NKLAM--A NOVEL GENE REQUIRED FOR NK FUNCTION

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Natural killer (NK) cells are large granular lymphocytes which lyse tumor and virus-infected cells without prior sensitization. Their lytic activity can be enhanced 20-30 fold in vitro by incubation with interferons (IFNs) alpha and beta or interleukin-2 (IL-2). An understanding of this activation process would be facilitated by examining changes in gene expression that occur upon IFN or IL-2 augmentation of NK function. We have isolated 56 cDNA clones with elevated expression in IFN-beta-activated NK cells compared with unstimulated NK cells, by differential screening of an IFN-B induced NK cDNA library. 46 of these cDNA clones encode previously undescribed genes. One of these novel genes, designated NKLAM, is induced by IL-2 in peripheral blood NK and T cells, but is not expressed either unstimulated or IFN-beta-and shows no significant homology to known genes. Of great excitement is the finding that treatment of NK cells with NKLAM antisense oligonucleotides completely abolishes both unstimulated and IL-2 activated cytolytic activity. This directly implicates NKLAM in the lytic process. We now propose to: 1) Assess the functional role of NKLAM in NK and T cell-mediated lysis using antisense oligonucleotides and gene transfer. Antisense oligonucleotide-treated cells and NK and CTL lines expressing bote sense and antisense gen constructs will be assessed for changes in target cell binding and lysis, granule exocytosis and cytosine production. Antisera to the NKLAM protein will be produced in order to determine the location and structure of NKLAM. We will study NKLAM RNA expression in NK and T cell subsets from a panel of normal donors using a variety of cytolytic enhancers and inhibitors. The genomic homolog of NKLAM will be isolated in order to define its genetic structure and ultimately, to study its regulation. 2) Examine the expression of other unique genes in the library in cytotoxic cells by RNA blot analysis. cDNA of expressed genes will be sequenced. Antisense oligonucleotides will be used to identify those genes potentially involved in the cytolytic process. These genes will be further characterized as described for NKLAM. This work will provide insight into the molecule mechanism of augmentation of NK cytolysis, which will ultimately aid the clinician in using these cells therapeutically in the most effective way.