Both in vitro and in vivo estradiol exposure promotes α-GalCer–induced IFN-γ production by iNKT cells. (A) E2 treatment in vitro enhances IFN-γ but not IL-4 production by α-GalCer–activated iNKT cells. After a first set of culture with IL-2, CD4⁺ spleen cells were incubated in the presence (▪) or not (□) of E2 (10^-8 M) for 20 hours, then harvested, and incubated with unloaded or α-GalCer–pulsed fixated APCs for an additional 60-hour period. IFN-γ and IL-4 concentrations in culture supernatants were expressed as means ± SEMs from 4 separate experiments. ^*P = .02, Mann and Whitney test. (B) E2 administration in vivo increases serum IFN-γ concentration induced by α-GalCer challenge. Placebo- (□) and E2-treated (▨) ovariectomized (Ovx) females received a single injection of α-GalCer (10 mice per group) or vehicle (5 mice per group) and were killed 90 minutes later. Serum IFN-γ and IL-4 concentrations were expressed as means ± SEMs. ^*P = .002. (C) E2 administration in vivo selectively promotes IFN-γ production by iNKT cells. Spleen cells from placebo- or E2-treated mice challenged with α-GalCer were analyzed for IFN-γ and IL-4 synthesis in the αβ-TCR⁺ NK.1.1⁺ cell gate by intracytoplasmic staining (bold line). Staining with irrelevant isotype-matched anti-IgG mAb was used as control (thin line). Histograms are representative of 3 separate experiments, each carried out with 2 mice per group. The proportion of IL-4– or IFN-γ–positive cells in the αβ-TCR⁺ NK.1.1⁺ gate was less than 0.5% in mice injected with vehicle alone.