The Influence Of HBV Persistent Infection On The CDR3 Spectratype Of αβT Cell Receptor β Chain In The Peripheral Blood Of Patients

BackgroundHepatitis B virus (HBV) infection is one of the public health events in the world. According to world health organization datum, more than 2 billions people have been infected with HBV, and approximately 350 millions people are chronically infected. It has been reported that chronically infected patients would become all kinds of chronic liver disease (CLD) and liver cirrhosis (LC) with higher risk, and the incidence of HCC in chronically infected patients was 100 times higher than that in popular crowds. Because of narrow host range of HBV and lack of routine cell culture systems, the study of pathogenesis responsible for HBV-induced hepatocelluar injury has been hampered. Recently, with the development of transgenic mice model system and improvement of new techniques, many evidences have demonstrated that cellular immunologic responses are involved in the pathogenesis of hepatocyte damage, and have indicated that weak cellular immunologic responses is one of the major reasons of HBV chronic infection. However, the little information of cellular immunologic responses is obtained in the HBV chronic infection, especially, about the relationship of viral load, HBV antigen concentration and T cellular immunologic response. Because previous data obtained from patients with HBV chronic infection is usually from expanded the T lymphocytes in vitro, it does not completely reflect the T cellular immunologic response in vivo. So it is very important to design the strategies of immunotherapy and to evaluate the patients' cellular immunologic response correctly. In present studies, we directly investigate the composition and T cell clonity of peripheral blood lymphocytes to understand the global picture of T cellular response to HBV in vivo in persistently infected patients and guide to reasonable treatment of HBV persistent infection.Objective1. To understand the role of T cellular immunologic response in the immunologic tolerance and the course of disease in chronic asymptomatic HBV carriers (AsC) through analysis of T cell receptor (TCR) complementarities determining region 3(CDR3) spectratype.2. Through TCR CDR3 spectratyping of patients with CHB, we want to explore the state of cellular immunologic response in vivo and the relationship between the pathogenesis and T cellular response; also to identify the HBV antigen specific T lymphocytes, and to provide the helps for developing the new individual immunological therapy.3. To understand the change of T cell immune response to HBV through comparing the TCR CDR3 spectratype of patients with CHB before and after antiviral treatment (Lamivudine therapy). The one aim is to investigate the changes of T cell clonity and relationship between the serum HBV DNA load, antigen concentration and T cellular immune response. The second aim is to guide the designation of clinical antiviral treatment strategies for CHB.4. To understand the homogeneous of the expanded T lymphocytes of peripheral blood lymphocytes in HBV chronically infected patients, the nucleotide sequences of TCRβchain CDR3 of expended T lymphocytes are further determined and the three dimensional structure were simulated and compared using the CPH models 2.0 Serve and IMGT database. These works are helpful to find new HBV specific CTL epitopes.Methods1.8 patients with chronic hepatitis B of active liver inflammation, 9 chronic asymptomatic HBV carriers, 4 patients with CHB receiving laminvudine antiviral treatment were enrolled this study, and 4 healthy individuals were as control group. The 10～20ml heparinized blood samples were collected from every patients and control group. Peripheral blood mononuclear cells (PBMCs) were isolated from every samples using Ficoll-Hypaque density gradient centrifugation.2. Total RNA was extracted from PBMCs using TRzol agents or total RNA extracted kit. The precipitated RNA was dissolved in RNase-free distilled water. The quantity of the total RNA was determined by spectrophotometry. The first strand cDNA was synthesized using 1μg total RNA, reversers transcriptase and oligo(dT)18 primers in a total volume of 50μl. Then the TCR CDR3 transcripts were amplified by polymerase chain reaction (PCR) using specific primer for 24 TCR BV families and one BC region primer (FAM fluorescence labeled).3. The PCR products were initially analyzed on 1.5% agarose gel by ethidium bromide staining. Then the products were run on 6% polyacrylamide denaturing sequencing gels containing 8M urea in an ABI 377 DNA sequencer. Fragment size was determined automatically using the GeneScan 672 software. The TCR CDR3 spectratype of samples were analyzed on the peak pattern and the monoclonal, oligoclonal expansion and skewed spectratype were judged for every BV families in every patient.4. The PCR products of the TCR BV families showing clonal expansion were amplified again by the same TCR BV family sense primers and TCR BC anti-sense primers (without FAM-labeled) at the same PCR condition. The PCR products purified were ligated to PMD 18 vectors and plasmids were transfected into Escherichia coli JM 109 using TA cloning kit. Nucleotide sequences of TCR CDR3 were determined on an ABI 377 DNA sequencer and were analyzed using DNAtools 6.0 and Vector NTI Suite 8.0 softeware.5. The three dimensional structure of TCR CDR3 were simulated and compared using the CPH models 2.0 Serve and IMGT database.Results1. In our study, the products of TCR BV 24 families were analyzed by ethidium bromide staining after 1.5% agarose gel electrophoresis. The analyzed results of TCR BV 24 families of healthy individuals and most BV families of patients with chronic hepatitis B (include 4 patients receiving antiviral treatment) showed a blur band in the predicted position of products size. A few BV families of CHB (include 4 patients receiving antiviral treatment) were not found the band. 2. When the fluorescent PCR products were analyzed on 6% acrylamide sequencing gel, every BV family of every healthy individual showed more than 8 bands. Most BV families of 9 chronic asymptomatic HBV carriers and 12 patients with CHB (include 4 patients