Fig. 6

Treatment of EAE mice with MIC_Cop increases CD4⁺CD25⁺FoxP3⁺ cell infiltration of peripheral lymphoid organs and the central nervous system. The infiltration with CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) of peripheral lymphoid organs (a and b) and the central nervous system (c and d) of cell-treated and control EAE mice was determined with FACS analysis in SPCs and immunohistochemistry in spinal cord sections, respectively. a For FACS analysis SPCs of animals treated with PBS (control), MIC_Cop and UVC-SPC_Cop were isolated 4 weeks after cell therapy and stained with fluorescence-labeled antibodies and the corresponding isotype controls. One representative FACS-plot is shown for each group representing the expression of CD25 and FoxP3 in CD4⁺ cells. b Tregs within SPCs of each treatment group were quantified and are depicted as percentage of CD4⁺CD25⁺FoxP3⁺ T cells within the CD4⁺ T-cell population (mean value ± SEM; n = 8 per group). Data were statistically compared with the unpaired Student’s t test (*p < 0.05). c Immunohistochemistry of the lumbar spinal cord for evaluation of infiltrating Tregs was performed on snap-frozen tissue of PBS- and MIC_Cop-treated animals. After incubation of tissue sections with anti-FoxP3-antibody, Tregs were visualized with biotin-conjugated rabbit anti-rat IgG and Neutravidin-Dylight549. Cellular nuclei were counterstained with Hoechst 33,342 (blue). Digital fluorescence images were obtained at ×40 magnification and the number of infiltrated Tregs (pink) was determined. Scale bars depict 50 µm. d Statistical analysis of evaluated sections based on values from single animals (n = 8 for each group) was performed using Mann–Whitney U test (*p < 0.05). Mean values of FoxP3⁺ cells per mm² ± SEM are presented