Colonic and splenic immune responses of SSP and non-SSP mice at 28 dpi. (A) Schematic representation of the experimental design. 129X1/SvJ mice (n = 10–50) were infected with Salmonella Typhimurium SL1344 (10⁸ CFUs; STm), and the fecal shedding (right) was monitored at the time indicated (7, 14, 21, 28 dpi). Each dot represents a single mouse; dark-pink circles indicate non-SSP mice, while light-blue circles indicate SSP mice. (B)STm CFUs/g of feces, spleen, MLN, and liver collected from 129X1/SvJ at 28 dpi (n = 10–15). Dark-pink circles indicate non-SSP mice, and light-blue circles indicate SSP mice. (C) Percentage of body change variation of uninfected (black circle), SSP (light-blue circle), and non-SSP (dark-pink circle) mice at 7, 14, 21, 28 dpi (n = 10). (D) Frequency of neutrophils, monocytes, and macrophages (MΦ) isolated from the colonic lamina propria (cLP) of uninfected (black circle), non-SSP (dark-pink circle), and SSP (light-blue circle) mice at 28 dpi (n = 5–10). (E) Frequency of neutrophils, monocytes, and macrophages (MΦ) isolated from the spleen of uninfected (black triangle), non-SSP (dark-pink triangle), and SSP (light-blue triangle) mice at 28 dpi (n = 5–10). (F and G) Frequency and numbers of CD4⁺ T (F) and CD8α⁺ T (G) cells isolated from the colonic lamina propria of uninfected (black circle), non-SSP (dark-pink circle), and SSP (light-blue circle) mice at 28 dpi (n = 10–15). (H) Frequency of CD4⁺ and CD8α⁺ T cells isolated from the spleen of uninfected (black triangle), non-SSP (dark-pink triangle), and SSP (light-blue triangle) mice at 28 dpi (n = 10–15). (I) Frequency and numbers of Th1 cells isolated from the colonic lamina propria of uninfected (black circle), non-SSP (dark-pink circle), and SSP (light-blue circle) mice at 28 dpi (n = 10–15). (J) Frequency and numbers of Th1 cells isolated from the spleen of uninfected (black triangle), non-SSP (dark-pink triangle), and SSP (light-blue triangle) mice at 28 dpi (n = 10–15). (K) Spearman’s correlation matrix showing association between Salmonella fecal CFUs with Th1, CD4⁺, and CD8α⁺ T cells (right) or with neutrophils, monocytes, and macrophages (left), isolated from the colonic lamina propria of SSP and non-SSP hosts (n = 10–15). Spearman’s correlation coefficient (r) and relative P values (*P < 0.05, **P < 0.01, ***P < 0.001) are shown. (L) Gating strategy for flow cytometry used to identify neutrophils, monocytes, and macrophages in the colonic lamina propria and spleen of uninfected, non-SSP, and SSP mice. (M) Gating strategy for flow cytometry used to identify T cell subsets in the colonic lamina propria and spleen of uninfected, non-SSP, and SSP mice. 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 (B) test was used to compare two groups. One-way ANOVA (D–K) followed by post hoc Tukey’s test was performed for multiple groups comparisons (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns = not significant). MLN, mesenteric lymph node.