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Images  Wikimedia Hematopoietic stem cells Hematopoietic progenitor cells Common lymphoid progenitorCommon myeloid progenitor In ThymusIn Bone marrow.

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Presentation on theme: "Images  Wikimedia Hematopoietic stem cells Hematopoietic progenitor cells Common lymphoid progenitorCommon myeloid progenitor In ThymusIn Bone marrow."— Presentation transcript:

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2 Images  Wikimedia Hematopoietic stem cells Hematopoietic progenitor cells Common lymphoid progenitorCommon myeloid progenitor In ThymusIn Bone marrow Acquired immunity Innate immunity Innate immunity Differentiation of hematopoietic cells

3 TH1 TH2 TH17 TReg TGF-  Cytokines (TNF-a, IL-6, IL-12, etc.) Naïve T-cell (TH0) Virus, bacteria, fungi, etc… Dendritic cell Images  Nature.com, もやしもん

4 Mature Helper T17 Naïve T-cell TFs IL17 gene TGF-  Images  RnDSystems.com  Donald P. Buckley

5 TGF-  IL-6  STAT3  Foxp3  IL17 TGF-   SMAD  ROR  t  +NFAT +Runx IL-6  STAT3  Foxp3  IL17 TGF-   SMAD  ROR  t  +NFAT +Runx

6 The cis elements regulating Il17 transcription

7 Interleukines Activated T cell How to activate a T cell Simulate T cell antigen receptor signals: 1a  Ca2+ intracellular levels increased by ionomycin 1b  Phorbol esters, ex. PMA simulating PKC The antigen receptor on a human T cell line initiates activation by increasing cytoplasmic free calcium. Imboden et al. J Immunol. 1985 Signal requirements for T lymphocyte activation. I. Replacement of macrophage function with phorbol myristic acetate. Rosenstreich et al. J. Immunol. 1979

8 Measuring a promoter activity using a vector

9 Transfection methods - Electroporation - Virus vector - Lipofection - Gene guns - Microinjection - Nucleofection (based on electroporation)

10 ~20kb The cis elements regulating Il17 transcription (cont.)

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12 Regulation of Il17 transcription by Runx1

13 Images  Molecularstation.com Site-directed mutagenesis

14 Runx1 is required for TH-17 differentiation

15 Flux cytometry Detection of all cells (Intensity  size) Ex. Detection of GFP+ cells Ex. Detection of IL-17+ cells (+IL-17 antibody with specific fluorochrome) FL-3 (IL-17+) GFP+ TH17+ GFP+ TH17- GFP- TH17- GFP- TH17+ FL-1 (GFP+) 1 dot = 1 cell

16 Figure 5 Runx1 acts together with ROR  t to induce IL- 17 production. (a) Flow cytometry of IL-17 production by purified naive CD4+ T cells transduced with retroviruses as described in Figure 4d and then cultured in the presence of various cytokines (above plots), followed by intracellular staining on day 4 after the first transduction (day 5 after activation). Numbers in quadrants indicate percent cells in each. (b) Flow cytometry of IL-17 expression in purified naive CD4+ T cells transduced with various combinations of retrovirus expressing GFP alone (MIG), Thy-1.1 alone (MIT), Thy-1.1-ROR  t, GFPRunx1 or GFP-Runx1DN, then cultured in TH0- or TH-17-polarizing conditions and analyzed as described in Figure 4a. Plots are gated on GFP+Thy-1.1+ cells. Numbers in outlined areas indicate percent IL-17+ cells. Data are representative of at least three independent experiments. Runx1 acts together with ROR  t to activate Il17

17 Runx1 acts together with ROR  t to activate Il17 (cont.)

18 Chromatin Immunoprecipitation assay DNA

19 Runx1 acts together with ROR  t to activate Il17 (cont.)

20 Foxp3 represses ROR  t- and Runx1- induced IL-17 expression

21 (a) Immunoblot (IB) analysis of Runx1 and ROR  t t in 293T cells transfected with Flag-Runx1 and Myc-ROR  t t constructs with or without ethidium bromide (EtBr), detected in lysates immunoprecipitated (IP) with anti-Flag or anti-Myc. (b) Immunoassay of purified CD4 + T cells cultured for 4 d in T H -17-polarizing conditions; lysates prepared in the presence of ethidium bromide were immunoprecipitated with IgG isotype-matched control antibody or anti-Runx1, then analyzed by immunoblot with anti-ROR  t t or anti-Runx1. (c,d) Immunoassay of 293T cells transfected with Myc-tagged ROR  t t and Flag-tagged wild-type Foxp3 (c,d) or mutant Foxp3 (d); lysates immunoprecipitated with anti-Flag or anti-Myc were analyzed by immunoblot with anti-Flag or anti-Myc. Results are representative of three independent experiments.


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