Fig. 6.

T_H17 polarized NTreg cultures contain distinct suppressive and nonsuppressive subpopulations. (A) Ex vivo sorted NTreg were stimulated in vitro with anti-CD2/CD3/CD28 microbeads in the presence of IL-1β, IL-23, TGF-β, and IL-2. Day 12 cultures were stained with mAb specific for IL-1RI and CCR6 and analyzed by flow cytometry. (B) NTreg were stimulated and stained as in A, and IL-1RI⁺CCR6⁻, IL-1RI⁺CCR6⁺, and IL-1RI⁻CCR6⁺ populations were sorted by flow cytometry. Cells from the total unsorted culture as well as sorted populations were stimulated in the presence of PMA/ionomycin, stained with mAb specific for FOXP3 and IL-17, and analyzed by flow cytometry. Dot plots for one donor and data for all donors (% FOXP3⁺ cells and mean fluorescence intensity (MFI) of FOXP3 staining in the FOXP3⁺ populations, mean ± SD, n = 4) are shown. **P < 0.01; ***P < 0.001. (C and D) CFSE-labeled conventional CD4⁺ T cells were stimulated with PHA in the absence or presence of IL-1RI⁺CCR6⁻, IL-1RI⁺CCR6⁺, and IL-1RI⁻CCR6⁺ populations sorted from T_H17 polarized NTreg cultures as in B or in the presence of ex vivo sorted Treg and conventional CD25⁻ cells. Growth of responder CD4⁺ T cells was assessed by flow cytometry analysis of CFSE dilution. Examples of CFSE dilution histograms, at 1:1 suppressor-to-responder cell ratio, are shown in C where numbers correspond to the proportion of undivided cells. Percent suppression is shown in D for all tested suppressor-to-responder cell ratios, populations, and donors (mean ± SD, n = 3).