Reduced IFN-γ production by NFAT1^−/−IL-4^−/− T-helper cells.

(A) NFAT1^−/− IL-4^−/− T cells show diminished levels of IFN-γ mRNA. CD4⁺ T cells from NFAT1^+/+ IL-4^−/− and NFAT1^−/−IL-4^−/−mice were cultured for 4 days with anti-CD3 and IL-2, rested for 48 hours, and restimulated with anti-CD3. Differentiation and restimulation were performed under neutral conditions, without the addition of antibodies and cytokines other than IL-2. Six hours after restimulation, total cellular RNA was isolated and analyzed by RNase protection assay for transcript levels of the indicated cytokines. (B) NFAT1^−/− IL-4^−/− T cells show diminished production of IFN-γ protein. NFAT1^+/+ IL-4^−/− and NFAT1^−/−IL-4^−/− CD4⁺ T cells were cultured as in panel A. Supernatants were collected 24 hours after restimulation, and levels of IFN-γ and IL-2 were determined by ELISA. Values are expressed as the means ± SEM of 9 (IFN-γ) or 3 (IL-2) independent experiments.