In vitro generation of CD101 ⁺ iTreg cells by the absence or blockade of costimulatory signal during their induction. (A) In vitro generation of iTreg cells and their CD101 expression. iTreg cells were induced by plate-bound anti-CD3e mAb with or without anti-CD28 mAb in the presence of TGF-β and IL-2 (n = 3). (B) iTreg induction and their CD101 expression by anti-CD3e (0.5, 1, 5, and 10 µg/ml) and anti-CD28 mAb (0, 0.5, 1, and 2 µg/ml) stimulation as in A (n = 3). (C and D) iTreg induction from CD28-deficient or CD28-intact CD4⁺ T cells (C) and their expression of CD101 (D) as in A (n = 3). (E and F) iTreg generation (E) and CD101 expression (F) by iTreg cells induced by coculturing DO naïve Tconv cells with SI-LP-derived cDC1 or cDC2 in the presence of OVA and CTLA4-Ig (n = 4). (G) Effects of inflammatory cytokines (10 ng/ml each) and CTLA4-Ig on antigen-specific iTreg induction and their CD101 expression as in E and F (n = 3). 1 and 2 mean cDC1 and cDC2, respectively. (H) iTreg induction from antigen-specific Tconv cells in antigen-sensitized DORe mice. Naïve or effector Tconv cells from dLNs of antigen-sensitized DORe mice were cocultured with SI-LP–derived cDC1 or cDC2 cells in the presence of OVA and CTLA4-Ig. (I and J) Frequencies of iTreg cells (I) and CD101 expression (J) of iTreg cells induced as shown in H (n = 6). Vertical bars indicate the mean ± SEM. Statistical significance was assessed by Tukey’s HSD test (B) and an unpaired t test or Welch’s t test (C–F, G, and J). P values were shown in the figures.