SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The immune control of hepatitis B virus (HBV) infection is essential for viral clearance. Therefore, restoring functional anti–HBV immunity is a promising immunotherapeutic approach to treatment of chronic infection. Plasmacytoid dendritic cells (pDCs) play a crucial role in triggering antiviral immunity through their ability to capture and process viral antigens and subsequently induce adaptive immune responses. We investigated the potential of pDCs to trigger antiviral cellular immunity against HBV. We used a human leukocyte antigen A (HLA–A)*0201+ pDC line loaded with HLA–A*0201-restricted peptides derived from hepatitis B core/hepatitis B surface (HBc/HBs) antigens to amplify specific CD8 T cells ex vivo from chronic HBV patients and established a Hepato-HuPBL mouse model to address the therapeutic potential of the strategy in vivo. Stimulation of PBMCs or liver-infiltrating lymphocytes from HLA–A*0201+ chronic HBV patients by HBc peptide-loaded pDCs elicited up to 23.1% and 76.1% HBV-specific CD8 T cells in 45.8% of cases. The specific T cells from the “responder” group secreted interferon-γ, expressed CD107 upon restimulation, and efficiently lysed HBV antigen-expressing hepatocytes. Circulating hepatitis B e antigen (HBeAg) was found to distinguish the group of patients not responding to the pDC stimulation. The therapeutic efficacy of the pDC vaccine was evaluated in immunodeficient NOD-SCID β2m−/− mice reconstituted with HBV patients' PBMCs and xenotransplanted with human HBV-transfected hepatocytes. Vaccination of Hepato–HuPBL mice with the HBc/HBs peptide–loaded pDCs elicited HBV-specific T cells able to specifically lyse the transfected hepatocytes and reduce the systemic viral load. Conclusion: pDCs loaded with HBV–derived peptides can elicit functional virus-specific T cells. HBeAg appears to be critical in determining the outcome of immunotherapies in chronic HBV patients. A pDC-based immunotherapeutic approach could be of interest in attempts to restore functional antiviral immunity, which is critical for the control of the virus in chronic HBV patients. (HEPATOLOGY 2012;56:1706–1718)

Despite increasing awareness and extensive vaccination campaigns, chronic hepatitis B infection remains a global health problem.1 Antiviral drugs such as interferon (IFN)-α and nucleoside/nucleotide analogues efficiently suppress viral replication and reduce hepatic symptoms. However, viral covalently closed circular DNA often persists in hepatocytes and, combined with viral escape mechanisms,2 may cause disease relapse. Unfortunately, antiviral therapies are not yet capable of definitive virus eradication.

Interestingly, the pathophysiology of hepatitis B virus (HBV) appears to be closely related to host immunity.3, 4 Patients who manage to clear the virus elicit vigorous and efficient multispecific T cell responses. In contrast, patients who evolve toward chronic infection mount only weak and inappropriate immune responses.5–7 Immune responses are directed toward epitopes located within the major HBV proteins:8 nucleoscapsid HBc and HBs. In particular, HBc-specific cytotoxic T cells play a critical role in controlling the viral infectious cycle through their ability to lyse persistently infected hepatocytes. Their activity has been shown to significantly contribute to virus clearance and resolution of infection.6, 9, 10 Resolution of chronic HBV infection has been achieved in patients after adoptive transfer of immunity to HBc antigen.11 Another approach, involving reversing T cell exhaustion, such as blocking the PD-1 pathway,12 could also restore functional antiviral immunity. Numerous immunotherapeutic approaches have been developed in attempts to restore functional anti-HBV immunity. In this context, different strategies have been developed to fulfill HBV-specific cellular immune responses, including the HBV-DNA vaccine,13, 14 lipopeptides,15 and peptide-pulsed myeloid dendritic cells (mDCs).16 Genetic engineering has also been used to redirect effector T cell specificity, either by transduction with a T cell receptor (TCR)-specific for the immunodominant human leukocyte antigen A (HLA-A)*0201-restricted HBc18-27 epitope,17 or by expressing a chimeric antigen receptor.18 Despite extensive efforts, most immunotherapeutic approaches are not yet clinically relevant. In addition, their preclinical development is limited by a lack of in vivo models addressing their efficacy in the context of a human immune system.19

