Expression And Functions Of TIGIT On T Cells Of Patients With Chronic HBV Infection

Hepatitis B is an infectious disease caused by hepatitis B virus(HBV),which endangers human health worldwide.In the process of HBV infection,adaptive immunity response plays an important role in liver damage and virus clearance.Some studies have shown that activation of CD4+ helper T cells and CD8+ cytotoxic lymphocyte(CTL)in peripheral blood can effectively eliminate viruses in acute HBV infection.When chronic HBV infection occurs,the expression of inhibitory receptors on virus-specific CD8+ T cells gradually increases,which participates in T cells exhaustion,making it unable to effectively eliminate the virus,leading to the persistence of virus and liver damage.Blocking the inhibitory receptor pathway by antibody can reverse exhausted function of T cell and enhance T cell immunity,thus enhancing the ability of virus clearance.T-cell immunoglobulin domain and immunoreceptor tyrosine suppressor motif(TIGIT)is a new co-inhibitory receptor found in genomic research,mainly expressed on the surface of T cells,NK cells and Treg cells.In the area of virus infection immunity,TIGIT plays a key role in T cell immunity as a co-inhibitory receptor such as lymphocyte choroid plexus meningitis virus(LCMV),hepatitis C virus(HCV)and human immunodeficiency virus(HIV).There are few researchs on the role of TIGIT as an inhibitory receptor in hepatitis B virus infection.Recently,it has been reported that in HBs Ag-tg mice,TIGIT plays a role in hepatic HBV-specific CTL,leading to cellular immune tolerance,and HBV is hardly effective to be cleared.Blocking TIGIT pathway or inhibiting TIGIT expression can restore the function of HBV-specific CTL,leading to liver inflammation activation and hepatitis B virus clearance.Programmed cell death protein-1(PD-1)is also a key inhibitory checkpoint involved in T cell exhaustion.The expression and function of TIGIT as an inhibitory receptor in T cells of patients with chronic HBV infection and its synergistic effect with PD-1 have not been reported yet.This study mainly explored the expression and functions of TIGIT on T cells of patients with chronic HBV infection,and revealed the synergistic effect of TIGIT and PD-1 on T cell exhaustion in patients with chronic HBV infection.This study is divided into three parts:Part ? Expression of TIGIT on peripheral blood T cells of patients with chronic HBV infection Objective: To investigate the expression of TIGIT on peripheral blood T cells,T cell differentiation subsets,HBVcore18-27-specific CTL in patients with chronic HBV infection and its relationship with the expression of PD-1.Methods: Peripheral blood plasma and peripheral blood mononuclear cells(PBMC)were collected and isolated from 73 patients with chronic HBV infection and 28 healthy donors(HD)and frozen in liquid nitrogen.Flow cytometry was used to detect:(1)the expression of TIGIT and PD-1 on peripheral blood CD4+ and CD8+ T cells of patients with chronic HBV infection group and HD group.(2)The expression of TIGIT and PD-1 on different stages of CD4+ and CD8+T cells differentiation of patients with chronic HBV infection group and HD group.(3)HLA-A*0201/FLPSDFFPSV(HBV core 18-27)and HLA-A*0201/NLVPMVATV(CMVpp65)pentamer was used to detect HBV-and CMV-specific CTL,and then the expression of TIGIT and PD-1 on HBV-and CMVpp65-specific CTL of patients with chronic HBV infection was detected.Results:(1)Compared with HD group,the expression of TIGIT on CD4+ and CD8+T cells in chronic HBV infected patients group was significantly increased(12.28±0.93% vs.7.98±0.86%,P=0.0083;30.77±2.00% vs.16.61±2.17%,P=0.0001).(2)Compared with HBe Ag negative group,the expression of TIGIT on CD4+ and CD8+T cells in HBe Ag positive chronic HBV infections group was significantly increased(13.79±1.31% vs.10.19±1.17%,P=0.0130;32.94±2.84% vs.27.75±2.64%,P=0.0043).(3)Compared with HD group,the expression of TIGIT on T cells differentiation subsets in patients with chronic HBV infection group was up-regulated(all of them,P<0.05),and the highest expression of TIGIT was found in CD28-CD45RA+CD4+and CD28-CD45RA+CD8+T cells in patients with chronic HBV infection group(P<0.01,P<0.001).