Natural Killer (NK) cells are a subset of lymphocytes which display cytotoxicity against tumour and virally infected cells. This cytolytic activity is controlled by activating and inhibitory receptors. NK activating receptors include FcRgIII and NKR-PI. These belong to the immunoglobulin- (FcRgIII) or to the C-type lectin (NKR-P1) superfamilies and trigger NK cytotoxicity when bound to an immunoglobulin Fc or to a carbohydrate ligand on target cells, respectively. NK inhibitory receptors turn off NK cytotoxicity when engaged with an MHC class I molecule on a target cell.
We will take advantage of the participants' expertise in the molecular biology of NK to achieve the following:
First, we will perform molecular cloning of NK receptor genes from human chromosomes 19 and 12. This includes isolation of YAC and cosmid clones containing Ig-SF and C-type lectin NK receptor genes, molecular cloning of new genes and analysis of the genomic organization of the identified genes by long-range mapping.
Secondly, we will perform functional characterization of new NK inhibitory receptors. We will establish a correlation between clonal expression of NK receptor genes and MHC specificity of NK cell clones. NK cell clones will be established from HLA-typed donors. The specificity of these clones will be determined by testing their cytotoxicity against a panel of MHC class I deletion mutants each transfected with distinct class I genes. NK clones with new MHC-related specificities will be analyzed for the expression of NK receptor genes by RT-PCR. We will perform transfection of NK receptor genes in NK cell clones. The correlation between clonal expression of new NK receptor genes and the MHC specificity of NK cell clones will be verified by transfecting the NK receptor genes into NK cell clones and by studying the specificity of the transfected cells. Novel monoclonal antibodies dependent upon peptides within the class I molecule groove will be employed in the analysis. We will produce such reagents also against soluble NK receptors. Chimeric genes in which the extracelluar domain of NK inhibitory receptors fused with the Fc portion of immunoglobulines will be produced and expressed in mouse myeloma cells. The resulting soluble molecules will be used to identify ligand specificities of NK receptors. In addition, these soluble molecules will be used to immunize mice for the production of monoclonal antibodies.
Funding SchemeCSC - Cost-sharing contracts