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Hematopoietic stem cells

Hematopoietic progenitor cells

Common lymphoid progenitorCommon myeloid progenitor

In Thymus

In Bone marrow

Acquired

immunity

Innateimmunity

Innateimmunity

Differentiation of hematopoietic cells

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

Mature Helper T17Naïve T-cell

TFs

IL17 gene

TGF-

Images RnDSystems.com Donald P. Buckley

TGF-

TGF-

IL-6

IL-6

STAT3

Foxp3

IL17TGF- SMAD RORt

+NFAT

+Runx +Runx

IL-6

STAT3

Foxp3

IL17TGF- SMAD RORt

+NFAT

+Runx +Runx

The cis elements regulating Il17 transcription

Interleukines

Activated T cell

How to activate a T cell

Simulate T cell antigen receptor signals:1a Ca2+ intracellular levels increased by ionomycin1b 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. 1985Signal requirements for T lymphocyte activation. I. Replacement of macrophage function with phorbol myristic acetate.Rosenstreich et al. J. Immunol. 1979

Measuring a promoter activity using a vector

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

~20kb

The cis elements regulating Il17 transcription (cont.)

Regulation of Il17 transcription by Runx1

Images Molecularstation.com

Site-directed mutagenesis

Runx1 is required for TH-17 differentiation

Flux cytometry

Detection of all cells(Intensity size)

Ex. Detection of GFP+ cells

Ex. Detection of IL-17+ cells(+IL-17 antibody with specificfluorochrome)

FL-3

(IL-

17+) GFP+

TH17+

GFP+ TH17-

GFP-TH17-

GFP-TH17+

FL-1 (GFP+)

1 dot = 1 cell

Figure 5 Runx1 acts together with RORt 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 RORt to activate Il17

Runx1 acts together with RORt to activate Il17 (cont.)

Chromatin Immunoprecipitation assay

DNA

Runx1 acts together with RORt to activate Il17 (cont.)

Foxp3 represses RORt- and Runx1-induced IL-17 expression

Foxp3 represses RORt- and Runx1-induced IL-17 expression

(a) Immunoblot (IB) analysis of Runx1 and RORtt in 293T cells transfected with Flag-Runx1 and Myc-RORt 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 TH-17-polarizing conditions; lysa

tes prepared in the presence of ethidium bromide were immunoprecipitated with IgG isotype-matched control antibody or anti-Runx1, then analyzed by immunoblot with anti-RORt t or anti-Runx1. (c,d) Immunoassay of 293T cells transfected with Myc-tagged RORt 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.