Surprisingly, plasmacytoid dendritic cells (pDCs), which are uniquely specialized in launching antiviral responses,20, 21 have not been used to stimulate antiviral responses against HBV. Due to their ability to detect the presence of single-stranded RNA and CpG-DNA and subsequently produce large quantities of type I IFN and induce adaptive immune responses, pDCs play a crucial role in immunity to viruses. pDCs can cross-present viral antigens following direct infection or after sensing infected cells,22, 23 induce virus-specific adaptive immune responses in vitro,24 and also elicit cytotoxic T lymphocytes (CTLs) in vivo following viral infection.25 Despite these outstanding properties, the potential of pDCs has not been harnessed to drive immunity against HBV. This is due in part to their scarcity and the difficulty of generating these cells from hematopoietic progenitors. If these difficulties could be overcome, pDCs would be a very promising means of restoring HBV-specific immune responses.

We developed a powerful tool in the form of a unique human HLA-A*0201+ pDC line that shares phenotypic and functional features of primary pDCs.26 This cell line has been used to promote immune responses toward viral- or tumor-specific antigens. The potential of irradiated peptide-loaded pDCs to induce antigen-specific responses in HLA-A*0201-matched settings has been shown to be effective in the context of melanoma27 as well as Epstein-Barr virus and cytomegalovirus infections.28 In the present study, we investigated the potential of pDCs in triggering functional antiviral cellular immunity against HBV ex vivo in a large cohort of chronic HBV patients and addressed their therapeutic potential in vivo using a Hepato-HuPBL mouse model. The results revealed that hepatitis B e antigen (HBeAg) is a key factor in inducing specific responses irrespective of overall clinical status.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

All procedures were approved by the local ethics committee of Grenoble University Hospital and the French Blood Service's Institutional Review Board. The Declaration of Helsinki Principles was followed, and all participants gave written consent for participation in this study. The studies in animals were conducted in accordance with European Union guidelines (86/609/CEE) and French National Chart guidelines, and protocols were approved by the local Ethics Committee for Animal Experimentation of Grenoble (ComEth).

HBV Patients and Healthy Donors.

Ninety-four HLA-A*0201+ chronic HBV-infected patients and one resolved control were studied. HBV patients (Table 1) were classified as inactive carriers (HBeAg-negative, HBV-DNA <2,000 IU/mL, and consistently normal alanine aminotransferase [ALT] for at least 1 year), HBeAg-negative hepatitis, and HBeAg-positive hepatitis. Forty-eight patients were treated (entecavir/tenofovir), and HBV-DNA was undetectable in 83% of these patients. Exclusion criteria included human immunodeficiency virus/hepatitis C virus/hepatitis D virus coinfection, other liver diseases, and treatment with IFN-α or immunosuppressive agents. Serum HBs antigen was quantified using the Abbott Architect HBsAg QT assay (Abbott Diagnostics). Samples were also obtained from HLA-A*0201+ healthy donors.

Table 1. Clinical Characteristics of the Subgroups of Chronic HBV Patients
CharacteristicsBlood SamplesLiver Biopsies (n = 6)
Inactive Carriers (n = 17)Untreated Chronic HBV PatientsTreated HBV Patients
HBeAg− (n = 24)HBeAg+ (n = 5)HBeAg− (n = 42)HBeAg+ (n = 6)
  1. Data are presented as the mean ± SD unless indicated otherwise.

  2. Abbreviations: ALT, alanine aminotransferase; F, female; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; M, male.

Age, years45 ± 943 ± 1331 ± 1055 ± 1349 ± 2152 ± 14
Sex, %, M/F59/4165/3560/4071/2983/1760/40
ALT level, IU/L22 ± 1544 ± 31217 ± 40035 ± 2249 ± 2327 ± 7
HBeAg statusNegativeNegativePositiveNegativePositiveNegative
Viral load, log IU/mL1.51 ± 1.836.66 ± 7.357.86 ± 8.032.86 ± 3.663.17 ± 3.543.79 ± 4.07
HBsAg, log IU/mL2.83 ± 1.063.78 ± 0.614.19 ± 0.383.23 ± 0.713.68 ± 0.3
Cirrhosis, %0007.116.70

Cell Preparation.

