Analysis Of T Cell Receptor Vβ Diversity In Peripheral Blood CD4+and CD8+T Lymphocytes Obtained From Patients With Persistent HBV Infection

Background：Hepatitis B virus (HBV) infects more than350million people worldwide.Hepatitis B is a leading cause of chronichepatitis,cirrhosis and hepatocellular carcinoma,accounting for1million deaths annually. The HBV replication cycle is not directlycytotoxic to cells. The fact accords well with the observation thatmany HBV carriers are asymptomatic and have minimal liverinjury,despite extensive and ongoing intrahepatic replication of thevirus. It is now thought that host immune responses to viral antigensdisplayed on infected hepatocytes are the principal determinantsof hepatocellular injury. This notion is consistent with the clinicalobservation that patients with immune defects who are infected withHBV often have mild acute liver injury but high rates of chroniccarriage. The immune responses to HBV and their role in thepathogenesis of hepatitis B are not completed understood.Correlative clinical studies show that in acute, self-limitedhepatitis B, strong T-cell responses to many HBV antigens are readilydemonstrablein the peripheral blood.By contrast,in chronic carriersof HBV, such virus-specific T-cellresponses are greatly attenuated, at least as assayed in cells from the peripheral blood.This patternstrongly suggests that T-cell responses, especiallythe responses ofcytotoxic T lymphocytes,play a central role in viral clearance. Tcell antigen receptor(TCR) recognize antigen epitope specifically,which can result in immune response.Those patientswith chronic HBVinfection may present in one of the four phases of infection: immunetolerance, immuneclearance, inactive carrier state,andreactivation. We study the TCR clonity of chronic asymptomaticcarriers and chronic hepatitis B to understand the status of cellimmune responses,which can guide to reasonable treatment of HBVpersistent infection.Objectives: To understand the clonity charateristic of CD4+and CD8+TCR in chronic asymptomatic carriers and to study the role of cellimmune response in the state of immune tolerance; To study the clonitycharateristic of CD4+and CD8+TCR in chronic hepatitis B and toinvestigate the role of cell immune response in the state of immuneclearance; To study if common nuleotide sequence of monoclonalTCR VβCDR3by nucleotide sequencing and comparison and to look forthe common HBV epitope in cell immune response. To clarify the roleof Th1/Th2cytokines in chronic HBV infection through detecting IL-4and IFN-γ level in supernatant of cultured PBMCs.Methods: Chronic asptomatic carriers and patients with chronic hepatitis B were enrolled in the study. RT-PCR-genescan-sequencewas used in our study.10-20ml heparinized blood samples werecollected from every patients. CD4+and CD8+T lymphocytes wereisolated using monoclonal antibody-coated magnetic beads and thenRNA was extracted. cDNA was synthesized using RNA, reversetranscriptase and oligo dT in a total volume of20ul. Using a two-stepPCR assay, CDR3length within the TCR Vβ chain was analyzed. ForwardV β and reverse C β primers of two rounds were designed toamplified the24TCR Vβ families. PCR products were analyzed usingaApplied Biosystems model373DNA sequencer. The data were analyzedand quantified using the ABIPRISM GeneScan analysis software todetermine the oligoclonal or monoclonal families.The amino acidsequence of the CDR3in monoclonal Vβ families were analyzedusing DNAMAN software version1.0. ELISA was used to detect IL-4and IFN-γ level in the supernatant of cultured PBMCs which werestimulated by HBsAg.Results: In this study, the results of genescan analysis showedthat the CD4+and CD8+TCR Vβfamilies of the healthy control donorsshowed Gaussian distribution.However, some CD4+and CD8+TCR Vβ families showed monoclnal and oligoclonal expansion in allthe12chronic asymptomatic HBV carriers. TCR Vβ2、3、4、5、7、8、9、11、12、15、18and21families showed clonal expansion. The expansion frequencies of TCRVβ7、9、12and15families were higherthan the others TCR Vβfamilies.In CD8+TCR Vβ families,6among12patients showed clonal expansion and the TCR Vβfamilies were Vβ3、5、7、9、11、12、16、18and21.The clonal expansion number ofCD4+TCR Vβ families (n=24) was significantly higer than that ofCD8+TCR Vβ families(n=9). The sequence results showed that nocommon amino acid exist in the monoclonal TCR Vβ CDR3. Clonalexpansion of CD4+and CD8+TCR Vβfamilies can also be seen in10patients with chronic hepatitis B. In CD4+TCR Vβfamilies,Vβ2、5、7、9、11、12、13、15families showed clonal expasion and theexpansion frequencies of Vβ2、9、11families were higher than theothers families. In CD8+TCR Vβfamilies,Vβ2、6、7、8、9、10、11、12、21、22showed clonal expasion and the expansion frequenciesof Vβ7、8、9、11、12were higher than the others families. Therewas no significant difference in cloanl expansion number betweenCD4+and CD8+TCR Vβfamilies. The sequence results showed that someCD8+TCR Vβ families shared the same motif. TCR Vβ12families ofcase2and case8, Vβ7family of case9shared the same motif “TL”and TCR Vβ8family of case3, TCR Vβ11family of case5, TCR Vβ7、11families of case9shared the same motif “TL”.The resultsof IL-4and IFN-γ levels in the supernatant of cultered PBMC showedthat IL-4level in asymptomatic HBV carriers was signigicantly higer than that in healthy controls and that IFN-γ level inasymptomatic HBV carriers was signigicantly lower than that inhealthy controls.In chronic active hepatitis B, the IFN-γ level wassignificantly higher than that in healthy controls and there was nosignificantly difference in IL-4level between the two groups.Conclusions: In chronic asymptomatic HBV carriers, the monoclonalexpansion of CD4+TCR Vβfamilies play an important role in cellularimmune tolerance. In addition, different TCR CDR3sequences inmonoclonal expansion families indicated that many HBV antigens wereinvolved in the celluar immune response. In patient with chronicactive hepatitis B, both CD4+and CD8+T cell are crucial incellular immune response. Common amino acid motif “LF” and “PR”in some monoclonal CD8+TCR VβCDR3suggested that they may recognizethe same regions of different antigen epitopes; Loss of Th1/Th2cytokines balance plays an important role in immunopathogenesis ofchronic HBV infection.The previous study in TCR Vβclonity of patients with HBV infectionwas focused on PBMCs,not CD4+and CD8+Tsubsets. The results maybe disurbed each other. In our study, CD4+and CD8+T subsets wereisolated using antibodies coated magnetic beads, and then the clonitycharacteristics of CD4+and CD8+TCR Vβfamilies were analyzedrespectively to investigate the role of the two subsets in the pathogenesis of chronic HBV infection.RT-PCR-Genescan-sequence wasused to analyze the TCR clonity，the length and the sequence ofTCR CDR3of patients with chronic HBV infection, which may helpdetermine the antigen-specific TCR clone. It is useful to guide tothe reasonable treatment of HBV persistent infection.