| BackgroundChronic infection with hepatitis B virus(HBV)currently affects about 400 million people,particularly in developing countries,and it is estimated that worldwide over 200,000 and 300,000 chronic HBV carriers die each year from cirrhosis and hepatocellular carcinoma(HCC),respectively.Natural history of chronic HBV infection and the disease is complex and highly variable.The natural history of HBV infection generally can be divided into four periods:Immune tolerant,immune clearance,inactive or low(non)replication and reactivation.Most patients become inactive carriers after spontaneous HBeAg seroconversion with good prognosis,but progression to HBeAg negative chronic hepatitis due to HBV variants not expressing HBeAg occurs at a rate of 1-3 per 100 person years following HBeAg seroconversion.The incidence of cirrhosis appears to be about 2-fold higher in HBeAg negative compared to HBeAg positive chronic hepatitis.In the cirrhotic patient the 5-year cumulative risk of developing hepatocellular carcinoma is 17%in East Asia and 10%in the Western Europe and the United States and the 5-year liver related death rate is 15%in Europe and 14%in East Asia.There is a growing understanding of viral,host and environmental factors influencing disease progression,which ultimately could improve the management of chronic hepatitis B.Circulating CXCR5+CD4+T cells,by producing IL21,may have a significant role in facilitating HBeAg seroconversion in patients with chronic HBV infection.Tfh cells to travel into the B cell follicle and deliver help to B cells,allowing maintenance of the germinal center reaction,generation of high-affinity antibody,and long-lasting plasma and memory B cells.HBeAg seroconversion is an important event in the evolution of a chronic HBV infection.It coincides with the development of the HBV-specific T cell repertoire that is believed to control viral replication,and is associated with a reduced risk of progressive liver inflammation,liver cirrhosis and liver cancer.Foxp3+Treg cells are a subset of CD4+T cells with immunosuppressive properties,including an ability to inhibit the proliferation and cytokine production of effector T cells.Notably,a series of recent studies have demonstrated that the suppression of each subset of helper T cell responses by Treg cells requires expression of a certain transcription factor together with Foxp3 in the Treg cells.Similar to Tfh cells,Foxp3+ regulatory T cells gain the ability to migrate into B cell follicles through their surface-expressed CXCR5.These CXCR5+ Treg cells in a chemotaxis assay and suppress antibody production by germinal center B cells and the function of Tfh cells in a coculture experiment in vitro.Thus,Tfr cells that are present in humans have an immunosuppressive capacity similar to that observed in murine Tfr cells.Achieving HBeAg seroconversion means acquire immune control,which is expected to achieve sustained low levels of HBV replication,the reverse process of liver fibrosis,reduce liver cirrhosis,liver cancer.Therapy that targets Tfr cells may be beneficial for the treatment of chronic HBV infection.Clarifying the characteristics of Tfr cells in different stages of HBV infection will have important clinical significance in discovery of new treatments,the timing of treatment options and programs,treatment and prognosis.ObjectiveCross sectional cohort of chronic HBV infection in natural history were enrolled in this study.We aimed to analyze the characteristics of Tfr cells in chronic HBV infection,explore the proportions of phenotypes and the cytokines production in circulating Tfr cells,the effect of IL21 on Tfr cells,the frequency of Tfr cells,and clarify its role in HBeAg seroconverson and immune tolerance.Methods1.SubjectsThere were fourty-one patients with chronic HBV infection collected for the study.They were classified into immune tolerant carrier(IT,n=5),HBeAg positive CHB(n=16),and inactive carrier(IC,n=13)groups.Seven healthy controls(HC)were also enrolled.IT was defined as HBeAg-positive,having normal ALT levels and being HBsAg-positi've