Naïve T Cell Differentiation Assay

Sorted naive T cells (CD62L^hiCD44^lo) were used for T cell activation and differentiation. Naïve T cells were activated with plate-bound anti-CD3 (10 μg/ml) plus soluble anti-CD28 (2 μg/ml) (BD Bioscience). For T cell differentiation, CD4⁺ naïve T cells were differentiated into Th0, Th1, Th2, Th17, or iTreg cells as previously reported.^{23 (link)–25 (link), 30 (link)} Briefly, CD4⁺ T cells were stimulated by anti-CD3/CD28 for 4 d with IL-2 (20 ng/ml) in the presence of anti-IFN-γ and anti-IL-4 (both 10 μg/ml, for Th0), anti-IL-4 and IL-12 (20 ng/ml, for Th1), anti-IFN-γ and IL-4 (20 ng/ml, for Th2). For iTreg and Th17 conditions, CD4⁺ T cells were stimulated by anti-CD3/CD28 for 4 d with anti-IFN-γ and anti-IL-4 (both 10 μg/ml) in the presence of IL-2 (20 ng/ml) plus TGF-β1 (5 ng/ml, for Treg), or TGF-β1 plus IL-6 (10 ng/ml, for Th17) (all from R&D Systems, Minneapolis, MN). Cells were restimulated with PMA (25 ng/ml) plus ionomycin (500 ng/ml) (Sigma, St Louis, MO) for 5 h with GolgiStop (BD Bioscience) in the last 2 h for intracellular cytokine staining; or without GolgiStop for cytokine assays in the culture supernatants by ELISA. Where indicated, CASIN (Chembridge Corporation, San Diego, CA) was added to the cultures.