RORγt enhances chemotaxis of Foxp3+ Treg cells into inflamed tissues. (A) CCR6 expression in CD4+ T cells of C57BL/6 mice and RORγt Tg mice. (B) CCR6 expression in Foxp3+ CD4+ Treg cells and Foxp3− CD4+ non-Treg cells from of C57BL/6 mice (open bars) and RORγt Tg mice (closed bars). (C) CD4+ cells were isolated by magnetic-activated cell sorting (MACS) from draining lymph-node (LN) cells of C57BL/6 mice (open bars) and RORγt Tg mice (closed bars), and added to the upper well of a Transwell system. The migration assay was performed in the presence of the indicated concentrations of CCL20 added to the bottom well. Data are numbers of respective cells that migrated to the bottom well counted by FACS. (D) CD4+ cells of RORγt Tg mice were subjected to intracellular Foxp3 staining, and then subjected to the migration analysis as in C. (C
D) Data are mean ± standard error of the mean. **P < 0.01, versus C57BL/6 mice, Student’s t-test. (E) Collagen-induced arthritis was induced in C57BL/6-Foxp3GFP and RORγt Tg-Foxp3GFP mice, and CD3+CD4+ T cells that infiltrated the ankle joints were harvested at day 30 post first type II collagen (CII) immunization and analyzed for green fluorescent protein (GFP) and CCR6 expression. Representative data of two independent experiments with similar results. MFI, mean fluorescence intensity.