Activity of p38 is essential for the induction of iTregs with suppressive properties. iTregs (R) and Teffs (E) were induced as described in “Materials and methods.” In some experiments, during the primary culture, the specific p38 inhibitor SB203580 was added. After primary culture, Teffs and iTregs were cocultured (ratio, 1:1) and activated by anti-CD3 mAb in suppressor assays. Coculture of Teffs or single T-cell populations served as controls. Proliferation was detected 72 hours after restimulation as incorporation of [³H]-thymidine and demonstrated as cpm ± SEM (A). (B) iTregs (R), SB203580-pretreated iTregs (RI) and Teffs (E) were CFDA labeled or left untreated to determine the proliferation of the T-cell populations after restimulation and in coculture experiments. The proliferative response of the CFDA-labeled population was assessed. FACS analysis was performed after 96 hours. One representative of 3 experiments is shown. IL-2 (C) and IFN-γ (D) production were measured by ELISA in supernatants harvested after 48 hours of culture. Similar results were obtained in 5 independent experiments. To discriminate Teffs, iTregs, and inhibitor-treated iTregs in coculture, the respective populations were CFDA labeled prior to the setup of the experiment (E-F). After 24 hours, intracellular IL-2 production was determined by FACS analysis in the unlabeled population. One representative is shown in panel E. (F) The IL-2 expression is normalized against cytokine expression in Teffs. A total of 5 independent experiments are demonstrated. Asterisks indicate statistical significance according to the Student t test (*P ≤ .05; **P ≤ .005; ***P < .001).