Splenic IL-17–producing iNKT cells are Nrp-1⁺ RTE iNKT cells. (A-B) Mice were treated as in Figure 5B and iNKT-enriched spleen cell suspensions were prepared for FACS analysis of IFN-γ and IL-17 production in iNKT cells. (A) Representative dot-plot analyses of IFN-γ and IL-17 expression in splenic iNKT cells of mice treated either with control diluent (left) or α-GalCer (middle), and histogram overlay showing Nrp-1 expression in total (shaded gray), IFN-γ⁺ (dotted line), and IL-17⁺ (thick black line) iNKT cells. (B) Nrp-1 relative MFI of total, IFN-γ⁺, and IL-17⁺ iNKT cells (n = 4, pooled data from 2 distinct experiments, *P < .05, ***P < .001 by unpaired Student t test). (C) IL-17 concentration in culture supernatants of FACS-sorted splenic Nrp-1⁻ and Nrp-1⁺ iNKT cells after 3 days of in vitro activation with irradiated spleen cells and α-GalCer. (D) Absolute numbers of IL-17⁺ splenic iNKT cells in sham-thymectomized (sTx, light gray) and thymectomized (Tx, dark gray) mice assessed by FACS analysis after in vitro activation of iNKT-enriched cell suspensions with PMA and ionomycin (n = 3 for sTx and n = 4 for Tx, **P < .01 by unpaired Student t test). (E) IL-17 concentration in culture supernatant of total spleen cells from sham-thymectomized (sTx, light gray) and thymectomized (Tx, dark gray) mice after 2 days of in vitro activation with α-GalCer (n = 3 for sTx and n = 4 for Tx, **P < .01 by unpaired Student t test).