2018 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-cell (Treg) / Tfollicular reg (Tfr) / Tfollicular helper (Tfh) / CyTOF |
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| Outline of Annual Research Achievements |
In the past year we have established the Mass Cytometry (CyTOF) technique in this laboratory. We have made substantial progress, designing, manufacturing, testing and establishing 36-40 marker antibody panels for comprehensive assessment of both human and mouse immune systems. While the panels are focused on regulatory T-cells and Tfh they also have sufficient breath to allow assessment of changes to other immune compartments such as B-cells, NK and DCs. We use a CD45 based barcoding strategy to mark individual samples before mixing them together for all processing, staining and data acquisition steps, this allows a high level of technical reproducibility.
This has allowed us to develop methods to allow comprehensive screening of several sites where Tox2 positive T-follicular helper Tfh and T-follicular regulatory (Tfr) cells can be found such as lymphoid organs following vaccination and the Peyer’s patches. We have also confirmed expression of Tox2 by Tregs in tissue sites such as the lamina propria of the small intestine and have developed methodology to screen these sites by CyTOF. Screening of samples from healthy human controls and autoimmune patients (Rheumatoid arthritis and Systemic lupus erythematosus) has also begun.
We have also established a technique to allow the in vitro differentiation of human Tfh cells from naive T-cells. This opens the possibility of manipulating Tox2 expression in human cells, to assess its role in the differentiation of Tfh cells in this in vitro system.
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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
While progress in the development of the CyTOF assays has been better than expected the production of the Tox2 floxed mouse has been slower than expected, meaning that screening of these mice is behind schedule. Despite this, we believe that overall the project is on schedule since we are confident that due to the good CyTOF progress we will be able to rapidly screen any phenotypic differences seen in Tox2 floxed mice and should complete that aspect of the work during the coming year.
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| Strategy for Future Research Activity |
The primary upcoming goal is to rapidly screen comprehensive immune changes in Tox2 deficient mice, since we have spent considerable effort in perfecting the CyTOF technique allowing us to rapidly screen a range of lymphoid sites and organs such as the lymph nodes, spleen, and lamina propria of mice. Our preliminary results suggest that as well as T-follicular regulatory cells certain tissue resident subsets of Tregs express high levels of Tox2.
In humans we intend to begin screening tonsil tissues containing Germinal center resident Tfh cells, which have high levels of Tox2 mRNA, to better characterize the distribution of Tox2 protein expression of Tox2 in these sites. We will also assess Tox2 protein expression in blood samples from healthy, Rheumatoid arthritis and Systemic lupus erythematosus donors.
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| Causes of Carryover |
The primary reason for carryover of some monies is that some CyTOF reagents, monoisotopic Cisplatin 195 and 196, could not be manufactured in FY2018 (manufacturing takes 4-6 months) so will instead be purchased in FY2019. This does not affect the overall research plan.
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[Journal Article] 濾胞性制御性T細胞2019
Author(s)
木本 富子, Wing James B.
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Journal Title
Journal of Clinical and Experimental Medicine (Igaku No Ayumi)
Volume: 268
Pages: 1106-1110
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