IL-17– and IFN-γ–producing Tregs. (A) The 3 Treg subpopulations and naive Tconvs were sorted and stimulated with anti-CD3/CD28 coated beads for 24 hours. mRNA expression of the indicated cytokines was determined by qRT-PCR using TaqMan low-density arrays. Mean values and SEM of 6 different donors are shown. (B-C) For intracellular cytokine staining, purified CD4 T cells were stimulated 5 hours with PMA/ionomycin (50 ng/mL, 1 μg/mL). (B) After 1 hour of stimulation, Brefeldin A was added to the culture media. After staining for extracellular antigens, cells were fixed, permeabilized, and intracellular staining of FOXP3, Helios, Ki-67, IL-17A, and IFN-γ was analyzed by flow cytometry. (C) Plots show the percentage of IFN-γ⁺ and IL17A⁺ T cells for conventional CD45RA⁻HLADR^- and CD45RA⁻HLADR⁺ T cells and the 3 Treg subpopulations. P values were obtained with the a-tailed nonparametric Mann-Whitney test. (D) For polarization assays, the 3 Treg subpopulations and conventional memory T cells were sorted and cultured for 2 weeks in Th0, Th1, or Th17 polarizing conditions. T cells were cultured in complete RPMI with CD3/CD28 beads (Invitrogen) and 100 U/mL IL-2. No additional cytokines or anti-cytokine antibodies were added to the Th0 condition. Th1 condition: IL-12 (10 ng/mL), anti–IL-4 (1 µg/mL). Th17 condition: IL-23 (50 ng/mL), IL-1β (10 ng/mL), IL-21 (10 ng/mL), TGF-β (10 ng/mL), anti–IL-4 (1 µg/mL), and anti–IFN-γ (1 µg/mL). Cytokines were added again to the media 48 hours after the first stimulation. Cells were restimulated with cytokines (day 7) and anti-CD3/CD28 beads (days 7 and 13). At day 14, the mRNA levels of IFNG, IL17F, FOXP3, and IKZF2 (Helios) were determined using 48.48 Dynamic Arrays.