Fig. 3.

Induction of Th17 cells in the SI-LP of SFB-positive NOD females. (A) Lymphocytes were isolated from the indicated lymphoid tissues of SFB-positive or SFB-negative female NOD mice at 6–8 wk of age. Intracellular cytokine expression was enhanced by a 4-h culture in the presence of phorbol ester and ionomycin. Cells were gated by side-scatter and as CD45⁺, CD19⁻, CD8⁻, TCRβ⁺, and CD4⁺. Shown is a representative plot for IL-17 and IFN-γ staining from six mice analyzed. (B) Frequencies of IL-17–producing CD4⁺TCRβ⁺ cells in various lymphoid organs of SFB-positive and SFB-negative NOD mice. Gating was done as per A. Statistical analysis was performed using the nonparametric Mann–Whitney test. (C) Correlation of relative SFB levels in fecal pellets (determined as per Fig. 1) and percentage of IL-17–expressing CD4⁺TCRβ⁺ cells (as per A and B). Statistical analysis was performed using the Pearson correlation. (D) Frequencies of IFN-γ–producing CD4⁺TCRβ⁺ cells in the same lymphoid organs of the NOD mice depicted in B. Gating was done as per A. (E and F) Representative flow cytometry plots of three mice analyzed and frequencies of Foxp3-expressing CD4⁺TCRβ⁺ cells for various lymphoid tissues of age-matched SFB-positive and SFB-negative NOD mice. Cells were gated by side-scatter and as CD45⁺, CD19⁻, CD8⁻, TCRβ⁺, and CD4⁺. Ns, not significant.