The aim of this proposal is to identify cDNA clones corresponding to genes preferentially expressed in CD3- NK cells and other cells that express NK function and to understand the function of the corresponding gene products. A subtracted cDNA library enriched for cDNA clones associated with NK cells and NK/LAK activity will be prepared from a cloned NK cell (CD3-,CD16+,Leu19+) that expresses high-level NK/LAK function. A variety of screening methods will identify cDNA clones of interest within this library. Our first priority is to identify cDNA clones encoding cell surface molecules associated with NK cells and/or NK and LAK function. Second, we are interested in identifying clones encoding cell surface molecules expressed in both NK and T cells; this may identify molecules important in the regulation of NK function. Third, we are interested in clones that encode other molecules expressed preferentially in NK cells and other cells that express NK function. Two types of probes will be used to identify cDNA clones that encode membrane-bound components or secreted products. First, the subtracted library will be screened with a probe derived from poly-A+ RNA from membrane-bound polysomes of the cloned NK cell. Second, antisera prepared against the NK cell clone and absorbed with LCL will be used to probe the subtracted cDNA library prepared in a lambda-based expression vector. A long-term goal of this project is to use information and reagents obtained by molecular techniques to understand further the cell biology of NK cells, especially the generation and expression of NK/LAK activity. Whereas all genes to be studied will be derived from the NK clone described above, probing for expression of these genes in other cells, e.g. CD3+ cells with NK or NK and LAK function may help understand the genetic basis of these functions. Newly-defined genes will be sequenced and their expression studied in a variety of cell types; antisera/moAb will be made to proteins of interest. Although more difficult, we also plan to perform anti-sense (anti- mRNA) inhibition and transfection studies. We anticipate that these studies will provide information that will be useful as activated NK cells are used in clinical protocols.
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