| BackgroundChronic hepatitis B(CHB)is a potentially life-threatening liver disease caused by chronic infection with hepatitis B virus(HBV)and is a major global health problem affecting approximately 250 million people worldwide.At present,there are two types of antiviral drugs recommended in the guidelines,namely interferon-?(IFN-?)and nucleoside(acid)analogues(NAs).There are many side effects of IFN-a and it must be administered by injection.The effector target of NAs is HBV polymerase,which has obvious effect of inhibiting virus replication and is convenient and safe to take.Hepatitis B surface antigen(HBsAg)seroconversion is a hallmark of functional healing of chronic hepatitis B(CHB).However,long-term treatment is needed because NAs cannot eliminate hepatic cccDNAand hardly ever achieve HBsAg seroconversion.For many patients,long-term treatment is not an ideal option considering the financial burden,side effects,and safety issues for women.Recent studies have shown that CHB patients treated with long-term nucleoside(acid)drugs may consider discontinuing NAs if appropriate discontinuation monitoring can be provided and treatment can be restarted as soon as needed.In the latest review of meta-analyses,the virological recurrence rate at 36 months after NA withdrawal was 38.2%,so how to predict long-lasting response after NAs discontinution,better to guide drug withdrawal and withdrawal of drag.The mechanism of sustained response is still not clear in CHB patients after treatment discontinution.The natural history of HBV infection and disease outcomes are diverse and depend primarily on the virus and the host's immune response.CXCL13 is a small cytokine belonging to the CXC chemokine family.Through its cognate receptor CXC chemokine receptor type 5(CXCR5),CXCL13 can attract properties for B cells and subsets of activated T cells.Importantly,CXCL13 promotes the co-migration of B cells and Tfh cells to B-cell follicles and germinal centers(GCs),where high affinity antibodies secrete memory B and plasma cells.Our previous studies found that increased levels of Kuffer cells(KC)and serum CXCL13 during treatment with telbivudine were associated with immune control of chronic HBV infection.In a recent study,CXCR5+CD8+ T cells were defined as a new subpopulation of cells that play an antiviral effect in chronic viral infections.In HIV infection,the frequency of CXCR5+CD8+ T cells is negatively correlated with viral load and has strong antiviral capacity.However,the association between the expression of CXCL13 and its receptor CXCR5 and long-lasting response after long-term nucleoside(acid)drug treatment discontinuation remains unclear.AimsThe plasma CXCL13 levels in patients with baseline withdrawal in the prospective withdrawal cohort and the CXCR5+CD8+ T cell levels in peripheral mononuclear cells(PBMC)were examined to observe their clinical prognosis,and possible and durable responses were explored.Materials and Methods1-SubjectsPatients were recruited at Nanfang Hospital from a prospective study.Patients were enrolled if they fulfilled the criterion.Enter criterion:For HBeAg positive patients,they were treated with NAs until HBV DNA undetectable with normal ALT levels after HBeAg seroconversion and continued the therapy at least for 12 months.For HBeAg negative patients,they were treated with NAs until HBV DNA undetectable with normal ALT levels and continued the therapy at least for 18 months.Clinical relapse(CR)defined as HBV DNA>2000 IU/mL with ALT?5 ULN or TBIL?2 ULN.HBsAg loss defined as HBsAg undetectable(<0.05 IU/ml).The study was approved by the Ethical of Nanfang Hospital.Written and informed consent was obtained from all subjects.2.Plasma Cytokine/Chemokine ELISA testThe plasma cytokine/chemokine expression levels were quantitated by enzyme-linked immunoassay(ELISA)according to the manufacturer's instructions.3.Detection of the frequency of peripheral CXCR5+T cellAfter resuscitation of cryopreserved PBMCs,CXCR5+ cells,CXCR5+CD4+ T cells,and CXCR5+ cells in peripheral blood of continuous response group(SR)and clinical relapse group(CR)after long-term nucleotide drug treatment were analyzed by flow cytometry.The frequency of CD8 + T cells and the expression of subsets of their memory cells.4.The function of CXCR5+CD8+T cells in CHB patients after stopping NAs treatment.Flow cytometry analysis was used to compare the expression levels of PD-1,Tim-3,and CD69 in peripheral blood CXCR5+CD8+ T cells in SR and CR.The expression levels of PD-1,Tim-3,and CD69 in peripheral blood CXCR5+CD8+ T cells and CXCR5-CD8+ T cells were analyzed.PBMCs were treated with PMA/ionomycin and cytokines secreted by CXCR5-CD8+T and CXCR5+CD8+ T cells were detected by ICS.Results1.End-of-treatment(EOT)CXCL13 levels can independently predict clinical relapse in CHB patients after NAs discontinuation.Of the 105 patients,47 had clinical relapse after stopping NA treatment for up to 5 years of follow-up.Multivariate analysis of COX risk proportional regression model showed that EOT CXCL13 levels(HR 0.26,95%CI 0.31-0.52,p<0.001)were independent predictors of clinical relapse.2.EOT CXCL13 levels can independently predict HBsAg loss in CHB patients after NAs discontinuation.Of the 105 patients,11 developed HBsAg loss after stopping NA treatment.Of 58 sustained response group,9(15.5%)cleared HBsAg and 2(4.3%)of clinical relapse group has HBsAg loss.Cox regression multivariate analysis showed lower EOT HBsAg(HR:0.25,95%CI 0.14-0.45,p<0.001)and higher EOT CXCL13 levels a(HR:3.01,95%CI 1.33-6.81,p = 0.008)is an independent predictor of HBsAg loss.3.Sustained response group have high expression of CXCR5+CD8 T cells.'The frequency of CXCR5+CD8+T cells in peripheral blood in patients with persistent response(n = 24)was significantly higher than that in patients with clinical recurrence(n = 19)(P = 0.012).CXCR5+CD8+T cells overexpressed CD69 and low-expressed Tim-3(P = 0.038,P = 0.005)in the sustained response group.4.CXCR5+CD8+T lymphocytes secrete cytokines and enhance cytotoxicityCompared with CXCR5-CD8+T cells,the proportion of central memory cells(CD45RA-CCR7+)and naive cells(CD45RA+CCR7+)in CXCR5+CD8+T lymphocytes increased,low expression of Tim-3(P = 0.005),enhanced secretion of cytokines(IFN-y and TNF-?)and enhanced cytotoxicity(CD107a).Conclusion1.Plasma EOT CXCL13 plasma levels independently predict clinical relapse and HBsAg loss after stopping long-term NAs treatment.2.Compared with the clinical relapse group,the frequency of CXCR5+CD8+ T lymphocytes in the peripheral blood of patients with sustained response after discontinuation was significantly increased.CXCR5+CD8+ T Cells have a stronger ability to secrete cytokines and cytotoxicity after discontinuation of treatment which is impotant to the control of HBV. |
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