Fig. 4

Colonic RORγt⁺ Treg cell-mediated immune responses are gut microbiota-dependent. a Representative flow cytometric analysis of colonic LP and spleen RORγt⁺ Treg cells (coexpression of RORγt and Foxp3) in WT and TLR4^−/− mice. Numbers in outlined areas indicate percent cells in each gated area. b Statistics results of Foxp3⁽⁺⁾ RORγt⁽⁻⁾ T cells, Foxp3⁽⁻⁾ RORγt⁽⁺⁾ T cells, Foxp3⁽⁺⁾ RORγt⁽⁺⁾ T cells in colonic LP in WT and TLR4^−/− mice. c Statistics results of Foxp3⁽⁺⁾ RORγt⁽⁻⁾ T cells, Foxp3⁽⁻⁾ RORγt⁽⁺⁾ T cells, Foxp3⁽⁺⁾ RORγt⁽⁺⁾ T cells in spleen in WT and TLR4^−/− mice. d Representative immunofluorescence double staining of RORγt and Foxp3 expression in colon tissue in WT and TLR4^−/− mice. e Quantification of the total number of double staining cells in ten high-power fields (HPFs) between WT and TLR4^−/− mice. f Representative flow cytometric analysis of colonic RORγt⁺ Treg cells among SiHo WT, CoHo WT, CoHo TLR4^−/−, SiHo TLR4^−/− groups. g Statistical analysis of CD4⁽⁺⁾ Foxp3⁽⁺⁾ RORγt⁽⁺⁾ T cells frequency in co-housing experiments. h Representative flow cytometric analysis of colonic RORγt⁺ Treg cells during FMT experiments. i Statistical analysis of CD4⁽⁺⁾ Foxp3⁽⁺⁾ RORγt⁽⁺⁾ T cells frequency in FMT experiments. a–e n = 6 mice per group. Data are shown as mean values ± SEM are presented, p-values were calculated using unpaired T-test, *p < 0.05, **p < 0.01, ***p < 0.001. f–i n = 6 mice per group. Data are shown as mean values ± SEM are presented, p-values were calculated using two-way analysis of ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.001. Data are pooled from three independent experiments with n = 6 mice per group