IL-17 secretion upon coculture of DR⁻ Tregs with Tresp cells inversely correlates with suppression. (A) CFSE-labeled CD25⁻ Tresp cells were cultured in RPMI medium containing 5% human AB serum, alone or with DR⁻ Treg or DR⁺ Treg (1:1 ratio) under 3 different stimulatory conditions: αCD3/αCD2 beads (left), or irradiated T-cell depleted APCs and plate-bound αCD3 at 0.1 μg/mL (middle) or 0.5 μg/mL (right). On day 4 of stimulation, proliferation of Tresp cells was analyzed by assessing CFSE dilution by FACS analysis. The numbers represent the percentage of Tresp cells that divided (CFSE dilution). (B) IL-17 production in these differentially stimulated Tresp cultures or DR⁻ or DR⁺ Treg cocultures was analyzed by ELISA from day 4 supernatants. Data shown are representative of 4 independent experiments. (C) The results of the same assay performed on 4 independent donors and represented as percentage suppression of proliferation and fold induction of IL-17 in DR⁻ and DR⁺ Treg cocultures stimulated with αCD3 (0.5 μg/mL) and APCs (mean + SEM). (D) Cultures containing CFSE-labeled Tresp cells, DR⁻ Tregs, or both cell types (1:1 ratio) were stimulated with high dose αCD3 and APCs. On day 4, PMA/ionomycin and GolgiStop were added to the cultures for a 4-hour pulse before the cells were harvested, surface stained with αCD2 to distinguish the CD4 T cell populations from the irradiated, T-depleted APCs, and then permeabilized and stained for IL-17. Data shown are representative of 3 independent experiments. **P < .01.