Antigen-dependent development of pTreg cells with Treg-type epigenome in oral tolerance. (A) Representative CD25 and Foxp3 expression by DO CD4⁺ T cells in mLNs and SI-LP from DORe mice maintained by a designated diet for 6 wk. (B and C) Frequencies of Foxp3⁺ cells among DO CD4⁺ T cells (B) and CD25 expression level of Foxp3⁺ cells (C) in mLNs and SI-LP in the mouse groups as shown in A (n = 5–8). AF, Ag-free. (D) Degrees of demethylation as percentage of demethylated CpG residues at Treg-specific demethylation regions in Foxp3-CNS2, Ikzf2, Ikzf4, and Ctla4 loci of pTreg cells in the mice as shown in A. (E) H3K27ac ChIP-seq signals at global Treg-specific or Tconv-specific enhancer regions in WT or DORe-derived naïve Tconv or Treg cells. (F and G) H3K27ac ChIP-seq and ATAC-seq peak call at the Foxp3 gene locus (F) and other Treg-specific enhancer loci at Ctla4, Il2ra, and Ikzf2 (G) in eFox-derived naïve Tconv and nTreg cells, and DORe-derived naïve Tconv and pTreg cells. Green-shading regions indicate Foxp3 transcription start site (TSS) and CNS in F and Treg-specific peaks in G. Vertical bars indicate the mean ± SEM. Statistical significance was assessed by the Tukey–Kramer test (B and C). P values were shown in the figures.