Figure 7. TSC1 ensures the iNKT-1/iNKT-17 dichotomy in part via by inhibiting ICOS expression.

(A) iNKT-17 cells are confined to the ICOS⁺ subset. CD1dTet-enriched WT thymic iNKT cells were stimulated with PMA and ionomycin for 5 hours in the presence of GolgiPlug followed by TCRβ, ICOS, NK1.1, CD4, and intracellular IFN-γ and IL-17A staining. Dot plots show IFN-γ or IL-17A and ICOS expression in live-gated CD1dTet⁺TCRβ⁺Lin^– (CD8, CD11b, CD11c, B220) iNKT subsets. (B) ICOS⁺ and ICOS^– iNKT cells in WT and TSC1KO thymi. (C) Expression of IFN-γ and IL-17A in ICOS⁺ and ICOS^– iNKT subsets from WT and TSC1KO thymi. (D and E) Overexpression of ICOS increases IL-17 production by iNKT cells. Thymocytes from WT mice were similarly stimulated with α-GalCer in vitro, infected with retrovirus expressing either GFP or GFP plus ICOS, and analyzed for intracellular cytokine expression, as in Figure 6F. (D) Dot plots show IFN-γ and IL-17A expression in GFP⁺CD1dTet⁺TCRβ⁺ iNKT cells. (E) Percentages of IFN-γ⁺, IFN-γ⁺IL-17A⁺, and IL-17A⁺ cells in GFP⁺ iNKT cells (mean ± SEM, n = 4). (F and G) Absence of ICOS partially reverted the iNKT-17 predominance over iNKT-1 caused by TSC1 deficiency. iNKT cells enriched from thymi of indicated mice were similarly stimulated by P + I and analyzed as in A. (F) Contour plots show IFN-γ and IL-17A expression in live-gated CD1dTet⁺TCRβ⁺Lin^– iNKT cells. (G) IL-17A⁺ to IFN-γ⁺ ratios in iNKT cells (mean ± SEM; n = 3). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t test (E) or 1-way ANOVA (G). Data shown represent 3 independent experiments.