Evaluation Of Host Immune Status And Research On TIGIT Molecule Regulating NK Cell Function In Chronic Hepatitis B Virus Infection

ObjectiveThis study was aimed to evaluate the host immune state in the stage of immune active and immune control of chronic HBV infection,as well as to investigate the expression of immunosuppressive molecule TIGIT（T cell immunoreceptor with Ig and ITIM domains）on the surface of NK cells and its role in regulating NK cell function in chronic HBV infection,so as to provide a new direction for the treatment of HBV infection.MethodIn this study,the heparin anticoagulant blood samples from patients in the immune-active（IA）stage and immune-control（IC）stage of chronic HBV infection,as well as healthy controls（HC）with age and sex matching in Tongji Hospital were collected.Flow cytometry was used to analyze the cell counts,phenotype and cytokine secretion capacity of peripheral lymphocyte subsets,as well as the expression levels of inhibitory receptors including Programmed death 1（PD-1）,T cell immunoglobulin domain and mucin domain-3（Tim-3）and TIGIT on the surface of CD4+T cells,CD8+T cells and NK cells.The immune status identification models between stage IA and HC,as well as stage IA and IC of chronic HBV infection were constructed by Logistic binary regression.Albumin-bilirubin（ALBI）score was used to evaluate the prognosis of hepatitis,Aspartate aminotransferase-to-platelet Ratio Index（APRI）were used to assess the degree of liver injury.The correlation between the expression level of inhibitory receptors on the surface of lymphocytes and lymphocyte function,HBV-DNA level,liver injury indexes were analyzed.The expression level of TIGIT on the surface of peripheral blood NK cells in IA group was analyzed by flow cytometry to explore its relationship with NK cell activation,cytokine secretion and cytotoxic activity.Non-specific stimulants and specific recombinant hepatitis B surface antigen（HBs Ag）and recombinant hepatitis B core antigen（HBc Ag）were administered in vitro.The cytokine secretion and cytotoxicity of NK cells were analyzed by flow cytometry.Anti-TIGIT antibody,recombinant TIGIT-Fc fusion protein and CD155-Fc fusion protein were used to observe the effects of blocking and activating TIGIT signaling pathway on the secretion of cytokines,degranulation ability and killing activity of NK cells.Results1.Compared with healthy controls,T lymphocyte counts,NK cell counts and NKT cell counts in peripheral blood of patients with chronic HBV infection were decreased.the percentage of CD28+CD8+T cells,central memory（CM）CD8+T cells,memory B cells（of lymphocytes）in IA group were decreased significantly,while the percentage of regulatory T（Treg）cells was increased.Compared with IC group,the percentage of CD4+T cells,CD28+CD8+T cells,EM（effector memory）CD8+T cells,memory B cells,unswitched B cells and NKT cells,as well as the ratio of CD4/CD8 were decreased,while the percentage of naive B cells and plasma cells were increased in CHB-IA group.Lymphocyte function tests found that,and the level of IFN-γsecreted by CD8+T cells and NK cells in CHB-IA group were decreased（P<0.05）.2.A five-index model for differentiating immune status between IA stage of chronic HBV infection and HC was established:P=1/[1+e-^{-6.290+0.138×CM CD4+T cells%+0.697×Treg cells%+0.256×Native B cells of lymphocytes%-0.459×CM CD8+T cells%-0.003×NK cell counts}],the sensitivity and specificity of the model were 79.59%and 87.50%.A four-index model for differentiating immune status between IA and IC of chronic HBV infection was established:P=1/[1+e-^{4.301-0.038×EM CD8+T cells%-0.046×CD28+CD8+T cells%-1.085×Memory B cells of lymphocytes%+17.414×Plasma cells of lymphocytes%}],the sensitivity and specificity of the model were 89.70%and78.79%.3.The expression levels of Tim-3 on CD4+T cells,CD8+T cells and NK cells（P<0.05）in peripheral blood of IA group were higher,while the expression levels of TIGIT on CD8+T cells and NK cells were lower than those in IC group,and the expression level of TIGIT on NK cells was lower than that in HC group.PD-1 and TIGIT levels on CD8+T cells were positively correlated with IFN-γsecretion level,respectively.PD-1 and Tim-3levels on CD4+T cells were significantly positively correlated with HBV-DNA levels,respectively.Tim-3 level on CD8+T cells was positively correlated with prognostic ALBI score,while TIGIT level on CD8+T cells and NK cells were negatively correlated with prognostic ALBI score（P<0.05）.4.The expression of TIGIT on NK cells was negatively correlated with APRI score,a non-invasive liver injury index.Compared with IC group and HC group,the activation level and cytotoxicity of NK cells in IA group were higher,but the reactivation and cytotoxicity potential after stimulation were lower.Compared with TIGIT+NK cells,TIGIT-NK cells had higher IFN-γsecretion capacity and cytotoxicity.IFN-γsecretion capacity and cytotoxicity of NK cells were increased by blocking TIGIT pathway,while down regulated by activating TIGIT pathway.Conclusion1.Compared to healthy controls,the lymphocytes number in chronic HBV infection patients was significantly reduced.The number of CD8+T cells and NK cells decreased and the function was impaired in IA group.The proportion of the activation and memory phenotypes decreased,while the proportion of Treg cells increased in IA patients.2.An effective immune model was established to distinguish between active and inactive HBV infection.3.TIGIT negatively regulates IFN-γsecretion and killing activity of NK cells in chronic HBV infection.TIGIT pathway might act as a protection factor in liver injury in chronic HBV infection.