(4)The expression of TIGIT on CD4+ and CD8+ T cells was positively correlated with the expression of PD-1 in patients with chronic HBV infection.TIGIT and PD-1 were co-expressed on the surface of CD4+ and CD8+T cells.(5)Compared with CMV pp65-specific CTL,TIGIT and PD-1 expressed higher on HBVcore18-27-specific CTL,respectively(P<0.0001,P=0.0003).(6)Compared with PENTA-CD8+T cells,the expression of TIGIT and PD-1 on HBVcore18-27-specific CTL were higher(P<0.0001,P<0.0001).(7)The percentage of double positive expression of TIGIT and PD-1 on HBV core 18-27 specific CTL was different from that of single positive expression of TIGIT or single positive expression of PD-1 on HBV core 18-27 specific CTL(P <0.001),and the percentage of double positive expression of TIGIT and PD-1 was the highest.Conclusions:(1)TIGIT expression on CD4+ and CD8+T cells was up-regulated in patients with chronic HBV infection,and the expression of effector T cell subsets was the highest.The expression of TIGIT on T cells of HBe Ag positive chronic HBV infection group was higher than that of HBe Ag negative group.(2)TIGIT and PD-1 were co-expressed on CD4+ and CD8+T cells in patients with chronic HBV infection.(3)The high expression of TIGIT and PD-1 on HBV-specific CTL was observed in patients with chronic HBV infection,and the co-expression of TIGIT and PD-1 was the main expression state.Part ? Functions of TIGIT on peripheral blood T cells of patients with chronic HBV infection Objective: To investigate the effect of TIGIT on the cytokines secretion,proliferation and apoptotic sensitivity of T cell in patients with chronic HBV infection,and to explore the synergistic effect of TIGIT and PD-1 in vitro.Methods: Plasma and PBMC were collected and isolated from 85 patients with chronic HBV infection and frozen in liquid nitrogen.Flow cytometry was used to detect:(1)T cells were stimulated by phorbol 12-myristate-13-acetate/Ionomycin(PMA/Ionomycin),CD69 expressed on TIGIT+ and TIGIT-CD8+T cell population.Granulase B(Gr B)and perforin secreted by TIGIT+ and TIGIT-CD8+ T cell populations were detected by intracellular staining.(2)T cells were stimulated with HBVcore18-27-specific peptides.Flow cytometry was used to detect:(1)the expression of CD69 on TIGIT+ and TIGIT-HBV-specific CTL,the secretion of Gr B,Perforin,IFN-?,TNF-? and IL-2 from TIGIT+ and TIGIT-HBV-specific CTL.(2)cell proliferation was detected by carboxy-fluorescein diacetate succinimidyl ester(CFSE)test.(3)The apoptotic ratio of TIGIT+ and TIGIT-CD8+T cells in untreated group,activation induced cell death(AICD)group and blockade TIGIT pathway group(4)T cells stimulated by PMA/Ionomycin,IFN-?,IL-2 and TNF-? were secreted by TIGIT+PD-1+and TIGIT-PD-1-CD8+T cells.(5)Blockade TIGIT and/or PD-1 pathways,stimulation with HBVcore18-27-specific peptides,the secretion of IFN-? and IL-2 were secreted by HBV-specific CTL.Results:(1)Compared with TIGIT-CD8+T cell population,the expression of activation marker CD69 on TIGIT+CD8+T cell population increased significantly(P=0.0095)and the percentage of cells secreting Gr B and Perforin in TIGIT+CD8+T cell group decreased significantly(P=0.0205,P=0.0377).(2)Compared with TIGITHBV-specific CTLgroup,the expression of CD69 in TIGIT+HBV-specific CTL group increased significantly(P=0.0014)and the percentage of cells secreting Gr B and Perforin in TIGIT+HBV-specific CTL group decreased significantly(P=0.0360,P=0.0205).(3)Compared with TIGIT-HBV-specific CTL,and the percentage of cells secreting IFN-?,TNF-? and IL-2 in TIGIT+HBV-specific CTL of patients with chronic HBV infection decreased(P=0.0407,P=0.0446,P=0.0087)and proliferation decreased(P=0.0029).Blockade TIGIT pathway and HBVcore18-27-specific peptides stimulation increased HBV-specific CTL proliferation(P=0.0023).(4)Compared with the non-stimulation group,the apoptotic sensitivity of TIGIT+CD8+T cell group was increased after activation induction cell death(AICD)(P=0.0004).Compared with TIGIT-CD8+T cell group after activation induction,the apoptotic sensitivity of TIGIT+CD8+T cell group increased after AICD(P=0.0070).Blocking TIGIT pathway,the apoptotic sensitivity of CD8+T cells decreased(P=0.0003).