| Objective: Acute-on-chronic liver failure(ACLF) associated withHepatitis B virus (HBV) develops an acute liver damage from chronic liverdiseases, resulting in the dysfunction or decompensation of synthesis,detoxification, excretion and biotransformation, arising a group of clinicalsyndromes such as coagulation disorders and jaundice, hepatic encephalopathyand ascites. It develops fast with high mortality and a poor prognosis. Thepathogenesis of ACLF associated with HBV is very complex. Theunanimously approved conclusion is that it is caused by the interactionbetween viral replication and protein antigens with host immune responses.Host immune response dysregulation induced by HBV in certain conditionsplays a crucial role in its pathogenesis, which has became research focus inrecent years. Previous studies about liver failure mainly focus on theregulation of “immunity positive”, holding the view that the excessiveproliferation of reactive T cells breaks the balance of host immunity systemand reactive T cells’(especially HBV specific CD8~+T cells) persistentattacking target cells infected by HBV causes a large number of target cells togo degeneration and necrosis, meanwhile it triggers the apoptosismechanism of liver cells， resulting in massive hepatocytes apoptosis.Programmed death1(PD-1) and its ligand as a negative costimulatory signalcan inhibit the HBV specific CD8~+T cells from activation and proliferation,which plays an important part in regulating reactive T cells. There is fewrelevant report about the expression of PD-1in patients of ACLF associatedwith HBV. This study aims to explore the role of PD-1in pathogenesis andprognosis of ACLF associated with HBV by determining serum concentrationof interferon γ (IFN-γ) and interleukin10(IL-10), and the expression of PD-1on T cell subsets and virus-specific CTL cell in peripheral blood ACLF patients associated with HBV.Methods: Subjects in the experimental group are patients in the ThirdHospital of Hebei Medical University.26cases of ACLF associated withHBV chosen as ACLF group,20cases with chronic hepatitis B (CHB) asCHB group,10cases with healthy cases as normal control group. The patientsand healthy cases were all identified HLA-A2positive by flow cytometry.Then liver function, blood coagulation function, HBV serological markers andHBVDNA load were detected. The expression of PD-1on peripheral bloodCD3~+CD4~+T lymphocyte, CD3~+CD8~+T lymphocytes and HBV-specificCD8~+T lymphocytes were detected by flow cytometry. The consentrations ofserum IFN-γ, IL-10were determined by enzyme-linked immunosorbent assays(ELISA). Group comparisons and correlation analysis were made by StatisticPackage for Social Science (SPSS)13.0.Results:1The cell frequency of HBV specific CD8~+T lymphocytes with antigenicpeptide epitopes (HBcAg18-27) in different groups (%)The cell frequency of HBV specific CD8~+T lymphocyte in ACLF group,CHB group and control group were3.640±0.390,0.629±0.010,0.002±0.001respectively. The cell frequency in ACLF group was significantly higher thanthat in both CHB group and control group. That in CHB group was also higherthan control group. The differences had statistical significance (P<0.05).2The expression of PD-1on peripheral blood CD3~+CD4~+T lymphocytes,CD3~+CD8~+T lymphocytes, HBV specific CD8~+T lymphocytes in differentgroupsThe expression of PD-1on CD3~+CD4~+T lymphocytes and CD3~+CD8~+Tlymphocyte in ACLF group, CHB group and control group were32.647±2.489,12.643±0.783,7.132±3.990;23.937±1.317,13.375±0.913,7.718±4.147. The PD-1expression in ACLF group was significantly higherthan those in both CHB group and control group. The expression in CHBgroup was also significantly higher than that in control group. The differenceshad statistical significance (P<0.05). The expression of PD-1on HBV specific CD8~+T lymphocytes in ACLFgroup and CHB group were79.216±13.410,69.950±15.582respectively. Theexpression in ACLF group was higher than that in CHB group. The differencehad statistical significance (P<0.05).The ACLF group was divided into MELD≥20group and MELD<20group by MELD score. The expression of PD-1on CD3~+CD4~+T lymphocytes,CD3~+CD8~+T lymphocytes and HBV specific CD8~+T lymphocytes inMELD≥20group and MELD<20group were32.431±2.519,32.992±2.533;23.819±1.390,24.112±1.241;77.780±13.179,81.483±14.168. The PD-1expression on different cells in MELD≥20group had no statistical differences,compared with MELD<20group (P>0.05).3The correlation between the frequency of HBV specific CD8~+T lympho-cytes and its PD-1expressionIn ACLF group, the frequency of HBV specific CD8~+T lymphocytes waspositively correlated with its PD-1expression(r=0.453, P<0.05), while CHBgroup showed negative correlation (r=-0.684, P<0.01).4The correlation between the PD-1expression on HBV specific CD8~+Tlymphocytes and HBVDNAPD-1expression were positively correlated with HBVDNA in bothACLF group(r=0.695, P<0.01) and CHB group(r=0.543, P<0.05).5The correlation between PD-1expression on HBV specific CD8~+Tlymphocytes and clinical laboratory indexPD-1expression was positively correlated with TBIL (r=0.583, P<0.05),while there was no significant correlation with ALT(r=-0.265, P>0.05) andINR (r=0.123, P>0.05).6The comparison of serum IFN-γ and IL-10concentration in different groupsand its correlation with PD-1expressionSerum IFN-γ and IL-10concentration in ACLF group in ACLF group,CHB group and control group were39.61±25.93,21.79±9.48,15.99±7.17;31.95±23.74,13.39±7.10,5.67±1.28. Serum IFN-γ concentration were inACLF group significantly higher than those in control group. The difference had statistical significance (P<0.05). Comparision of ACLF group and CHBgroup, CHB group and control group had no statistical differences (P>0.05).Serum IL-10concentration was higher in ACLF group than that in both CHBgroup and control group. That in CHB group was also higher than that incontrol group. The differences all had statistical significance (P<0.05).IFN-γ/IL-10ratio in ACLF group was higher than that in CHB group andcontrol group, but no statistical differences (P>0.05). There was no significantcorrelation between serum IFN-γ and IL-10concentration and PD-1expression (P>0.05).Conclusions:1HBV specific CD8~+T cell frequency was significantly increased inperipheral blood of ACLF associated with HBV patients. It is suggested thatHBV specific CD8~+T cells play a crucial role in the immunopathologicaldamage of ACLF associated with HBV.2The expression of PD-1on CD3~+CD4~+、CD3~+CD8~+T cells and HBVspecific CTL in ACLF patients were significantly higher. The expression onHBV specific CTL was positively correlated with HBV specific CTL. It issuggested that PD-1plays an important role through regulating CTL inimmune response of ACLF associated with HBV patients.3Significantly increased serum IFN-γ and IL-10concentration andimbalanced ratio of IFN-γ/IL-10in the ACLF patients indicated that thereexisted immune disorders in ACLF patients. Cytokines play an important roletogether with other regulatory molecules including PD-1. |
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