NIK is dispensable for naive CD4 T-cell proliferation but is required for the induction of Th17-specific genes. (A) Naive CD4 T cells from NIK^+/+ and NIK^−/− mice were labeled with CFSE and stimulated for 72 hours with plate-bound anti-CD3 and anti-CD28 under Th0 or Th17 conditions. Cell proliferation was measured by flow cytometry and determined based on the dilution of CFSE during cell division. The intensity of CFSE is reduced to one-half after each cell division (indicated by numbers). Data are representative of 3 independent experiments. (B) Naive NIK^+/+ and NIK^−/− CD4 T cells were stimulated with plate-bound anti-CD3 and anti-CD28 (1 μg/mL) under Th0 or Th17 conditions for 18 hours. Real-time quantitative RT-PCR assays were performed to determine the relative expression of the indicated genes (fold to the NIK^+/+ Th0 sample). Data are representative of 3 independent experiments. (C) Naive NIK^+/+ and NIK^−/− CD4 T cells were either directly subjected to Th17 differentiation assays as described in panel B (left) or preinfected with pCLXSN(GFP) or pCLXSN(GFP)-NIK retroviral vectors (right). The infected cells were enriched by cell sorting based on GFP expression and then subjected to differentiation assays. Relative expression of IL-17A was determined by real-time PCR and presented as fold relative to NIK^+/+ Th0 sample (left panel) or to the Th0 GFP sample (right panel).