Lymphokine production in CD4⁺ cells cultured under Th-1 and Th-2 polarizing conditions. (A) Naive CD4⁺CD62L^high T cells, isolated from spleens of TG and WT littermate mice, were differentiated under Th-1 and Th-2 skewing conditions (described in “Materials and methods”). After 7 days, the cells were washed and restimulated by anti-CD3 Ab for 6 hours. (B) Naive T cells were cultured for 7 days under nonskewing conditions with anti-CD3/anti-CD28 Ab in the presence of anti–IL-4 antibody for Th-1 or anti–IFN-γ and anti–IL-12 Abs for Th-2. Cells were washed and restimulated with anti-CD3 antibody for 6 hours. The amounts of cytokines secreted into the supernatants were measured by ELISA. Averages ± 1 SEM of 6 independent experiments are shown. ^*P < .05, TG vs WT. (C) Naive CD4⁺CD62L^high T cells from WT and TG littermates were differentiated for 7 days under Th-1 and Th-2 skewing conditions and restimulated with PMA (50 ng/mL) and inomycin (1 μM) for 5 hours. Intracellular cytokine staining was performed using anti-CD4–FITC antibody together with either anti–IFN-γ–PE or anti–IL-4–PE Abs. Control isotypes are omitted for clarity. Data are representative of 4 independent experiments. Numbers represent the percent of cells positive (upper right corner) or negative (lower right corner) for IL-4 or IFN-γ.