Establishment of oral tolerance in antigen-presensitized mice by CD80/CD86 blockade and subsequent antigen feeding. (A) Experimental protocol. DORe mice were OVA-sensitized at the right ear four times, i.p. injected with CTLA4-Ig or saline on day 11, fed with EWP chow or NC for 4 days, OVA-challenged at the left ear four times from day 22, and assessed for ear swelling. (B and C) Changes in body weight (B), left ear thickness, and histological scores (C) (n = 5–6). Arrowheads indicate OVA challenges. (D and E) Frequencies of pTreg cells in mLNs, dLNs, and the right ear at day 15 (D) and day 33 (E) (n = 5–6). (F and G) Frequencies of IFN-γ⁺, IL-4⁺, and IL-17A⁺ cells in dLNs at day 15 (F) and day 33 (G) (n = 5–6). (H) CD101 expression by pTreg cells in dLNs and the right ear at day 15. Numbers in the histogram are representative of the frequency of CD101⁺ cells. Barplots indicate the frequency of CD101⁺ pTreg cells (n = 4–10/group). (I) Frequencies of CD101⁺ cells within pTreg cells in dLNs and the left ear at day 33 (n = 5–6). (J and K) Effects of Treg depletion after CTLA4-Ig injection and EWP feeding, After CTLA4-Ig injection and EWP feeding, DORF mice were i.p. treated with PBS or DTx at days 15 and 18, and rechallenged with OVA (arrowheads). Changes in left ear thickness at day 33 in DORF mice (J) (n = 6–7). CD101 expression by pTreg cells in mLNs, dLNs, and the left ear (K). Barplots indicate the frequencies of CD101⁺ pTreg cells (n = 3). Vertical bars indicate the mean ± SEM. Statistical significance was assessed by the Tukey–Kramer test (C–I) and unpaired t test or Welch’s t test (J and K). P values were shown in the figures.