Generation of tissue-specific effector-type pTreg cells by antigen feeding. (A) scRNA-seq of DO CD4⁺ T cells in mice fed with NC or EWP food for 4 wk from weaning. (B) HTO classification of DO CD4⁺ T cells in SI-LP in NC- or EWP-fed mice (left) with proportion of cells in each cluster (right). (C and D) Volcano plot of differentially expressed genes (C) and gene ontology (GO) analysis (D) between Cluster 3 and 5 pTreg cells. (E) UMAP plots of cells reclustered from Clusters 3, 4, 5, 11 shown in A, with projection of specifically expressed genes. (F) UMAP plots with NC- or EWP-derived HTO and Foxp3 expression, as shown in E. (G) Pseudotime trajectory of reclustered cells in F. (H and I) Contour plots and MFI of 4-1BB and OX40 (H), TIGIT and GITR (I) staining of pTreg cells in SI-LP of NC- or EWP-fed mice (n = 3–6). (J) Frequencies of T-bet⁺, GATA3⁺, or RORγT⁺ pTreg cells in NC- or EWP-fed mice (n = 4–6 each). (K) Ki-67 and KLRG1 expression of SI-LP–derived pTreg cells in DORe mice kept fed with NC or EWP, or ceased to be EWP-fed for 4 wk (EWP [−]4 wk) as shown in Fig. 1 H (n = 4–5). (L) PCA of transcriptomes of pTreg cells from mLNs, dLNs, SI-LP, and ear tissue (n = 3 each). (M and N) Transcripts Per Million (TPM)-normalized Bcl2 (M), and Ccr9 and Ccr5 (N) expression by pTreg cells from designated tissues. (O) CCR9 and CCR5 expression by SI-LP– and ear tissue–derived pTreg cells. Vertical bars indicate the mean ± SEM. Statistical significance was assessed by an unpaired t test or Welch’s t test (H–J), and the Tukey–Kramer test (K). P values were shown in the figures. HTO, hashtag oligonucleotide. UMAP, Uniform Manifold Approximation and Projection.