FoxP3⁺/IL-17⁺ clones remain suppressive after they have been induced to secrete IL-17. The clones derived from the DR⁻ Treg population were stimulated for 5 days with αCD3/αCD28 and IL-2 in the presence of IL-1β/IL-6, and analyzed for IL-17 secretion by ELISA (A), RORC mRNA expression by real-time PCR (B), and FoxP3 expression by intracellular staining (C). (D) Intracellular IL-17/IFNγ staining of IL-1β/IL-6 treated clones. (E) FoxP3⁺/IL-17⁺ clones were stimulated in the presence of IL-1β/IL-6 or TGFβ, and analyzed for IL-17 secretion by ELISA. (F) IL-1β/IL-6 treated clones were expanded for 2 weeks and tested for suppressive function in cocultures stimulated with αCD3/αCD2 beads, as compared with freshly isolated DR⁻ and DR⁺ Tregs. One representative clone from each pattern is shown.