1460. Differential Role of Liver-targeted Regulatory T Cell to Hepatitis B and C Virus in Chronically Infected Patients

H. Miyaaki; T. Ichikawa; K. Nakao; H. Shibata; M. Akiyama; S. Miuma; Y. Motoyoshi; M. Fujimoto; K. Eguchi

 

Regulatory T cells play a critical role in chronic virus infection. The role of regulatory T cell in chronic hepatitis B and chronic hepatitis C is unknown. We examined the distribution and frequency of Foxp3 positive regulatory T cell in the liver tissues and compared the clinicopathlogical characters of HBV and HCV.

 

Methods

Liver needle biopsies were collected from 26 patients with hepatitis B surface antigen positive (mean age:33.6±10.4, male: female=23:3) and 27 patients with hepatitis C virus antibody positive (mean age 57.8±10.6, male: female=15:12). In each liver tissues, T cells were examined with anti-CD3 antibody (Novocastra, Newcastle, UK) and regulatory T cells were examined with anti-Foxp3 antibody (eBioscience, San Diego, CA, USA). The average proportions of FoxP3+Tregs among the total number of CD3+T cells in each portal tracts was determined. The ratio of Foxp3 in CD3 was similar in HBV and in HCV cases(HBV:HCV=0.09±0.04:0.09±0.05).

 

In HBV cases, there was significant correlation between the ratio of Foxp3 in CD3 and serum ALT level (P=0.025, r=0.402). The ratio of Foxp3 in CD3 was significantly increase in severe activity group than in mild activity group (mild:severe=0.08±0.03:0.11±0.04, P=0.04). There were no significant differences in the ratio of Foxp3 in CD3 in HBV cases among the other clinical factors (age, gender, platelet, fibrosis, HBV-DNA viral load). In HCV cases, the variables that were significantly associated with the ratio of Foxp3 in CD3 were serotype2 (serotype1: serotype2=0.07±0.04:0.15±0.05, P=0.0002) and male gender (male:female = 0.07±0.03:0.11±0.05, P=0.04). These factors were easy sensitivity factors for interferon therapy. There were no significant differences in the ratio of Foxp3 in CD3 in HCV cases among the other clinical factors (age, ALT level, platelet, activity, fibrosis, HCV viral load).

 

Conclusion

In conclusions, these data provided the impaired of regulatory T cell function cause acute exacerbation in chronic hepatitis B. On the other hand, regulatory T cell may be related with action of interferon in chronic hepatitis C.

 


1467. Quantitative Detection of Integrated HBV Surface Gene Sequences in the Liver by Real-Time PCR

A. Laras; A. Kostamena; S. J. Hadziyannis

 

Introduction

Chronic hepatitis B (CHB) patients under long-term nucleos(t)ide analogue treatment may achieve profound HBV suppression with non-detectable serum HBV DNA and low intrahepatic cccDNA levels. Continuing surface antigen expression observed in these patients could be attributed to transcribed integrated HBV DNA harbouring intact surface antigen (S) gene sequences.

 

Aim

To develop a quantitative method for the detection of integrated HBV DNA harbouring S sequences in the liver of CHB patients under suppressed HBV replication and to evaluate its performance in liver biopsy samples of patients under long-term antiviral therapy.

 

Methods

Liver biopsy specimens from 20 CHB patients in profound HBV suppression under nucleos(t)ide analogue treatment were tested for integrated HBV S gene sequences using specifically designed real-time PCR assays that simultaneously measure intrahepatic HBV DNA in the S and the core promoter-precore (CP-preC) regions. HBV integration occurs predominantly in the CP-preC region of the viral genome leaving S sequences frequently intact. Cloned and serum HBV DNA were used as controls. Intrahepatic HBV cccDNA was also measured. HBsAg and HBcAg expression was also assessed in the same biopsy samples by immunohistochemical methods.

 

Results

Low HBV cccDNA levels were found in all patients, median 0.31 (range 0.05-0.84) copies/cell. In fact, cccDNA was the predominant form of viral DNA representing on the average 79% of total intrahepatic HBV DNA. These conditions permitted quantitation of low copy integrated HBV sequences. Total intrahepatic HBV DNA levels measured in the S region were significantly higher than in the CP-preC region, 0.70 (0.10-2.65) vs 0.38 (0.05-1.74). In 11/20 patients, as well as in cloned and serum HBV DNA controls, there was no discrepancy between the two measurements. In 9/20 patients S levels were significantly higher (2-fold to 13-fold) than the corresponding CP-preC measurments (0.71 vs 0.20). In these patients, integrated HBV S sequences were detectable in significant levels, 0.51 (0.17-1.4) cp/cell. Consistent with these findings were the results of histochemical demonstration of HBsAg proteins in the same liver samples.

