NIK knockout mice are resistant to EAE induction and defective in Th17 differentiation. (A) Age- and sex-matched NIK^−/− and NIK^+/+ mice (7 +/+ and 5 −/−) were immunized with MOG_35-55 peptide on day 0 and day 8 and monitored daily for EAE disease symptoms. Data are representative of 3 experiments. (B,C) MOG_35-55-immunized mice were killed on day 14. Splenic T cells were stimulated for 4 hours with PMA plus ionomycin and subjected to ICS and flow cytometry to determine the frequency of Th1 and Th17 cells among CD4⁺CD44⁺ cells based on their production of IFN-γ and IL-17A, respectively. Data are presented as a representative flow cytometry graph (B) and mean value of multiple mice (each circle or square in this and all the subsequent figures represents an individual mouse). (D,E) MOG_35-55-immunized mice (7 NIK^+/+ and 5 NIK^−/−) were killed on day 35. The frequency of Th1 and Th17 cells in the spleen was analyzed as described in panels B and C. (F) Splenic T cells from day 35 MOG_35-55-immunized mice were stimulated for 4 hours with PMA plus ionomycin. RNA was prepared from the cells and subjected to real-time PCR assays to detect the relative expression of the indicated genes (fold relative to one of the NIK^+/+ samples). Data are representative of 2 experiments and are presented as mean value of multiple (7 NIK^+/+ and 5 NIK^−/−) mice.