on at least three occasions in one year,with HBV DNA higher than 107 copies/mL.CHB was defined as being HBsAg-positive,HBeAg-positive,with HBV DNA higher than 104 copies/mL,and having a flare ALT levels.IC was defined as being HBsAg-positive on two occasions at least six months apart,HBeAg-negative,anti-HBe-positive with persistently normal ALT levels and HBV DNA<104 copies/mL.Seven students with normal ALT levels who were negative for HBsAg were recruited as healthy controls.None of the patients were coinfection with hepatitis A virus,hepatitis C virus,hepatitis D virus,hepatitis E virus,or human immunodeficiency virus.Patients with primary biliary cirrhosis,primary hepatocellular carcinoma,autoimmune diseases,and any form of antiviral therapy were also excluded.Written,informed consent was obtained from all subjects.2.The frequencies of Treg and Tfr cells and the ratio of Tfr/TfhThe frequencies of Treg,Tfr cells and the ratio of Tfr/Tfh within HC,IT,CHB and IC groups were analyzed with BD FACSDiva and FlowJo softwar.3.The proportions of phenotypes in circulating Tfr cellsThe proportions of GITR,CTLA-4,PD-I,ICOS and Bcl-6 in circulating Tfr cells of CHB patients were analyzed with BD FACSDiva and FlowJo softwar.4.Cytokines production of circulating Tfr cellsIntracellular staining(ICS)analysis was performed to detect the cytokines(GranzymeB,IL17A,IL4,IL10,IL21 and IFN-y)production of circulating Tfr cells in CHB patients.5.The effect of IL21 on Tfr cellsThe frequencies of Treg and Tfr cells and the ratio of Tfr/Tfh stimulated by IL21 for three days in patients with chronic HBV infection were analyzed with BD FACSDiva and FlowJo softwar.6.Statistical AnalysisContinuous data are shown as median and 10%-90%percentile.The unpaired t test and the Chi-squre test were used when 2 groups were compared.The statistical analysis was performed by using either GraphPad Prism 6.0 or SPSS 19.0 software.All statistical analyses were based on a two-tailed hypothesis test with a significance level of P<0.05.Results1.The proportions of GITR and CTLA-4 were higher in circulating Tfr cells of CHB patients,and there was almost no proportion of Bcl-6.The proportions of GITR and CTLA-4 in Tfh cells were significantly lower than Tfr cells in patients with chronic HBV infection(P<0.0001).2.The proportions of GranzymeB and IL4 producing cells within Tfr cells were significantly higher than other cytokines in patients with chronic HBV infection(P<0.001).The proportion of IL21 producing cells within Tfr cells was significantly lower than other cytokines in patients with chronic HBV infection(P<0.01).3.Compared with control group,the proportions of GranzymeB,IL17A,IL21 and TFN-y producing cells within Tfr cells were significantly higher in CD3/CD28 group of patients with chronic HBV infection(P<0.05).4.Compared with IL21(100ng/ml)group,the frequency of Treg cells were significantly higher in IL21(10ng/ml)group(P<0.05).There were no significance about the frequency of Tfr cells and the ratio of Tfr/Tfh between three groups.5.Compared with IC group,the frequency of Tfr cells were significantly higher in IT(P<0.001)and CHB group(P<0.05).Compared with IC group,the frequency of Treg cells were significantly higher in IT(P<0.001)and CHB group(P<0.001).Compared with IC group,the ratio of Tfr/Tfh were significantly higher in IT(P<0.001)and CHB group(P<0.01).Conclusions1.The proportions of GITR and CTLA-4 were higher in circulating Tfr cells of CHB patients,and there was almost no Bcl-6.2.By the high level of IL10 and the low level of IL21,circulating Tfr cells regulate negatively HBeAg seroconversion and promote immue tolerance.3.The human peripheral blood mononuclear cells is a complex mixture of cells system.IL21 may cause changes in various groups and factors over the entire system,which may mask the direct regulation of IL21 on Tfr cells.4.The increaced circulating Tfr cells may be related with immue tolerance and inhibit B cells immunity and HBeAg seroconversion in patients with chronic HBV infection. |
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