Conceived and designed the experiments: PNH PJC. Performed the experiments: HTT HFT HJL YJL. Analyzed the data: PJC HFT PNH. Contributed reagents/materials/analysis tools: LC HFT. Wrote the paper: HTT PNH.
The authors have declared that no competing interests exist.
Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. High PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T cells in mice with HBV persistence, but PD-1 upregulation was resolved in mice which had cleared HBV. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Blockade of PD-1/PD-L1 interactions increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes. Furthermore, blocking the interaction of PD-1 with PD-L1 by an anti-PD-1 monoclonal antibody (mAb) reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV
Hepatitis B virus (HBV) causes acute and chronic inflammatory liver diseases and subsequent hepatic cirrhosis and hepatocellular carcinoma (HCC). During chronic HBV infection, a dynamic balance between viral replication and the host immune response is pivotal to the pathogenesis of liver disease. It is widely accepted that adaptive immune responses, particularly cellular immune responses, mediate the clearance of HBV
The immunologic receptor, programmed death (PD)-1, a 55-kDa transmembrane protein containing an immunologic receptor tyrosine-based inhibitory motif, was originally isolated from a T-cell line exhibiting a high sensitivity to apoptosis
A. C57BL/6 or BALB/c mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. Four weeks post-injection, intrahepatic lymphocytes were isolated and PD-1 expression was analyzed by flow cytometry. B and C. C57BL/6 mice were hydrodynamically injected with pAAV/HBV1.2. Four weeks after hydrodynamic injection, intrahepatic lymphocytes from mice which were HBsAg-positive (carrier) or HBsAg-negative (cleared) were isolated, and the PD-1 expressions by CD8+ (B) and CD4+ (C) T-ells were analyzed by flow cytometry. The data were representative of at least 12 independent experiments. D. C57BL/6 mice were injected with pAAV/HBV1.2 plasmid hydrodynamically. HBsAg-positive carrier were sacrificed at 4 weeks post-injection. Intrahepatic lymphocytes and splenocytes were isolated and the populations of CD4+PD-1+ or CD8+PD-1+ were determined by flow cytometry analysis. *
Recent studies using animal models of viral infection in both mice and macaques indicated that the interaction between the inhibitory PD-1 on lymphocytes and its ligand (PD-L1) plays a critical role in T-cell exhaustion by inducing T-cell inactivation, and high PD-1 expression levels by peripheral T-lymphocytes and the possibility of improving T-cell function by blocking PD-1-mediated signaling confirm the importance of this inhibitory pathway in inducing T-cell exhaustion
This study was thus undertaken to further define the role of T-cell exhaustion in chronic HBV infection in a mice animal model, by comparing the phenotype and function of intrahepatic infiltrating T lymphocytes in mice with HBV persistence or HBV clearance, and the effect of PD-1/PD-L1 blockade in restoring immune dysfunction and clearance of HBV. It is the first report to demonstrate PD-1/PD-L1 blockade could reverses immune dysfunction and HBV viral persistence
A and B. HBV core mutants induced upregulation of PD-1 expression in intrahepatic T lymphocytes. C57BL/6 mice were hydrodynamically injected with WT pAAV/HBV1.2 or HBV core mutant DNA, including HBV-175 or HBV-38 constructs. Four weeks after the injection, intrahepatic lymphocytes were isolated, and the PD-1 expressions by CD8+ (A) and CD4+ (B) T-cells were analyzed by flow cytometry. C. C57BL/6 mice were hydrodynamically injected with wild type hepatitis B virus (HBV-wt) and HBV core mutants. Intrahepatic lymphocytes were isolated at 4 weeks, and PD-1 and CD127 expressions by CD8+ T cells were analyzed by flow cytometry. D. C57BL/6 mice were hydrodynamically injected with HBV core mutant DNA, HBV-175. Intrahepatic infiltrating lymphocytes and splenic lymphocytes were isolated at 4 weeks, and PD-1 and CD127 expressions by CD8+ T cells were analyzed by flow cytometry. *
