CD25 deficiency on TRCs promotes in vitro Th17 differentiation. Purified, naive CD4⁺ T cells (2 × 10⁵) were cocultured with or without WT or CD25-deficient TRCs (2 × 10⁴) with irradiated APCs (1 × 10⁵) in the presence or absence of soluble anti-CD3 and CD28 antibodies (3 and 1 μg/ml) for 72 h. (A) The expression of TFs (T-bet for Th1 cells, GATA-3 for Th2 cells, and RORγt for Th17 cells) in CD4⁺ T cells cocultured with WT or CD25-deficient TRCs was detected by Western blot analysis. β-Actin is shown as a loading control. (B) Cytokine production (IFN-γ for Th1 cells, IL-4 for Th2 cells, and IL-17A for Th17 cells) in culture supernatants was quantified by ELISA. ND, not detectable; *, P < 0.05 compared with antibody-stimulated CD4⁺ T cells cultured with WT TRCs or CD25-deficient TRCs. Error bars indicate the mean ± SEM (n = 5 per each group). (C) Representative flow cytometric plots of intracellular IFN-γ⁺, IL-4⁺, and IL-17A⁺ cells in the CD4⁺ T cell population. (D) Representative flow cytometric plots of intracellular IFN-γ⁺, IL-4⁺, and IL-17A⁺ cells in the CD4⁺ T cell population cultured in a trans-well system. Data are representative of three independent experiments.