Sensitivity of CD4⁺CD25^bright Tregs to ROS-induced cell death compared with CD4⁺CD25^−/low T cells (T4 conv). Tregs and T4 conv were (A) treated with increasing concentrations of H₂O₂ (5-20 μmol/L) or (Bi) cocultured with autologous granulocytes (granulocyte to T-cell ratio 1:2 to 1:4) in (Bii) the presence or absence of 100 U/mL human catalase (hCAT) and cell viability was assessed by flow cytometry and 7-AAD labeling. Viable cells were identified using dual criteria: no shift in the forward and/or side scatter and no staining of 7-AAD. Survival data are shown for Tregs, both purified by magnetic beads (Ai and Bi; solid line) and T4 conv (dashed line) and “untouched” negatively selected gated Tregs (Aii; solid line) and T4 conv (dashed line). (C) CD4⁺CD25^bright Tregs, CD4⁺CD25^−/lowCD45RA⁺ naive and CD4⁺CD25^−/lowCD45RA⁻ memory T cells were treated with increasing concentrations of H₂O₂ (5-20 μmol/L) for 18 hours, and cell viability was assessed as described previously. (D) Bead-isolated naive CD45RA⁺ CD4⁺CD25^bright Tregs (representative FACS data, Di) and CD4⁺CD25^−/low T4 conv were treated with H₂O₂ (40 and 80 μmol/L, respectively) for 18 hours and cell viability was assessed as described previously (Dii). (E) CD4⁺CD25^bright Tregs were treated with increasing concentrations of H₂O₂ (5-20 μmol/L) for 18 hours, stained with anti-CD45RA antibody, and a fraction of naive Tregs among viable population and its percent increase was measured. Cell viability was assessed as described previously. The initial proportion of CD45RA⁺ among the untreated Tregs served as the baseline value. Bars indicate standard error mean based on data from 6 (Ai), 4 (Aii), 4 (Bi), 5 (Bii), 6 (C), 3 (Dii), and 5 (E) different healthy donors in each and every panel. P values were calculated using a 2-tailed paired t test. Regression analyses were evaluated with analysis of variance testing. *P < .05, **P < .01.