HBV Specific CD4~+T Cells Are Associated With Virus Clearance And Liver Damage In Chronic Virus Infection

According to the report of World Health Organization,There are about 240 million people who are carriers of HBV surface antigen(HBsAg)in the world.There is a significant regional difference between low level(2%)and high level(8%)of patient who are positive HBsAg.Some studies have reported that about 2%~6%of patients with chronic hepatitis B(CHB)patients develop into cirrhosis and hepatocellular carcinoma every year and almost 650,000 people die of chronic hepatitis B every year.Chronic HBV infection is also the most important chronic infectious disease in China.There are about 93 million people who are chronic HBV carriers in China,and most the patients are transmitted from mother or from childhood.Chronic hepatitis B is a major disease in China.Liver cirrhosis and hepatocellular(HCC)can be induced easily by chronic liver inflammation.Short-term fulminant inflammation of hepatitis B can lead to HBV related acute-on-chronic liver failure(ACLF),which is usually defined as acute liver injury based on chronic liver disease in China and asia-pacific region.ACLF is accompanied by systemic cytokine storm in the early stage and this disease develops rapidly with a high short-term mortality rate.The mortality of ACLF is more than 30%within 28 days.The pathogenesis of chronic hepatitis B is complicated and has not been fully elucidated.HBV virus is a kind of hepatotropism virus which don’t directly cause liver damage.Some studies show that HBV specific CD8~+T cells play a leading role in liver damage and HBV virus clearance.On aspect,CD8~+T cells can secrect IFN-γand TNF-αwhich mediate a virus clearance of noncytotoxic degradation.On the other hand,CD8~+T cells can directly mediate the liver damage and HBV clearance through Perforin-granulase and Fas-FasL pathways.The role of CD4~+T cell is not yet clear in liver damage although it is an important helper T cell.A clinical research about acute limited hepatitis B finds strong HBV specific CD4~+T cell response.These researches suggest that HBV specific CD4~+T cells may be involved in self-limitted HBV clearance in acute HBV infection.Our group have conducted the genome-wide association study(GWAS)on HBV-related ACLF in the early stage,and analyzed the influence of host genetic variation on the aggravation of hepatitis B on the perspective of the whole genome.The results show that the rs3129859 is significant correlation locus associated with HBV,especially the HLA-DRB1*1202 allele is the most significant risk associated with HBV related ACLF,while the HLA-DRB1*0803 allele is the protective allele associated with HBV related ACLF.This result suggests that HBV specific CD4~+T cells may be involved in the liver injury in HBV infection patients.Peripheral blood of patients with chronic HBV infection and PBMCs are isolated and purified in this study.We stimulate PBMCs by HBsAg and HBcAg derived peptides and analyze HBV specific CD4~+T cell response of IFN-γand TNF-α.In this research,we analyze HBV specific HBV specific CD4~+T cell response of 33 chronic HBV infection patients with no/low inflammation chronic HBV infection(ALT<2×ULN)and15 hepatitis B flare with high inflammation(ALT>5×ULN).In addition,patients with HLA-DRB1*0803 and HLA-DRB1*1202 genotype are used to identify CD4~+T cells epitopes.Our findings are as follows:1.199 patients with no/low inflammation(ALT<2×ULN)diagnosed as chronic HBV infection were collected in our outpatient department of Southwest hospital from January 2016 to February 2019.We selected 33 samples from these 199 samples to analyze HBV specific CD4+T cell response.33 samples include 19 HBeAg positive patients,7 HBeAg sero-conversion patients and 7 HBsAg sero-conversion patients.In addition,HBV specific CD4+T cells response of 15 hepatitis B flare patients(ALT>5×ULN)are analyzed,including 11 HBeAg-positive patients and 4HBeAg-negative patients.06#,09#,10#,11#,15#patients are collected at several time points.2.TNF-αsecreted function