PKC-θ is required for ITAM-dependent IFN-γ secretion. PKC-θ^−/− and WT splenocytes were stimulated ex vivo with plastic-coated anti-NK1.1 and anti-Ly49D either in the absence (A) or in the presence (B) of suboptimal concentrations of IL-12 and IL-18. Intracellular IFN-γ content was measured by FACS, gating on DX5⁺CD3⁻CD19⁻ NK cells. (C) IL-2–cultured PKC-θ^−/− and WT NK cells were stimulated with plastic-coated anti-NK1.1, anti-NKG2D, and anti-Ly49D. After 16 hours of stimulation, cell culture supernatants were assayed by cytometric beads array for IFN-γ and TNF-α production. (D) PKC-θ^−/− and WT splenocytes were stimulated ex vivo with PMA (50 ng/mL) and ionomycin (5 μg/mL). IFN-γ production was measured in NK1.1⁺CD3⁻CD19⁻ NK cells by intracellular staining. Results are representative of at least 6 independent experiments.