Lymphocytes prepared from lymph nodes and/or spleens of mice were stimulated with soluble anti-CD3 mAb (0.5 mg/ml) in the presence of various cytokines. Unless indicated, cells were cultured at 1610 6 cells/ml in 24-well tissue culture plates (2 ml/well) and IL-12 was used at 1 ng/ml. To analyze cell proliferation, cells were labeled with CFSE (2 mM, Invitrogen) before culture. To detect intracellular IFN-c or IL-2 production by T cell subpopulations, PMA (50 ng/ml, Sigma), ionomycin (1 mg/ml, Sigma) and Golgi plug (1 ml/ml, BD Biosciences) were added for the last 4 hr of culture.