Human Th17 cells are preferentially generated from naive FOXP3⁺ Tregs on costimulation via CD28 or CD5. (A) CD4⁺ T cells isolated by magnetic cell sorting from healthy donors' PBMCs were stained with CD4-, CD45RA-, CCR7-, CD25-, and CD127- specific mAb and naive (N, CD45RA⁺CCR7⁺) CD4⁺ T cells were sorted into NTreg and Nconv populations according to CD25 and CD127 expression, as shown. Aliquots of sorted populations were stained with anti-FOXP3 mAb and analyzed by flow cytometry. (B) Sorted populations were stimulated in vitro with anti-CD2/3/28– or anti-CD2/3/5– coated beads in the absence or presence of Th1 (IL-12, 10 ng/mL) or Th17 (TGF-β, 5 ng/mL; IL-1β, 10 ng/mL; IL-23, 100 ng/mL) polarizing factors and cultured in the presence of IL-2. Aliquots of day-12 cultures were stimulated in the presence of PMA (100 ng/mL) and ionomycin (1 μg/mL) for 4 hours (the last 3 in the presence of brefeldin A, 10 μg/mL), stained with IL-17A and RORγt-specific mAb and analyzed by flow cytometry. The proportions of IL-17⁺ and RORγt⁺ cells in the cultures are shown (mean ± SEM, n = 4). (C) Aliquots of day-7 cultures, obtained as in panel B, were stained with fluorochrome-labeled anti–IL-1RI mAb or with unlabeled anti–IL-23R antibodies followed by fluorochrome-labeled goat anti–rabbit Ig antibodies and analyzed by flow cytometry. The proportions of IL-23R⁺ and IL-1R⁺ cells in the cultures are shown (mean ± SEM, n = 4).