NIK deletion in myeloid cells results in reduced Il23a, which is partially responsible for the resistance against EAE. (A–F) CD11b⁺ cells were isolated from the dLNs of NIK^ΔCX3CR1 and littermate controls and were cultured with or without anti-(α)CD40 or LPS for 1 or 4 h. Relative mRNA levels (ΔΔCt) of (B) Il6, (C) Il1b, (D) Il12a/p35, (E) Il23a/p19, and (F) Il10, and mRNA levels of each sample were normalized against the housekeeping gene HPRT and the medium control of the corresponding cytokine. Dotted line = medium control. (G) NIK^ΔCX3CR1 and littermate controls 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 Rag−/− recipient mice. (H) Flow cytometry gating for CD44/GM-CSF and CD44/IL-17A of the donor cells before being injected into recipient mice, pregated on single/live/CD90⁺/CD4⁺ cells. (I) Passive transfer EAE disease course of recipients receiving control cells (Ctrl → Rag^−/−) or NIK^ΔCX3CR1 cells (NIK^ΔCX3CR1 → Rag^−/−). (J) AUC. (K) Day of onset. (L) Maximum disease score. Data in graphs are shown as the mean ± SEM and analyzed using two-way ANOVA with Šídák’s multiple comparisons test or two-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Each dot represents one mouse. Data are from at least two independent experiments. dpi = days after immunization. AUC, area under the curve.