(5)Compared with TIGIT-PD-1-CD8+T cells,and the percentage of TIGIT+PD-1+CD8+T cells secreting IFN-?(P<0.001),IL-2(P<0.001)and TNF-?(P<0.001)in patients with chronic HBV infection were significantly down-regulated.Compared with the control group,and the percentage of HBV-specific CTL secreting IFN-? and IL-2 in blockade TIGIT and/or PD-1 pathways were significantly higher(P<0.001,P<0.001).Compared with blocking TIGIT pathway alone,blockade TIGIT combined with PD-1 pathways significantly increased the percentage of cells secreting IFN-?(P<0.001)and IL-2(P<0.001)by HBV-specific CTL of patients with chronic HBV infection.Conclusions:(1)CD8+T cells and HBV-specific CTL activity were increased in PBMC of chronic HBV infected patients expressing TIGIT,but their function of releasing effector molecules was inhibited.(2)TIGIT could inhibit CD8+T cell cytokine secretion and cell proliferation,increase cell apoptosis sensitivity in patients with chronic HBV infection.(3)After blockade TIGIT pathway,the secretion of cytokines by CD8+T cell was up-regulated,cell proliferation was enhanced and cell apoptosis sensitivity was decreased in chronic HBV infected patients.(4)Blockade TIGIT and PD-1 pathways could reverse the secretion of cytokines in exhausted CD8+T cells.Part III The correlation between the expression of TIGIT on peripheral blood T cells and clinical parameters in patients with chronic HBV infection Objective: To explore the correlation between the expression of TIGIT on peripheral blood T cells,HBVcore18-27-specific CTL in patients with chronic HBV infection and clinical indicators.Methods: Plasma and PBMC were collected and isolated from 141 patients with chronic HBV infection and 20 HD and frozen in the liquid nitrogen.We tested the specimens as follows:(1)Alanine aminotransferase(ALT)and gamma-glutamyl transpeptidase(GGT)were detected by automatic biochemical analyzer.(2)The titers of hepatitis B surface antigen(HBs Ag)and HBe Ag were detected by chemiluminescence.(3)HBVDNA load was detected by real-time fluorescence quantitative PCR.(4)Flow cytometry was used to detect:(1)the expression of TIGIT and PD-1 on CD4+ and CD8+T cells.(2)the expression of TIGIT and PD-1 on HBVcore18-27-specific CTL.(3)the expression of TIGIT on CD4+ and CD8+T cells in chronic hepatitis B group,HBV-related liver cirrhosis group,HBV-related cirrhosis with hepatocellular carcinoma group and HD group.(4)TIGIT was expressed on CD4+ and CD8+T cells of patients with chronic HBV infection treated with lamivudine(LAM),adefovir dipivoxil(ADV),telbivudine(LDT),entecavir(ETV)group,and untreated with antiviral therapy group.Results:(1)TIGIT expression on CD4+ and CD8+T cells was positively correlated with ALT level and HBVDNA viral load logarithms,respectively.(2)TIGIT+PENTA+CD8+T cells were positively correlated with ALT level and HBVDNA viral load logarithms,while PD-1+PENTA+CD8+T cells were positively correlated with ALT level and HBVDNA viral load logarithms,respectively.(3)The double positive expression of TIGIT and PD-1 on CD4+ and CD8+T cells was positively correlated with ALT level and HBVDNA viral load logarithms,respectively.(4)The expression of TIGIT on CD4+ and CD8+T cells of chronic HBV infected patients treated with four nucleoside(acid)analogues was significantly lower than that of patients with chronic HBV infection utreated with antiviral therapy(P<0.001,P<0.001),and the lowest expression was found in ETV group.(5)With the progression of chronic HBV infection,the expression of TIGIT on CD4+ and CD8+T cells increased(P<0.001,P<0.001),and the co-expression of TIGIT and PD-1 on CD4+ and CD8+T cells also increased(P<0.001,P<0.001).Conclusions:(1)In patients with chronic HBV infection,viral replication and liver inflammation environment could induce up-regulation of TIGIT and PD-1 expression on T cells and HBVcore18-27-specific CTL,and the co-expression of TIGIT and PD-1 on T cells increases with the progression of the disease.(2)All four nucleoside(acid)analogues could inhibit the expression of TIGIT on CD4+ and CD8+ T cells of patients with chronic HBV infection.