Expression And Functional Research Of Activating Receptor CD226 On NK Cells

As first line of defense against invading organisms,the innate immune system provides an immediate and non-specific response to eliminate the pathogen.Natural killer(NK) cell is one of the important components of innate immune cells,which plays an important roles in immune response to viral infection and tumor cells.The immune response of NK cell has been regulated by both activating receptors and inhibitory receptors which expressed on NK cell surface.In recent years,the activating NK receptors have been investigated widely because of their various functions and recognition mechanisms.CD226 is an important activating NK receptor,which takes part in various immune responses of NK cell,including cell transfer,cytotoxic response to tumor cells and interaction with dentritic cells.CD226 was also determined to be concerned with different diseases.The functions of CD226 has been studied widely,however,the mechanism of recognition and activation of CD226 is still unclear.In this study,two different extracellular fragments of CD226 protein were acquired for further investigation.The fragment extracellular domain 1(ECD1) includes the first N terminal IgV-like domain,19-129aa.The fragment extracellular domain 2(ECD2) includes the first N terminal IgV-like domain,19-243aa.The two ECD proteins were interacted with CD226 ligands expressed on target cell surface, and then the recognition and activation of NK cell has been studied.In the following,we summarize the major results of our study and the conclusion.1.The expression of recombinant extracellular domain(ECD) of human CD226The two different extracellular fragments of CD226,CD226-ECD1 and CD226-ECD2 were expressed.After renaturation and purification,the purity of recombinant proteins was up to 95%.Further,a CHO-K1 cell line which expresses the soluble extracellular fragments of CD226 effectively and persistently was constructed for further investigation.2.Recombinant CD226-ECD reduced the NK cell cytotoxicity to tumor cellsBoth the ECD1 and ECD2 of CD226 could recognize and bind with the ligands expressed on tumor cell lines and block the binding of antibodies of these ligands. Recombinant CD226 could reduce NK cell cytolysis to tumor cell effectively,addition of the ECD1 only could decrease the NK92 cell cytolysis to tumor cell lines significantly,and the influence to cytolysis has no significant difference between ECD1 and ECD2 of CD226.Addition of recombinant protein could influence not the survival and proliferation of target cells,but the combination between the effect cells and target cells.This result indicated that soluble CD226 could reduce NK cell cytolysis to tumor cell effectively via the block of the interaction of NK cell and target cell,and suggest the ECD1 might the functional domain of CD226.3.CD226 induced NK cell activation via phosphorylation of ERK1/2After stimulated by CD226 mAb,the activation of NK cell was detected.The expression of CD69 was up-regulated,and secretes of cytokine and granzyme were increased on NK cell.Target cell K562 could also induced the activation of NK cell via phosphorylation of ERK1/2,and the ERK1/2 signal could be blocked by Recombinant CD226-ECD.This result indicated that CD226 could induce NK cell activation and ERK1/2 might be involved in this process.4.Rapid immunosorting of transmembrane proteins of lymphocytes from a cDNA expression library of COS-1 cellsThe proteins on lymphocyte surface play important roles in a wide range of immunological processes,but the profile and characterization of surface proteins remain to be further investigated,among which the method for fast screening of surface proteins needs to be established.In this study,we constructed a cDNA library of mouse hepatic lymphocytes for screening transmembrane proteins.To enrich these membrane proteins,an expression vector containing a signal peptide and a poly-His tag was constructed,which facilitated rapid immunodetection and fluorescence screening.COS-1 cells expressing these membrane proteins on their surface were sorted and highly enriched to facilitate easy screening of the cDNA library.