IL-10 is not critical for regulatory activity of iTregs. Expression of IL-10 mRNA and protein levels of iTregs (R), Teffs (E), and SB203580-pretreated iTregs (RI) and Teffs (EI) was quantified at day 5 of primary culture and day 4 after restimulation by ELISA and qRT-PCR, respectively (A). Relative IL-10 mRNA expression was normalized to GAPDH. Secretion of IL-10 in cocultures of iTregs and Teffs (E+R) or SB203580-pretreated iTregs and Teffs (E+RI) was determined by ELISA (n = 4) (B). In cocultures of iTregs and Teffs, IL-10 and TGF-β bioactivity was neutralized by anti–IL-10, anti–IL-10R, or anti–TGF-β antibodies (C). Proliferation was measured in triplicate 72 h after restimulation as incorporation of [³H]-thymidine. *Statistical significance according to the Student t test (P ≤ .05). n.s. indicates not significant. One representive experiment of 3 is shown.