pTreg cell development at different antigen doses and after depletion of preexisting pTreg cells in NC-fed DORe mice. (A and B) CD62L and CD44 expression by pTreg cells (A) and frequencies of CD44^hiCD62L^lo effector pTreg cells (B) in mLNs and SI-LP from DORe mice fed with designated diets for 6 wk. AF, Ag-free. (C and D) Foxp3⁺CD4⁺ T cells in the thymus and peripheral lymphoid tissues of NC-fed DORe or WT (eFox) mice. Representative staining for Foxp3/CD25 and the frequencies of CD25⁺Foxp3⁺ T cells in the thymus and Foxp3⁺ T cells in various tissues (n = 3 for thymus, spleen, pLNs, mLNs, and PP; or n = 6 for SI-IE, SI-LP, and LI-LP). (E) Methylation status in TSDRs of DORe-derived pTreg cells compared with eFox-derived nTreg cells. Empty and black circles indicate demethylated and methylated CpG residues, respectively. (F) Helios and Nrp-1 expression levels of eFox-Tconv, DORe-Tconv, eFox-nTreg, and DORe-pTreg cells. Barplots indicate MFI of Helios and Nrp-1 expression on eFox-nTreg cells and DORe-pTreg cells (n = 3). (G)De novo pTreg development after depletion of pTreg cells preexisting in NC-fed mice. DORF mice having been maintained with NC feeding were DTx-treated and then fed with NC or EWP chow for 4 wk. (H and I) Foxp3⁺CD4⁺ T cells immediately after DTx treatment (H) and after EWP or NC feeding for 4 wk (I). Representative flow cytometry plots and the ratios of Foxp3⁺ pTreg cells in mLNs (n = 3–4). Vertical bars indicate the mean ± SEM. Statistical significance was assessed by an unpaired t test (I). P values were shown in the figures. PP, Peyer’s patches.