TGFβ signaling is required for RORγt⁺ iNKT development. (A) Flow cytometry analysis of the presence of iNKT cell subsets in the thymus and pLNs of CD4-Cre; TgfbRII^flox/flox mice (TGFβRII°) and WT littermate control. Mice were analyzed at 2 weeks old, before the onset of T cell–mediated autoimmunity.^22,41  (B) Mean absolute numbers ± SEM of CD4⁻ RORγt⁺ and conventional RORγt⁻ iNKT cells from the thymus and pLNs. (C) Flow cytometry analysis of the expression of RORγt in CD4 single-positive (CD4 SP), DP, and total iNKT cells (iNKT) in the thymus. Invariant NKT cells were identified by excluding B220⁺ and CD8⁺ cells and gating on CD1d tetramer⁺ TCRβ⁺ cells. Representative plots and combined data of 6 experiments totaling 8 to 10 mice in each group. P values were calculated using an unpaired Student t test (*P < .05; **P < .01; ***P < .001).