Specific expression of CD101 by pTreg cells generated by antigen feeding. (A) Irradiated CD45.1 Rag2KO mice reconstructed with CD3e⁺ cell–depleted BM cells isolated from WT (Thy1.1-eFox) and DORe mice. Mixed BMC mice were analyzed at 4–8 wk after BM cell transfer. (B–D) Representative flow cytometry profiles of WT- and DORe-derived CD4⁺ T cells in mLNs of BMC mice fed with NC or EWP chow for 4 wk (B). Frequency of WT- and DORe-derived Foxp3⁺ cells in designated tissues (C) (n = 10–12) and in the thymus (n = 8) (D). (E) Volcano plot of differentially expressed genes between DORe-pTreg cells and WT-nTreg cells assessed by RNA-seq analysis. (F and G) TPM-normalized differentially expressed genes (F) and genes encoding known tTreg markers (G) in BMC mice as shown in B (n = 3). (H) CD101 expression by DORe-pTreg cells and WT-nTreg cells in BMC mice as shown in B. Barplots indicate MFI of CD101 expression and frequency of CD101⁺ cells (n = 6). (I) CD101 expression by WT-Treg or Tconv cells, and DORe-pTreg or Tconv cells in untreated mice. Barplots indicate MFI of CD101 expression and frequency of CD101⁺ cells (n = 3). Vertical bars indicate the mean ± SEM. Statistical significance was assessed by an unpaired t test or Welch’s t test (C and H). P values were shown in the figures.