The Affects Of Chronic HBV Infection On TCRβ Chain CDR3 Spectratype Of T Cells Subpopulation In Peripheral Blood

Background:HBV infection is a global problem. Epidemiological datum show that about 2 billion people worldwide have been infected with HBV, of which about 350 to 400 million present with chronic infection. Once infected by HBV, the interaction of the virus, liver cells and human immune response determine the prognosis of the disease. The cellular immune response mediated through the antigenic component of the virus is the primary cause of liver inflammation, and T cell dysfunction is the primary cause of the persistence of the virus and chronic hepatitis. Depending on the characteristics of the immune response, chronic HBV infection manifests in four clinically identifiable periods, and the immune characteristics, virus levels and extent of liver injury are different in each respective period. There are 2 types of antiviral drugs available, interferon a and nucleoside (acid) analogs cannot eliminate covalently closed circular viral DNA in liver cell nuclei completely. The virus can thus reproduce in some patients after discontinued medication. Studies have shown that sustained virological response and HBeAg seroconversion after antiviral treatment were related to the state of the immune system. Therefore, the immune status affects not only the natural progression of hepatitis B, but also the virological response on anti-viral treatment.Immune responses to HBV infection is mainly cell mediated, where T cells are the major effector cells. The T lymphoid cellular immune response affects the immune status through the processes of TCR recognition, T cell proliferation, differentiation, cell-mediated effects and other aspects. There are many ways to monitor the immune status of the human body, including detection of cytokines, cell frequency, cell toxicity experiments, proliferation experiments and so on. Recently, TCR has become an object of fervent research. The CDR3 spectratype of TCRβchain variable region (BV) is pinned to the physiological and pathological status of human body. Studies have reported that the core antigen-specific TCR genes when transduced into T cells can contribute to HBV-specific immune reconstitution, which indicates that TCR plays an important role in the effective control of the virus. Research on TCR may contribute to the understanding of the cellular immune status of CHB patients.In different T lymphoid clones, the length and sequence of the CDR3 region are different. The recognition of specific epitomes by T cells via its TCR leads to the activation and proliferation of T cell clones with a specific length of the CDR3 region. These clones constitute a unique TCR CDR3 pedigree, with a specific TCR CDR3 fingerprint. Analyzing TCR CDR3 length distribution of various T cell clones can contribute to an understanding of the composition of specific samples of T cells preliminarily, and lead to an understanding of the role of cellular immunity in immune pathology of the disease. Objectives:1. To learn more about the characteristics of the TCR BV CDR3 spectratype of a T cell subpopulation in peripheral blood of CHB patients by utilizing methods of cell subsets sorting and the TCR immune fingerprint technology. To minimize the interference of spectratypes of different sub-groups in order to determine the true immune status of patients with CHB.2. To provide an important clue to the mechanism of cellular immunity and its role in the natural progression of disease in CHB patients by analyzing the characteristics of TCR BV CDR3 spectratype of T cell subpopulation in peripheral blood of chronic HBV patients at different stages of infection. Dynamic observation of changes in TCR CDR3 spectratype will be valuable in discerning the basis for the development and prognosis of the disease.3. Attempt to find immunological indicators related to immune reconstitution and to further explore the immune mechanism of the virological response and HBeAg seroconversion in patients with CHB after antiviral treatment by studying changes in the TCR BV CDR3 spectratype in CD8+T cell subsets in CHB patients before and after antiviral treatment.Methods:1. Five immunotolerant chronic asymptomatic HBV carriers,8 patients with CHB,5 inactive HBsAg carriers, and 20 patients with CHB receiving laminvudine or Telbivudine antiviral treatment were enrolled in this study. Three healthy individuals were also participated, serving as a control group.2. The 10-30ml heparinized blood samples were collected from each patient and the control group. PBMCs were isolated from each sample using Ficoll-Hypaque density gradient centrifugation. All PBMCs were frozen in a cryopreservation medium with 90% fetal cattle serum and 10% dimethyl sulphoxide, and were stored in isopropanol boxes overnight at-80℃in a freezer prior to long-term storage in liquid nitrogen. When measurements started, the thawed cells were rested for over 2 hours, and only cells with more than 75% viability were made available for the subsequent CD8+cell sorting by magnetic beads. The purity of the CD8+cells was identified by flow cytometry.3. Total RNA was extracted from the 1×106 sorted CD8+and CD8-T cells respectively, using a commercial 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, reverse transcriptase and oligol8 primers in a total volume of 100μl. Then the TCR CDR3 transcripts were amplified by PCR using a specific primer for 24 TCR BV families and one BC region primer.4. The PCR products were initially analyzed on a 1.5%agarose gel by ethidium bromide staining. Then the products were run on an ABI 3730 DNA sequencer. Fragment size was determined using the GeneMarker software. The TCR CDR3 spectratype of the samples were analyzed based on the peak pattern and the monoclonal, oligoclonal expansion and skewed spectratypes were discerned for 24 BV families in each patient. The relative fluorescence intensity (RI) was calculated as:RI [%]=100%×clonal peak area/total peak area of corresponding family. According to the previous reports, we used the following criteria to determine whether or not TCR BV CDR3 family with perturbation which represents T cell expansion. (1) Single peak:RI of dominant peak was more than 35%. (2) Twin peaks: each peak’s RI was more than 25%. (3) Dominant peak differed from