CD48 inhibition is nonredundant with MHC class I and regulates NK cells from MHC class I-deficient mice and NK cells that lack self-MHC class I inhibitory receptors. (A) WT and 2B4^-/- NK cells activated with IL-2 were tested for lysis of CD48^-H-2b^- RMA-S, CD48⁺H-2b^- RMA-S, and CD48⁺H-2b⁺ RMA target cells. (B) IL-2-stimulated NK cells from WT and β2m^-/- mice were tested for lysis of CD48^-H-2b^- RMA-S, CD48⁺H-2b^- RMA-S, and CD48⁺H-2b⁺ RMA target cells. (C) Ly49C/I⁺ NKG2A/C/E⁺ and Ly49C/I^- NKG2A/C/E^- NK cells from polyI:C-treated B6 mice were sorted by MoFlo (DakoCytomation, Fort Collins, CO) for DX5-phycoerythrin-positive (PE⁺) and either Ly49C/I(clone 5E6)/NKG2A/C/E(clone 20d5)-fluorescein isothiocyanate (FITC)-positive or -negative NK populations resulting in 99% purity. PolyI:C stimulation in vivo was used instead of IL-2 stimulation in vitro, because IL-2 may reverse a potential hyporesponsive phenotype of these populations.12 NK-cell populations were tested for lysis of CD48⁺ and CD48^- RMA-S targets at an effector-target (E/T) ratio of 20:1. Error bars in all experiments indicate standard deviation from triplicate wells within 1 experiment. Data shown are representative of at least 3 independent experiments.