scRNA-seq of colonic CD4 ⁺ T cells reveals the upregulation of granzyme gene expression in ΔTreg SSP mice. (A) UMAP plot of colonic CD4⁺T cell clusters (0–8) generated by merging F1-SSP and ΔTreg SSP mice at 28 dpi. (B) Barplot showing the proportion of each cluster of CD4⁺T cell from F1-SSP and ΔTreg SSP mice. (C) Dot plot representing a list of genes manually curated to identify the different gene expression signature that characterizes 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. (D) Dot plot representing the expression of Gzmb, Gzma, Prf1, Ccl5, Nkg7 Pdcd1, Ifitm1-2-3, Itgb1, and Nrp1 in clusters 6 and 4 of F1-SSP and ΔTreg SSP mice. 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) Frequency of Gzmb⁺ and Gzma⁺ CD4⁺ T cells isolated from the colonic lamina propria of F1-SSP and ΔTreg SSP mice at 28 dpi (n = 10–12). Results are representative of at least 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). (F and G) Representative immunofluorescence images of colonic tissues (magnification: 40×; scale bar: 50 μm) collected from F1-SSP (F) and ΔTreg SSP (G) mice, showing the close localization of Gzmb⁺ CD4⁺ T cells (white arrowheads) to the epithelial layer (E-cadherin). Sections were stained with E-cadherin (white), CD4 (red), Gzmb (green), and DAPI (blue). (H) Quantification of the localization of Gzmb⁺ CD4⁺ T cells as distance between them and the colonic epithelium. Results are representative of 5–10 areas per section per condition (F1-SSP and ΔTreg SSP), with at least three sections per condition. Each dot represents a Gzmb⁺ CD4⁺ T cell, and the data are 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 < 0.005). See also Fig. S4.