| BackgroundHepatitis B virus(HBV)chronic infection is characterized by damaging the host immune response,especially the branch of adaptive immune response,which results in the inability of the host to completely eliminate virus,and persistence of hepatic inflammatory pathology and eventually leads to liver cirrhosis and hepatocellular carcinoma.HBV is a hepatotropic virus that does not directly damage hepatocytes.During the chronic HBV infection,persistent exposure of T cells to high concentrations of HBV antigen cause a hierarchical loss of antiviral function with cytotoxicity and non-cytotoxic effects.In general,the reduced absolute number and disfunction of HBV-specific T cells are the main immune causes that lead to chronic infection.Following a phase ? study,PegIFNa was licensed for antiviral treatment in pediatric patients with CHB between 2-18 years old.Nucleoside or nucleotide analogues(NA)are relatively safety but treatment duration may be lifelong time.Furthermore,HBsAg serological conversion rate is less than 1%in NA therapy at week 48.Despite the side effects of PegIFNa,it shows the most favorable benefit of achieving a higher HBsAg serological conversion rate in adults(around 4%).PegIFNa therapy for pediatric CHB could have an higher HBsAg serological conversion rate and a better prognosis compared with adult patients but the mechanism of immune response is not clear.ObjectiveThe aim of this study was to explore adaptive immunity especially HBV-specific T cell responses in PegIFNa treated pediatric patients with HBeAg-positive especially who experienced HBsAg loss.Methods1.Patients enrollment and sample collectionA total of 37 patients with HBeAg-positive CHB were treated with PegIFNa from 2015 to 2017,20 patients were enrolled in this trial.Eleven patients experienced HBsAg serological conversion during week 48-96 and were defined as complete response group(CR),and nine patients who either failed treatment(inability to obtain a 1 log10 IU/mL decrease in viral load from baseline to week 12 of treatment)or relapsed(inability to maintain a successful response after achieving a viral load<2000 IU/mL after week 24 of treatment)were defined as non-response group(NR).Assessments of HBV DNA,HBsAg,HBeA,HBeAb,ALT and thyroid stimulating hormone(TSH)were taken every 12 weeks.2.Serological assays and HBV DNA assaysFresh serum was tested for HBsAg,HBeAg using chemiluminescent microparticle immunoassay.HBV DNA was quantified with COBAS TaqMan HBV test.3.Flow cytometry analysisDead cells were excluded by staining with LIVE/DEAD.Then anti-CD8 FITC,anti-CD4 PERCP,anti-CD56 APC,anti-CD3 FITC,anti-TRAIL PE,anti-PD-1 APC-CY7,anti-CD 127 PE,anti-CD69 APC,were added to analyze activated T cells and NK cells on BD FACS Canto ?.4.In vitro expansion of HBV-specific T cellsResuscitated cells were stimulated with 15-mer overlapping synthetic peptides(2ug/mL)of genotype B covering core(35),envelope(78),and X(29)at day 0 and 10 to boost T cells proliferation.At the last day of stimulation(day 10),cells were stained with anti-IFN ? PE-CY7 for intracellular cytokines staining.All flow data was analyzed by FlowJo V10.0.Results1.Patients characteristicsClinical characteristics and P value of two treated groups at baseline showed miner difference,only HBsAg was significantly different when compared between the CR and NR groups,however,HBV DNA,HBeAg,ALT showed no differences.For the treated patients,titer of HBV DNA,HBsAg and HBeAg showed a continuous decrease during treatment(median HBV DNA 1.7E+08 IU/mL vs.10 IU/mL,HBsAg 23339.500 IU/mL vs.0.92 IU/mL,HBeAg 1260.690 COI vs.2.17 COI,ALT 110.5 U/L vs.69 U/L;P<0.01).2.T cell phenotype differs between the CR and NR groupAfter treatment,significance differences between the CR and NR group were found in the frequency of CD69+CD8 T cells(1.82%vs.0.61%,P=0.0003)and TRAIL expression on CD4+/CD8 T cells(1.30%vs.0.19%,P=0.001,1.34%vs.0.34%,P=0.002).3.Dynamic alteration of T cell and NK cell in pediatric CHB patients experiencing HBsAg lossAs these patients responded to PegIFNa heterogeneously,we chose the occurrence of maximum fold change(MFC)of log10 HBV DNA as the time point at which pairwise analysis was performed for comparisons among baseline,MFC time and the end of treatment.These patients expressed evidently higher levels of CD69 on CD4+/CD8 T cells over time,when baseline was compared with and MFC time(1.19%vs.0.69%,P=0.01;2.45%vs.1.18%,P=0.003).In addition,the frequency of TRAIL expressed on CD4+/CD8+T cells were comparable at baseline and MFC time(2.81%vs.1.92%,P=0.009;2.75%vs.1.89%,P=0.002).The expression of TRAIL was remarkably increased on CD56hi NK cells at MFC time(2.00%vs.0.92%,P=0.003)when compared with baseline.Interestingly,we noticed that ALT levels was positively correlated with the frequency of TRAIL+CD56hi NK cells at baseline in all patients(r=0.663,P=0.002).3.Multispecific HBV T cell responses in pediatric CHB patientsT cell expansion in the overall pools of three peptides(core,envelope and X)was not only observed in the CR group,but also in the NR group,which showed a lower frequency.At baseline,the magnitude of core-specific T cell responses was considerably higher in the CR than NR group in CD4+/CD8 T cells(mean 0.68%vs.0.26%,P=0.001).Furthermore,a significant increase in HBV-specific T cell responses in the CR group was observed at end of treatment compared with NR group.Next,HBV-specific T cell responses continued to increase as therapy progressed.Core-specific CD4+ T cell response was increased to a significant high level at the end of treatment than at baseline,and the frequency of core-specific IFN-? CD8+ T cell was also significantly increased over baseline value at MFC time and at the end of treatment(0.67 vs.1.04,P=0.009;0.5 vs.0.91;P=0.041).The frequency of core-specific IFN-? CD4+ T cell at baseline was positively correlated with the decline in log10 HBV DNA from baseline to week 12.A similar correlation was observed in the frequency of envelope-specific IFN-? CD4+ T cells at baseline and the decline in log10 HBV DNA from baseline to week 12.Overall,PegIFN? therapy could result in incresed responsiveness of HBV-specific T cell and lead to reduce DNA and antigen load in individual patients(r=0.639,P=0.002;r=0.680,P=0.001).ConclusionBy analyzing the frequency of CD69/TRAIL on T cell and TRAIL on CD56hi NK cell and HBV-specific T cell responses in PegIFNa treated pediatric patients with CHB,we have showed that PegIFNa can triggered T/NK cells activation and HBV-specific T cell responses thus contribute to successful virus control.Furthermore,the magnitude of HBV-specific T cell responses is positively related with the decline of virus thus results to different progosis. |
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