Natural killer (NK) cells are capable of lysing both virally infected cells and tumor cells by a mechanism termed spontaneous cytotoxicity. NK cell spontaneous cytotoxicity differs from T cell cytotoxicity in two important ways: NK cells do not require prior exposure to target cell antigens and the specificity of killing is not restricted by MHC antigens. Both the receptors on NK cells as well as the target cell ligands responsible for this killing are unknown. Ly-49, a type II integral membrane protein with homology to the C-type lectin superfamily, is selectively expressed on a subset of murine NK cells. Functional studies indicate the Ly-49 interacts with H-2 Dd on target cells leading to global suppression of NK cytotoxicity. My research demonstrates that ly-49 binds to h-2 Dd directly. We constructed a cell line expressing Ly-49 at high levels by the use of gene amplification techniques. This cell line is able to specifically rosette target cells expressing H-2Dd. To quantitate this result a cell-cell binding assay was developed. The Ly-49high cell line bound Dd targets specifically. Mabs had no effect. This is the first demonstration of a receptor selectively expressed on NK cells binding a known target cell ligand with functional significance. I plan to investigate the molecular requirements for the binding of Ly-49 to H-2Dd, focusing on the role of carbohydrate, cation and processed peptide. Since Ly-49 has homology with the C-type lectin superfamily carbohydrate may be involved. To test this hypothesis I will employ several approaches, including neuraminidase digestion, competitive monosaccharides and inhibitors of glycosylation. I will determine the need for divalent cations using divalent cation-free media supplemented with EDTA. I will assess the effects of these treatment with the cell-cell binding assay. Processed peptide may participate in the interaction of Ly-49 to H-2Dd because mAbs to its alpha1, alpha2 domain can block binding. To examine the role of peptides. I will use embryonic mouse cells that express empty class I antigens on the cell surface. Class I antigens complexed with peptide can be produced by incubating the cells in media that contains Dd specific peptides. I will compare the ability of empty Dd and Dd complexed with peptide to bind Ly-49 using the cell-cell assay. A second complementary approach will use affinity-purified class I antigens absorbed onto latex microbeads.
