Dose effects of CTLA-4-Ig, TGF-β, and IL-2 on iTreg generation. (A) Synergistic effect of TGF-β supplementation and CD28 signal deprivation on in vitro iTreg induction and CD101 expression by naïve CD4⁺ Tconv cells (n = 4). (B) Experimental design. DORe-derived naïve Tconv cells and splenic DCs were cocultured with or without CTLA4-Ig in the presence of OVA, TGF-β, and IL-2. (C) Response to various concentrations of CTLA4-Ig (0, 2, 10, and 50 µg/ml) in in vitro iTreg induction (n = 4). (D) Response to various concentrations of IL-2 (0, 1, 10, and 100 U/ml) in in vitro iTreg induction and CD101 expression (n = 4 [0 U/ml] and n = 7 [1–100 U/ml]). (E) Degrees of demethylation as percentage at Foxp3-CNS2 regions of iTreg cells induced with or without CTLA4-Ig. (F) Gating strategy for isolating classic dendritic cells from SI-LP of WT mice. Vertical bars indicate the mean ± SEM. Statistical significance was assessed by an unpaired t test or Welch’s t test (A and C). P values were shown in the figures.