Simultaneous activation of Tconvs and Tregs in the presence of APCs promotes IL-17 production. A–C, Tconvs from C57BL/6 mice were cultured with or without Tregs from Foxp3-GFP knock-in mice with anti-CD3 mAb and APC, supernatants were harvested on day 3, and cytokines were measured by ELISA. Representative results of five experiments are shown. D, 2D2 Tconvs were cultured with OT-II Tregs and APCs in the presence of MOG_35–55, OVA_323–339, or both peptides. Representative results of two experiments are shown.

Conversion of Tregs into IL-17–producing cells requires TGF-β and IL-1β. A, Tconvs and Tregs were cultured with anti-CD3 mAb and APCs. TGF-β neutralizing Ab or isotype control was added, and production of IL-17A was measured on day 3. *p = 0.003. Representative results of four experiments are shown. B, Tconvs and Tregs were cultured with anti-CD3 mAb and APCs, either individually or mixed at a 1:1 ratio, and IL-6 was measured. C, Tconvs and Tregs were mixed at a 1:1 ratio, cultured with anti-CD3 mAb and APCs in the presence of media, anti-IL-6 neutralizing Ab, anti-IL-6 receptor blocking Ab, or their combination, and IL-17A was assessed. Representative results of three experiments are shown. D, Tconvs and Tregs were cultured with anti-CD3 mAb and APCs in the absence or presence of IL-1β neutralizing Ab. Results are representative of four experiments. E, Tc^WT or Tc^KO were cultured with Tregs from Tr^WT or from Tr^KO with anti-CD3 mAb and APCs, and production of IL-17A was assessed on day 3. Representative results of two experiments are shown. Tc^KO, Tconvs from IL-1R1^−/− mice; Tc^WT, Tconvs from wild type mice; Tr^WT, Tregs from wild type mice; Tr^KO, Tregs from IL-1R1^−/− mice.

Tregs display increased IL-1β signaling and IL-17 production. A, Naive Tconvs (CD4⁺CD62L⁺), effector memory Tconvs (CD4⁺CD62L⁻), and Tregs (CD4⁺ CD25⁺Foxp3⁻GFP⁺) were cultured with anti-CD3 mAb and APCs with or without IL-1β, and IL-17A was measured by ELISA. B, Expression of IL-1R1 the same cell populations as in A was assessed by flow cytometry. C, Naive Tconvs and Tregs were cultured with IL-1β and phosphorylated IκB; p38 and JNK were examined in cell lysates by SDS-PAGE and immunoblot analysis. D, Tregs were cultured for 3 d with anti-CD3 and CD28 mAb in the presence of IL-1β (10 ng/ml) alone or with the indicated concentrations of inhibitors, and IL-17A was measured by ELISA.

Generation of RORgt⁺IL-17⁺ Tregs in the presence of Tconvs and APCs. A, Thy1.1⁻ Tconvs and Thy1.1⁺ Tregs were cultured with APCs and anti-CD3 mAb either independently (left and middle panels) or mixed at a 1:1 ratio (right panel), and production of IL-17A was assessed by intracellular staining on day 3. B, Thy1.1⁻ Tconvs and Thy1.1⁺ Tregs were cultured at a 1:1 ratio as in A (right panel), and production of IL-17A was assessed by intracellular staining on day 3. Representative results of three experiments are shown. B–D, After gating on Thy1.1⁻IL-17⁻ Tconvs, Thy1.1⁺IL-17⁻ Tregs and Thy1.1⁺IL-17⁺ Tregs, as indicated in the colored boxes in panel B, expression levels of Foxp3 (C) and RORγt (D) were analyzed on day 3 of culture.

Tregs are capable of producing IL-17A in vivo after immunization. IL-1R1^−/− or B6.PL congenic (Thy1.1⁺) Tregs were transferred to IL-1R1^−/− mice that were subsequently immunized with KLH in IFA. A and B, Three days later, IL-17A–producing cells in the draining lymph nodeswere assessed by intracellular staining. Plots represent three individual mice per group. C and D, Five days later, splenocytes were harvested and restimulated in vitro, and levels of IL-17A and IFN-γ were assessed by ELISA.