C57BL/6 mice were hydrodynamically injected with WT pAAV/HBV1.2 or HBV core mutant DNA, including HBV-175 or HBV-38 constructs. Ten days after the injection, splenocytes were isolated and HBcAg-specific IFN-γ responses were analyzed by an ELISpot assay. The frequency of HBcAg-specific IFN-γ-secreting cells in the presence of an anti-PD-1 or control antibody were measured. Spot-forming cells per million splenocytes are shown. *
A. C57BL/6 mice were intraperitoneally treated with an anti-PD-1 blocking mAb or isotype control Ab. Antibody administration was initiated every 2 days on day 6 before being injected with an HBV core-truncated (HBV-175) mutant construct. Thereafter, Abs (200 µg) were repeatedly administered every 3∼4 days for a period of 8 weeks. The rates of positive serum HBsAg in the mice receiving anti-PD-1 blocking mAb (•, n = 12) were compared with those in mice receiving isotype control Ab (○, n = 11), The HBsAg level in mice serum was determined by an ELISA. HBsAg-positive mice were defined as having a signal-to-noise (S/N) ratio of ≥2. B. The ALT levels in the mice receiving anti-PD-1 blocking mAb, isotype control Ab, and untreated. The ALT level in mice serum was determined by an ELISA. *
Male C57BL/6 and BALB/c mice were obtained from the Animal Center of National Taiwan University and maintained under specific pathogen-free (SPF) conditions. All animal experiments were performed according to regulations approved by the Animal Ethical Committee of National Taiwan University. The HBV constructs and pAAV/HBV1.2 mutants were generated by site-directed mutagenesis as previously described
C57BL/6 mice and BALB/c (male, 6∼7 weeks old) were anesthetized with ketamine and xylazine. Ten micrograms of HBV plasmid DNA in a volume equivalent to 8% of the mouse body weight was injected via a tail vein in 5 s. Serum HBsAg, HBeAg, anti-HBs, and anti-HBc levels were assayed as indicated to monitor the state of HBV persistence.
Livers were perfused with 0.2% bovine serum albumin (BSA)/phosphate-buffered saline (PBS), passed through a nylon mesh, and digested in collagense-IV and DNase-I (Sigma-Aldrich, St. Louis, MO) for 30 min. Hepatocytes were removed by centrifugation for 5 min at 100
Serum levels of HBsAg, HBeAg, anti-HBc, and anti-HBs Abs were determined using the AXSYM system kit (Abbott Diagnostika, Wiesbaden, Germany). The cutoff value for determining HBsAg positivity was a signal-to-noise (S/N) ratio of ≥2 and a signal-to-cutoff (S/CO) ratio of ≥1. To detect serum HBV DNA, each serum sample was pretreated with 25 units of DNase I (Roche Diagnostics, Mannheim, Germany) at 37°C overnight, and total DNA was extracted and HBV DNA was detected by a real-time PCR as previously described
At indicated time points after the hydrodynamic injection, mice were killed, and splenocytes were cultured and assayed for the frequencies of antigen-specific IFN-γ- secreting cells using an ELISPOT kit (BD Biosciences, San Jose, CA). Briefly, 106 splenocytes were co-cultured with 1 µg/ml of rHBcAg (ID Labs, London, Canada) in 200 µl RPMI 1640 supplemented with 10% fetal calf serum (FCS). Cell suspensions were incubated for 20 h. Spot-forming cells were revealed with a biotin-conjugated antibody, streptavidin-horseradish peroxidase (HRP) and AEC substrates (Sigma-Aldrich) and were analyzed using the ImmunoSpot series 5 analyzer (Cellular Technology Limited, Cleveland, OH).
For flow cytometry, allophycocyanin (APC)-conjugated anti-mouse CD3 (BD Biosciences, Palo Alto, CA), phycoerythrin (PE)-conjugated anti-mouse PD-1, fluorescein isothiocyanate (FITC)-conjugated CD127, and PE-Cy5.5-conjugated anti-mouse CD4 or CD8 (BD Biosciences Pharmingen) monoclonal (m)Abs were used for flow cytometry. For the flow cytometric analysis, 105 cells were labeled in a fluorescence-activated cell sorter (FACS) buffer (PBS/2% FCS/0.1% sodium azide), fixed in 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and analyzed on a FACSCalibur using the CellQuest software (Becton Dickinson, Mountain View, CA).
An anti mouse PD-1 Ab was purified from hybridoma (clone G4) culture supernatant (kindly provided by Dr. Lieping Chen, Yale University). C57BL/6 mice were intraperitoneally (i.p.) treated with an anti-PD-1 blocking Ab or isotype control Ab. Ab administration was initiated every 2 days on day 6 before injection with the HBV core-truncated (HBV-175) mutant construct. Thereafter, Abs (200 µg) were repeatedly administered every 3∼4 days for a period of 8 weeks.
