CD94 identifies functionally distinct human CD56^dim NK-cell subsets with regard to IFN-γ production. (A left panel) Enriched NK cells were costimulated with IL-12 and IL-18 for 24 hours followed by intracellular staining for IFN-γ. After gating on CD56^dimCD3⁻ cells, density of CD94 surface expression and intracellular IFN-γ production were simultaneously assessed as shown in the representative dot plot. (A right panel) Equal numbers of the 3 NK-cell subsets were stimulated with IL-12 plus IL-18, followed by measurement of secreted IFN-γ using ELISA. (B) Relative comparison of each of the 3 NK-cell subsets (left) costimulated by IL-12 and IL-18 for 24 hours in ability to produce IFN-γ using intracellular flow-cytometric analysis (right). Cells on the right side of the dashed vertical line are positive for IFN-γ. The figures are representative of at least 3 separate experiments. (C) Equal numbers of the 3 NK-cell subsets were stimulated with IL-12 plus IL-15 for 24 hours, followed by measurement of IFN-γ mRNA by real-time PCR (left) as well as secreted IFN-γ by ELISA (right). Bar graphs (A,C) represent mean ± SD for at least 3 experiments. Enriched NK cells under no monokine stimulation were used as a negative control for IFN-γ ELISA assay (A,C).