 

Conclusions

1. We have developed a method for quantitative detection of integrated HBV DNA harbouring S gene sequences in liver biopsy samples. 2. This method can only be used in situations of highly suppressed viral replication permitting detection of low copy integrated S sequences. 3. Integrated HBV S sequences were detectable in approximately 50% of treated patients with compatible findings in immunohistochemical observations of HBsAg expression in the liver.

 


146. Chronic HBV Evolution Is Associated with Numeric and Phenotypic Changes in Peripheral HBV-core Epitope-Specific CD4+ T-cells: A Study Using a Novel HBV-core-Specific HLA-DRB1*0101 Tetramer for the Analysis of Antiviral CD4+ T-cell Responses During Acute and Chronic Hepatitis B

M. H. Heeg; A. Ulsenheimer; N. H. Gruener; H. M. Diepolder; W. Schraut; M. Waechtler; R. Zachoval; G. R. Pape; M. C. Jung

 

A strong HBV-specific CD4+ T cell response is associated with spontaneous viral clearance in acute hepatitis B but is weak or short-lived in patients developing chronic disease. So far, this CD4+ T-cell response could just be investigated by functional assays which lack the possibility of further T-cell phenotyping and that are unable to identify dysfunctional cells. HBV-specific HLA-class-II tetramers now can negotiate those limitations.

 

Method

To establish a new HBV-core MHC-class-II tetramer we screened 33 patients with acute HBV infection by overlapping HBV-core peptides and identified a HLA-DRB1*0101 restricted HBV-core epitope. This epitope was recognized by all DRB1*0101 patients with acute HBV infection as well as by 77 % (17/22) of all patients with acute HBV. Using this epitope we assembled a new DRB1*0101 MCH-class-II tetramer. The tetramer specifically stained a corresponding CD4+ T cell clone. To proof the tetramer sensitivity dilution experiments of Tet+ clone cells into PBMC were performed and a strong correlation between added and measured frequencies was found. In six HBV-negative DR1+ individuals and nine patients with acute hepatitis B lacking DR1, no CD4+ cells were tetramer positive.

 

In three HLA-DR1+ patients with acute hepatitis B, HBV-specific CD4+ T cells were detectable during the acute phase of disease, ranging from 900 to 1680/106 CD4+ T cells during the peak phase. The peak phase was associated with an ALT peak and viral clearance as well as with a peak level of IFN-γ secretion. The frequencies of HBV specific CD4+ T-cells decreased rapidly after viral clearance but were detectable in considerable levels over several months. In contrast, at different time points of disease 15 investigated patients with chronic hepatitis B showed no or only very low frequencies of tetramer positive cells. During the course of acute self limiting hepatitis B, phenotypic characterization demonstrated a rapid increase of IL7R and CD62L expression on Tet+/CD4+ T-cells.

 

Conclusion

In conclusion, using a new MHC-class-II tetramer containing one immunodominant HLA-DRB1*0101 restricted epitope within HBV-core, we were able to quantify and characterize HBV specific CD4+ T cell responses patients with acute hepatitis B and chronic hepatitis B. In patients with chronic disease almost no Tet+/CD4+ T-cells were found indicating a complete loss of antiviral CD4+ T-cells rather than a loss of function of those cells. Furthermore those cells showed an impaired capacity to develop a memory phenotype. So far, tools that allow to distinguish between the absence of specific cells or their non-function were not available.

 


87. Dual Over-expression of Insulin Receptor Substrate-1 and Hepatitis Bx Gene Causes Pre-malignant Changes in Liver

L. Longato; S. de la Monte; N. Kuzushita; M. Horimoto; B. Slagle; A. Rogers; J. R. Wands

 

Background

The cellular changes that contribute to the development of hepatocellular carcinoma (HCC) are poorly understood. Activation of the IN/IRS-1/Ras/Raf/MAPK and the Wnt Frizzled/β-catenin signaling cascades is frequent in HCC related to persistent hepatitis B viral (HBV) and hepatitis C viral (HCV) infection. The aims of this study were to recapitulate these findings in an animal model system by providing a proliferative stimulus via IRS-1 in the context of HBx protein over-expression in transgenic mice. The purpose was to determine if alteration in these genes is sufficient to produce dysplasia and cellular transformation in the normal liver.

 

Methods

We generated transgenic mice in which the HBx (ATX; N=16), IRS-1 (N=15) or both (ATX+/IRS-1+; N=23) genes were over-expressed under a liver specific promoter. Wild-type litter mates were included as controls (N=11). We also assessed oxidative damage as well as upregulation of signaling molecules related to these signal transduction cascades in the liver by “real time” RT-PCR. Histologic analysis included an assessment of lobular and periportal inflammation, macro and microsteatosis, polyploidy, dysplasia and tumor formation.