Although the chronicity of HBV infection is the result of impaired HBV-specific immune responses that cannot efficiently eliminate or cure infected hepatocytes, many issues remain unsettled. We established a mouse model of HBV persistence in immunocompetent mice by a hydrodynamic injection of replication-competent HBV DNA. This approach generated HBV persistence in C57BL/6 mice, but not in BALB/c mice
Furthermore, to further define the role of the HBV core in HBV persistence in this mice animal model, C57BL/6 mice were injected with wild-type (WT) pAAV/HBV1.2 or HBV core mutant DNA, including HBV-175 or HBV-38
Recent studies in animal models of viral infection indicated that the interaction between PD-1 on lymphocytes and its ligands plays a critical role in T-cell exhaustion by inducing T-cell inactivation. HBV-specific PD-1-positive CD8 cells of patients with chronic HBV infection also displayed lower levels of the interleukin (IL)-7 receptor, CD127, which was previously described in association with the exhausted phenotype
We then asked whether the PD-1/PD-L1 interaction of intrahepatic T-cells was associated with the impaired immune response, resulting in a defective T-cell response to HBV in mice with HBV persistence after an infection. The HBV core-specific IFN-γ T-cell response in mice hydrodynamically injected with WT pAAV/HBV1.2 (HBV-wt), HBV-175, or HBV-38 in the presence and absence of anti-PD-1 mAb treatment were analyzed by an ELISpot assay. Results in
To further define the role of T-cell exhaustion in the pathogenesis of persistent HBV infection in this mice animal model, and determine the effect of PD-1/PD-L1 blockade on restoring immune dysfunction and clearance of HBV, C57BL/6 mice were intraperitoneally treated with an anti-PD-1 blocking mAb or a control isotype Ab. Ab administration was initiated every 2 days on day 6 before an injection with the HBV core-truncated (HBV-175) mutant construct. Thereafter, Abs (200 µg) were repeatedly administered every 3∼4 days for a period of 8 weeks. The HBsAg level in mice serum was determined by an ELISA. The results in
In this study, we demonstrated that there were increased intrahepatic PD-1-expressing CD8+ and CD4+ T-cells in mice with HBV persistence. Intrahepatic-infiltrating CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in mice with HBV persistence. Furthermore, PD-1/PD-L1 blockade partially restored the function of intrahepatic T-cells, leading to viral clearance. Although there were studies with similar observations from HBV transgenic mice
Our results also demonstrated there were increased PD1-expressing CD8+ and CD4+ T-cells in mice infected with core mutants with viral persistence, indicating the HBV core protein, in particular the terminal 10 amino acids, is critical for inducing immunity to HBV. In a previous study, we isolated liver-infiltrating lymphocytes (IHL) from liver tissues of chronic hepatitis B patients and analyzed their immune phenotype. We found a significantly increased CD8+ T-cell population with increased PD-1 and decreased CD28
Studies in animal models of viral infections show that persistent exposure to high antigen concentrations can affect the antiviral T-cell function by causing different degrees of functional impairment, up to physical T-cell deletion, as a function of the quantity of antigens to which T-cells are exposed
The outcome of HBV infection, recovery or persistence, is controlled by the interplay between viral proteins and host factors. The genetic background of recipients, which correlates with the strength of immune responses against HBV antigens during primary activation, also determines the outcome after hydrodynamic injection. In our results, the CD8+ T cells from BALB/c mice had significant lower PD-1 expression compared to C57BL/6 mice. Moreover, in C57BL/6 mice, the PD-1-expressing CD8+ intrahepatic T-cells significantly decreased in mice which had cleared the injected HBV DNA (cleared mice), compared to mice with HBV persistence (carrier mice). These results indicate a significant increase in PD-1 expression in intrahepatic T cells of mice with HBV persistence. In HBV carrier C57BL/6 mice, impaired HBcAg-specific immune responses failed to clear HBV DNA
In conclusion, in this study, we demonstrated that blockade of the PD-1/PD-L1 interaction increased IFN-γ production in response to the HBV core by intrahepatic lymphocytes. Furthermore, blocking the interaction of PD-1 with its ligand, PD-L1, by an anti-PD-1 mAb reversed the viral persistence to clearance of the core-null HBV viral construct in this mouse animal model. Our results indicate that PD-1 blockage reverses immune dysfunction and viral persistence to HBV infection in this mouse animal model, suggesting that anti-PD-1 might be a good therapeutic candidate for chronic HBV infection.
We thank Dr. Mung-Lu Yen for critically reviewing the manuscript; and Ms. W.-L. Wang and Y.-L. Chen for assistance. We also thank the Department of Medical Research and core laboratory of National Taiwan University Hospital for facility support.