| Background and AimsHepatitis B virus (HBV) infection leads to a wide spectrum of liver disease ranging from acute to chronic viral hepatitis, which often progresses to liver cirrhosis and development of hepatocellular carcinoma (HCC). More than 93 million people are chronically infected worldwide and about 1 million people die per year due to HBV-associated liver pathologies. Untill now, HBV infection remains a globe public health problem.Long-term therapy with NAs is an effective strategy for patients with chronic hepatitis B virus (HBV) infection. Although nucleos(t)ide analogs seem to have few side effects, they still have to prove their safety in the long term treatment.Yet, the possible disadvantages must be considered as well. Long-term treatment may result in a considerable financial burden on our healthcare systems, and in many countries, patients are not fully reimbursed for the costs of treatment with nucleos(t)ide analogs. Whether nucleos(t)ide analogs are able to induce a sustained off-treatment response is therefore an important focus of research. Unfortunately, it is still unclear for how long treatment using nucleos(t)ide analogs should be continued and whatif any criteria can be used to stop therapy.The sustained response meaned persistently not detectable HBV DNA or 2000 IU/mL combined with persistently normal ALT values (upper limit of normal-ULN,40 U/L) at least more than 1 year follow-up.In recent years, some retrospective studies have focused on the clinical indicators about the sustained response after stopping NAs therapy.Consistent conclusions are:the younger patients, the lower titers of HBsAg at the baseline of stopping drugs, the lower level of HBV DNA at the time of HBeAg seroconversion, the lower level of HBV covalently closed circular DNA (cccDNA) in the liver tissue, the lower level of serum HBcrAg, the shorter time when patients obtained complete response, the longer time to consolidate the treatment after achieved complete response, the chance of relapse are less.The sustained response after discontinuation of treatment with nucleos(t)ide analogue (NAs) in chronic hepatitis B (CHB) patients is a satisfactory expectation. At present the factors predictive of the sustained response after stopping drug are only limited to clinical indicators,however, the immunological parameters hidden behind these routine clinical indexes and mechanism of its immune control the sustained response still need to be studied.Interleukin-21 (IL-21) is produced by activated CD4+T cells, NKT cells, and Th17 cells and has pleiotropic actions on a range of lymphoid lineages. IL-21 is a type Ⅰ cytokine that like IL-2, IL-4, IL-7, IL-9, and IL-15 shares the common cytokine receptor y chain, γc. IL-21 regulates immunoglobulin production and drives B cell terminal differentiation into plasma cells, cooperatively expands CD8+T cells and drives Th17 differentiation, has inhibitory effects on antigen presentation by dendritic cells, and can be pro-apoptotic for B and NK cells. Moreover, IL-21 has potent anti-tumor effects and is implicated in the development of infection diseases.In the past few years, it has become clear that not only does IL-21 regulate normal lymphoid development and function, but it also serves crucial roles in inflammatory responses, allergy, and infection diseases, and it has anti tumor actions. The current studies have showed that, IL-21 can promote the proliferation of CD8+T cells and enhance the responses of CD8+T cells, and plays an important role in helping CD8+T cells to control of virus in chronic infection.But whether IL-21 plays an important role in the sustained response after stopping NAs, and the mechanism by which to maintain sustained response after discontinuation of drugs need to be elucidated. With the method of detecting the virological and biochemical parameters, we investigate the clinical predictors of sustained response after stopping drugs; By detecting the serum level of IL-21, we investigate the correlation between IL-21 and the sustained response; By detecting the function of HBV-specific CD8+T cells, we analysis the relationship between the level of serum IL-21 and the responses of CD8+T cells, investigate the possible mechanism of the sustained response after stopping NAs, and provide scientific basis for clinical application of IL-21 and the related immune cells in CHB infection.Materials and MethodsDetection of clinical indicators after stopping drugsIn HBeAg-positive patients, treatment can be stopped when HBeAg seroconversion with undetectable HBV DNA and normal serum alanine aminotransferase (ALT) has been maintained for at least 12 months. At the same time, in HBeAg-negative patients, treatment discontinuation when patients had been treated for at least 18 years with undetectable HBV DNA and normal serum alanine aminotransferase (ALT). Patients were excluded if they were co-infections with hepatitis A,C,D,E or other viral infection, or presence of autoimmune hepatitis, liver cirrhosis or hepatocellular carcinoma. According to the criteria,we followed up the patients and took the close observation.The Institutional Review Board of Nanfang Hospital, Southern Medical University had approved the study (ID:NFEC-201209-K3). Each enrolled patient provided informed consent.All subjects at each follow-up point were extracted of 30ml venous blood in the morning:Peripheral blood mononuclear cells were isolated with density gradient centrifugation from 20ml anticoagulant and stored at -80℃; Serum from 10ml coagulation were isolated and stored at -20℃ until used. The presence of HBsAg, HBeAg, anti-HBs, anti-HBc, anti-HBe, anti-HCV, and anti-HDV was determined using commercial AxSYM MEI kits (Abbott Laboratories, North Chicago, IL). Serum HBV DNA levels were measured by using a real-time PCR quantification, the Cobas Ampliprep/Cobas TaqMan, version 2.0 (Inc, Pleasanton, CA, USA). Serum ALT levels were measured by automated