Overexpression of RORγt augments the suppressive capacity of Foxp3+ CD4+ Treg cells. (A) At day 10 post first CII immunization, the draining lymph-node (LN) cells were harvested from C57BL/6 mice (n = 3 or 4) and RORγt Tg mice (n = 3 or 4) and subjected to flow cytometry. (B) Percentage of Foxp3+ cells among CD4+ T cells and mean fluorescence intensity (MFI) of RORγt in Foxp3+ CD4+ cells. (C) Cells were analyzed by staining for intracellular IL-17 and Foxp3. Data represent gated CD4+. (D) Expression of CD25 on gated CD4+Foxp3+ cells. (E) Expression of surface markers glucocorticoid-induced tumor necrosis factor receptor (GITR) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) on Foxp3+CD4+ cells. Flow cytometric data (left) and MFI data of C57BL/6 mice and RORγt Tg mice. (F) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+CD25−GFP− cells from C57BL/6-Foxp3GFP mice cultured with or without CD4+GFP+ cells from C57BL/6-Foxp3GFP mice or RORγt Tg-Foxp3GFP mice and stimulated with anti-CD3/28 beads for 72 h. Left, representative data of two independent experiments with three mice per group; right, percentage of inhibition rate. (G) IL-10 production by CD4+ GFP+ cells of splenocytes stimulated for 72 h with anti-CD3/28 beads from C57BL/6-Foxp3GFP and RORγt Tg-Foxp3GFP mice. Representative data of two independent experiments with similar results, and are mean ± standard error of the mean values. *P <0.05, **P <0.01 versus C57BL/6 mice, Student’s t-test.