PBMCs were purified via Ficoll-Hypaque density-gradient centrifugation (Eurobio). Liver tissues, obtained from six HLA-A*0201+ HBV patients (Table 1), were processed to prepare liver-infiltrating lymphocytes (LILs). From all liver biopsies, we obtained 0.45 × 106 to 2.6 × 106 cells, among which 14.2%-58.2% were CD3+ T cells and 8.3%-43.3% were CD8+ T cells.

Cell Lines and Peptides.

The GEN2.2 pDC line was cultured as described.26 The HLA-A*0201+ hepatocyte line HepG2 was cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 50UI/ml penicillin/streptomycin (Invitrogen), 1 mM sodium pyruvate (Sigma). HepG22.15 (HepG2 transfected with HBV-DNA) was cultured in William's E, 10% fetal bovine serum, 50 IU/mL penicillin/streptomycin, 2 mM Glutamine (Invitrogen), 5 μg/mL insulin (Sigma) and 5.10−5 hydrocortisone hemisuccinate (Roche). All other cultures were performed in RPMI1640-Glutamax, 1% nonessential amino acids, 100 μg/mL gentamycin, 10% fetal bovine serum (Invitrogen), and 1 mM sodium pyruvate (Sigma). T2 and K562 lines were purchased from American Type Culture Collection (LGC Standards). We used the following HLA-A*0201-restricted peptides (NeoMPS) and corresponding HLA-A*0201 tetramers (Beckman): HBc18-27 (FLPSDFFPSV; core), HBs335-343 (WLSLLVPFV; surface), pol575-583 (FLLSLGIHL; polymerase), and FluM158-66 (GILGFVFTL; influenza matrix).

In Vitro Induction of a Specific T Cell Response.

The pDC line was loaded with peptides as described.26 PBMCs or LILs were cocultured with peptide-loaded pDCs at a 1:10 ratio and restimulated weekly in presence of 200 IU/mL IL-2 (Proleukine, Chiron). In some experiments, PBMCs were directly stimulated with the peptide (1-10 μM final) for 10 days. Specific T cell responses were measured via tetramer labeling prior to analysis on a FACSCalibur flow cytometer (BD).

Proliferation Assay.

PBMCs resuspended at 1 × 106 /mL in 96-well plates were stimulated with phytohemagglutinin (PHA) (1 μg/mL) or OKT3 (0.1 μg/mL) for 5 days. 3H-thymidine was then added to each well. 3H-thymidine incorporation was measured on a liquid scintillation counter (TopCount NXT, PerkinElmer) 18 hours later.

IFN-γ Secretion and CD107 Surface Expression by HBV-Specific CD8 T Cells.

T cells were labeled with tetramer before restimulation with peptide-pulsed T2 cells (10:1 ratio) for 5 hours and 30 minutes. To measure IFN-γ secretion, 1 μL/mL brefeldin A (BD) was added for the last 3 hours. Cells were then labeled with anti-CD3/CD8 antibodies (Beckman) and stained for intracellular IFN-γ (BD). To detect CD107, anti-CD107a/b antibodies (10 μL/1 × 106 cells) (BD) were added in the culture, and GolgiSTOP (0.67 μL/mL) was added for the last 4 hours. Cells were then labeled with anti-CD3/CD8 antibodies. IFN-γ production was also assessed via cytometric bead array (BD) in culture supernatants 24 hours after stimulation of T cells with T2 cells.

51Cr Cytotoxicity Assay.

Cytotoxicity was measured by performing a standard 51Cr release assay. Effector T cells were sorted from the coculture using an EasySep human T cell enrichment kit (StemCell) and plated in 96-well plates with 51Cr-labeled target cells (peptide-pulsed T2 cells, K562) at the indicated E:T ratio. Radioactivity was measured 4 hours later in supernatants on a scintillation counter Top-Count-NXT (PerkinElmer). Measurements were performed in triplicate and mean values were expressed as a percentage of specific lysis using the following formula: 100 × (sample release − spontaneous release)/(maximal release − spontaneous release).

Carboxyfluorescein Succinimidyl Ester–Based Cytotoxicity Assay Toward Human-Infected Hepatocytes.

