Double restriction in NK cell recognition is linked to transmethylation and can be triggered by asparagine-linked oligosaccharides on tumor cells.

The determinants of tumor cell susceptibility to NK cell-mediated cytolysis were analyzed in a two stage model. The binding of tumor cells to NK effectors was measured by target-effector conjugation and cold target competition in 51Cr-release assays, whereas triggering was measured by assaying phospholipid methylation in NK cells stimulated by intact targets. Representative targets could be grouped into three phenotypes based on the data. Those such as YAC 1.2 could bind and trigger NK cells whereas the mutagenized variant, YAC 6.28.8, could bind but was unable to trigger NK cells and therefore resisted lysis. The third phenotype was represented by HL-60 which could neither bind nor trigger NK cells and was therefore completely NK resistant. The oligosaccharide nature of the triggering molecules was demonstrated by showing that purified, high mannose containing, asparagine-linked oligosaccharides from tumor cell targets were potent stimulators of NK transmethylation at submicromolar levels. Tunicamycin pretreatment of target cells inhibited their triggering capacity but not their NK binding function. These results suggest a double restriction in NK specificity involving two independent but sequential stages in recognition represented in binding and triggering by asn-linked oligosaccharides on the tumor cell surface.