2019 Fiscal Year Research-status Report
The role of the transcription factor Tox2 in Treg and Tfh biology
| Project/Area Number |
18K07175
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| Research Institution | Osaka University |
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Principal Investigator |
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)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | Regulatory T-cells / T-follicular regulatory / T-follicular helper / Mass Cytometry |
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| Outline of Annual Research Achievements |
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.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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.
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| Strategy for Future Research Activity |
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.
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