2018 Fiscal Year Research-status Report
Elucidation of the cause of allergy by identifying somatic mutations in human IgE+ memory B cells.
| Project/Area Number |
18K16162
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| Research Institution | National Center for Global Health and Medicine |
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Principal Investigator |
NguyenTien Dat 国立研究開発法人国立国際医療研究センター, その他部局等, 上級研究員 (50750270)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | Allergy / B cells / IgE |
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| Outline of Annual Research Achievements |
It has been largely unknown how IgE production is regulated and how IgE B-cell memory is generated in allergic patients. However, there are technical limitations in identifying rare IgE+ B cells, especially in humans. Adopting the Fas-mediated Ag-specific iGB cell selection (FAIS) system, I have developed a system to selectively expand IgE+ B cells by a four-step B cell culture procedure. 1) Culture with IL-21 on feeder cells expressing exogenous CD40L and BAFF (40LB). 2) Stimulation culture on 40LB cells expressing FcεR1 to stimulate IgE+ iGB cells. 3) Selection culture on 40LB cells expressing the Fas ligand: all but IgE+ iGB cells undergo apoptosis. 4) Recovery culture on 40LB cells. Starting with blood B cells of hay fever allergic donors, I successfully obtained almost pure population of IgE+ B cells with this procedure in combination with cell sorting. I will search for such mutations in genomic genes of mIgE+ Bm cells by comparing with non-B leukocytes from the same patients of various allergic diseases, using whole exome sequencing
I have also identified 2 species of mRNA of membrane-bound εH chain: one encoding the conventional εH chain with an extracellular membrane-proximal domain (EMPD) and transmembrane domains, and the other lacking these domains but containing C-terminal peptide with a shifted reading frame, termed IgE-NET. The IgE-NET facilitated spontaneous plasma-cell differentiation and apoptosis of IgE+ B cells more than conventional IgE, and thus may contribute to the regulation of IgE+ B cells and of IgE synthesis in allergic disease.
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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
The project progressing is smooth as the plan.
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| Strategy for Future Research Activity |
The research is focusing on two subjects:
1 Finding and analyzing gene abnormality in mIgE+ Bm cells from allergic patients.
I will continue searching for causal somatic gene abnormalities in allergic patients that enable mIgE+ B cells to survive and to become memory B (Bm) cells or long-lived plasma cells (LLPCs), and verify the functions of such abnormal genes in the autonomous mIgE signaling that forces IgE+ germinal center (GC) B cells to be short-lived plasma cells, and in pathogenesis of allergy.
2 Analyzing the function of IgE-NET
I am elucidating the function of a novel IgE εH chain isoform, namely IgE-NET, an alternative splicing variant that I have identified. I will examine which form of εH chain mRNA is expressed in IgE+ B cells induced with IL-4 from IgM+ B cells of healthy donor. I will also examine the IgE-NET function by check ER stress pathway gene expression in mIgE+ B cells from patients and IL-4 induced IgE+ from healthy donors. If IgE-NET has a function, the next question is how RNA splicing is regulated to synthesize the IgE-NET mRNA.
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