NIK-deficient T cells are defective in mediating EAE induction when adoptively transferred to Rag2^−/− mice. (A,B) T cells isolated from NIK^+/+ and NIK^−/− mice were mixed with B cells derived from NIK^+/+ mice and adoptively transferred into Rag2^−/− mice. One day after the cell transfer, recipient mice were immunized with MOG_35-55 peptide as described in Figure 1A. EAE incidence (A) and disease scores (B) were monitored daily. Mice transferred with NIK^+/+ and NIK^−/− donor T cells are indicated as filled circles and squares, respectively. (C) Rag2^−/− mice were transferred with the indicated donor T cells together with wild-type B cells. MOG_35-55-immunized mice were killed on day 14 or day 24. Splenic T cells were stimulated for 4 hours with PMA plus ionomycin and subjected to ICS and flow cytometry to determine the frequency of Th17 cells among CD4⁺CD44⁺ cells based on IL-17A production. Data are mean value of the indicated number of mice (each circle or square represents 1 individual mouse). (D) Rag2^−/− mice were transferred with the indicated donor T cells together with wild-type B cells. After 14 or 24 days of MOG immunization, flow cytometry was performed to determine the frequency of CD4⁺CD45⁺ cells infiltrating to the brain and spinal cord. Data are mean value of the indicated number of mice. (E) MOG-immunized Rag2^−/− recipients were killed on day 24 for isolating RNA from total CNS tissue. Real-time PCR was performed to determine the relative expression of the indicated genes as described in Figure 1F.