From R D Systems) and 25 ng/ml human IL-21 (Cell Sciences). On day 3, cells have been expanded with extra PPAR medchemexpress medium and half-concentration of cytokines. Cells have been harvested for analysis on day 5. Transfection of siRNA–siRNAs targeting Twist1 or TWIST1 have been bought from Santa Cruz Biotechnology. For mouse Th17 cell transfection, CD4 T cells have been transfected with siRNA on day two applying Amaxa Nucleofector kit (Lonza), rested overnight with hIL-2, and restimulated with anti-CD3 for 24 hVOLUME 288 Quantity 38 SEPTEMBER 20,EXPERIMENTAL PROCEDURES Mice–C57BL/6 mice were bought from Harlan SpragueDawley (Indianapolis, IN). Twist1fl/flCD4-Cre and Stat3fl/flCD4Cre mice were described previously (17, 33). Twist1fl/flCD4-Cre mice were backcrossed to C57BL/6 mice for six generations with Cre-negative littermates as wild kind mice for in vivo experiments. Mice had been maintained below precise pathogen-free conditions. All experiments were performed using the approval on the Indiana RSV Synonyms University Institutional Animal Care and Use Committee. In Vitro T Cell Differentiation–Na e CD4 CD62L T cells had been isolated from spleen and lymph nodes using MACS beads and columns (Miltenyi Biotec). CD4 T cells had been activated with plate-bound anti-CD3 (two g/ml 145C11) and soluble anti-CD28 (0.5 g/ml BD Pharmingen) with further cytokines (all from PeproTech) and antibodies (Bio X cell) to produce Th1 (5 ng/ml IL-12; and ten g/ml anti-IL-4, 11B11), Th2 (ten ng/ml IL-4; and 10 g/ml anti-IFN- XMG), Th9 (20 ng/ml IL-4; two ng/ml TGF- ; and 10 g/ml anti-IFN- , XMG), Th17 (one hundred ng/ml IL-6; ten ng/ml IL-23; ten ng/ml IL-1 ; 2 ng/ml TGF;ten g/ml anti-IL-4, 11B11; and ten g/ml anti-IFN- , XMG) or regulatory T (Treg; two ng/ml TGF- , and ten g/ml anti-IL-4, 11B11) culture conditions. Cells have been expanded right after three days with half-concentration on the original cytokines in fresh medium. Cells were harvested on day five for evaluation. To inhibit STAT3 activation, doses of cucurbitacin I (JSI-124, Sigma Aldrich) were added into WT and Twist1-deficient Th17 cell cultures. Phosphorylated STAT3 and cytokine production have been measured applying intracellular staining and ELISA, respectively. For receptor-blocking experiments, Th17 cells were cultured as above within the presence of handle antibody or blocking27424 JOURNAL OF BIOLOGICAL CHEMISTRYTwist1 Represses IL-6-STAT3 Signalingfor gene expression and cytokine production analyses. For human Th17 cell transfection, day 5-differentiated Th17 cells were transfected with siRNA working with a human T cell nucleofector kit (Lonza), rested overnight with hIL-2, and restimulated with anti-CD3 for 24 h for gene expression analyses. Luciferase Reporter Assay–The IL6RA promoter reporter was bought from SwitchGear Genomics. For analyzing the effect of Twist1 on IL6RA promoter activity, Jurkat T cells had been grown in RPMI 1640 with 10 FBS and transfected with 2 g on the IL6RA luciferase reporter plasmid and handle or escalating concentration of plasmid expressing Twist1 by means of FuGENE reagent (Roche Diagnostics). Following 24 h, transfected cells have been stimulated with PMA and ionomycin for 6 h ahead of analyzing using the Dual-Luciferase system (Promega). Evaluation of Gene Expression, ELISA, and Flow Cytometry– Quantitative RT-PCR and ELISA were performed as described previously (36). For surface staining, resting T cells had been stained for IL-2R -FITC and IL-6R -phycoerythrin (BD Pharmingen) and fixed with 2 paraformaldehyde for 10 min just before evaluation. For cytokine staining, CD4 T cells w.
