Generation and immunophenotype of Tr17 cells.

<p>(<i>A</i>) Tregs isolated from Foxp3.GFP knock in mice were stimulated with anti-CD3/CD28 mAbs alone or in the presence of IL-1β, IL-2 or their combination. Production of IL-17 was measured by ELISA (eBioscience). Data was representative of five independent experiments. (<i>B</i>) Tregs were cultured with anti-CD3/CD28 alone or in the presence of IL-1β and IL-2 (polarizing conditions), and were subsequently stimulated with PMA and ionomycin for additional 4 h, followed by intracellular staining with anti-IL-17 antibody. Numbers indicate percent IL-17⁺ cells (upper box) and IL-17⁻ cells (lower box) within total live cells. (<i>C</i>) Expression of RORγt and Foxp3 in freshly isolated Tregs (Unstimulated) and in Tregs cultured under polarizing conditions (Stimulated) was assessed by intracellular staining. (<i>D</i>) Expression of the indicated transcription factors and surface markers was analyzed on gated IL-17⁺ and IL-17⁻ Tregs. The data (<i>B–D</i>) were representative of three independent experiments.