Less cytokine-producing T cells in secondary lymphoid organs before EAE onset. (A) Scheme of the adoptive transfer EAE model: Ly5.1 wild-type mice were immunized with MOG_35–55/CFA and PTx; isolated cells from dLNs and spleen were cultured for 4 days with MOG_35–55, IL-23, and anti-IFNγ before being injected into NIK^ΔCX3CR1 and littermate controls. (B and C) Passive transfer EAE disease course and (C) maximum EAE score of NIK^ΔCX3CR1 and littermate controls after adoptive transfer of MOG-activated wild-type T cells. (D–F) Experimental scheme: NIK^ΔCX3CR1 and littermate control mice were immunized with MOG_35–55/CFA and PTx, and cells were isolated from the spleen and dLN at 8 dpi and restimulated with MOG for 6 h. (E) Representative flow cytometry plots and total cell number of CD4⁺CD90⁺ T cells and (F) MOG-reactive (CD44⁺CD40L⁺) T cells. (G) Representative flow cytometry plots and percentages of IL-17A, GM-CSF, and IFNγ-producing T cells. (H and I) 2D2 T cells were transferred into NIK^ΔCX3CR1 and littermate controls 1 day before MOG_35–55 immunization. Six dpi, cells were isolated from the spleen and dLN and restimulated with PMA, ionomycin, and brefeldin A. (I) Representative flow cytometry plots of the spleen and the total cell number of transferred CD90.1⁺CD4⁺ 2D2 cells. (J) Percentages of 2D2 T cells that produce IL-17A, GM-CSF, or IFNγ in the dLNs and spleen and cytokine-producing CD4⁺ host cells, pregated on live/single cells. Data in graphs are shown as the mean ± SEM and analyzed using two-tailed unpaired Student’s t test or two-way ANOVA with Šídák’s multiple comparisons test. *P < 0.05, **P < 0.01, ****P < 0.0001. Each dot represents one mouse. Data are from at least three independent experiments. dpi = days after immunization.