of HBV specific CD4+T cells is relatively preserved in chronic HBV infection patients with no/low inflammation compare to IFN-γsecreted function of HBV specific CD4+T cells.HBV Core specific TNF-α~+CD4~+T cells are detected in 52%(17/33)the patients,while HBV Core specific IFN-γ~+CD4~+T cells are detected in 15%(5/33)the patients.The mean and standard deviation of HBV Core specific TNF-α~+CD4~+T cells frequency is 0.202%and 0.451%,while the mean and standard deviation of HBV Core specific IFN-γ~+CD4~+T cells frequency is 0.032%and0.106%,with statistically significant difference(p=0.013).HBV S specific CD4~+T cell response is similar to that of HBV Core specific CD4~+T cells.HBV S specific TNF-α~+CD4~+T cells are detected in 52%(17/33)the patients,while HBV S specific IFN-γ~+CD4~+T cells are detected in 39%(13/33)the patients.The mean and standard deviation of HBV S specific TNF-α~+CD4~+T cells frequency is 0.171%and 0.368%,while the mean and standard deviation of HBV S specific IFN-γ~+CD4~+T cells frequency is0.038%and 0.09%,with statistically significant difference(p=0.037).3.Dominance of HBV specific IFN-γ~+CD4~+T cells is associated with HBV virus clearance in chronic HBV infection patients with mo/low inflammation.HBV Core specific IFN-γ~+CD4+T cells or HBV Core specific TNF-α~+CD4+T cells can be detected in 19 chronic HBV infection patients with no/low inflammation.In these 19patients,the mean and standard deviation of HBV Core specific IFN-γ~+CD4~+T cells are 1.6%(5.4%),4.5%(5.2%)and 66.7%(57.7%)in the patinets with positive HBeAg,negative HBeAg and HBsAg sero-conversion respectively.There is a negative correlation between HBV Core specific IFN-γ~+CD4~+T cells and serum HBsAg level in chronic HBV infection patients with no/low inflammation(Rs=-0.442,P=0.058).HBV S specific IFN-γ~+CD4+T cells or HBV S specific TNF-α~+CD4+T cells can be detected in 23 chronic HBV infection patients with no/low inflammation.In these 23patients,the mean and standard deviation of HBV S specific IFN-γ~+CD4~+T cells are11.4%(26.8%),54.8%(52.8%)and 70.1%(42.8%)in the patinets with positive HBeAg,negative HBeAg and HBsAg sero-conversion respectively.There is a significant negative correlation between HBV S specific IFN-γ~+CD4~+T cells and serum HBsAg level in chronic HBV infection patients with no/low inflammation(Rs=-0.563,P=0.005).4.HBV specific TNF-α~+CD4~+T cells is associated with the degree of liver damage in hepatitis B flare patients(ALT>5×ULN).The frequency of HBV specific TNF-α~+CD4~+T cells in HBV flare patients(n=15,ALT>5×ULN)is higher than in chronic HBV infection patients with no/low inflammation(n=33,ALT<2×ULN),with statistically significant difference(p<0.001).There is a positive correlation between the frequency of HBV Core specific TNF-α~+CD4~+T cells and the serum ALT level in HBV flare patients(Rs=0.661,P=0.007).There is a positive correlation between the frequency of HBV S specific TNF-α~+CD4~+T cells and the serum ALT level in HBV flare patients(Rs=0.661,P=0.007).While the frequency of HBV specific IFN-γ~+CD4~+T cells is not correlated with the serum ALT level of HBV flare patients(P>0.05).5.12 HBV core derived peptides and 8 HBV S derived peptides/pairs were targeted by at least 1HLA-DRB1*0803 patient.11 HBV core derived peptides and 17HBV S derived peptides/pairs were targeted by at least 1 HLA-DRB1*1202 patient.Cells from T-cell lines of interest were stimulated with peptides or peptide-pulsed HLA-DRB1 matched allogeneic B lymphoblastoid cell lines(BLCLs)to test the HLA class II restriction of candidate peptides.This method was used to examine the HLA-DRB1 restriction of the peptides targeted by at least 2 patients.Peptide core46-60,core56-70,core66-80,core136-150,core161-175,B&C_S81-95,B&C_S156-170,B&C_S186-200 were verified to stimulate peptide-specific CD4 T cells in a HLA-DRB1*0803 restricted way.Peptide core116-130,core166-180,B&C_S6-20,B&C_S36-50,B&C_S51-65,B&C_S56-70,B&C_S76-90,B&C_S141-155,C_S191-205 were verified to stimulate peptide-specific CD4 T cells in a HLA-DRB1*1202 restricted way.