Previous attempts during the past two decades to identify recognition structures exclusive to NK cell–tumor interaction have been unsuccessful (reviewed in reference 8
), though important components on both NK cells and on tumor cells that contribute to cellular adhesion and regulation of the cytolytic process have been revealed (5
). However, these receptor–ligand interactions do not appear to be unique to NK cells, because they also occur between T lymphocytes and respective target cells (10
). The limited number of NK cells available for biochemical studies (<5% in peripheral blood) has undoubtedly contributed to the difficulty in analysis of NK cell–specific receptor–ligand interactions. Many investigators have addressed this issue by the expansion of freshly isolated NK cells in vitro using a variety of culture systems employing growth factors and/or feeder cells (11
). Such techniques have provided sufficient numbers of cells for immunologic and biochemical studies but at the expense of altering the phenotype of the freshly isolated NK cells from a naive state to an activated one, as demonstrated by induction of the Lag 3 protein (17
). It is possible that activation results in decreased expression of naive NK cell recognition structure(s), thereby preventing their detection. In the present study, several strategies were used to overcome the difficulties of heterogeneity and the low frequency of NK cell in HPBL preparations. Only freshly isolated NK cells were used. Cells from individuals that demonstrated activity against NK-resistant LAK-sensitive tumor cell lines were not used, because the activated NK cells in these fresh preparations might mask the detection of naive NK cell–specific ligands. Finally, a tagged ligand–cell adsorption technique (13
) was used with enriched NK cell preparations to enhance the likelihood of detection of tumor proteins that selectively bound to the naive killer cells.
Using the above approaches and additional biochemical and immunologic techniques, we demonstrate a novel 38.5- kD protein on the plasma membrane of certain tumor cell lines that preferentially reacts with a surface component of naive human NK cells. The interaction appears to be unique to NK cells, because T lymphocytes did not bind p38.5. However, binding studies have not been conducted with B cells, monocytes, or polymorphonuclear leukocytes. In further experiments, a 70-kD protein on the plasma membrane of NK cells was identified as a p38.5 binding molecule. Consistent with p38.5 cellular binding studies, a 70-kD receptor was not detected on T lymphocytes. Addition of purified p38.5 to fresh lymphocyte preparations before incubation with K562 target cells substantially decreased NK cell lytic activity. Preferential interaction of p38.5 with NK cells and its blocking activity in functional assays are properties consistent with a role in an early recognition event in NK cell–mediated tumor cytolysis.
Additional evidence of a role for p38.5 as a target ligand in naive NK cell–mediated cytotoxicity is provided by data demonstrating an association of the expression of this molecule and susceptibility to cytolysis of different tumor cell lines. Flow cytometry and immunoprecipitation studies (of surface labeled cells) revealed that p38.5 is expressed on NK susceptible targets such as K562, MOLT-4, and Jurkat, whereas this molecule was not detected on the plasma membrane of NK-resistant LAK-sensitive targets such as Raji, A549, and MDA-MB-231, suggesting that p38.5 is not involved in LAK-mediated cytotoxicity. The functional role of p38.5 in NK cell–mediated cytolysis was also demonstrated in studies of p38.5 loss variants. After long-term culture of wild-type, NK-sensitive Jurkat and Molt-4 cell lines, variants were isolated that exhibited decreased levels of p38.5 and reduced susceptibility to lysis by NK cells. This property was not due to a phenotypic alteration in the cells as a result of culture conditions, because resistant clones were obtained at limiting dilution (Norin, A.J., unpublished data). These studies clearly establish a strong association between the expression of p38.5 on the tumor plasma membrane and susceptibility to NK cell–mediated cytolysis.
Recent studies suggest that cytolytic activity is affected by recognition of HLA class I polymorphisms on target cells by NK cell receptors of the C-lectin family (CD94) (18
) or members of the immunoglobulin multi gene family (p50/ p58) (19
). These receptors, which we note are detected on both T cells as well as NK cells, may downregulate or upregulate cytolytic activity (10
) depending on the subtype of receptor and/or the presence of cytosol associated clonotypic signaling molecules (18
). Inhibitory receptors apparently interfere with proximal signaling events such as Ca2+
flux, phospholipase Cγ2 activity, and inhibition of specific tyrosine kinases activities (lck and ZAP-70), whereas the activity of other tyrosine kinases may remain unchanged (18
). Experimental results in the current study are likely not influenced by the above families of molecules, because K562 cells do not express HLA class I or class II molecules (11
Current concepts regarding the mechanism of lymphocytemediated cytolysis have focused on three alternative pathways: (a
) Fas–FasL-induced apoptosis of tumor cells (28
) extracellular ATP-mediated osmotic lysis (13
), and (c
) granule exocytosis of effector molecules such as perforin and granzymes (29
). Clearly, NK cell–induced cytolysis is not mediated by Fas–FasL interaction, because K562 tumor cells do not express Fas and naive NK cells do not express FasL (41; Das, B., unpublished data). Previous reports by us and others have suggested that NK cells use the ATP–osmotic lysis pathway (13
) and the granule exocytosis pathway (28
). Further studies are likely to reveal which lytic pathway is mediated by the interaction of tumor surface ligand p38.5 with NK cell plasma membrane proteins.