Gaussian patterns:RI of the peak was more than 25%.5. Statistical analysis:Continuous data were expressed as mean±standard deviation (SD). Independent t test or Paired t test were used to determine the significance of the rate of perturbed spectratype within two groups. Correlations were performed using the Spearman rank correlation. Differences were considered statistically significant at P<0.05.The statistical analysis was performed by using SPSS 13.0 software.Results:1. In order to balance the impact of cell frequency, three groups (1×105,1×106,3×106)were selected for detection of BV families via spectratyping. The overall shape of the graphs for each group was very similar. The main peak of the relatively intensity of fluorescence of each family were 36.23±0.55%,25.70±4.36%,38.20±2.19% and 30.83±1.53%, and the coefficients of variation were 0.015,0.170,0.057 and 0.050, which signify small variations. In addition, results of double testing the same samples showed good reproducibility.2. Different extents of shifting occurred in the TCR BV family’s CDR3 spectratype of cell subsets in the peripheral blood of the eight cases of CHB, in forms of monoclonal, oligoclonal and partial peaks of clonal proliferation. On the other hand the TCR BV family’s CDR3 spectratype of cell subsets in peripheral blood of the 3 normal control cases displayed a roughly normal distribution.3. Due to the existence of CD4+T cell subsets in total PBMCs, the dominant peak formed in TCR BV CDR3 spectratypes by this family, may overlap with the dominant peak that corresponds to the family of CD8+T cell subsets. Therefore when TCR BV-family CDR3 spectratype of CD4+T cell subsets shows a normal distribution it covers up the dominant peak that corresponds to the family of CD8+T cell subsets.4. The total number of TCR BV families with perturbation of CD8+T cell subsets of the eight CHB cases was higher than the number of CD8-T cells. (Paired t test,10.63±4.75 Vs.4.13±3.09, T=6.62, P<0.001).5. In the immuno-tolerant AsC group, CHB group and the HBsAg-inactive carrier group, the number of perturbation family that occurred in TCR BV CDR3 spectratype of CD8+T cell subsets were significantly more than the number of CD8-T cell (Paired t test:t=-3.69, P=0.034; t=6.62, P<0.001;t=7.51, P=0.005).6. The number of shifting family that occurred in TCR BV CDR3 spectratype of CD8+T cell subsets in immuno-tolerant AsC group was less than the number in the CHB group and the HBsAg-inactive carrier group significantly (LSD-t test: P=0.033; P=0.038). The number of shifting family in the HBsAg-inactive carrier group was more than that in the CHB group, but there was no statistical difference between them (P=0.863).7. In the immune tolerant phase, immune active phase and inactive stage of chronic HBV infection, shifting family that occurred in TCR BV CDR3 spectratype of CD8-T cells which represent CD4+T cells were rather few in number and were 1.50±1.30,4.13±3.09,4.75±3.60 respectively, and there were no statistical differences between groups.8. After antiviral therapy for 48weeks and according to the virological response, the subjects were divided into 2 groups:serum HBV DNA< 2.48 lg copies /ml were virological response group (VR), the rest belonged to the non-virological response group (NVR). There was no significant difference in the profile of the number of perturbed TCR families between the VR group and the NVR group before antiviral treatment (Independent t test:5.77±2.62 vs.7.71±4.19,t=-1.285, P= 0.319). In addition, neither ALT level nor serum HBV DNA level showed significant correlation with the number of CD8+TCR BV families with perturbatation (Spearman correlation:RR= 0.138, P=0.561; RR= 0.017, P= 0.944). 9. Within the first 12 weeks of treatment, the number of TCR BV families with perturbation in the VR group and NVR groups were more than that at the baseline. However, with the progress of anti-viral treatment, the number of TCR BV families with perturbation in the VR group at week 24 was significantly higher than that at week 12 (P= 0.028), while the NVR group was significantly lower (P= 0.012). Based on the changes in the perturbed TCR BV families from week 12 to 24, 3 distinct patterns were identified such as an ascendant pattern, a descendent pattern and a horizontal pattern, respectively. Six patients with a descendent pattern were persistently positive for serum HBV DNA at week 48. Further more, the number of descendent pattern in the NVR group was higher than that in the VR group. On the contrary,9 patients with ascendant pattern showed negative serum HBV DNA at week 48, and the number of ascendant pattern in VR group was higher than that in NVR group.10. At baseline,70% of the patients were PCR amplification negative in BV15 family, while 65% patients were with Spectratype Perturbation in BV24 family. During antiviral treatment, comparing the baseline with the week 12 and 24, the majority of BV families in the VR group showed newly emerging perturbation. In BV2, BV4, BV5 and BV9 especially,46% of patients showed newly emerging perturbed families. while the percentage of the NVR group patients with newly emerging perturbed families in BV21Conclusions:1. The TCR CDR3 Spectratype of healthy individuals show gaussian distribution, while the chronic hepatitis B patients showed obvious clonal expansion of T cells. The number of perturbed families in the CD8+subpopulation was significantly more than that of CD8-subpopulation. Overlapping interference between CD8+and CD8-T cell could be avoided through sorting of the T cell subpopulation.2. The number of total perturbed families in CHB group and HBsAg-inactive carrier group were significantly more than that in immuno-tolerant AsC group. This suggests that CHB patients and inactive carriers might have a higher degree of T cell expansion than immuno-tolerant AsC, and that T cell expansion might be involved in the immunopathogenic mechanism of hepatitis B virus.3. The dynamic pattern of TCR BV spectratype perturbation between week 12 and 24 could be a novel immunological parameter to predict the prognosis of antiviral treatment of CHB. Furthermore during antiviral treatment the restored antiviral immunity plays a pivotal role for further control of HBV replication, and additional immunoregulative therapy may be required for maintaining sufficient CTL levels.