FIGURE 1. Co-expression of Foxp3 and RORt in vitro and in vivo.

a, Naive CD4⁺ T cells were stimulated with anti-T-cell receptor (TCR) and 5 ng ml^-1 TGF- for 48 h, and were stained with 4,6-diamidino-2-phenylindole (DAPI; blue nuclear stain; UV channel), anti-ROR (red; Cy3 channel) and anti-Foxp3 (green; Cy5 channel) monoclonal antibodies. All panels are of the same section. The bottom-left image shows Foxp3-only, bottom-right shows ROR-only, top-right shows overlay of Foxp3 and ROR channels, and top-left shows overlay of all three channels. Foxp3, RORt and double-expressing (DP) cells are indicated with coloured arrows. b, Analysis of Foxp3⁺RORt⁺ cells from the small intestinal lamina propria. CD4⁺GFP^int and CD4⁺GFP^- cells were sorted from lamina propria of Rorc(t)^+/gfp and Rorc(t)^gfp/gfp mice, and Foxp3 expression was examined by intracellular staining. Results are representative of three experiments. The numbers indicate the percentage of total cells in each gate. c, Expression of IL-17 in Foxp3⁺RORt⁺ and Foxp3^-RORt⁺ T cells from small intestine. Foxp3 and IL-17 expression was examined by intracellular staining of sorted TCR⁺CD4⁺GFP^int cells from the lamina propria of Rorc(t)^+/gfp mice.