Characterization of the cytolytic trigger molecules G7/PNK-E as a molecular complex on the surface of porcine phagocytes

G7 and PNK-E mAbs recognize distinct porcine NK cell and granulocyte function-associated molecules that enhance and induce a significant cytolytic response against tumor cell targets through a mechanism of redirected cytotoxicity. The present study shows that the G7 and PNK-E molecules are present on the surface of porcine neutrophils, monocytes, and pulmonary alveolar macrophages as a physically and functionally associated cytolytic trigger molecular complex. Two-color flow cytometric analysis demonstrates that most, if not all, neutrophils are G7 and PNK-E antigen positive. In contrast, monocytes and PAM contain both G7 and PNK-E positive as well as G7 positive and PNK-E negative subpopulations. mAb binding competition experiments indicate that pretreatment of phagocytes with G7 mAb can block subsequent binding by PNK-E mAb, suggesting that these antigens are physically associated on the surface of porcine phagocytes. Fluorescent co-capping experiments utilizing G7 and PNK-E mAbs clearly demonstrate a physical association between G7 and PNK-E antigens present on the surface of porcine neutrophils. Pretreatment of phagocytes with F(ab’)₂ fragments of G7 and PNK-E mAbs shows that F(ab’)₂ G7 mAb blocks subsequent induction of phagocyte-mediated tumor cell cytotoxicity by whole PNK-E mAb but pretreatment with F(ab’)₂ PNK-E does not block subsequent induction of phagocyte-mediated tumor cell cytotoxicity by whole G7 mAb. In addition, pretreatment of neutrophils and mononuclear phagocytes with F(ab’)₂ fragments of G7 mAb blocks subsequent whole PNK-E mAb-dependent activation of a phagocytic cell intracellular oxidative burst response but pretreatment with F(ab’)₂ PNK-E does not block subsequent G7 mAb-dependent activation of a phagocytic cell intracellular oxidative burst response. These data reinforce a physical and functional association between the G7 and PNK-E molecules. Recent identification of the G7 antigen as the porcine homolog of FcγRIII indicates that the G7 and PNK-E mAbs recognize a unique and previously uncharacterized FcγR cytolytic trigger molecular complex present on the surface of porcine neutrophils, monocytes, and PAM.