In light of the potential importance of the natural killer (NK) and killer (K) cell system in the first line of host defense against neoplastic growth and metastasis, as well as viral, bacterial and parasitic infections, it has been our long term objective to investigate ontogenic development and regulation of NK/K cells using the unique gnotobiotic and specific pathogen-free (SPF) miniature swine model. Germfree (GF), colostrum- deprived, immunologically """"""""virgin"""""""" piglets have absolutely no NK activity but have well-developed K cells mediating antibody-dependent cellular cytotoxicity (ADCC). The NK activity develops at two week sand three to four weeks of age in SPF and GF piglets, respectively. This unique gnotobiotic miniature swine model presents a unique opportunity for investigation of the development and regulation of NK cells. We have developed important monoclonal antibodies (mAbs) that enhance NK activity without affecting ADCC (PNK-E), enhance NK activity but block ADCC (G7), and inhibit NK activity without affecting ADCC (PNK-I). Utilizing these mAbs, we propose to investigate the following five specific aims: (1) Further characterization of porcine NK function-associated PNK-E and G7 molecules: determine cell and tissue distribution of PNK-E and G7 mAb enhanced NK activity and of PNK-E+ and G7+ cells; biochemical characterization of PNK-E and G7 molecules; (2) Characterization of the PNK-E/G7 molecular complex and define PNK-E and/or G7 as """"""""triggering"""""""" and/or """"""""signal transducing"""""""" molecules: establish molecular association of PNK-E and G7 on the surface of porcine LGL, granulocytes, monocytes and macrophages; determine the role of PNK-E and/or G7 molecules in the NK second messenger system; determine effects of various proteolytic enzymes on PNK-E and g7 molecules; (3) Determine mechanisms of enhancement of NK activity by PNK-E and G7 mAbs: further characterization of PNK-E and G7 mAb in """"""""redirected cytotoxicity"""""""" and """"""""reverse"""""""" ADCC; determine if stimulation of LGL with PNK-E and G7 mAb activates IL-2R expression and cytokine production; (4) Cloning and sequence analysis of genes encoding PNK-E and G7 molecules; and (5) Ontogenic development of NK cells: Ontogenic development of PNK-E+ and/or G7+ cells in GP piglets of various ages by two color flow cytometric analysis; ontogenic development of NK cell function and PNK-E and/or G7 mAb enhanced NK activity; and ontogenic development of mRNA expression of PNK-E and/or G7 by dot blot analysis using newly developed cDNA probes. These investigations on the cellular and biochemical characterizations of PNK-E and G7 molecules, defining their role in NK and ADCC function, defining the mechanism of enhancement/activation of NK activity by these mabs, and defining the ontogenic development of expression of these molecules in relation to ontogeny of NK and ADCC function may lead to the manipulation and/or regulation of this important host defense mechanism against neoplastic diseases as well as microbial and parasitic diseases.
