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.)

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.