 

Results

ATX+/IRS-1+ double transgenic livers had increased frequency of hepatocellular dysplasia and several developed HCC (N = 3). IRS-1+ and ATX+/IRS-1+ livers had more extensive hepatic steatosis compared to the other groups. All three transgenic lines had significantly increased IRS-1, IGF-1, proliferating nuclear cell antigen, Wnt 1 and Wnt 3 mRNA levels, and increased immunoreactivity for 8-OHdG and HNE indicative of DNA damage and oxidative stress. The ATX+/IRS+ double transgenic mice were distinguished by having the highest level of activation of Wnt 3 and Frizzled 7 and selectively increased expression of IGF-II and aspartyl (-asparaginyl)-β-hydroxylase (AAH) a downstream insulin responsive gene associated with increased cell motility and migration.

 

Conclusion

These results suggest that over-expression of HBx or IRS-1 can contribute to hepatocyte transformation by promoting cell turnover and DNA damage but these effects are not sufficient to trigger neoplastic changes in the liver. However, chronic over-expression of HBx together with IRS-1 is sufficient to cause hepatocellular dysplasia and HCC in vivo suggesting that upregulation of both the IN/IRS-1/MAPK and Wnt/β-catenin signaling cascades are important in the transformation of normal hepatocytes to the malignant phenotype.

 


109. In Vitro and In Vivo Inhibition of HBV Replication by Peptide Aptamers Via Disruption of the HBX-Proteasome Interaction

U. Zaid; Z. Zhang; T. Liang

 

The X protein (HBX) of the hepatitis B virus (HBV) is important for productive infection in vivo. Although this viral protein is not essential for viral replication in vitro, HBV negative for HBX replicates less efficiently in transfected cells. Our previous studies have suggested that the interaction between HBX and the proteasome complex may underlie the pleiotropic functions of HBX (Hu et al. JVI 2006; 8-: 1405-1413, Zhang et al. JCI 2001; 108: 1523-1531, Zhang et al. JBC 2000; 257: 15157-15165, Zhang et al. JVI 2004; 78: 4566-4572). We have further demonstrated that inhibition of cellular proteasome activities enhances hepadnavirus replication in an HBX-dependent manner.

 

Aim

In this study, we aim to develop potential anti-HBV agents by targeting the functions of HBX using a novel screening process.

 

Methods

A modified yeast two-hybrid disruptor system was developed to screen a randomly generated library of peptide aptamers displayed on the bacterial protein Thioredoxin A (TrxA). By screening for a disruption of the interaction between HBX and the proteasome subunit PSMA7, 367 yeast transformants were isolated from 1.5 x 10^7 independent yeast colonies of the peptide aptamer library. On secondary screen against nonspecific interacting pairs, 23 colonies were confirmed to show specific disruption of the HBX-PSMA7 interaction. The peptide aptamers from these colonies were isolated, sequenced, and cloned into a plasmid expression construct for transfection into HepG2 cells for functional studies. Transcriptional activation assays by HBX demonstrated that most of these peptide aptamers interfered with HBX transactivation by decreasing the reporter activities to 20-80% of the control level. When co-transfected with an HBV replication competent construct, most of the peptide aptamers which inhibited HBX transactivation also suppressed HBV viral replication by 50-80%. Very preliminary in vivo murine studies via intravenous injection of one of the peptides suggest a beneficial effect on inhibition of HBV DNA in HBV transgenic mice. Further studies are required to confirm this observation.

 

Conclusion

Our results demonstrate that selection of random peptide aptamers based on disruption of the HBX-proteasome interaction using a modified yeast two-hybrid system may identify peptide aptamers as potential therapeutic drugs for HBV infection. Also, this system may provide a molecular and structural basis for novel antiviral drug development.

 


110. Induction of Antigen-Specific Humoral and Cellular Immune Responses by Antigen-Pulsed Human Blood Dendritic Cells in Hepatitis B Vaccine Nonresponders and Patients with Chronic Hepatitis B

S. Akbar; O. Yoshida; S. Furukawa; Y. Hiasa; N. Horiike; M. Onji

 

Dendritic cells (DCs) loaded with HBV-related antigens induce HBV-specific immunity in HBV-transgenic mice. However, the utility of antigen-pulsed DCs have not been assessed in human in the context of HBV infection. This study was conducted (1) to prepare hepatitis B surface antigen (HBsAg)-pulsed human blood DCs, (2) to evaluate their safety and (3) to assess their prophylactic and therapeutic efficacies against HBV.

 

Methods

Six hepatitis B vaccine nonresponders, who never developed antibody to HBsAg (anti-HBs) due to immunization with commercial vaccines for 6-9 times and 5 patients with chronic hepatitis B were enrolled in this study. Immature blood DCs were prepared by culturing peripheral blood mononuclear cells with human grade granulocyte-macrophage colony stimulating factor and interleukin-4 for 7 days. HBsAg-pulsed DCs were prepared by culturing immature DCs with HBsAg (Hepatvex II, Banyu Pharmaceutical Co, Tokyo, Japan) for 8 hours. Five million HBsAg-pulsed DCs were administered to each individual and parameters of generalized inflammation, liver functions, kidney functions, and autoantibodies were checked at day 0, 1, 3, 7, 14, 28, 90, and 180 and after 2 years. HBsAg-specific T cell responses and anti-HBs were assessed in all subjects before and after administration of HBsAg-pulsed DCs.

