SMAD4-dependent signaling is required for IL-17 production and proliferation of RORγt⁺ iNKT cells during an inflammatory response. Ex vivo intracellular flow cytometry analysis of IL-17 producing RORγt⁺ iNKT cells in the pLNs of CD4-Cre; Smad4^flox/flox (SMAD4°) and WT littermate control mice 10 to 16 weeks old. (A) Representive flow cytometry plots of the percentage of IL-17⁺ RORγt⁺ iNKT cells in SMAD4° and WT mice with and without PMA/ionomycin stimulation. Invariant NKT cells were identified by excluding B220⁺ and CD8⁺ cells and gating on CD1d tetramer⁺ TCRβ⁺ cells. (B) The mean percentage ± SEM of RORγt⁺ iNKT cells that produce IL-17. Combined data of 3 experiments. P values were calculated using a paired Student t test (*P > .05). (C) Number of RORγt⁺ and (D) percentage of Ki67⁺ RORγt⁺ iNKT cells in the draining inguinal lymph nodes with and without subcutaneous injection of CFA at the base of the tail. Invariant NKT cells were identified as previously described. One experiment representive of 3 to 5 unimmunized mice and 6 to 8 immunized mice of each genotype in total, 10 to 16 weeks old. Mean ± SEM are shown. The 1-way ANOVA was used to determine overall significance between groups and P values were calculated using Bonferroni multiple comparison test. (P < .01; ***P < .001; NS indicates not significant).