FIGURE 3.

CD4 T cells deficient in TTP produce more IL-17 than wild-type (WT) T cells at the single-cell level. (A) Naive CD4⁺ T cells from CD4^CreTTP^f/f and WT mice aged 6–8 months were cultured under T_H1 or T_H17 polarizing conditions in 96-well plate coated with anti-CD3 (2 μg/mL) and anti-CD28 (2 μg/mL) Abs for 3 days before stimulation with PMA and ionomycin for 4 h. Intracellular IL-17A and IFN-γ in CD4⁺ T cells were analyzed by fluorescence-activated cell sorting (FACS). (B) Supernatants of the above cells were collected to measure IL-17A protein levels by ELISA (means ± SD from three independent experiments). (C,D) Total CD4⁺T cells from WT and TTP^{– /–} mice were stimulated with anti-CD3/CD28 Abs under T_H17 polarizing conditions for 3 days, rested for 3 days, and then treated with P/I for 4 h, followed by FACS analysis (C). IL-17A levels in the supernatants of the above cells were detected by ELISA and summarized from four independent experiments (D). (E) Mean fluorescence intensity (MFI) of intracellular IL-17A in CD4⁺ T cells from WT and TTP^{– /–} mice under T_H17 cell differentiation. Each dot represents one experiment. (F) IL-17A, IL-6, IFN-γ, Rorc, Stat3, and Tbx21 mRNA expression in CD4 T cells under T_H1 and T_H17 cell differentiation for 4 days. Data shown are means ± SD from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 between groups.