪大学, 免疫学フロンティア研究センター, 特任准教授(常勤) (00648694)

研究期間 (年度) 2018-04-01 – 2021-03-31
キーワードRegulatory T-cells / T-follicular regulatory / T-follicular helper / Mass Cytometry
研究実績の概要

We have determined the epigenomic signature of Tox2 in humans by assessing Assay for transposase-accessible chromatin sequencing (ATAC-seq). In highly activated Tfh and effector like Tfr Tox2 shows increased open chromatin regions correlating with its greater expression. Additionally, Tfr circulating in human blood have a Naïve like subgroup which also has increased chromatin accessibility peaks within the Tox2 gene. By mass cytometry (CyTOF) we have more fully assess the heterogeneity of T-follicular regulatory cells (Tfr) in both the blood and tonsils of healthy and autoimmune human donors. Tox2 is highly expressed by Tfh and Tfr in both the circulation and the tonsils themselves in both healthy and autoimmune individuals. From this phenotypic analysis it is now clear that Tox2 is widely expressed by both human and mouse T-follicular helper and T-follicular regulatory cells.
We have also recently established the methodology that will allow us to CRISPR delete the Tox2 gene from humans T-cells and are now proceeding to do this. In mice the Tox2 floxed mice have been made and are now crossing with Foxp3-IRES-Cre in order to produce Mice with a conditional loss of Tox2 only in Tregs, the first mice have been confirmed not to suffer from severe autoimmunity and the colony is now expanding for more in depth study. These two approaches will allow us to assess the contribution of Tox2 to the biology of both human and murine Tregs.

現在までの達成度 (区分)
現在までの達成度 (区分)

2: おおむね順調に進展している

理由

CyTOF method establishment. We are confident that we have established excellent procedures for CyTOF screening of both humans and mice. We have also developed methods for the assessment of Tregs at tissue sites. This methodological section of the work is complete.
Screening of Tox2 floxed mice. This has taken longer than expected due to several attempts being needed to successfully generate the Flox inserts. However, these mice have now been made and crossed with Foxp3-IRES-Cre and are now breeding for in depth assessment.
Screening of humans. Donors with SLE reveal some abnormalities in the proportions of Tfr cells but no clear change in Tox2 expression. We have found that Tfh/Tfr cells in the tonsils have higher levels of Tox2 expression than their circulating counterparts.

今後の研究の推進方策

The Tox2 floxed mice have been bred and crossed with Foxp3-IRES-Cre in order to produce Treg specific Tox2 deficient mice. While this process is at an early stage the mice do not develop overt autoimmunity although they will be monitored over time for the development of abnormalities. Further in depth assessment of these mice by CyTOF is about to begin.
However, demonstrating a functional role for Tox2 in already differentiated Tfr cells is complex. We have established an assay for the induction of Tfh/Tfr like cells expressing CXCR5 from Naive T-cells in vitro. We will use this model system to better understand which factors, such as Cytokines, are responsible for the induction of Tox2 expression. We are using this assay as a model system for CRISPR knockout of Tox2 and its related protein Tox in human T-cells to assess its contribution to both the induction of CXCR5 and other Tfh and exhaustion related molecules such as PD-1. Particular attention will be paid to ATAC-Seq since Tox2 is believed to function as a chromatin remodeler. Partly due to the delay in the production of the mice the project includes more human data than was initially planned, however we feel this is beneficial since we can both perform in vivo studies in mice and confirm them in humans.

  • 研究成果

    (9件)

すべて 2020 2019 その他

すべて 雑誌論文 (4件) (うち国際共著 3件、 査読あり 2件) 学会発表 (4件) (うち招待講演 3件) 備考 (1件)

  • [雑誌論文] Dynamics of effector and naive Regulatory T cells throughout pregnancy2020

    • 著者名/発表者名
      Shigeta Naoya、Kumasawa Keiichi、Tanaka Atsushi、Badger Wing James、Nakamura Hitomi、Sakaguchi Shimon、Kimura Tadashi
    • 雑誌名

      Journal of Reproductive Immunology

      巻: 140 ページ: 103135~103135

    • DOI

      https://doi.org/10.1016/j.jri.2020.103135

    • 査読あり / 国際共著
  • [雑誌論文] Regulatory T Cells and Human Disease2020

    • 著者名/発表者名
      Sakaguchi Shimon、Mikami Norihisa、Wing James B.、Tanaka Atsushi、Ichiyama Kenji、Ohkura Naganari
    • 雑誌名

      Annual Review of Immunology

      巻: 38 ページ: 541~566

    • DOI

      https://doi.org/10.1146/annurev-immunol-042718-041717

    • 査読あり / 国際共著
  • [雑誌論文] Control of Regulatory T Cells by Co-signal Molecules2019

    • 著者名/発表者名
      Wing James Badger、Tay Christopher、Sakaguchi Shimon
    • 雑誌名

      Co-signal Molecules in T Cell Activation

      巻: 1189 ページ: 179~210

    • DOI

      https://doi.org/10.1007/978-981-32-9717-3_7

    • 国際共著
  • [雑誌論文] Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)2019

    • 著者名/発表者名
      Cossarizza A,…150 authors…,Wing, JB….16 authors… Zychlinsky A
    • 雑誌名

      European Journal of Immunology

      巻: 49 ページ: 1457~1973

    • DOI

      doi: 10.1002/eji.201970107

  • [学会発表] High dimensional analysis of T-follicular regulatory cells2020

    • 著者名/発表者名
      James Wing
    • 学会等名
      IMSUT young investigators symposium “Rising stars in cutting edge Immunology research”
    • 招待講演
  • [学会発表] Exploring regulatory T cell function and phenotype with mass cytometry2019

    • 著者名/発表者名
      James Wing
    • 学会等名
      2019 Japanese society for Immunology, lunch time technical seminar
    • 招待講演
  • [学会発表] High dimensional analysis of T-follicular regulatory cells2019

    • 著者名/発表者名
      James Wing
    • 学会等名
      2019 Japanese society for Immunology
  • [学会発表] Exploring regulatory T cell function and phenotype with mass cytometry2019

    • 著者名/発表者名
      James Wing
    • 学会等名
      8th Annual Fluidigm Mass Cytometry Summit
    • 招待講演
  • [備考]

    • URL

      http://www.ifrec.osaka-u.ac.jp/jpn/laboratory/wing_james_badger/

URL: 

公開日: 2021-01-27  

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