The objective of this proposal is to isolate and characterize genes encoding serine esterases (SEs) from human natural killer (NK) and cytotoxic T-lymphocytes (CTL) in order to investigate the role of these molecules in cellular cytotoxicity. The mechanisms by which NK cells and CTLs destroy their cellular targets is of fundamental importance for our understanding of cell mediated immunity. Cell killing involves contact and binding between killer and target cells followed in most instances by secretion of the contents of large cytoplasmic granules from mature cytotoxic lymphocytes. These granules contain several polypeptides with potential involvement in cytolysis including a pore-forming protein, perforin, another cytotoxin which is antigenically related to tumor necrosis factor and lymphotoxin, nucleolytic factor(s) as well as several different serine proteases (SEs). The functions of SEs in cytotoxic cells are unknown, but one possibility is that they may constitute an intracellular activation pathway for other cytotoxic molecules (e.g., perforin) analogous to that seen extracellularly with the SEs of the complement pathway. So far, as many as eight different but structurally related SEs have been isolated from murine cytotoxic lymphocytes, however, little if any information is available regarding the corresponding molecules in man. Thus we propose to: #1) Clone and characterize cDNA and genomic clones encoding SEs from human NK cells and CTLs; #2) characterize SE proteins using prokaryotic expression vectors, purify hybrid proteins and produce rabbit antisera; #3) Investigate expression of SE genes in human lymphocyte subsets, their differential expression during activation of cytotoxic and non-cytotoxic lymphocytes, and factors involved in the regulation of these genes; #4) Express individual SE in a biologically active form for detailed functional studies.
