| Project/Area Number |
20K07538
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Review Section |
Basic Section 49070:Immunology-related
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| Research Institution | Osaka University |
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Principal Investigator |
ディエス ディエゴ 大阪大学, 免疫学フロンティア研究センター, 准教授 (90597741)
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| Project Period (FY) |
2020-04-01 – 2024-03-31
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| Project Status |
Granted (Fiscal Year 2022)
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| Budget Amount *help |
¥4,420,000 (Direct Cost: ¥3,400,000、Indirect Cost: ¥1,020,000)
Fiscal Year 2022: ¥520,000 (Direct Cost: ¥400,000、Indirect Cost: ¥120,000)
Fiscal Year 2021: ¥260,000 (Direct Cost: ¥200,000、Indirect Cost: ¥60,000)
Fiscal Year 2020: ¥3,640,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥840,000)
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| Keywords | Single Cell Genomics / Immunology / Immune repertoire / Systems Biology / Computational Biology / Systems Immunology / Bioinformatics / Systems immunology / Computational biology |
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| Outline of Research at the Start |
In this project we want to get insight into how TCR binding strength, weakened in the SKG mice due to a mutation in the downstream molecule ZAP70, alters the differentiation of NKT cells in the thymus. We will characterize the different NKT populations in the thymus of WT and SKG mice using single cell genomics approach. We will combine single cell RNA sequencing with TCR immune repertoire profiling and protein quantification using feature barcoding using 10x genomics platform. This combined dataset will provide unprecedented resolution into the mechanisms driving differentiation of NKT cells.
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| Outline of Annual Research Achievements |
We have reanalyzed the single cell transcriptome data and classify NKT cells in the thymus into several populations including CD4+CD8+ double positive progenitor cells, proliferating cells and NKT1, NKT2 and NKT17 cells. Analysis of the inferred lineage suggests NKT subpopulation phenotype already exists in proliferating cells and is determined during the DP->Proliferating step. A group of pro-apoptotic cells have signatures matching the NKT subpopulations and show increased expression of markers of activation suggesting that overactivation marked them for deletion (negative selection). Most of the cells have a canonical receptor with alpha chain made of TRAV11 and TRAJ18 genes. A fraction of the cells have non-canonical receptors. Although this could represent contaminating cells, non-canonical receptors exist also in differentiated NKT cells. Contaminating cells like developing T cells would show as a distinct clusters, based on our experience with other datasets. NKTs harboring a non-canonical TCR could represent type 2 NKTs, which carry a more diverse TCR.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
We have performed additional experiments to analyze chromatin accessibility changes associated with ZAP70 mutation in SKG mice. The purpose is to identify epigenetic changes associated with lineage commitment, in particular in the double positive to proliferating population. We need to finish the analysis of this data and integrate the results with the original experiment. Writing of a manuscript reporting the results has started.
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| Strategy for Future Research Activity |
Analysis of multiome (RNA+ATAC) data and integration with the previously obtained single cell transcriptome and repertoire data. Finishing writing the manuscript.
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