Figure 6. Tregs and IFN-γ production.

EAN mice were sacrificed at nadir of EAN (day 28 p.i.) and CE infiltrating cells were analyzed by flow cytometry. A. Gating for Treg in CE infiltrating cells was shown. R1 represents CD4⁺ T cells. R2 represents CD4⁺CD25⁺ cells, which were confirmed by FoxP3 staining. B. In TNF-α KO mice, the number of FoxP3-expressing cells (blue histogram) is slightly increased compared to WT mice (green). The red histogram denotes the staining with FITC-conjugated isotype antibody. C. The percentage of Tregs (CD4⁺CD25⁺FoxP3⁺) relative to CD4⁺ CE infiltrating cells was higher in TNF-α KO than in WT mice with EAN. D. Representative flow cytometric data show that infiltrating cells from TNF-α KO mice (blue histogram) expressed lower levels of IFN-γ than from WT mice (green). The red histogram denotes the staining with FITC-conjugated isotype antibody. E. The percentage of IFN-γ⁺ cells relative to CD4⁺ CE infiltrating cells was significantly lower in TNF-α KO than in WT mice with EAN. F. EAN mice were sacrificed at nadir of EAN (day 28 p.i.) and single cell suspensions of splenic MNCs were analyzed by flow cytometry. The percentage of IFN-γ expressing cells relative to CD4⁺ splenic MNCs was lower in TNF-α KO than in WT mice with EAN. Data are presented as mean value ± SD of one representative out of three independent experiments (n = 5 in each group). * p<0.05.