 

Results

HBsAg-pulsed human blood DCs expressed DC-related markers such as HLA DR, CD86 and CD40. In vitro, HBsAg-pulsed DCs induced proliferation of HBsAg-specific T cells and anti-HBs from HBsAg-specific B cells. Hepatitis B vaccine nonresponders or patients with chronic hepatitis B did not develop any adverse effects due to administration of HBsAg-pulsed DCs. All vaccine nonresponders and patients with chronic hepatitis B did not show anti-HBs or HBsAg-specific T cells before administration of HBsAg-pulsed DCs. Anti-HBs and HBsAg-specific T cells were detected in the peripheral blood of all vaccine nonresponders within 28 days after single administration of HBsAg-pulsed DCs. HBsAg-pulsed DCs were safe for all patients with chronic hepatitis B. Three and 2 of these patients exhibited HBsAg-specific T cells and anti-HBs in the peripheral blood, respectively.

 

Conclusions

This study has opened a new field of immune intervention strategy against HBV infection in human in which human consumable HBsAg-pulsed DCs have been prepared in vitro and their safety and efficacy have been confirmed in vivo. The principle of this study may be used for development of cell-based prophylactic and therapeutic vaccines against hepatitis C virus and other viruses when those cannot be achieved by traditional approaches.

 


111. Powerful Inhibition of Hepatitis B Virus Replication by CpG-induced Cytokines in Combination with Lamivudine In Vitro

I. E. Vincent; J. Lucifora; I. Chemin; D. Durantel; F. Zoulim; C. Trepo

 

Background and Aim

CpG oligodesoxynucleotides (CpG ODN) are synthetic agonists of Toll-like receptor 9 (TLR9) and potent inducers of innate and acquired immunity. Antiviral mechanisms of CpG ODN include induction of antiviral and pro-inflammatory cytokines (INF-α, IL-6), activation of natural killer cells (INF-γ), thereby promoting strong TH1, CD8 T cell and humoral responses. Current therapy of chronic hepatitis B infection, either interferon alpha or nucleoside analogs, fail to control hepatitis B virus (HBV) replication in most patients. This limitation is mainly due to the slow kinetic of viral clearance, HBV genetic variability and weak or absent anti-viral cellular immunity. This provides the rational for the development of immunotherapeutic approaches combining nucleosid analogs and TLR9 agonists to accelerate viral clearance and restore anti-HBV specific immune responses.

 

Our aim was therefore to evaluate the antiviral effect of CpG-induced cytokines combined with lamivudine on HBV replication in vitro.

 

Methods

HepaRG cells were infected with purified HBV virions or transduced with HBV recombinant baculovirus. Peripheral blood mononuclear cells from naïve individuals were stimulated with CpG ODN and cytokine secretion (INF-α, INF-γ, IL-6) determined by Elisa. HBV-infected or -transduced HepaRG cells were treated with CpG-induced cytokines in combination with lamivudine. Antiviral effects were evaluated 48h post-treatment by quantification of antigen secretion (HBsAg, HBeAg) and HBV DNA intermediates of replication by Southern blot.

 

Results

CpG-induced cytokines inhibited HBsAg and HBeAg production from infected HepaRG cells. The antiviral effect was demonstrated by up to 90% inhibition of intracellular HBV DNA intermediates in HBV transduced-cells. More importantly, the combination of CpG-induced cytokines with lamivudine reduced by one hundred fold the lamivudine IC50. Overall, combination was more effective than CpG-induced cytokines or lamivudine treatment alone.

 

Conclusion

CpG-induced cytokines with lamivudine represent a powerful combination to inhibit HBV replication in vitro, most likely by inhibiting different steps of viral replication. The use of TLR9 agonists combined with nucleoside analogs should be evaluated in vivo in order to assess restoration and duration of anti-HBV specific immune responses. The development of such novel immunotherapeutic combinations provides promising approaches to decrease the risk of liver disease progression in chronic hepatitis B patients.

 


112. Long Term Inhibition of Hepatitis B Virus Replication Using the Non Viral Episomal Vector pEPI-1

A. C. Jenke; V. Orth; H. Lipps; S. Wirth

 

Introduction and Aim

In the past, it has been shown that RNA interference (RNAi) has the potential to suppress hepatitis B virus (HBV) replication. For further gene therapy an optimal vector system free of common side effects as for example induction of mutagenesis has still to be found. The aim of this study was to test the non viral episomal vector system pEPI-1 as a tool for inhibition of HBV replication.

