Sorted DR⁻ Tregs, but not DR⁺ Tregs, produce IL-17 in response to short-term mitogenic activation. (A) Gating strategy for FACS sorting of CD4⁺ peripheral blood T cells into populations of Tregs (CD45RA⁻CD25^high), memory Tresps (CD45RA⁻CD25^med), and naive Tresps (CD45RA⁺CD25⁻). (B) Percentages of HLA-DR⁺ cells within the 3 different gates (n = 9). (C) FoxP3 intracellular staining of FACS-sorted naive Tresps (dashed line), memory Tresps (thin solid line), DR⁻ Tregs (bold line), and DR⁺ Tregs (gray line). (D) The percentages of FoxP3⁺ cells is shown (n = 4). (E) FACS-sorted naive Tresps, memory Tresps, DR⁻ Tregs, and DR⁺ Tregs (10⁴ cells/well) were stimulated in serum-free X-Vivo medium for 4 hours with PMA/ionomycin and GolgiStop, and stained for intracellular IL-17 (n = 4). (F) IL-17 levels measured by ELISA in the same samples after 20 hours of stimulation with PMA/ionomycin. (G) One representative staining of intracellular FoxP3 and IL-17 in the 4 populations ex vivo or after 4 hours of stimulation with PMA/ionomycin and GolgiStop. **P < .01; *P < .05.