pTreg generation and maintenance by antigen feeding in establishing oral tolerance. (A) Antigen feeding to DORe mice and assessment of ear swelling upon antigen challenge. (B) Changes in right ear thickness in DORe mice treated as shown in A. Arrowheads indicate OVA challenges. (C) Histology of the right ear and histological scores in NC- or EWP-fed DORe mice. Bars in histology indicate 100 µm. (D) CD25 and Foxp3 expression by DO CD4⁺ T cells in mLNs, dLNs, and both ears. Barplots indicate frequencies of Foxp3⁺ cells (pTreg cells) (n = 10 and 11). (E–G) Treg cell depletion in EWP-fed DORF mice. Mice were treated twice with DTx followed by OVA challenge (E). Ear thickness and histological scores (F). Frequencies of pTreg cells in the right ear (G) (n = 5–7). (H–J) Cessation of EWP feeding for 4 wk (designated EWP[−]4 wk) or 8 wk (EWP[−]8 wk) before OVA challenge (H). Ear thickness and histological scores (I). Frequencies of pTreg cells in the right ear (J) (n = 4–6). (K–N) Effects of skin OVA sensitization and subsequent OVA feeding on ear swelling upon OVA challenge (K). Ear thickness and histological scores (L). Frequencies of pTreg cells in the right ear (M) (n = 5 and 6). Changes in body weight of DORe mice (N). Vertical bars indicate the mean ± SEM. Statistical significance was assessed by an unpaired t test or Welch’s t test (B–D, F, G, and L–N), and Tukey–Kramer’s test (I and J). P values were shown in the figures.