The Dynamic Changes Of T-cell Receptor Repertoires Of Intrahepatic Lymphocytes In Patients With Chronic Hepatitis B During 2-year Antiviral Therapy

BackgroundHepatitis B virus(HBV)infection can cause acute and chronic hepatitis,liver failure,liver cirrhosis,liver cancer and other diseases,seriously affecting the global human health.After antiviral treatment with nucleos(t)ide analogue(NUC),the patients' condition can be improved obviously.At the same time,the emergence of high throughput sequencing technology can make TCR repertoire research more comprehensive and in-depth.However,the changes of intrahepatic lymphocytes TCR repertoire undergoing NUC treatment were not elucidatedObjectiveIn this study,5'-terminal rapid amplification of cDNA ends(5'-RACE)was used to obtain the fragments of TCR ? chains and the fragments were sequenced by Illumina platform.The changes of TCR repertoire,the relationship between HBeAg seroconversion and TCR repertoire were in-depthly analyzedMethods1.Research objectA total of 12 patients with chronic hepatitis B(CHB)were recruited.The specimens of liver biopsy before and after 2-year NUC treatment and the serum of patients were collected.Patients were divided into seroconversion group(SR)and non seroconversion group(NSR)2.Construction and sequencing of TCR libraryThe serological marker of HBV,aminotransferase and HBV DNA were measured.Total RNA was extracted from liver tissue samples and TCR ? chains were amplified by 5'-RACE combined with nest-PCR.Libraries of TCR ? chains were constructed and high throughput sequencing was performed by using Illumina Hiseq platform.3.Sequencing data processing and analysisThe productive V,D and J gene segments and CDR3 sequence of TCR? chain were identified by MiXCR.The same CDR3 sequence is called a clonotype.Results1.Usage of TCR VDJ gene combination in intrahepatic lymphocytesA total of 791 non repetitive VDJ combinations were obtained in this study After statistical analysis,33 non repetitive VDJ combinations were found to have statistical differences at baseline and week 104.Among them,15 combinations frequencies in baseline were higher than those in week 104,and 18 of them were lower than those in week 1042.Diversity of TCR ? chain library in CHB patients before and after 2-year antiviral treatment.In all CHB patients,the number of CDR3 clonotypes was higher at baseline than that at week 104(P=0.045).Furthermore,the number of CDR3 clonotypes in SR group at baseline was significantly higher than that at week 104(P=0.046),while there was no significant difference in the number of CDR3 clonotypes at two time points in the NSR group.At the same time point,there was no significant difference in the number of CDR3 clonotypes between SR group and NSR group.Using normalized Shannon diversity entropy(NSDE)as the index of TCR repertoire diversity,there was no significant difference in the diversity of TCR repertoire between SR group and NSR group at the same time point.Besides,no significant difference was found in TCR repertoire diversity from baseline to week 104 in either group.3.Similarity analysis of TCR ? chain Library in CHB patients before and after antiviral treatmentThe same CDR3 clonotype that appeared at baseline and week 104 was called share clone.The proportion of share clones in most patients was low,except for patient 15(P15),whose proportion was 22.5%.After statistical analysis,there was no significant difference in the proportion of share clones between SR group and NSR group.As the index of TCR repertoire similarity in baseline and week 104,the MHSI(Morisita-Horn similarity index)of SR group was lower than that of NSR group.4.Correlation analysis of the dynamic changes of TCR CDR3s with HBV antigen level and viral loadPositive correlation was found between the proportion of ablated clonotype and the decrease of HBsAg level from baseline to week 104 in SR group(r=0.886,P=0.019),but it was not found in NSR group;the proportion of ablated clonotype was not correlated with the decrease of HBV DNA and HBeAg level from baseline to week 104.However,there was no significant correlation between the proportion of ablated clonotypes and the decrease of HBV antigen level and viral load.ConclusionThe clonal expansions might exist in intrahepatic lymphocytes in CHB patients with HBeAg seroconversion,the increase of ablated clonotypes proportion might benefit favorable outcome.To our speculation,oligo and vigorous cloanl expansion might appear in intrahepatic T cells during 2-year NUC treatment,which may play a positive role in treatment outcomes.In the future,we may be able to predict prognosis by high throughput sequencing.