technniques. HBsAg, antibody against HBsAg (HBsAb), HBeAg, antibody against HBeAg (HBeAb) were determined using a commercially available radioimmunoassay (Abbott Laboratories).Measurement of serum IL-21Serum IL-21 levels were measured by ELISA Kit (eBioscience, San Jose, CA, USA).The lower detection limit of the kit was 20 pg/ml. All operations were in strict accordance with kit.The samples of serum and the standard each 100ul were added in enzyme label plates, at the same time the standard were double dilution.After the reaction was terminated, the microplate reader instrument (FACSCalibur, BD, USA) were used to determine the absorbance (OD) level, the serum IL-21 level were measured by OD numerical value.The detection of HBV-specific T cells responsesTo determine the frequency of HBV-specific T cell, thawed PBMC were stained with fluorescent antibody. First at all the PBMC were divided into HBV-core peptide pools for 72 h at 37℃ with 5% CO2. After 24 h, anti-CD28 and anti-CD49d (final concentration 5 lg/ml, BD Pharmingen,San Diego, CA) were added to the cultures. At the fourth day IL-2 were added into the plate, then at the tenth day the cells were tested by the Intracellular cytokine staining (ICS).Brefeldin A (1 1g/ml, BD Pharmingen, San Diego, CA) was added in the last 5h. The cells were stained with surface marked antibodies (consisted of 7AAD-Percp, anti-CD3-FITC, anti-CD8-APC) and intracellular marked antibodies(anti-IFN-y-PE), after washing with 300ul 2%paraformaldehyde, the stained cells were analyzed on a BD Canto Ⅱ flow cytometer (Becton Dickinson, San Jose, CA, USA), and analyzed with BD FACSDiva (BD Bioscience, San Jose, CA) and FlowJo (Tree Star Inc., Ashland, OR) software.ResultsClinical indicators to predict the sustained response after discontinuation of treatment with NAs60 patients according to the current stopping rule guidelines stopped NAs. After 51.3±1.9(48-56) weeks of follow-up,24 patients (40%) were virologic relapse, including 13 cases(21.6%) of clinical relapse (6 cases relapsed at the 12th week follow-up; 7 cases relapsed at the 24th week follow-up); 36 patients(60%) were sustained responses.Clinical relapse patients were treated with NAs again.The age and HBsAg titres in baseline in the sustained response group were significantly lower than those in the virological relapse group (respectively p=0.03,p=0.001), but there were no significant difference about sex ratio, HBeAg status before treatment, total treatment time, response time, and consolidated treatment time between the sustained response group and the virological relapse group.The dynamics of serum levels of IL-21 in the patientsAbout all the 60 patients, the serum levels of IL-21 at baseline,12th week,24th week,48th week were 40.90 pg/ml (24.50-182.44 pg/ml),42.71 pg/ml (24.50-156.16.17 pg/ml),42.68 pg/ml (25.92-215.48 pg/ml),42.90 pg/ml (27.35-200.85 pg/ml). There were no significant difference about the serum levels of IL-21 between the baseline and 12th week,24th week,48th week.In the virological relapse group, the serum levels of IL-21 at the baseline,12th week,24th week,48th week were 42.68 pg/ml (25.92-182.44pg/ml),41.48pg/ml (24.50-150.16pg/ml), 45.68 pg/ml (28.77-215.48 pg/ml),42.64 pg/ml (27.35-200.85pg/ml), there were no significant difference about the serum levels of IL-21 between the baseline and 12th week,24th week,48th week; in the sustained response group.the serum levels of IL-21 at the baseline,12th week,24th week,48th week were 39.59 pg/ml (24.50-117.16pg/ml),42.71pg/ml(25.92-114.53pg/ml),40.84pg/ml(25.92-118.48p g/ml),43.52 pg/ml (28.77-128.65pg/ml),and serum IL-21 levels at 12th week,24th week,48th week were significantly higher than those at baseline (respectively p=0.0035,p=0.0124,p=0.0002).The correlations of serum levels of IL-21 with HBsAg titers and HBV DNA levelsIn the virological relapse group,the serum HBV DNA levels increased continuously with the follow-up weeks prolonging, and the titers of HBsAg and the serum IL-21 levels had no obvious change; Serum HBV DNA levels and HBsAg titers in the sustained response group is continuous and stable low level with the follow-up weeks prolongs, but the serum IL-21 levels maintained a high state.There were no correlation between serum IL-21 levels and HBsAg titers, HBV DNA levels after discontinuation of NAs in all 60 patients; There were no correlation between serum IL-21 levels and HBV DNA levels after discontinuation of NA(s) in the virological relapse group and the sustained response group; The serum levels of IL-21 correlated with the titers of HBsAg in the virological relapse group (r=0.290,p=0.01) positively, while correlated with the titers of HBsAg in the sustained response group (r=-0.328,p=0.01) negatively.The responses of HBV-specific T cellsThe responses of HBV-specific T cells were detected at baseline after discontinuation of NAs therapy in the 20 patients with chronic hepatitis B.The detected rate of the responses of HBV- specific T cells was 30%(6/20),and the frequency of IFN-γ+CD8+T cells was 0.133%±0.062%(0.054%-0.228%).There were 2 cases (2/11)detected in the virological relapse group,while 4 cases (4/9) in the sustained response group. The detected rates of the two groups were not significant difference.ConclusionThe age and HBsAg titers at the withdrawal baseline can be good clinical indicators to predict the sustained response after discontinuation of treatment with NAs inCHB patients; the serum levels of IL-21 maintained a high state during the follow-up weeks in the sustained response patients after stopping NAs; the serum levels of IL-21 correlated with the titers of HBsAg in the virological relapse group positively, while negatively in the sustained response group.These may suggest that IL-21 plays an important role in the immune control of CHB patients after stopping NAs drugs as a kind of inflammatory and immune regulatory factors. |
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