Drosha- and Dicer-dependent pathways are required for efficient induction of Foxp3 expression and function of T reg cells. (A) Reduced TCRβ⁺Foxp3⁺ T reg cell populations in the thymus and spleen of Drosha^F/Δ CD4-cre and Dicer^F/F CD4-cre mice. (B) Foxp3 expression in peripheral T reg cell populations in the absence of Drosha or Dicer. (C and D) Drosha and Dicer are required for efficient differentiation of induced T reg cells. Naive CD62L^hi44^lo25⁻ CD4⁺ T cells from Drosha^F/Δ CD4-cre, Dicer^F/F CD4-cre, and control Drosha^F/+ CD4-cre mice were activated in vitro with anti-CD3/28 antibodies plus 10 U/ml IL-2, and TGF-β (C) or TGF-β plus 10 nM RA (D) for 3 d. The cells were restimulated with PMA/ionomycin plus GolgiStop for 3 h, and Foxp3 and IL-17A expression was analyzed by intracellular FACS. Percentages of cells are shown in A–D. (E) FACS-purified naive CD4⁺ cells from Cd45.1 mice were loaded with CFSE and mixed with purified CD4⁺25⁺ cells from Drosha^F/Δ CD4-cre or Drosha^F/+ CD4-cre (Cd45.2) mice at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 antibody. After 4 d, the CD45.1⁺ cells were analyzed for CFSE dilution.