 

Methods

Recombinant vectors pEPI/HBV1 and pEPI/NonSense were constructed and transfected into HepG2.2.15 cells. After selection of transfected clones, cells were grown for 3 months in the absence of selection pressure and harvested at 2 and 12 weeks. The episomal status of the plasmid was confirmed by Southern analysis. The level of HBsAg mRNA was measured by RT-PCR and the concentration of HBsAg in the supernatant was determined using ELISA.

 

Results

Transfection of HepG2.2.15 cells with pEPI/HBV1 resulted in more than 90% suppression of HBsAg mRNA 2 weeks after transfection compared with the control plasmid pEPI/NonSense. This effect was even more pronounced after 12 weeks, when almost no HBsAg mRNA could be detected. Likewise, the secretion of HBsAg was inhibited by more than 95%. Also the number of DNA copies within the culture medium was significantly decreased.

 

Conclusion

The non viral episomal vector system pEPI-1 allows efficient long term suppression of HBV replication in the absence of selection pressure and without the risk for insertional mutagenesis or expression of additional viral proteins.

 


113. Adeno-associated Virus Rep78 Protein Inhibits Human Hepatitis B Virus Replication in Human Cells by Binding to X Promoter and C Promoter of HBV

T. Liu; H. You; M. Cong

 

Rep78, the large rep gene product of adeno-associated virus (AAV), is a highly multifunctional protein, which is required for AAV DNA replication and the trans-regulation of AAV gene expression. Apart from these essential functions prerequisite for the life cycle of AAV, Rep78 is able to inhibit the replication and gene expression of some DNA virus by binding to the promoter, such as HPV and HIV. The aim of this study was to determine whether Rep78 can bind to the promoter of HBV DNA and then inhibit the replication of HBV DNA.

 

Methods and Results

Here, we demonstrated that Rep78, as a chimeric with the maltose binding protein, binds to both the full length of HBV X promoter and the full length of HBV C promoter, as analyzed by electrophoretic mobility shift assay. To map the key region of binding, sequentially smaller substrates from the full length of promoter were synthesized and tested for recognition by Rep78. It is demonstrated that the binding between Rep78 and the full length of HBV X promoter was specific, but not with Rep78 and the smaller substrates from the full length. It was therefore possible that multiple binding sites were needed or the secondary structure of the full length of HBV X promoter was needed for specifically binding with Rep78. The smaller substrates from the full length, B1, the active region of HBV C promoter can bind to Rep78 specifically. Furthermore, investigating the biological implications of these interactions, Rep78 inhibited both the HBV X promoter and HBV C promoter activity in vitro transcription assays in Hela nuclear extracts, and the inhibition of X promoter was stronger than that of C promoter. In addition, to investigate whether the binding between Rep78 and HBV DNA promoters could inhibit the replication of HBV DNA, we constructed the plasmid containing the AAV2 rep78 gene and transfected the plasmid to the HepG2.2.15 cell line, the concentration of HBV cccDNA was decreased indicating that the replication of HBV was inhibited.

 

Conclusion

The ability of Rep78 to interact with the HBV X promoter and HBV C promoter and down-regulate corresponding transcription and replication suggests the mechanism by which AAV may modulate the HBV life cycle.

 


114. Construction of the shRNA Plasmid of Human fgl2 Prothrombinase Gene and Its Effect on hfgl2 Expression In Vitro

D. Xi; S. Gao; L. Li; C. Zhu; J. Guo; X. Luo; Q. Ning

 

Background and Aims

Our previous studies have shown an anti-sense plasmid to mouse fgl2 prothrombinase (mfgl2) gene significantly reduced mfgl2 gene expression in vivo, markedly ameliorated inflammatory infiltration, fibrin deposition and hepatocyte necrosis, prolonged the survival time period and elevated the survival rate in Balb/cJ mice with murine hepatitis virus type 3 (MHV-3) induced fulminant hepatitis. This study was designed to explore the opportunity of RNA interference technique in the inhibitory application of human fgl2 prothrombinase (hfgl2) expression, which has been reported to be involved in a variety of disease development including fulminant viral hepatitis, acute rejection of allo/xeno transplantation and fetal loss syndrome.

 

Methods

A plasmid named p-hfgl2shRNA complementary to the sequence of hfgl2 was constructed, meanwhile irrelevant shRNA plasmid with a random combination of the hfgl2shRNA sequence was used as control. A plasmid named pEGFP-hfgl2 expressing hfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-hfgl2shRNA on the hfgl2 expression. By cotransfection of p-hfgl2shRNA and pEGFP-hfgl2 or pcDNA3.1-hfgl2 expression construct into CHO cells, the inhibition of hfgl2 expression by hfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, real time PCR and immunohistochemistry staining. Results: The experiments showed the significant inhibitory effect of p-hfgl2shRNA on hfgl2 expression at 48h post-transfection by observation of green fluorescent cells, FACS, real time PCR and immunohistochemistry staining as well in CHO cell lines with the inhibitory efficiency reached as high as 85.5%.

