Abstract
CD226, an activating receptor that interacts with the ligands CD155 and CD112, activates natural killer (NK) cells via its immunoreceptor tyrosine-based activatory motif (ITAM). There are two extracellular domains of CD226; however, the comparative functional relevance of these domains remains unknown. In this study, two different deletion mutants, rCD226-ECD1 (the first extracellular domain) and rCD226-ECD (full extracellular domains), were recombinantly expressed. We observed that rCD226-ECD1, similar to rCD226-ECD, specifically bound to ligand-positive cell lines and that this interaction could be competitively blocked by an anti-CD226 mAb. In addition, rCD226-ECD1 was able to block the binding of CD112 mAb to tumor cells in a competitive binding assay. Importantly, based on surface plasmon resonance (SPR), we determined that rCD226-ECD1, similar to rCD226-ECD, directly bound to its ligand CD155 on a protein chip. Functionally, NK cell cytotoxicity against K562 or HeLa cells was blocked by rCD226-ECD1 by reducing the expression of CD69 and granzyme B, indicating the critical role of ECD1 in NK cell activation. We also examined the role of rCD226-ECD1 in effector/target interactions by using rCD226-ECD to block these interactions. Using flow cytometry, we found that the number of conjugates between IL-2-dependent NKL cells and HeLa cells was reduced and observed that the formation of immune synapses was also decreased under confocal microscopy. In addition, we prepared two anti-rCD226-ECD1 agonistic antibodies, 2E6 and 3B9. Both 2E6 and 3B9 antibodies could induce the phosphorylation of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain.