CD4⁺CD25^bright Tregs display a lower Bcl-2 expression, a greater antioxidative capacity compared with CD4⁺CD25^−/low T cells, and maintain suppressive activity after overnight incubation with H₂O₂. (A) Bcl-2 expression was measured in freshly isolated CD4⁺CD25^bright Tregs (□ −), CD4⁺CD25^−/low T cells (T4 conv; ■) and in Tregs (□ +) after treatment with H₂O₂ (10 μmol/L) for 18 hours. (B) Tregs (□) and T4 conv (■) were exposed to increasing concentrations of H₂O₂ (5-20 μmol/L) for 18 hours, and thiol expression was determined by the use of Alexa Fluor 488-coupled maleimide (ALM-488). Dead cells were excluded in the analysis to avoid artefacts caused by the release of intracellular thiols resulting from membrane instability. (C) The intracellular antioxidative capacity for Tregs (□) and T4 conv (■) was measured by staining the cells with CM-H₂-DCFDA and exposing them to H₂O₂ (20 μmol/L) for 45 minutes. The results are presented as the MFI of Bcl-2-FITC (Ai), ALM-488 staining (ALM FL-2; Bi), and DCF fluorescence (DCF FL-1; Ci) and as representative histograms (Aii,Bii,Cii). (D) Purified Tregs and T4 conv were analyzed for their relative gene expression of human catalase (hCAT), manganese superoxide dismutase (MnSOD), zinc superoxide dismutase (ZnSOD), and FOXP3 as an internal control by real-time PCR by the use of gene-specific primers for each gene. (E,F) Tregs were incubated in the absence or presence of 10 μmol/L H₂O₂ for 18 hours. Subsequently, cells were washed thoroughly and cocultured (ratio 1:2) with CFSE-labeled T4 conv in the presence of stimulatory beads coated with anti-CD2, anti-CD3, and anti-CD28 Abs. After 3 or 5 days the proliferation of CD4⁺CD25^−/low T cells was assessed with the use of flow cytometry. Dead cells were excluded by 7-AAD labeling. (E) Percentage of inhibition of proliferation on day 3 by non- and H₂O₂-treated Tregs. (F) Two representative and independent experiments with cells from 2 different healthy donors, each evaluated at day 3 or day 5. The unlabeled CFSE negative autofluorescent Tregs were gated out during fluorescence-activated cell sorting (FACS) analysis. Bars indicate standard error of the mean based on data from 4 (Ai), 5 (Bi), 4 (Ci), 8 (D), and 4 (E) different healthy donors in each and every panel. P values were calculated using a 2-tailed paired t test. *P ≤ .05, **P ≤ .01, ***P ≤ .001.