scRNA-seq of colonic SSP Tregs reveals their distinct profile compared with Tregs from uninfected hosts. (A) UMAP plot of colonic Treg clusters (0–5) generated by merging uninfected and SSP conditions. (B) UMAP plot of colonic Treg clusters (0–5) represented in each condition separately (uninfected/SSP). (C) Barplot showing the proportion of each Treg cluster of F1-uninfected or F1-SSP mice. (D) Dot plot representing a list of genes manually curated to identify the different gene expression signatures defining each cluster. The size of the dot represents the percentage of the cells in each cluster expressing a specific marker, while the color is a measure of the average expression of that gene in a specific cluster. (E) UMAP plots showing the expression of Gata3, Rorc, and Tbx21 among all the identified Treg clusters. (F and G) Representative FACS plot and frequency of T-bet⁺ Tregs (F) and Ki-67⁺ T-bet⁺ Tregs (G) detected in the colonic lamina propria of F1-uninfected or F1-SSP mice (n = 5–10). Results are representative of two independent experiments and presented as means ± SEM. Normality was assessed by the D’Agostino–Pearson test. The Mann–Whitney U test was used to compare two groups (****P < 0.0001). (H) Violin plots showing the expression levels of Il10, Tgfb1, Areg, Gzmb, and Tbx21 among clusters 0 and 1, only present in F1-SSP mice, versus clusters 2 and 3, only represented in uninfected F1 mice. See also Fig. S3.