Fig. 3

ALX impairs antigen-specific CD4+ T cell responses and allogeneic T cell proliferation. MOG-loaded BMDCs were stimulated with LPS (1 μg/ml) alone or together with the indicated doses of ALX for 48 h. Different groups of cells were cultured with splenic T cells at the indicated ratios for 3 days. The effect of ALX on TBK1, IRF3, and AKT expression and phosphorylation in the different groups of cells was determined by western blot assays. Similarly, splenic DCs from C57BL/6 mice were loaded with MOG antigen peptide and stimulated with LPS in the presence or absence of ALX at the indicated dose for 2 days. Some cells were pre-treated with mitomycin for 30 min. The different groups of DCs were co-cultured with splenic CD4⁺ T cells from MOG-immunized mice at 1:5 for 4 days. The frequency of CD4+CD25+ and CD4+CD69+ T cells was determined by flow cytometry, and the levels of IFNγ and IL-2 in the supernatants of co-cultured cells were measured by ELISA. The proliferation of T cells was determined by CCK-8 assay. a Flow cytometry analysis of CD4+CD25+ and CD4+CD69+ T cells. b Quantitative analysis of activated T cells. c The levels of IFN-γ and IL-2. d Proliferation of antigen-specific CD4+ T cells. e BMDC-stimulated allogeneic T cell proliferation. f Western blot analysis of TBK1, p-TBK1(S172), IRF3, p-IRF3(S396), AKT, and p-AKT (S473 and T308) expression following co-cultured with allogenic cells. ^※p < 0.05, *p < 0.01, ^#p < 0.001 the cells treated with LPS alone