The Functional Study Of Nectin-related Immune Receptor PVRIG

In recent years,immunotherapies targeting immune checkpoints have achieved great success in the treatment of a variety of solid tumors.Many checkpoint inhibitors have been approved clinically efficient in treating solid and hematological tumors,and due to the contributions of checkpoint immunotherapy in the area,professor James Allison and Tasuku Honjo have been awarded the Nobel Prize in Physiology or Medicine in the year of 2018.With the advancement in clinical research,scientists are urged to find new immune checkpoints or new combinatorial strategies.As a recently discovered inhibitory receptor on T cells and NK cells,PVRIG binds to its ligand to inhibit TCR signaling,leading to the inhibition of T cell effector functions and T cell exhaustion,which results in the progression of tumors.However,whether PVRIG influences the anti-tumor immunity and exhaustion of NK cells,another important antitumor subset besides T cells,merits further research.In the present study,we assessed the role of PVRIG in the tumor microenvironment by generating a monoclonal antibody specifically blocking the interaction between PVRIG and its ligand,further,we evaluated the importance of NK cells in PVRIG-targeted immunotherapy.The results were mainly obtained in the following six aspects:1.Construction of PVRIG gene expression vector and expression of the fusion protein.We constructed pLVX-EF1?-mPVRIG lentiviral vector,pLVX-mPVRIG-hFc lentiviral vector and pLVX-mCD 112-hFc lentiviral vector respectively,and overexpressed mPVRIG protein,mPVRIG-hFc fusion protein and mCD112-hFc fusion protein in 293T cell line respectively.We then evaluated the purity and sequence obtained by protein G affinity chromatography,which proved that the mPVRIG-hFc fusion protein generated can be used for the preparation of monoclonal antibodies.2.Generation and screening of anti-PVRIG monoclonal antibodies.We used mPVRIG-hFc fusion protein to immunize rats,and then screened the supernatant of hybridoma cells and found that clone 12D9 successfully blocked the interaction between PVRIG and its ligand CD112.We then cultured the hybridoma cell,and purified monoclonal antibody 12D9 through protein G chromatograph of 12D9 hybridoma cell supernatant.We further verified the blocking function and specificity of monoclonal antibody 12D9,and determined the CDR sequence of 12D9 and the affinity of 12D9 binding to mPVRIG-hFc fusion protein.These results indicated that a blocking monoclonal antibody 12D9 was successfully prepared and screened,which could be used for the in vivo therapeutic experiments and provided a foundation for our study.3.PVRIG was highly expressed on tumor-infiltrating NK cells.To determine the importance of PVRIG-CD112 in the tumor microenvironment,we analyzed the TCGA database and found that CD 112 was highly expressed in various tumor tissues.In addition,IHC staining showed high CD 112 expression in the tumor tissues of colon adenocarcinoma patients,and high PVRIG expression on the surface of infiltrated lymphocytes.Furthermore,a positive correlation was observed between the gene expression of PVRIG and the gene expression of other immune checkpoints such as PDCD1,TIGIT,CD96,etc.PVRIG expression was negatively correlated to the survival prognosis of colon adenocarcinoma patients.Consistent with our results in patients,we also found high PVRIG expression on the surface of tumor-infiltrating NK cells in various mouse xenograft models,and PVRIG+tumor-infiltrating NK cells exhibited a more exhausted phenotype.The above results suggested that PVRIG was closely associated with the exhaustion of NK cells.4.PVRIG deficiency inhibits the exhaustion of tumor-infiltrating lymphocytes and tumor growth.In order to further investigate the effect of PVRIG on lymphocytes anti-tumor functions,we used subcutaneous tumor models constructed by Pvrig-/-mice and wildtype mice respectively,to compare the tumor growth in these two mouse strains.We found that deficiency of PVRIG significantly inhibited the tumor growth of various subcutaneous tumors and prolonged the survival of tumor-bearing mice.In addition,the effector functions of tumor-infiltrating NK cells and CD8+T cells were also restored.5.Blockade of PVRIG reverses the exhaustion of tumor infiltrating lymphocytes and inhibits tumor growth.We used PVRIG-specific blocking antibody 12D9 to intervene the subcutaneous tumor constructed by wildtype mice,and observed the tumor growth in tumor-bearing mice.We found that blockade of PVRIG significantly inhibited the growth of MC38 subcutaneous tumor and prolonged the survival of tumor-bearing mice.The therapeutic effect achieved by blocking PVRIG was mainly due to the reversed exhaustion of tumor-infiltrating NK cells and CD8+T cells,and the promotion of CD4+T cells differentiating into Th1 cells,thereby enhancing the systemic anti-tumor immune responses.In addition,combined blockade of PVRIG and PD-L1 resulted in better curative effects,which further inhibited tumor progression.6.NK cells and CD8+T cells are essential for the anti-tumor efficacy of PVRIG blockade.We depleted NK cells and/or CD8+T cells respectively in MC38 subcutaneous tumor model constructed by wildtype mice,and found that the therapeutic effect of PVRIG blockade was significantly impaired and tumor growth was significantly accelerated.In addition,PVRIG blockade significantly inhibited tumor growth in the subcutaneous tumor 1odel constructed by Ragl-/-mice(those that lack adaptive immune system),suggesting that the therapeutic effect of anti-PVRIG monoclonal antibody depends on the presence of NK cells and CD8+T cells,and more importantly,NK cells may respond to PVRIG blockade independent of the adaptive immune system.Conclusions:The inhibitory receptor PVRIG was involved in the exhaustion of NK cells,and the use of anti-PVRIG monoclonal antibody enhanced the anti-tumor immune responses by acting on both NK cells and CD8+T cells,in which the two cell subsets were both indispensable.Our study revealed the importance of NK cells in PVRIG-targeted immunotherapies and provided important theoretical foundation for PVRIG-targeted drugs in clinical application.