Nrp-1⁺ RTE iNKT cells are biased toward IL-4 secretion after in vitro activation but contain few rapid IFN-γ and IL-4 producers. (A) IFN-γ (top) and IL-4 (bottom) concentrations 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. (B) Representative analysis of IFN-γ and IL-4 production in gated splenic Nrp-1⁻ and Nrp-1⁺ iNKT cells 2 hours after IP injection of α-GalCer in 8-week-old mice. The mean ± SEM percentage of cells in defined gates or quadrants is indicated in FACS dot plots (n = 4, pooled data from 2 distinct experiments). (C-D) Mice were injected IP with 2 μg of α-GalCer or control diluent without prior injection of brefeldin A and killed 2 hours later for analysis. (C) CD69 expression in splenic Nrp-1⁻ (blue) and Nrp-1⁺ (red) TCRβ⁺ CD1d Tet⁺ iNKT cells from control (shaded) and treated (bold line) animals. Three levels of CD69 expression (CD69⁻, CD69^int, and CD69^high) were determined, as indicated by the dotted lines. (D) Intracellular staining for IFN-γ and IL-4 in Nrp-1⁻ (blue) and Nrp-1⁺ (red) iNKT cell subsets separated according to CD69 expression levels. These data are representative of 3 distinct experiments. ND indicates not detected.