First extracellular domain of CD226 in Natural Killer cells activation CD226 involves in immune synapse formation and triggers NK cell activation via its first extracellular domain

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 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M113.498253 The latest version is at JBC Papers in Press. Published on January 22, 2014 as Manuscript M113.498253 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on A uust 5, 2017 hp://w w w .jb.org/ D ow nladed from First extracellular domain of CD226 in Natural Killer cells activation of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain. Natural killer cells recognize and eliminate virus-infected or abnormally transformed cells via cytotoxic effects (1-3). Multiple receptors have been identified that play important roles in the biological functions of natural killer cells (4, 5). NK cell cytotoxicity is triggered by the cooperation of activating receptors and adhesion molecules (6), and activating signals activate NK cells to secrete cytotoxic granules (7, 8). CD226 is an activation receptor on NK cells and T cells. It was initially discovered by Burns and named T lineage-specific activation antigen (TLiSA) (also called DNAM-1). Previous studies have revealed that CD226 plays an important role in the NK cell-mediated cytotoxicity of tumor cells (9-14). Previously, CD226 was mainly considered to be an adhesion molecule that is involved in immune synapse formation in T cells and NK cells during cell cytotoxicity. During the formation of the immune synapse, CD226 transmits a signal and then induces the aggregation of LFA-1 (15-17). CD226 has two ligands, CD155 and CD112 (18), both of which belong to the immunoglobulin super family (IgSF) and are type I transmembrane proteins. Many members of the immunoglobulin superfamily have been reported to be involved in cell adhesion and activation in the immune system (19, 20). CD155 and CD112 have three IgV-like domains in their N-terminal extracellular regions. CD155 is also called the poliovirus receptor (PVR) and binds poliovirus via the first IgV-like domain at its N-terminus (21). CD112 is also known as nectin-2, a member of the nectin family, which is composed of four members, nectin-1 to -4. There are two extracellular domains of CD226 (22), and a recent structural study implies that CD226 binds its ligands via its first N-terminal Ig V-like domain (23); however, which extracellular domain is functionally important remains