Ovariectomy leads to decreased IFN-γ production by iNKT cells. Female mice were ovariectomized or sham-operated at 4 weeks of age, then challenged with α-GalCer at 8 weeks of age, and killed 90 minutes later. Spleen cells were analyzed in the αβ-TCR⁺ NK.1.1⁺ cell gate for IFN-γ and IL-4 synthesis by intracytoplasmic staining (bold line). As controls, cells were stained with irrelevant isotype-matched anti-immunoglobulin G (IgG) mAb (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. MFI indicates mean fluorescence intensity.