HepG2 (control target) and HepG22.15 (specific target) cells were first labeled with low (0.1 μM) and high (2.5 μM) carboxyfluorescein succinimidyl ester (CFSE) concentrations, respectively (Invivogen). The two cell lines were mixed and cultured in control conditions or with HBV-specific T cells elicited by the pDCs at a 1:15 to 1:60 ratio for 24 hours. Cell suspensions were analyzed via flow cytometry (FACSCalibur, BD). The percentage of specific lysis was calculated using the formula: % lysis=1-(R1/R2)*100 where R1=%specific target/%control target after incubation with effectors and R2=%specific target/% control target in absence of effectors.

In Vivo Functional Assays in Humanized Mice.

Irradiated (120 cGy) immunodeficient NOD-SCID β2m−/− mice (NOD.Cg-PrkdcSCIDβ2mTm1Unc/J, Jackson-ImmunoResearch Laboratories) were transplanted intraperitoneally with 50 × 106 PBMCs from a resolved HLA-A*0201+ HBV patient and further vaccinated with 5 × 106 irradiated HBc/HBs peptide-pulsed pDCs once a week. A total of 25 × 106 human hepatocyte lines were implanted subcutaneously into the flank of the HuPBL mice either 3 days after (prophylactic setting) or 3 days before (therapeutic setting) the first vaccination. Response to vaccination was analyzed in notified organs upon digestion with collagenase D (Roche Diagnostics) and tetramer staining. Tumor size was monitored every 2-3 days and tumor volume was calculated using the following formula: ((short diameter)2 × long diameter/2). Viral load was measured in the serum using the COBAS Ampliprep/Taqman HBV test version 2.0 (Roche Diagnostics).

Statistical Analysis.

Statistical analyses were performed using a Mann-Whitney nonparametric U test, Wilcoxon matched pairs test, and unpaired t test using Prism software.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Human HLA-Matched Allogeneic pDCs Induce Strong HBc-Specific T Cell Responses Ex Vivo from Chronic HBV Patient PBMCs and LILs.

pDCs have never been used to stimulate HBV-specific T cells. As autologous pDCs are rare and difficult to purify or generate in vitro, we used a pDC cell line and a protocol that we validated in the context of tumor and viral antigens.27, 28 To investigate the ability of the HLA-A*0201+ pDC line to trigger HBc-, HBs-, and pol-specific T cells, PBMCs (n = 94) and LILs (n = 6) purified from HLA-A*0201+ chronic HBV patients were stimulated once a week with the irradiated pDC line loaded with the HLA-A*0201-restricted HBV peptide. Antigen-specific T cell expansion was evaluated after labeling cells with HBV tetramers. No amplification of HBs- and pol-specific T cells could be observed (data not shown). However, potent amplification of the HBc-specific T cells was obtained in 45.8% (PBMCs) and 66.6% (LILs) of cases (Fig. 1A, one representative patient for each condition; Fig. 1B,C, all patients). Thus, we distinguished two groups of patients: the “responders,” who are able to respond to the HBc-loaded pDC stimulation, and the “nonresponders,” who are unable to amplify HBc-specific T cells upon stimulation (level of HBc-specific T cells at day 14 <0.24%). In the responder group, the level of HBc-specific T cells averaged at 3.2% (range, 0.24%-23.1%) for PBMCs (Fig. 1B) and 16.6% (range, 4.5%-76.1%) for LILs (Fig. 1C) over the 14 days of culture. Up to now, the usual method to generate specific T cells from HBV patients consisted in direct culture with 1-10 μM peptides.6, 8, 12 Comparison of the two methods reveals that peptide-loaded pDCs elicited HBc-specific T cells from PBMCs significantly more effectively than peptide alone (Fig. 2). This difference was observed both in terms of percentages (Fig. 2A) and amplification of absolute numbers (Fig. 2B) of HBV-specific T cells. Thus the peptide-loaded pDCs elicit strong HBc-specific T cell responses ex vivo from one part of chronic HBV patients.

thumbnail image

Figure 1. HBc-loaded pDC line induces HBc-specific T cells from HLA-A*0201+ chronic HBV patients in vitro. PBMCs or LILs from chronic HBV patients were stimulated once a week with the pDC line loaded with HLA-A*0201–restricted HBc peptide. HBc-specific T cells were analyzed via flow cytometry using HLA-A*0201–specific tetramers. (A) Tetramer labeling of HBc-specific T cells at day 0 (D0) and at day 7 (D7) and day 14 (D14) of culture (dotplots gated on CD8 T cells). One representative responder and nonresponder patient. (B) Evolving percentage of HBc-specific T cells obtained from PBMCs from 94 chronic HBV patients (44 responders and 52 nonresponders) (two patients were performed twice, see Fig. 4) or (C) LILs from six patients (four responders, two nonresponders). ns, not significant.

