CD4+ T cell differentiation in vitro. Purified CD4+ T cells were cultured at 106/ml in the presence of IL-2 (50 U/ml), plate-bound anti-CD28 mAb (2 μg/ml), and anti-CD3 mAb (2 μg/ml) without (a, Th0, left column) or with (b, Th1, right column) IL-12 (10 ng/ml) and anti-IL4 mAb (20 ng/ml). Five days later, cells were washed and restimulated with plate-bound anti-CD3 mAb and anti-CD28 mAb for up to 72 hours. Culture supernatants were collected at 24, 48, and 72 hours, and cytokine concentrations were determined by ELISA. Results are representative of two experiments. Only those bars for SEM that are wider than the symbol are shown.