Figure 5. Impaired in vitro response of IRF4^−/− CD4⁺ T cells.

CD4⁺ T cells from WT (CD90.1⁺) and IRF4^−/− mice (CD90.2⁺) were mixed with a 1:1 ratio. Cells were stimulated in vitro with anti-CD3 mAb, anti-CD28 mAb and IL-2 in the presence of IL-12 and anti-IL-4 mAb to induce T_H1 differentiation. (a) After 4d, cells were stimulated for 4 h with PMA/ionomycin and IFN-γ and CD40L expression was determined by intracellular staining. Spleen cells from naive WT and IRF4^−/− mice were stimulated and stained in parallel (d0). (b) T-bet expression in WT and IRF4^−/− CD4⁺ T cells without stimulation and at d4 of stimulation (numbers give the MFIs). (c) Ratio of WT and IRF4^−/− CD4⁺ T cells in co-culture at indicated days of stimulation. (d) Loss of CFSE staining intensity on viable WT and IRF4^−/− CD4⁺ T cells. (e) Expression of Cdkn2a at d0 and d2 of purified WT and IRF4^−/− CD4⁺ T cells stimulated in individual cultures. Blots and histograms in (a,b) show representative results for CD4-gated cells. Results are representative for 3 (a–c) or 2 (d) independent experiments. Bars in (e) represent the mean ± SEM of 4 individual samples from 2 independent experiments.