Download figure to PowerPoint

thumbnail image

Figure 2. Comparison of in vitro HBc-specific T cell induction from chronic HBV patients using a peptide-loaded pDC line or peptide alone. PBMCs from chronic HBV patients (n = 9) were exposed to the pDC line loaded with HBc peptide (1-10 μM) or directly to the HBc peptide (1-10 μM). HBc-specific T cells were analyzed via flow cytometry using HLA-A*0201–specific tetramer at day 0 and at day 10 of culture. (A) Percentage of HBc-specific T cells at day 10 of culture. (B) Fold amplification of HBc-specific T cell absolute numbers from day 0 to day 10.

Download figure to PowerPoint

Nonresponse to pDC Stimulation Is Related to the Presence of HBeAg.

To determine the basis for responsiveness of chronic HBV patients to the HBc-loaded pDC stimulation, we first studied the response of PBMCs from our cohorts of responder and nonresponder patients to mitogeneic stimulation. The overall proliferative potential, as assessed by 3H-thymidine incorporation, following TCR-independent (PHA) or TCR-dependent (OKT3) stimulation was similar for responders, nonresponders, and healthy donors (Fig. 3A). We then analyzed whether the difference between the groups of patients was specific to the HBc antigen. To do so, we used the protocol described above, but with the pDC line loaded with the HLA-A*0201-restricted influenza peptide. Interestingly, amplification of influenza-specific T cells observed in healthy donors and the responder group was significantly higher than in the nonresponder group of chronic HBV patients (Fig. 3B). This suggests that T lymphocytes from the nonresponder group are generally compromised in their ability to respond to a specific antigen following major histocompatibility complex–dependent presentation by an antigen-presenting cell.

thumbnail image

Figure 3. Proliferative potential and clinical parameters of responder and nonresponder chronic HBV patients. (A) PBMCs from healthy donors (n = 5) and chronic HBV patients (five responders and five nonresponders) were cultured for 5 days in presence of PHA or OKT3. Proliferation was measured using a 3H-thymidine assay. (B) PBMCs from healthy donors or chronic HBV patients were stimulated once a week with the pDC line loaded with HLA-A*0201–restricted influenza peptide. Influenza-specific T cells were analyzed using HLA-A*0201–specific tetramer at day 14 of culture in healthy donors (n = 20) and chronic HBV patients (28 responders and 28 nonresponders to the pDC stimulation). (C-E) Comparison of clinical parameters between chronic HBV patients classed as responders and nonresponders to HBc-loaded pDC stimulation. (C) Blood viral load, (D) HBsAg level in serum, (E) ALT level in blood. (F) Levels of HBc-specific T cells at day 14 for untreated and treated HBV patients. ns, not significant.

Download figure to PowerPoint

No significant correlation was found between the inability to respond to the HBc-loaded pDC stimulation and HBV-DNA levels (Fig. 3C), HBs antigen level (Fig. 3D), ALT measurements (Fig. 3E), or antiviral treatment (Fig. 3F). In contrast, the presence of HBeAg in the serum appeared to differentiate between responder and nonresponder chronic HBV patients (Fig. 4). The HBc-specific T cell response was much greater in inactive carriers and treated or untreated HBeAg-negative hepatitis patients than in HBeAg-positive patients (Fig. 4A). After pooling patients according to HBeAg status alone, this difference appeared clearly significant (Fig. 4B). This interesting observation was corroborated by data for two patients in whom HBeAg status changed over a 6-month interval (Fig. 4C,D). One HBeAg-positive patient, unexpectedly capable of responding to pDC stimulation, achieved HBeAg loss followed by HBeAg seroconversion 6 months later. The other patient, initially HBeAg-negative and capable of responding to HBc-loaded pDC stimulation, became unresponsive 6 months later during a transient HBeAg-positive peak. Thus, HBeAg status distinguishes between chronic HBV patients capable of responding, or not, to HBc-loaded pDC stimulation.

