Effector cytokine production at the site of inflammation is insufficiently controlled, because of resistance of effector cells to suppression. (A) SFMCs were stained for cytokine expression by flow cytometry after 4.5 hours of PMA and Ionomycin stimulation. Dotplots showing the percentage of IL-10, IL-17, TNFα, and IFNγ-positive cells in CD4⁺ (top panel) and CD8⁺ cells (bottom panel), 1 representative of n = 4. Percentages indicate the percentage of positive cells from representative data, or, in upper line, average percentage ± SD from accumulative data of n = 4. (B-F) CD4⁺CD25⁺CD127^low Tregs were sorted by flow cytometry and co-cultured with effector cells. At day 4, cytokine production in the culture supernatant was analyzed. (B-D) IL-5, IL-13, IL-10, IL-17, TNFα, and IFNγ levels in the absence (eff) or presence of Tregs at 1:8 and 1:4 ratios or additional effector cells (+eff) at a 1:4 ratio for PBMCs from HCs (B), PBMCs from JIA patients (C), and SFMCs from JIA patients (D). Data represent mean cytokine levels in pg/mL ± SEM of n = 4 PBMC HC, n = 4 PBMC JIA and n = 8 SFMC JIAs, *P < .05, ***P < .001 compared with effector cells (eff). (E-F) Percentage suppression of IL-5, IL-13, IL-10, IL-17, TNFα, and IFNγ production in the presence of Tregs at a 1:4 ratio relative to effector cells alone. (E) Percentage suppression in cocultures of Tregs and effector cells from PBMCs of HCs (black bars), PBMCs of JIA patients (gray bars), and SFMCs of JIA patients (white bars). Bars represent mean ± SEM of n = 4 PBMC HC, n = 4 PBMC JIA and n = 8 SFMC JIA, **P < .01, ***P < .001. (F) Percentage suppression in cocultures of PB derived Tregs and PBMC (black bars), PB Tregs and SFMCs (white bars), or SF derived Tregs and PBMCs (gray bars). Bars represent mean ± SEM of n = 4, **P < .01, ***P < .001 compared with PB-Tr + PBMCs.