Journal of Immunology Research / 2015 / Article / Fig 6

Research Article

Infusion of Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate-Conjugated MOG35–55-Coupled Spleen Cells Effectively Prevents and Reverses Experimental Autoimmune Encephalomyelitis in Mice

Figure 6

MOG-SPs treatment induces MOG-specific CD4+ T cell response with increased MOG-specific Foxp3+ Tregs and decreased MOG-specific Th17 cells and Th1 cells. (a) Spleen cells from each group were labeled with CFSE; then CFSE-labeled spleen cells (5 × 105/well) were stimulated with MOG35–55 (10 μg/mL) for 4 days. Then, the cells were harvested and stained with anti-CD4-PerCp and anti-Foxp3-APC following the protocol from the manufacturer (eBioscience). Foxp3+ CD4+ T cells in the proliferating and nonproliferating CD4+ T cells were analyzed by flow cytometry in gated CD4+ T cells (Gate 1 and Gate 2, resp.). (b) The summary of Foxp3+ CD4+ T cells in proliferating CD4+ T cells of each group (10 mice/group) and statistical analysis data were shown. (c) Spleen cells from each group were labeled with CFSE; then CFSE-labeled spleen cells (5 × 105/well) were stimulated with MOG35–55 (10 μg/mL) for 4 days. Then, leukocyte-activation cocktail (0.7 μL/mL) (BD Bioscience) was added to the cultures for 4 h. Thereafter, the cells were harvested and stained for anti-CD4-PerCp and then stained intracellularly for IL-17 by anti-IL-17-PE. IL-17+ CD4+ T cells (Th17) in proliferating and nonproliferating CD4+ T cells were analyzed in gated CD4+ T cells (Gate 1 and Gate 2, resp.). (d) The summary of IL-17+ CD4+ T cells in proliferating CD4+ T cells of each group (10 mice/group) and statistical analysis data were depicted. (e) Spleen cells from each group were labeled with CFSE; then CFSE-labeled spleen cells (5 × 105/well) were stimulated with MOG35–55 (10 μg/mL) for 4 days. Then, leukocyte-activation cocktail (0.7 μL/mL) (BD Bioscience) was added to the cultures for 4 h. Thereafter, the cells were harvested and stained for anti-CD4-PerCp and then stained intracellularly for IFN-γ by anti-IFN-γ-PE. IFN-γ+ CD4+ T cells in proliferating CD4+ T cells were analyzed in gated CD4+ T cells. (f) The summary of IFN-γ+ CD4+ T cells in proliferating CD4+ T cells of each group (10 mice/group) and statistical analysis data were depicted. This experiment was repeated twice with similar results.
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