Anti mil 4

Flow cytometry monoclonal antibodies against mouse antigens (CD4, clone RM4-5; CD8a, clone 53–6.7; CD62L, clone MEL-14; IFN-γ, clone XMG1.2; IL-2, clone JES6-16E3; Ifnar1, clone MAR1-5A3; PD-1, clone RMP1-30; T-bet, clone 4B10) were obtained from Biolegend. Tim-3 monoclonal antibody (clone 5D12) was a gift from V. Kuchroo. Carboxyfluorescein succinimidyl ester (CFSE) was obtained from Biolegend. The following recombinant cytokines and antibodies were used for in vitro T cell cultures: mIFN-β (PBL InterferonSource, indicated concentrations), mIL-12 (R&D Biosystems, 10 ng mL^-1), mIL-2 (Miltenyi, 5 ng mL^-1), hTGF-β (Miltenyi, 3 ng mL^-1), mIL-6 (Miltenyi, 20 ng mL^-1), mIL-23 (R&D Biosystems, 20 ng mL^-1), anti-mIL-4 (BioXCell; clone 11B11, 10 μg mL^-1), anti-CD3 (eBioscience; Functional Grade Purified, clone 145-2C11, 2 μg mL^-1), anti-CD28 (Biolegend; LEAF-purified, clone 37.51, 2 μg mL^-1). Western blotting primary antibodies were obtained from BD Transduction Laboratories (Stat1, clone 42/Stat1, dilution 1:1000; Stat1 pY701, clone 14/P-STAT1, dilution 1:1000; Stat4 pY693, clone 42/Stat1, dilution 1:500), Cell Signaling (Stat4, clone C46B10, dilution 1:500) or Millipore (GAPDH, mAb374, dilution 1:1000). Anti-mouse or-rabbit secondary antibodies were obtained from Jackson Immunoresearch and were used at 1:10000.

Purified CD4⁺ T cells were prepared using immunomagnetic negative selection (Miltenyi Biotec). Briefly, lymphocytes were allowed to react with CD4⁺ T cell biotin-antibody cocktail (biotin-conjugated monoclonal antibodies against CD8a, CD11b, CD11c, CD19, CD45R (B220), CD49b (DX5), CD105, anti-MHC class II, Ter-119, and TCRγ/δ) and then incubated with antibiotin microbeads, followed by magnetic separation. The purity of the resulting CD4⁺ T cell populations was examined using flow cytometry, and was consistently above 95%.
T-helper cell differentiation was conducted as previously described [26 (link)]. Briefly, naive CD4 T cells (0.4 × 10⁶) in 24-well plates (Costar) precoated with 5 μg/ml of anti-CD3 (154-2C11, BioXcell) and 2 μg/ml of anti-CD28 (37.51, BioXcell) were cultured in RPMI containing 10% FCS and polarizing cytokines at the various concentrations of ECF. The cytokines used were Th1, mIL-12 (10 ng/ml, Biolegend), and anti-mIL-4 (11B11, 2 μg/ml, BioXcell). Cells stimulated in “neutral” conditions (anti-mIL-4 and anti-mIFN-γ without cytokines) were considered Th0 cells. The differentiated cells were harvested after 3 days and restimulated for 4 h with 50 ng/ml of PMA (Sigma-Aldrich) and 750 ng/ml of ionomycin (Sigma-Aldrich) in the presence or absence of Brefeldin A for flow cytometry.