CD226 protein is involved in immune synapse formation and triggers Natural Killer (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 of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain.
Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027.
Shengke
S
Hou
Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027.
Kuikui
K
Ge
Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027.
Xiaodong
X
Zheng
Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027; Hefei National Laboratory for Physical Sciences at the Microscale, Hefei, Anhui 230027, China.
Haiming
H
Wei
Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027; Hefei National Laboratory for Physical Sciences at the Microscale, Hefei, Anhui 230027, China. Electronic address: sunr@ustc.edu.cn.
Rui
R
Sun
Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027; Hefei National Laboratory for Physical Sciences at the Microscale, Hefei, Anhui 230027, China. Electronic address: tzg@ustc.edu.cn.
Zhigang
Z
Tian
D000595:Amino Acid Sequence; D000818:Animals; D000945:Antigens, Differentiation, T-Lymphocyte / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018958:CD55 Antigens / Q000378:metabolism; D016466:CHO Cells; D003412:Cricetulus; D003602:Cytotoxicity, Immunologic; D006367:HeLa Cells; D006801:Humans; D054992:Immunological Synapses / Q000378:metabolism / Q000502:physiology*; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology*; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance; D000098862:T Lineage-Specific Activation Antigen 1
D000945:Antigens, Differentiation, T-Lymphocyte; D000098862:T Lineage-Specific Activation Antigen 1; D018958:CD55 Antigens; D011994:Recombinant Proteins
Cell Biology; Cell Junctions; ERK; Natural Killer (NK) Cell; Phosphorylation