thumbnail image

Figure 4. HBeAg status distinguishes chronic HBV patients according to their ability to respond to HBc-loaded pDC stimulation. PBMCs were stimulated once a week with the pDC line loaded with HLA-A*0201–restricted HBc peptide and cultured for 14 days. HBc-specific T cells were analyzed using HLA-A*0201–specific tetramer at day 0 and day 14. (A) Percent HBc-specific T cells at day 0 (d0) and at day 14 (d14) of culture in different subgroups of chronic HBV patients: inactive carrier, treated or untreated patients, HBeAg-negative (HBe-), and HBeAg-positive (HBe+). Bars indicate the mean. (B) Levels of HBc-specific T cells at day 14 in HBeAg-negative (HBe-) and HBeAg-positive (HBe+) HBV patients. (C,D) For two patients, analysis was performed on two different blood samples taken at a 6-month interval, during which HBeAg status changed. (C) Patient positive for HBeAg for the first sample but negative for the second. (D) Patient negative for HBeAg for the first sample but positive for the second.

Download figure to PowerPoint

The HBc-Specific T Cells Elicited by the pDCs Are Functional and Exhibited In Vitro HLA-A*0201-Restricted Cytotoxic Activity Toward HBV-Transfected Hepatocytes.

To investigate the functionality of HBV-specific T cells generated from responder chronic HBV patients we examined T cell exhaustion and cytotoxic potential. PD1 expression, a marker of T cell exhaustion, was not detected on the HBc-specific T cells elicited by the pDC line (Supporting Fig. 1). The cytotoxic potential of expanded HBV-specific T cells was determined by performing a 51Cr release assay using peptide-loaded HLA-A*0201+ T2 cells as targets. As expected, HBc-specific T cells exhibited a strong cytotoxicity toward T2 cells loaded with HBc peptide but not with an irrelevant peptide, showing the specificity of the HBV-specific T cells function (Fig. 5A). Next, we tested the ability of these specific T cells to lyse a more relevant target, such as HBV-transfected HLA-A*0201+ hepatocytes. Due to the lack of P3 facilities necessary to perform radioactive experiments with virus-producing cells, a CFSE assay was used. This assay consisted of culturing specific T cells with a mixture of two targets labeled with distinct CFSE intensities. The disappearance of the CFSE pic, as measured via flow cytometry, indicates killing of the corresponding cells. For all patients tested, HBc-specific T cells were able to specifically lyse the HBV-transfected HLA-A*0201+ hepatocyte cell line HepG22.15, but not the HBV-free HepG2 line (Fig. 5B). Specific lysis was measured at between 15% and 95% with T cells containing 0.99%-23.1% HBc-specific T cells. Moreover, when cocultured with peptide-loaded T2 cells, HBc-specific T cells expressed CD107 (Fig. 5C,D) and secreted IFN-γ (Fig. 5E,F) only in the presence of HBc but not control peptide. These results reinforce the full functionality of HBc-specific T cells elicited by peptide-loaded pDCs.

thumbnail image

Figure 5. Functionality of HBc-specific T cells elicited by the pDC line. PBMCs were stimulated once a week with the pDC line loaded with HLA-A*0201–restricted HBc peptide and cultured for 14 days. (A,B) Cytotoxic activity of HBc-specific T cells was measured toward (A) peptide-loaded T2 cells or K562 cells using a 51Cr release assay; one representative experiment (left panel) and eight experiments (right panel) and (B) HBV-infected human hepatocytes. HBc-specific T cells were incubated in control conditions, or with a mix of CFSElow-labeled HepG2 and CFSEhigh-labeled HepG22.15; the percentage of each CFSE peak was measured via flow cytometry 24 hours later (upper panel) and the percentage of HepG22.15 specific lysis and absolute numbers of HBc-specific T cells calculated (lower panel). (C,D) CD107 surface expression on HBc-specific T cells after restimulation with peptide-loaded T2 cells. (C) One representative patient (upper panel) and (D) 10 patients. (E) IFN-γ production assessed by intracellular staining within tetramer+ CD8 T cells upon restimulation with peptide-loaded T2 cells. (F) IFN-γ secretion measured in culture supernatants 24 hours after restimulation of HBc-specific T cells with peptide-loaded T2 cells (n = 11 experiments).

