Images Wikimedia Hematopoietic stem cells Hematopoietic progenitor cells Common lymphoid...
Transcript of Images Wikimedia Hematopoietic stem cells Hematopoietic progenitor cells Common lymphoid...
Images Wikimedia
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.