Mil 2

Naive CD4 1 T cells (CD4 1 CD62L hi CD44 2 ) were sorted from a single-cell suspension drawn from spleens with the Na€ ıve CD4 1 T cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany), and purity levels were greater than 95%, as determined by using the BD FACSCelesta. Naive T cells were used for in vitro cultures in RPMI 1640 medium (Corning, Manassas, Va) supplemented with b-mercaptoethanol, 10% FBS, and 1% penicillin-streptomycin. For T-cell activation, proliferation, and viability detection, naive T cells were stimulated with or without anti-CD3 (precoated, 5 mg/mL) and anti-CD28 (1 mg/mL; BD Biosciences, San Jose, Calif) for 3 days. For T H 2 cell differentiation, naive CD4 1 T cells were differentiated under T H 2-skewed conditions: mIL-2 (10 ng/ mL), mIL-4 (50 ng/mL), and anti-IFN-g (10 mg/mL; Miltenyi Biotec) with anti-CD3 (precoated, 5 mg/ml) plus anti-CD28 (1 mg/mL; BD Bioscience) for 7 days. In pharmacologic inhibition experiments T cells were pretreated with mock stimulant or 2-deoxy-D-glucose (2-DG; 1 mmol/L; Santa Cruz Biotechnology, Santa Cruz, Calif), rapamycin (100 nmol/L; Sigma-Aldrich, St Louis, Mo), and 5-aza-29-deoxycytidine (1 mmol/L; MedChemExpress, Monmouth Junction, NJ) and then stimulated with anti-CD3 (precoated, 5 mg/mL) and anti-CD28 (1 mg/mL; BD Biosciences) or T H 2-skewed conditions.