 

Conclusion

The study demonstrated that the construct of p-hfgl2shRNA successfully interferred hfgl2 expression in vitro and this provides the foundation for further investigation of this construct’s application in vivo and further more as a therapeutic strategy for a targeting intervention in the disease control to which the gene fgl2 contributed. This work was supported by the NSFC30571643,30672380 and National Key Basic Research Program of China(2005CB522901).

 


146. Chronic HBV Evolution Is Associated with Numeric and Phenotypic Changes in Peripheral HBV-core Epitope-specific CD4+ T-cells: A Study Using a Novel HBV-core Specific HLA-DRB1*0101 Tetramer for the Analysis of Antiviral CD4+ T-cell Responses During Acute and Chronic Hepatitis B

M. H. Heeg; A. Ulsenheimer; N. H. Gruener; H. M. Diepolder; W. Schraut; M. Waechtler; R. Zachoval; G. R. Pape; M. C. Jung

 

A strong HBV-specific CD4+ T cell response is associated with spontaneous viral clearance in acute hepatitis B but is weak or short-lived in patients developing chronic disease. So far, this CD4+ T-cell response could just be investigated by functional assays which lack the possibility of further T-cell phenotyping and that are unable to identify dysfunctional cells. HBV-specific HLA-class-II tetramers now can negotiate those limitations.

 

To establish a new HBV-core MHC-class-II tetramer we screened 33 patients with acute HBV infection by overlapping HBV-core peptides and identified a HLA-DRB1*0101 restricted HBV-core epitope. This epitope was recognized by all DRB1*0101 patients with acute HBV infection as well as by 77 % (17/22) of all patients with acute HBV. Using this epitope we assembled a new DRB1*0101 MCH-class-II tetramer. The tetramer specifically stained a corresponding CD4+ T cell clone. To proof the tetramer sensitivity dilution experiments of Tet+ clone cells into PBMC were performed and a strong correlation between added and measured frequencies was found. In six HBV-negative DR1+ individuals and nine patients with acute hepatitis B lacking DR1, no CD4+ cells were tetramer positive. In three HLA-DR1+ patients with acute hepatitis B, HBV-specific CD4+ T cells were detectable during the acute phase of disease, ranging from 900 to 1680/106 CD4+ T cells during the peak phase. The peak phase was associated with an ALT peak and viral clearance as well as with a peak level of IFN-γ secretion. The frequencies of HBV specific CD4+ T-cells decreased rapidly after viral clearance but were detectable in considerable levels over several months. In contrast, at different time points of disease 15 investigated patients with chronic hepatitis B showed no or only very low frequencies of tetramer positive cells. During the course of acute self limiting hepatitis B, phenotypic characterization demonstrated a rapid increase of IL7R and CD62L expression on Tet+/CD4+ T-cells.

 

Conclusion

In conclusion, using a new MHC-class-II tetramer containing one immunodominant HLA-DRB1*0101 restricted epitope within HBV-core, we were able to quantify and characterize HBV specific CD4+ T cell responses patients with acute hepatitis B and chronic hepatitis B. In patients with chronic disease almost no Tet+/CD4+ T-cells were found indicating a complete loss of antiviral CD4+ T-cells rather than a loss of function of those cells. Furthermore those cells showed an impaired capacity to develop a memory phenotype. So far tools that distinguish between the absence of specific cells or their non-function were not available.

 


1031. Higher Percentages of Intra-Hepatic Regulatory T Cells Are Present in Chronic Hepatitis B Patients with a High Viral Load

H. L. Janssen; J. N. Stoop; A. M. Woltman; R. S. Binda; J. G. Kusters; P. E. Zondervan; E. J. Kuipers; R. G. van der Molen

 

CD4+CD25+FoxP3+ regulatory T cells (Treg) play a key role in the impaired immune response observed in patients with a chronic hepatitis B virus (HBV) infection. Most studies concerning Treg and HBV are performed with cells isolated from peripheral blood, while the liver is the main site of infection. To gain more insight in the role of Treg in chronic HBV we have monitored intra-hepatic Treg.

 

Methods

Peripheral blood and liver biopsy samples were obtained from 40 patients with a chronic HBV infection. The liver tissue was collected in culture medium and was digested with collagenase for 15 minutes to create a single cell suspension. The percentage of Treg was determined by flowcytometry using antibodies to CD4, CD25 and FoxP3.

