Study On The Immunomodulatory Of G-Protein-Coupled Receptor GPR54 On NK Cells And The Mechanisms

Natural killer（NK）cells are cytotoxic lymphocytes of the innate immune system.When the body is invaded by pathogens,NK cells are important component of first line of defense.NK cells express a variety of activating and inhibitory receptors,through which NK cells recognize target cells and respond immunologically.When NK cells recognize tumor cells and the infected cells,they exert a cytotoxic effect to induce target cell apoptosis.The cytotoxic effects of NK cells are mainly through the secretion of cytotoxic granules such as perforin,Gzm B,and INF-γ.Furthermore,NK cells can also secret tumor necrosis factor（TNF）and induce apoptosis of target cells through death receptors such as FAS.NK cells can also release chemokines and cytokines that recruit other immune cells to exert antitumor effects directly or indirectly.Since recognition of tumor cells by NK cells is not MHC restricted,NK cells can recognize tumor cells expressing lower amounts of MHC I.Based on this property,the research of NK cells has great potential in tumor immunotherapy.G protein-coupled receptors（GPCR）,as the largest family of membrane receptors,mediate a variety of cellular responses by sensing various signals in the extracellular environment.GPR54 receptor belongs to the rhodopsin family,the Gαq/11 subfamily,and its ligand is Ki SS-1,which is expressed in many tissues and organs in the body.Studies have shown that GPR54 receptor plays multiple significant functions in vivo.It is a key gene in the initiation of puberty,and it also plays an essential role in tumor initiation,progression,and metastasis.However,its role in anti-tumor immunity is poorly understood.In order to explore the role of Ki SS-1/GPR54 in anti-tumor immunity,we compared the proportion of NK and T cells in WT and GPR54^-/-mice and found that the loss of GPR54 receptor impaired the formation of immune cells in vivo.In addition,to further explore the role of GPR54 receptor in antitumor immunity,we constructed subcutaneous tumor models and found that tumors grew faster in GPR54^-/-mice.Then we analyzed the infiltration of immune cells in tumor tissues by flow cytometry,and found that the proportion of NK cell infiltration in tumor tissues of GPR54^-/-mice was reduced,and its function was also weakened,suggesting that GPR54 receptor may be related to the function of NK cells.Subsequently,NK cells were stimulated by IL-15 and the expression of GPR54 receptor was detected.It was found that the expression level of GPR54 receptor was significantly up-regulated after the activation of NK cells.In order to verify the effect of GPR54 receptor on cytotoxicity ability of NK cells,we designed and constructed the interference plasmid to down-regulate the expression level of GPR54 receptor in NK92 cell line and detect the changes in the killing ability of NK92 cells.Besides,NK cells from spleens of WT and GPR54^-/-mice were sorted,and NK cell killing capacity was examined.It was found that the killing capacity of NK cells was positively correlated with the expression level of GPR54 receptor.Furthermore,the mechanism analysis that kisspeptin-10/GPR54 signaling axis enhanced the ability of NK92 cells to kill tumor cells by activating the NF-κB signaling pathway.Finally,we demonstrated that the expression of GPR54 receptor can improve the anti-tumor metastasis ability of NK cells in vivo with mouse melanoma lung metastasis model.In summary,our results demonstrated that kisspeptin-10/GPR54 promotes the anti-tumor ability of NK cells and inhibits tumor metastasis in vivo by activating the NF-κB signaling pathway.