Download figure to PowerPoint

The HBc- and HBs-Loaded pDC Line Elicited In Vivo HBV-Specific T Cell Responses in Humanized Mice.

We further evaluated the capacity of the peptide-loaded pDCs to elicit virus-specific T cell responses against HBV antigen in vivo by using a humanized mouse model constructed by xenotransplanting PBMCs from a patient with resolved HBV infection into immunodeficient NOD-SCID β2m−/− mice (HuPBL mouse model, Fig. 6A). HBc- and HBs-specific CD8 T cells could be amplified in vitro with the HBc- and HBs-loaded pDC line from PBMCs from the patient with resolved HBV infection (Fig. 6B). Treatment of HuPBL mice with the irradiated HBc- and HBs-loaded pDC line led to the induction of HBc- and HBs-specific T cells at the site of immunization, in the draining lymph nodes but also in the circulation and spleen (Fig. 6C,D). Thus, the HBc- and HBs-loaded pDC line elicited widespread HBc- and HBs-specific T cell responses in vivo.

thumbnail image

Figure 6. Induction of HBc- and HBs-specific T cells upon vaccination with the pDC line in vivo in a humanized mouse model. (A) Immunodeficient NOD-SCID β2m−/− mice were humanized with PBMCs from a resolved HLA-A*0201–positive patient and treated with the pDC loaded with HLA-A*0201–restricted HBc/HBs peptides or control peptide once a week. (B) PBMCs from the resolved HBV patient were stimulated once a week by the pDC line loaded with HLA-A*0201–restricted HBc and HBs peptides. HBc- and HBs-specific T cells were analyzed via flow cytometry using HLA-A*0201–specific tetramer at day 0 (d0) and at day 14 (d14) of culture (dotplots gated on CD8 T cells). Tetramer Pol was used as a control. Data are representative of one experiment. (C,D) After two vaccinations, HBc- and HBs-specific T cells were analyzed via tetramer labeling on cell suspensions at the injection site, in the draining lymph nodes, spleen and blood. Tetramer Pol was used as a control. (C) One representative experiment in HuPBL mice and (D) percentage of HBc- and HBs-specific T cells obtained from 11 vaccinated HuPBL mice.

Download figure to PowerPoint

Treatment with HBc- and HBs-Loaded pDCs Protected Hepato-HuPBL Mice Against HBV Antigen-Expressing Hepatocyte Development.

We next investigated the therapeutic potential of the pDC treatment in humanized mice further xenotransplanted with a HLA-A*0201+ hepatocyte cell line transfected with HBV, also referred as Hepato-HuPBL mice. HuPBL mice were weekly treated with the irradiated pDC line loaded with HBc/HBs or control peptides before (Fig. 7) or after (Supporting Fig. 2) being challenged with human hepatocyte cell lines transfected (HepG22.15) or not (HepG2) with HBV. In the prophylactic setting, HBc- and HBs-loaded pDCs inhibited the development of HepG22.15 cells compared with the control pDCs whereas the HepG2 cell development was similar in the two conditions (Fig. 7B,C). Importantly, the HBV viral load in the serum of Hepato-HuPBL mice treated with HBc- and HBs-loaded pDCs was significantly lower than in mice receiving the control pDCs (Fig. 7D). Notably HBV-specific T cells were found at the HepG22.15 site of treated Hepato-HuPBL mice (Fig. 7E), suggesting that the HBV-specific T cells induced by the pDCs were able to migrate to the site of virus expression and kill HBV antigen-expressing hepatocytes. These findings were reproduced in a therapeutic setting (Supporting Fig. 2) demonstrating the efficacy of the pDC vaccine against established HBV infection.

