Anti-PD-1 Therapy Reverses TIGIT + CD226 + NK Depletion in Immunotherapy Resistance of Hepatocellular Carcinoma through PVR/TIGIT Pathway

Abstract Background: Immunotherapy combined with targeted therapy significantly improved the prognosis of patients with hepatocellular carcinoma (HCC). Immunotherapy resistance conducts the main reason for the failure of PD-1-based immune checkpoint inhibitors (ICIs). The gist of this study was to clarify the mechanism of Nature kill cells (NK) depletion in immunotherapy resistance of HCC. Methods: Cancerous /paracancerous tissues and peripheral blood (PB) of 55 patients with HCC treated in our hospital from Sep. 2019 to Sep. 2021 were collected. Patients were grouped according to the degree of differentiation, FCM, IHC, and in vitro lymphocyte culture drug intervention experiments were used to determine the degree of NK cell depletion. Furthermore, a novel mouse model of HCC in situ was constructed and divided into different groups according to the various intervention measures of ICIs. Immunofluorescence thermography was used to observe the changes in tumor burden for survival analysis. Results: NK cells in cancerous tissues significantly up-regulated the expression of TIGIT compared with paracancerous tissues (P < 0.001). FCM for PB indicated a more severe depletion of NK cells and higher expression of TIGIT and PD-1 in poorly differentiated HCC (P < 0.001). Intervention experiments in vitro revealed that expression of TIGIT and PD-1 decreased gradually with the increase of PD-1 inhibitor dose in moderately-highly differentiated patients (P < 0.05), while there was no change in poorly differentiated patients. Animal experiment showed that the proliferation of tumors in the experimental group was significantly inhibited compared with the control group after being blocked by PD-1 through immunofluorescence thermography, meanwhile, WB results of tumor tissues indicated that ICIs effectively decreased the expression of TIGIT, increased the expression of CD226, decreased the expression of PVRL1 protein and increased the expression level of PVRL3 protein. Conclusion: TIGIT + NK cells competitively bind to PVR with activator receptor CD226, thus promoting NK cell depletion. Anti-PD-1 efficiently decreases the expression of PVRL1 through the PD-1/PD-L1 pathway, promoting the independent endocytosis of PVRL3 and PVR binding, reducing the PVR/TIGIT inhibitory signal pathway, and enhancing the function of PVR/CD226 activation signal, which provides a theoretical basis for improving the immunotherapy resistance of HCC.