Depletion of CD4 ⁺ T cells, but not CD8 ⁺ T cells, ameliorates intestinal immune pathology of SSP hosts in the absence of Tregs. (A) Schematic representation of the experimental design. F1-Foxp3^DTR mice (n = 10) were infected with Salmonella Typhimurium SL1344 (10⁸ CFUs; STm), treated with streptomycin to induce SSP at 14 dpi, and injected (i.p.) with DT and anti-CD4 or isotype antibody to deplete CD4⁺ T cells, as indicated. (B) Percentage of body change variation (left) and probability of survival (right) of ΔTreg SSP (orange circle) and CD4-depleted ΔTreg SSP (purple circle) mice starting at 21 dpi (n = 10). (C) Representative images (left) and H&E staining of colon collected from ΔTreg SSP and CD4-depleted ΔTreg SSP at 28 dpi, as indicated. Magnification: upper panels = 1.25×; lower panels = 20×. Scale bars: upper panels = 1.0 mm; lower panels = 50 μm. Inflammation in the lamina propria (black asterisks) and the submucosa (red asterisk). Luminal cell debris (black arrowheads), goblet cells (black arrows). (D) Pathology scores assigned to colonic tissue sections of ΔTreg SSP (orange circle) and CD4-depleted ΔTreg SSP (purple circle, n = 4–8). (E) FITC-dextran detected in the serum of ΔTreg SSP and CD4-depleted ΔTreg SSP at 28 dpi (n = 6–8). (F)Salmonella CFUs measured in the feces of ΔTreg SSP and CD4-depleted ΔTreg SSP at 28 dpi (n = 8–15). (G) Volcano plot of differentially expressed genes between ΔTreg SSP and CD4-depleted ΔTreg SSP colonic tissues at 28 dpi. Orange dots indicate the genes that were differentially upregulated in the colon collected from ΔTreg SSP mice compared with CD4-depleted ΔTreg SSP. (H) Representative immunofluorescence images of colonic tissues (magnification 40×; scale bar: 50 μm) collected from ΔTreg SSP (left) and CD4-depleted ΔTreg SSP (right) mice, showing the localization of cCasp-3⁺ cells (white arrowheads) mostly in epithelium layer (E-cadherin⁺). Sections were stained with E-cadherin (white), cCasp-3 (red), Gzmb (green), and DAPI (blue). Numbers of cCasp-3⁺ E-cadherin⁺ cells localized in the epithelium in at least five areas per section per condition (ΔTreg SSP and ΔTreg SSP + anti-CD4), with at least three sections per condition. Each dot represents a cCasp-3⁺ E-cadherin⁺ 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). Results in panels B and D–F 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 (D–F and H) was used to compare two groups. Two-way ANOVA (B) followed by post hoc Sidak’s test was performed for multiple groups comparisons (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns = not significant). A log-rank (Mantel–Cox) (B) test was used to compare survival probability between the two groups. See also Fig. S5; and Tables S1 and S2.