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Images  Wikimedia

Differentiation of hematopoietic cells. Hematopoietic stem cells. Hematopoietic progenitor cells. Common myeloid progenitor. Common lymphoid progenitor. Acquired immunity. Innate immunity. In Bone marrow. In Thymus. Innate immunity. Images  Wikimedia. Virus, bacteria, fungi, etc….

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Images  Wikimedia

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  1. Differentiation of hematopoietic cells Hematopoietic stem cells Hematopoietic progenitor cells Common myeloid progenitor Common lymphoid progenitor Acquired immunity Innate immunity In Bone marrow In Thymus Innate immunity Images  Wikimedia

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

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

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

  5. The cis elements regulating Il17 transcription

  6. 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 Activated T cell Interleukines 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

  7. Measuring a promoter activity using a vector

  8. Transfection methods • Electroporation • Virus vector • Lipofection • Gene guns • Microinjection • Nucleofection (based on electroporation)

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

  10. Regulation of Il17 transcription by Runx1

  11. Site-directed mutagenesis Images  Molecularstation.com

  12. Runx1 is required for TH-17 differentiation

  13. 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) 1 dot = 1 cell GFP- TH17+ GFP+ TH17+ FL-3 (IL-17+) GFP- TH17- GFP+ TH17- FL-1 (GFP+)

  14. Runx1 acts together with RORt to activate Il17 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.

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

  16. Chromatin Immunoprecipitation assay DNA

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

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

  19. Foxp3 represses RORt- and Runx1-induced IL-17 expression (a) Immunoblot (IB) analysis of Runx1 and RORtt in 293T cells transfected with Flag-Runx1 and Myc-RORtt 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 TH-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tt or anti-Runx1. (c,d) Immunoassay of 293T cells transfected with Myc-tagged RORtt 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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