Colonic and splenic immune responses of F1-SSP and ΔTreg SSP mice at 28 dpi. (A and B) Frequency and numbers of CD4+ T (A) and CD8α+ T (B) cells isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 10–15). (C) Frequency of neutrophils, monocytes, and macrophages (MΦ) isolated from the colonic lamina propria of F1-uninfected (black circle) and F1-SSP (green circle) mice at 28 dpi (n = 5–10). (D) Frequency of neutrophils, monocytes, and macrophages (MΦ) isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 5–10). (E) Frequency and numbers of Tregs isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 10–15). (F) Representative FACS plots and frequency of Ki-67+ Tregs isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 10–15). (G) Representative FACS plot of tetramer+ (2W:I-Ab) Foxp3+ CD4+ T cells isolated from the spleen of F1-SSP mice at 28 dpi. (H) Frequency and numbers of Tregs (left) and Ki-67+ Tregs (right) 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). (I) Frequency and numbers of Tregs (left) and Ki-67+ Tregs (right) 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). (J and K) Representative FACS plots and frequency of Tregs in the colonic lamina propria (J) and spleen (K) of uninfected F1-Foxp3DTR (F1-uninfected) and ΔTreg after DT treatment, at 28 dpi (n = 5). (L) Frequency and numbers of Th1 cells detected in the colonic lamina propria of F1-SSP (green circle) and ΔTreg SSP (orange circle) at 28 dpi (n = 8–10). (M)STm CFU counts in the spleen and MLNs of F1-SSP (green triangle) and ΔTreg SSP (orange triangle) mice at 28 dpi (n = 6–10). (N) Frequency of neutrophils (left panel) and monocytes (right panel) isolated from the colonic lamina propria of uninfected ΔTreg (open circle) and ΔTreg SSP (orange circle) mice at 28 dpi (n = 8–10). 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 (A–F and J–N) test was used to compare two groups (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns = not significant). One-way ANOVA (H and I) followed by post hoc Tukey’s test was performed for multiple groups comparisons. MLN, mesenteric lymph node.
Figure S2.

Colonic and splenic immune responses of F1-SSP and ΔTreg SSP mice at 28 dpi. (A and B) Frequency and numbers of CD4+ T (A) and CD8α+ T (B) cells isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 10–15). (C) Frequency of neutrophils, monocytes, and macrophages (MΦ) isolated from the colonic lamina propria of F1-uninfected (black circle) and F1-SSP (green circle) mice at 28 dpi (n = 5–10). (D) Frequency of neutrophils, monocytes, and macrophages (MΦ) isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 5–10). (E) Frequency and numbers of Tregs isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 10–15). (F) Representative FACS plots and frequency of Ki-67+ Tregs isolated from the spleen of F1-uninfected (black triangle) and F1-SSP (green triangle) mice at 28 dpi (n = 10–15). (G) Representative FACS plot of tetramer+ (2W:I-Ab) Foxp3+ CD4+ T cells isolated from the spleen of F1-SSP mice at 28 dpi. (H) Frequency and numbers of Tregs (left) and Ki-67+ Tregs (right) 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). (I) Frequency and numbers of Tregs (left) and Ki-67+ Tregs (right) 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). (J and K) Representative FACS plots and frequency of Tregs in the colonic lamina propria (J) and spleen (K) of uninfected F1-Foxp3DTR (F1-uninfected) and ΔTreg after DT treatment, at 28 dpi (n = 5). (L) Frequency and numbers of Th1 cells detected in the colonic lamina propria of F1-SSP (green circle) and ΔTreg SSP (orange circle) at 28 dpi (n = 8–10). (M)STm CFU counts in the spleen and MLNs of F1-SSP (green triangle) and ΔTreg SSP (orange triangle) mice at 28 dpi (n = 6–10). (N) Frequency of neutrophils (left panel) and monocytes (right panel) isolated from the colonic lamina propria of uninfected ΔTreg (open circle) and ΔTreg SSP (orange circle) mice at 28 dpi (n = 8–10). 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 (A–F and J–N) test was used to compare two groups (****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns = not significant). One-way ANOVA (H and I) followed by post hoc Tukey’s test was performed for multiple groups comparisons. MLN, mesenteric lymph node.

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