 

Results

The results show that patients with a high viral load of >1 x 105 geq/mL have a significantly higher percentage of intra-hepatic Treg as compared to patients with low viral load <1 x 105 geq/mL (8.20 % ± 0.50% vs. 6.18% ± 0.54% respectively; p< 0.05), while the percentage of peripheral blood Treg was similar in both groups (9.38% ± 0.39% vs. 8.94% ± 0.43% respectively). When patients were divided into two groups based on liver inflammation, indicated by their serum ALT levels (ALT < 2 x ULN and ALT > 2 x ULN) no differences in percentages were observed between the two patient groups for intra-hepatic Treg (7.61% ± 0.56% vs. 7.03% ± 0.52% respectively) as well as for peripheral blood Treg (9.34% ± 0.31% vs. 8.89 ± 0.60% respectively). There was also no relation observed between the proportion of Treg and the Metavir Fibrosis score. Next to CD4+CD25+FoxP3+ Treg the liver also contained a large population of CD4+CD25-FoxP3+ cells (2.40% ± 0.21% (intra-hepatic) vs. 0.89 % ± 0.06% (peripheral blood); p < 0.05). Additionally, approximately 40% of liver Treg expressed the immunoregulatory marker programmed death-1 (PD-1) while peripheral blood Treg hardly express PD-1. PD-1 expression does not seem to be specific for Treg, since the liver contains more PD-1+CD4+ T cells compared to peripheral blood.

 

Conclusion

Liver CD4+ T cells have a higher expression of the immunoregulatory receptor PD-1. There was no relation between previous liver damage or liver inflammation and the presence of Treg. On the other hand, an increased proportion of intra-hepatic Treg was observed in patients with a high viral load, suggesting the importance of Treg in HBV tolerance and persistence of viral replication.

 


1032. Antigen-pulsed Dendritic Cells Are Capable of Inducing Both Innate ad Adaptive Immunity: A Lesson Learned from Cross-Talk Between Natural Killer Cells and Dendritic Cells and Their Application for Immune Therapy of Chronic Hepatitis B Virus Infection

O. Yoshida; S. Akba; T. Miyake; M. Hamada; Y. Hiasa; K. Michitaka; M. Onji

 

Purpose

The magnitudes of both innate and adaptive immunities are decreased in chronic infections and cancers. Accordingly, immune therapy against these diseases have been formulated to activate innate immunity targeting natural killer (NK) cells and adaptive immunity targeting dendritic cells (DC). This study was conducted to develop insights about crosstalk between NK cells and DCs and to assess if antigen-pulsed DCs can activate both innate and adaptive immunity.

 

Methods

Both normal C57BL/6 mice and hepatitis B virus transgenic mice (that express hepatitis B surface antigen (HBsAg), HBV DNA, and hepatitis B e antigen (HBeAg) are used. NK cells were depleted by administrating anti-asialo GM1 antibody, 100 microliter/mouse, intraperitoneal, twice at an interval of 48 hours. Normal C57BL/6 mice and NK-depleted mice were immunized with hepatitis B vaccine containing HBsAg, intraperitoneally and the levels of antibody to HBsAg (anti-HBs) and HBsAg-specific T cells were estimated 28 days after immunization. Spleen DCs from normal C57BL/6 mice and hepatitis B virus transgenic mice were cultured with HBsAg to prepare HBsAg-pulsed DCs and administered to NK-depleted mice, twice, at an interval of 2 weeks. We assessed if HBsAg-pulsed DCs activated innate immunity in NK-depleted mice by inducing proinflammatory cytokines (interleukin-12p70, interferon-gamma, tumor necrosis factor-alpha, and interleukin-6), 3 days after administration of HBsAg-pulsed DCs. Also, we evaluated if HBsAg-pulsed DCs induced anti-HBs and HBsAg-specific lymphocytes in NK-depleted mice.

 

Results

Flow cytometric analyses revealed that injection with anti-asialo GM1 in mice resulted in depletion of >90% NK cells from liver, spleen and bone marrow. Compared to normal C57BL/6 mice, DCs from NK-depleted normal C57BL/6 mice produced significantly lower levels of all proinflammatory cytokines (p<005) and could not activate HBsAg-specific lymphocytes in vitro indicating that NK cells are essential for proper functioning of DCs. High levels of anti-HBs were detected in the sera of all normal mice (10 of 10, 100%), but very low levels of anti-HBs were found in only 2 of 10 NK-depleted mice due to immunization with HBsAg (p<0.05). Also, the levels of HBsAg-specific T cells were significantly lower in NK-depleted mice compared to those in normal C57BL/6 mice (p<0.001). Administration of HBsAg-pulsed DCs induced higher levels of all proinflammatory cytokines, anti-HBs and HBsAg-specific lymphocytes in NK-depleted mice.

 

Conclusion

This study provides the rationale for using antigen-pulsed DCs for treatment of chronic infections when both innate and adaptive immunities are impaired.

 


1042. Expression of Notch Signaling and Antigen Processing Molecules in Progressive Pathogenenic Biopsies of Chronic Hepatitis B Infection

N. T. Pati; S. Bose; S. Baveja; H. Syed; S. K. Sarin

 

Background

The Notch signaling plays a key role in cell-fate determination and differentiation, functioning in a cell- and tissue-specific manner. Both up-regulated and down-regulated Notch 1 signaling is seen in carcinogenesis of different tissues. Whereas, in stem cells, notch activity inhibits differentiation and promotes carcinogenesis but in primary epithelial cells high notch activity leads to exit from the cell cycle and commitment to differentiation. Notch 1 exerts specific protective effects against HPV-virus and down-modulation of Notch 1 was observed in the late stages of HPV-induced carcinogenesis.

