The Influences Of Dynamic Changes Of T-cell Receptor Repertoires On Antiviral Therapy Outcome In Patients With Chronic Hepatitis B

BackgroundT cells play crucial roles in hepatitis B virus(HBV)infection,with multiepitope-specific and vigorous CD4+ and CD8+ T-cell response in patients with acute infection for viral clearance,while weak or exhausted T cells activity in chronic HBV infection.Studies have shown that restored function of T cells was detected in patients who underwent nucleos(t)ide analogues(NUC)therapy.Up to now,antigen specific T cells have been measured by stimulating PBMCs with antigen peptide pools and staining cytokines.Whether and how the dynamic changes of T-cell receptor repertoires can influence on antiviral therapy outcome in patients with chronic hepatitis B remain to be clarified.ObjectiveThis study investigates the characteristics and the dynamic change of CD4+ and CD8+ TCR repertoires during early antiviral treatment by high thoughtput sequencing,and the differences of TCR repertoires derived from HBeAg seroconversioners and HBeAg non-seroconversioners.Methods1.Patients enrollment and sample collectionA total of 22 chronic hepatitis B patients with 2-year telbivudine treatment were enrolled from a clinical trial,including 10 with complete response(CR)and 12 with non-complete response(NCR)based on HBeAg seroconversion and HBV DNA level on week 52 and week 104 of treatment.Serum and heparinized blood were collected on 0,12,24,52,104 weeks after receiving telbivudine treatment.Patients in the CR group achieved HBeAg seroconversion and had a level of serum HBV DNA<300 copies/mL at week 52,which was sustained at week 104,while patients in the NCR group maintained HBeAg positivity and an HBV DNA level>300 copies/mL at weeks 52 and 104.All the patients achieved normal ALT levels at weeks 52 and 104.2.Peripheral CD4+ and CD8+ T cells on baseline,week 12 and week 24 were sorted by flow cytometer.The total RNA was isolated and the TCRβ chain complementarity determining region 3(CDR3)was amplified by 5’Rapid amplification of cDNA ends(RACE)PCR combined with nest-PCR.The high-throughput libraries of TCRβ chain products were constructed and sequenced on Illumina Hiseq platform.3.Data analysisThe V/D/J gene segment and CDR3 identification were used by MiXCR,a software which is specific for analyzing immune repertoire.Identical CDR3 sequences represent one clonotype.To further determine how the frequency of CD4 and CD8 TCRβ repertoires changed between any two of three timepoint,the clones were classified into four classes(1+ to 4+)based on the frequency of the clonotype.Clonotypes in these four classes in each patient were assigned to one of 5 categories:ablated,depleted,persistent,expanded or new.Results1.Differing diversity but similar patterns of change in CD4 and CD8 T cells in CR and NCR patientsThe numbers of unique VDJ combinations,aa clonotypes,as well as the Shannon entropy values,were relatively lower in CD4 subset but were higher in CD8 subset in the CR patients at baseline,week 12 and week 24 of treatment,although statistically significant differences were observed only at week 12 for CD4 cells and at baseline for CD8 cells.This result indicates that there was a relatively higher diversity in CD8 T cells,while the CD4 cells exhibited reduced diversity in CR patients compared with the NCR patients.Compared with NCR group,the relatively lower level of diversity in CD4 cells in the CR patients at baseline reached significance at week 12,as indicated by the number of VDJ combinations(1423 vs 1570;P=0.035),aa clonotypes(17,255 vs 27,840;P=0.03),and Shannon entropy values(10.80 vs 12.78;P=0.041).At baseline,higher number of aa clonotypes was observed in CD8 cells in the CR patients than in the NCR(20,710 vs 14,600;P=0.041).The diversity of CD4 TCRβ repertoire significantly decreased from baseline to week 12 during treatment in the CR patients(12.30 vs 10.80;P=0.022).2.CR patients were characterized by greater perturbations of both CD4 and CD8 TCR repertoiresThe percentage of persistent clonotypes was lower in the CR than in the NCR group between any two time points in both CD4 and CD8 subsets(all P<0.001).In contrast,patients in the CR group had much higher combined percentages of new and expanded clonotypes than those in the NCR group between any two time points in both CD4 and CD8 cells(all P<0.05).When clone size was considered,the CR group exhibited a higher cumulative frequency of persistent clones(all P<0.05)and a lower combined cumulative frequency of new and expanded clonotypes in CD4 cells during the first 12 weeks of treatment(70.14%vs 89.11%;P=0.021),while no difference was observed in the cumulative frequency of any category clones among the CD8 cells.These results indicate that a turnover of the TCRβ repertoire occurred in both CD4+ and CD8+ T cells of the CHB patients undergoing NUC treatment.Patients with a CR underwent more profound perturbations and broader clonal expansions of both the CD4 and CD8 T cell repertoires,but the clone size of each new and expanded clonotype was more restricted.3.Correlation between the decline of HBV antigens and T cell expansionWith the rapid decline in HBV DNA,HBeAg and HBsAg levels from baseline to week 12,a correlation between viral antigen and T cell expansion was observed for both CD4 and CD8 T cells.The percentage of unique new and expanded clonotypes exhibited a negative correlation,while persistent clonotypes were positively correlated with antigen levels at week 12.In addition,the amount of HBeAg reduction from baseline to week 12 was consistently and significantly associated with the percentage of new and expanded clonotypes.These results suggest the importance of viral antigen suppression in inducing an effective T cell response.ConclusionBy analyzing the clonotypic composition and diversity of peripheral CD4 and CD 8 TCR repertoires and tracking dynamic changes in the different clonotypes in CHB patients upon NUC treatment,we have shown that substantial clonal expansions of the CD4 and CD8 T cell repertoires are associated with early treatment-induced HBeAg seroconversion.Effective T cell responses are closely associated with a decline in viral antigen and viral load.Therefore,the robust inhibition of HBV replication complemented by immunomodulatory strategies in NUC monotherapy,such as the adoptive transfer of HBV-specific TCR-redirected T cells targeting broad HBV epitopes,may improve T cell responses and achieve complete control of HBV infection.