Enriched Th17 cells express high levels of FOXP3 on activation. (A) Sorting procedure for human Th1 (CD4⁺CD25⁻CXCR3⁺CCR4⁻CCR6⁻) and Th17 (CD4⁺CD25⁻CXCR3⁻CCR4⁺CCR6⁺) cells. CD4⁺ T cells were isolated from human PBMCs and depleted of CD25⁺ cells before sorting. (B) Phenotype of Th1-enriched and Th17-enriched cells after 2 weeks of expansion with APC and anti-CD3 Abs. T cells were restimulated and IL-17A and IFN-γ production were determined by intracellular cytokine staining. The cytokine profiles from 1 representative donor of 6 are shown. (C) Representative plots of FOXP3 expression in Th1-enriched and Th17-enriched cells from one donor of 4 on days 1, 3, and 12 after reactivation with anti-CD3/anti-CD28–coated beads (1 bead: 32 cells) are shown. (D) Average FOXP3 expression in Th1-enriched and Th17-enriched cells after reactivation with anti-CD3/anti-CD28–coated beads (1 bead: 32 cells) over 14 days. Each time point represents the average FOXP3 expression from 2-5 different donors. **Significant difference in FOPX3 expression between Th1-enriched and Th17-enriched cells 14 days after reactivation (P = .0028). There is no difference at day 0 (P = .1080). †Significant difference in FOXP3 expression between day 0 and 14 in Th17-enriched cells (P = .0126). (E) Representative (left panel) and average (right panel; n = 3 for IL-17, n = 2 for IFN-γ) FOXP3 expression in IL-17A⁺, IL-17A⁻, IFN-γ⁺, and IFN-γ⁻ subsets within Th17-enriched cultures 12 days after restimulation with anti-CD3/anti-CD28–coated beads (1 bead: 32 cells).