 

Aim

To study notch signaling in different stages of HBV induced liver disease and its role in liver disease progression. Patients and Methods: PBMCs of chronic hepatitis B (CHB) (n=10) (age 29.4±13.2 yr., M:F::8:2) patients and healthy controls (n=10) (age 27.9 ± 4.6 yr., M: F ::7:3) and liver biopsies from normal liver – 3 (age 49±3.8 yr.M:F::2:1), CHB patients (age 39±12.8, M:F::3:1) HBV cirrhosis -4 (age-36.2±20.0, M:F::3:1) and HBV related HCC –3(age-55.5±7.2 yr., M:F::3:0) were studied. Total RNA was extracted from PBMCs and biopsies and Notch1, Hes1, Jagged1, and NFKB genes were analyzed by quantitative RT-PCR.

 

Results

Compared to PBMCs of CHB patients, the liver biopsies showed significantly lower expression of Notch1 (p=0.034), Jagged 1 (p=0.021) but not Hes 1 (Table 1). The reduction in expression of these genes was significantly greater in HCC patients compared to CHB and controls. In comparison to control biopsies and PBMCs in CHB, cirrhotic tissue showed significantly lower expression of NFKB (p=0.02).

 

Conclusion

In different stages of liver disease due to HBV, the PBMCs show higher expression of notch signaling molecules, but in the target liver tissue, the expression of these molecules is significantly inhibited, maximum being in CHB and HCC. This expression pattern of Notch1 signaling suggests that this protein may play a permissive or tumor-promoting function in chronic HBV infection.

 

CHB PBMCs

CHB Bx

P

Cirrhotic Bx

P

HCC Bx

P

Notch1

0.39

-0.006

0.034

-0.03

NS

-0.06

0.05

Hes1

0.82

-0.26

0.4

-0.29

NS

-0.5

NS

Jagged1

0.63

0.057

0.021

0.02

NS

-0.34

0.034

NFKB

2.2

-0.3

0.027

-0.42

0.021

-0.25

0.034

 


1043. Intrahepatic Status of Regulatory T cells in Autoimmune Hepatitis, Primary Biliary Cirrhosis, Chronic Hepatitis C, and Chronic Hepatitis B

M. Sakaki; K. Hiroishi; T. Baba; M. Kushima; M. Imawari

 

Background/Aims

Regulatory T cells (Tregs) maintain immunological tolerance and suppress autoreactive immune responses. We evaluated the intrahepatic status of Tregs in patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), chronic hepatitis C (CH-C), or chronic hepatitis B (CH-B).

 

Methods:

We analyzed 85 patients (20 AIH, 22 PBC, 27 CH-C, and 16 CH-B) and 14 controls. Using liver tissue samples obtained by needle biopsy or from marginal parts of resected metastatic liver tumors in controls, immunohistochemical analyses of forkhead box P3+, which is a specific marker for Tregs, CD4+, and CD8+ cells were performed.

 

Results

Intrahepatic Tregs were significantly infiltrated in patients with liver diseases than in controls. There were significantly fewer intrahepatic Tregs in AIH patients than in PBC patients (p=0.037). Patients with a low frequency of intrahepatic Tregs were detected significantly less in the AIH and CH-B groups than in the PBC and CH-C groups. We found significantly less infiltration of CD4+ T cells in AIH than in other diseases (p<0.05). Liver-infiltrating CD8+ T cells were detected more frequently in the CH-B group than in other groups (p<0.003).

 

Conclusion

Intrahepatic Tregs were increased in both patients with autoimmune liver diseases and those with viral hepatitis. In autoimmune liver diseases, intrahepatic Tregs in AIH patients were fewer than in PBC patients.

Frequency of intrahepatic FOXP3+, CD4+, and CD8+ cells in patients with each liver disease.

 

AIH

PBC

CH-C

CH-B

Control

%FOXP3+

5.2±2.4*,**

7.2±3.5 *,**

6.2±3.7 **

6.3±1.6 **

1.1±1.1 **

%CD4+

37.8±11.0†

47.4±11.0 †

44.0±9.3 †, ¶

50.6±8.7 †, ¶, Ω

41.0±10.9Ω

%CD8+

32.2±12.2‡

33.3±10.4 ‡

30.5±7.0 ‡

44.2±9.3 ‡

32.0±1.9‡

The values are means ± SD. *p = 0.037 (AIH vs PBC); **p < 0.001 (Patients vs Control) †p = 0.007, 0.045, 0.001 (AIH vs PBC, CH-C, CH-B); ¶p = 0.026 (CH-C vs CH-B); Ω p = 0.013 (CH-B vs Control); ‡p = 0.003, 0.002, 0.001, 0.001 (CH-B vs AIH, PBC, CH-C, Control).