SMAD4-dependent signaling pathway controls survival of RORγt⁺ iNKT thymocytes. (A) Flow cytometry analysis of the presence of iNKT cell subsets in the thymus and pLNs of CD4-Cre; Smad4^flox/flox (SMAD4°) mice and WT littermate controls. Invariant NKT cells were identified by excluding B220⁺ and CD8⁺ cells and gating on CD1d tetramer⁺ TCRβ⁺ cells. (B) Mean absolute numbers ± SEM of RORγt⁺ and conventional RORγt⁻ iNKT cells from the thymus and peripheral lymph nodes (pLNs) of 6- to 8-week-old mice. Representative plots and combined data from at least 6 experiments totaling 8 to 10 mice in each group. P values were calculated using unpaired Student t test (*P < .05; **P < .01; NS indicates not significant). (C) RORγt⁺ iNKT cells costained for annexin V and RORγt in the thymus after 36 hours incubation. (D) Annexin V staining on CD4⁻ CCR6⁺ iNKT thymocytes (RORγt⁺ iNKT) and CD4⁻CCR6⁻ iNKT thymocytes (conventional iNKT). Annexin V data includes 4 experiments totaling 8 mice in each group. The 1-way ANOVA was used to determine overall significance between groups and P values were calculated using Bonferroni multiple comparison test. (*P < .05; NS indicates not significant).