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, Methods Mice All animals were maintained in specific pathogen free (SPF) facility at Dijon Zootechnical Centre. Animal use and care were approved by the Burgundy University Animal Experiments Ethics Committee (approved protocol #2212), in accordance with the Federation of Laboratory Animal Science Associations (FELASA), Female C57BL/6J were purchased from Charles River Laboratories

. Flavell, OT-II mice and Irf8 f/f Cd4 cre mice were provided by the CDTA (Cryopréservation, Distribution, Typage et Archivage animal) and distributed by EMMA (European Mouse Mutant Archive, a service funded by the EC FP7 Capacities Specific Programme)

, Naive CD4 + T cells (CD4 + CD62L hi ) were obtained from spleens and lymph nodes of C57BL

, Miltenyi Biotec), then were further sorted as naive CD4 + CD62L hi T cells. The purity of the isolated naive T cell population routinely exceeded 95%. Naive T cells were cultured for three days with anti-CD3 (2µg/mL) and anti-CD28 (2µg/mL) antibodies in the presence of anti-IFN? (10µg/mL) and anti, WT) or Irf8 f/f Cd4 cre mice. CD4 + T cells were purified from spleen and lymph nodes with anti-CD4 microbeads, pp.130-093

, Th0, anti-IL-4 (10µg/mL) and IL-12 (10ng/mL) to obtain Th1, anti-IFN? (10µg/mL) and IL-4 (10ng/mL) to obtain Th2, anti-IFN? (10µg/mL), IL-4 (20ng/mL) and TGF-? (2ng/ml) to obtain

, Anti-CD3 (Armenian Hamster IgG, clone 145-2C11, #BE0001-1), anti-CD28 (Armenian Hamster IgG, clone PV-1, #BE0015-5), anti-IL-4 (Rat IgG1, clone 11B11, #BE0045) and anti-IFN-? (Rat IgG1, clone XMG1.2, #BE0055) antibodies were obtained from BioXcell, Th9, anti-IFN? (10µg/mL), anti-IL-4 (10µg/mL), IL-6 (20ng/mL) and TGF-? (2ng/ml) to obtain Th17 and anti-IFN? (10µg/mL)

, JNK inhibitor (SP600125, 10µM, 1h), and the ROCK inhibitor (Y27632, 10µM, 1h) were obtained from Merck Chemical France, SMAD3 inhibitor (SIS3, 10µM, 1h), TGF-? receptor kinase inhibitor (SB431542, 5µM, 1h), p38 inhibitor (SB203580, 10µM, 1h)

, USA). 300ng of RNA were reverse-transcribed into cDNA using M-MLV reverse transcriptase (28025-013, Invitrogen), Random Primers and RNAseOUT inhibitor, Total RNA from T cells was extracted using Trizol, pp.10777-10796

, ELISA After polarization for 72 hours, cell culture supernatants were assayed by ELISA for mouse IL-9 (442704, BioLegend), BD Biosciences), vol.4, issue.555232, p.5

, IFN? (555138, BD Biosciences) according to the manufacturer's protocol, eBiosciences), IL-17A (432501, Biolegend)

, Western Blotting and immunoprecipitation assays Cells were lysed in boiling buffer [1% SDS, 1 mM sodium orthovanadate, and 10 mM

. Tris, containing protease inhibitor cocktail for 20 minutes at 4°C. Cell lysates were subjected to sonication (10 seconds at 10%) and protein concentration was assessed using the Bio-Rad DC Protein Assay Kit, After blocking with 5% non-fat milk in Phosphate-Buffered Saline containing 0.1%, vol.5000112

, Supplementary Table 2) diluted (1 ?g/ml) in PBST containing 5% BSA, washed and incubated for 1 hour with secondary antibody diluted in PBST-5% non-fat milk. After additional washes, membranes were incubated with luminol reagent, PBST), membranes were incubated overnight with primary antibody, vol.20

, Immunoprecipitation assays were done with at least 50x10 6 Th9 cells. Briefly, cells were lysed in 1 ml lysis buffer (20 mM Tris (pH 7.5), 15 mM KCl, 1% CHAPS, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA and CPIM

. Sepharose-6b-(6b100 and ). Sigma-aldrich, After centrifugation at 1000

, ChiP-IT of pSmad3, IRF8, PU.1, BATF or IRF4 antibodies, or 3 ?g of negative control immunoglobulin at 4°C overnight. After chromatin elution, cross-links were reversed and samples were analysed by quantitative PCR. The sequences of the oligonucleotides used are described in Supplementary Table 1. siRNA transfection siRNA knockdown experiments were performed with validated control, or Irf8 (s68003), or Smad3 (s69494) or Etv6 (s65718)-specific siRNAs (Life technologies), vol.53009

, Fragments were amplified by high-fidelity PCR with C57BL/6 mice DNA as the template and specific primers, The Il9-luc and Irf8-luc luciferase constructs were obtained by inserting 2390 pb of Il9 and 1634 pb of Irf8 mouse promoters in the multicloning site of the pGl3 basic vector

, Mouse NIH3T3 cells (cultured in DMEM 4.5 g/l + glutamine + FBS at 37°C, 5% CO2) were transiently transfected for 48 hours with reporter plasmid and the pCMV-SPORT6-IRF8

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, Liste des publications issues de travaux collaboratifs, vol.4

, Sirtuin-1 Activation Controls Tumor Growth by Impeding Th17 Differentiation via STAT3

E. Limagne, M. Thibaudin, R. Euvrard, H. Berger, P. Chalons et al., Cell Rep, vol.4, pp.746-759, 2017.

M. Bruchard, C. Rebé, V. Derangère, D. Togbé, B. Ryffel et al., The receptor NLRP3 is a transcriptional regulator of TH2 differentiation, Nat. Immunol, vol.16, pp.859-870, 2015.