receiving antiviral treatment) also showed more than 8 bands. However, some BV families presented a single or double fluorescent bands, another families were not received any fluorescent signals.3. The results of GeneScan analysis indicated that the patterns of CDR3 of all the TCR BV families in 4 healthy control donors showed Gaussian distribution. However, the results of 9 chronic asymptomatic HBV carders and 12 patients with CHB (include 4 patients receiving antiviral treatment) indicated that partial BV families presented a single or double dominant peaks, some BV families presented one dominant peaks at the background of multiple peaks differed from Gaussian distribution. In addition, a few families showed absent signals.4. when we analyzed the TCRβchain CDR3 sequences of BV8, BV13 of case 1, BV22 of case 3, BV8 of case 5, BV13, BV19 of case 9, BV14 of case 6, BV12of case7, BV10 of case8 in chronic asymptomatic HBV carriers, we found that they shared different nucleotide and amino acids sequences. Similar analysis was performed in patients with CHB. We also obtained the TCRβchain CDR3 sequences of 9 clonal expanded T cell clones in patients with CHB of inflammatory active stages. Although they also had not same CDR3 sequences, but TCR BV9 of case 1, BV5 of case 2, TCR BV12 of case 3, TCR BV4, TCR BV16 of case 5 shared same motif "QY"; TCR BV9 of case 1 and TCR BV12 of case 3 shared same motif "YE"; TCR BV13 of case 2; TCR BV16 of case 5 shared same motif "QT"; TCR BV17 of case 4 and TCR BV18 of case 5 shared same motif "GELF". We did not find the expanded T cell clones sharing the same as CDR3 sequences in patients with CHB before and after lamivudine treatment. However, patient-3-BV3 and patient-1-BV20 shared same motif "QH"; patient-2-BV9, patient-4-BV1, patient-1-BV6, BV8 shared same motif "QY".5. We simulated the three dimensional structure of TCR CDR3 of clonal expanded T cells using the CPH models 2.0 Serve and IMGT database in the 8 patients with CHB of active liver inflammation and 4 patients with CHB before and after antiviral treatment. However, we did not find any same structures of TCR CDR3 in diffenent patients and diffenent BV families. 6. According to the judgment of the relative florescence intensity, there was significant skewed spectratypes before and after lamivudine treatment in 24 BV families in 4 patients with CHB. Although there was no statistical difference in the total rate of skewed patterns before and after antiviral treatment(t=0.513, p＞0.05), but remarkably changes for TCR CDR3 spectrtypes in some individual BV families in 4 patients were found pre- and post- therapy. We could find that some BV families with skewed spectatypes before treatment have restored to the Gaussian distribution after antiviral treatment. Other BV families with normal patterns presented skewed spectatypes, consisting monoclonal expansions, oligoelonal expansions and absence.Conclusion:1. By CDR3 sepctratyping, we have demonstrated that there are significantly restricted TCRβchain CDR3 spectratypes of peripheral blood in chronic asymptomatic HBV carders. This result suggests that there are many T cell clone expansions in chronic asymptomatic HBV carriers and these clonal expansions may be HBV antigen-driven expansion. Expanded T cells maybe play an important role in inducing the cellular immunological tolerance. In addition, expended T cells with different TCR CDR3 sequences further indicates that many antigen epitopes are involved in the T cellular immunologic responses. This maybe related to the difference of HLA genotype and HBV mutation in different patients.2. The results of TCR CDR3 spectratyping revealed clonal expansion in all 8 patients with CHB of active liver inflammation, most of which showed more than two T cell clones. The number ofHBcAg specific-r IFN secreting cells in the patients with CHB was higher than that in the health control. These data indicated the T cell immunologic responses were involved in the immunopathogenesis of CHB. Active liver inflammation may be caused by the T cell clonal expansions. T cell clonal expansions are stimulated by several HBV epitopes, because expanded T cell clones have different CDR3 sequences. The different three dimensional structure of TCR CDR3 of clonal expanded T cells further demonstrated the T cell responses were polyclonal and multispecific in CHB patients with active liver diseases. Moreover, common motifs of expanded T cell clones suggest that they might recognize the same regions of different HBV peptides, but have different due to HBV mutational changes.3. By analyzing the TCR CDR3 spectratyping of patients of CHB before and after lamivudine treatment, we can draw following conclusions:①After antiviral treatment, the total rate of skewed spectratypes of TCR CDR3 is similar to that of before treatment. It maybe associated with slow-moving decrease of HBV antigen.②The changes of spectratypes of partial BV families before and after antiviral treatment indicate viral load can directly affect on the T cell immunologic response in patients with CHB. On the one hand, with the decrease of viral load, HBV antigens gradually declined, and the expanded T cells, which were stimulated by these antigens, will lose activation. The TCR CDR3 pattern restored Gaussian distribution. On the other hand, with the decrease of viral load, some T cell clones which were exhausted by high viral load might present again.③The changes of spectratype before and after lamivudine treatment further indicate the expanded T cell clones in patients with CHB of peripheral blood maybe HBV antigen-specific.④The different three dimensional structures of TCR CDR3 of clonal expanded T cells further reveal that these T cell clones were stimulated by different antigens before and after antiviral treatment.4. Our results also indicate the CDR3 spcetratyping technique is sensitive method to assess the T cell responses in vitro. This method can make us not only understand the composition of T cell repertoire in vivo but also find out the antigen selective clonal expanded T cell clone in complex T cell population. The combination of simulating the TCR CDR3 structure and Tetramer technique maybe help us to discover the new antigen epitopes.