thumbnail image

Figure 7. Therapeutic efficacy of the pDC vaccine in vivo in a humanized mouse model toward HBV infection. (A) Immunodeficient NOD-SCID β2m−/− mice were humanized with HLA-A*0201+ PBMCs and vaccinated once a week with the pDC loaded with HLA-A*0201-restricted HBc/HBs or control peptides. After one vaccination, HuPBL mice were transplanted subcutaneously with a HLA-A*0201+ hepatocyte cell line infected (HepG22.15) or not (HepG2) with HBV. (B) Evolution of the HepG2 or HepG22.15 growth in Hepato-HuPBL mice. (C) Tumor size measured at day 15 (d15). (D) Blood viral load measured at day 15 (d15). Pool of two experiments (n = 7-8 Hepato-HuPBL mice). (E) Analysis of HBc/HBs-specific T cells at the tumor site by tetramer labeling (dotplots gated on CD8 T cells). One representative Hepato-HuPBL mouse.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Current antiviral treatments for chronic HBV infection cannot definitively clear the virus. Resolution of HBV infection would require the lysis of persistently infected hepatocytes through the action of HBV-specific T cells. pDCs are important antigen-presenting cells, particularly in the context of infectious diseases. However, they have never been used in an experimental setting to induce functional HBV-specific T cells. We demonstrate that an irradiated pDC line loaded with HLA-A*0201-restricted peptides can efficiently trigger functional HBV-specific T cell responses ex vivo from HBeAg-negative chronic HBV patients.

High levels of functional HBc-specific T cells that display efficient antigen-restricted functions and are able to lyse HBV-infected hepatocytes could be elicited from both PBMCs and LILs of chronic HBV patients. Intrahepatic HBV-specific T cells are known to be in an exhaustion state.12 Despite this, specific T cells were strongly amplified from LILs, underlining the potency of the pDC-based strategy. Compared with current strategies developed to amplify HBV-specific T cells (peptides, mDCs), peptide-loaded pDCs induced greater numbers of specific T cells and faster immune responses.5, 16

HBeAg is known to have an immunoregulatory function in promoting viral persistence through the modulation of the immune response to HBc antigen.29–31 Indeed, here HBeAg status was found to be a critical factor determining patients' ability to elicit anti-HBV immune responses upon pDC stimulation. Two patients in our cohort switched their ability to respond to pDC stimulation within a 6-month interval. This switch was in line with modification of their HBeAg status. These observations highlight the major role of HBeAg in regulating specific T cell function. In accordance with our findings, mDCs pulsed with HBV-derived peptides elicited a stronger anti-HBV immunity in HBeAg-negative patients than in HBeAg-positive patients.32 In addition, HBeAg seroconversion has been shown to be associated with the restoration of pDC function in chronic HBV patients underlying IFN-α treatment.33 The fact that immunity to influenza antigen is also abrogated in nonresponder HBeAg-positive chronic HBV patients suggest that HBeAg not only modulates HBc antigen–specific responses but has wide-ranging effects on an individual's ability to respond to specific immune stimulation. Our observations confirm that HBeAg is a critical factor determining the outcome of immunostimulation which should be taken into consideration when optimizing future approaches to HBV treatment. Moreover, our results demonstrate that other clinical parameters such as viral load, ALT levels, HBs antigen levels, or antiviral treatment are not related to the ability of chronic HBV patients to respond to the pDC stimulation. These observations therefore support the hypothesis that treatment with nucleoside/nucleotide analogues is not associated with reinforced antiviral T cell responses.

In addition to allowing the study of critical parameters of successful immune responses in the context of chronic HBV infection, the pDC cell line used as antigen-presenting cells is an interesting new tool to elicit HBV-specific T cells. It could also be used as a potential cell-based immunotherapeutic strategy in which its potent efficacy and simple design would be ideal. Virus-specific T cell responses are thought to be responsible not only for viral clearance but also for disease pathogenesis during HBV infection. Nevertheless, a strong cytotoxic response has been described in the absence of liver damage,5 and the quantity of virus produced within hepatocytes has been shown to influence virus-specific CD8 T cell function.34 Regulating the viral load using antiviral drugs may help control the balance between the cytotoxic and inflammatory effects of virus-specific T cells. Interestingly, specific T cell responses might even be restored in this setting.35 In addition, a reduction of HBeAg load could be observed upon antiviral treatments in some patients,36 and HBs seroconversion has been achieved following mDC-based vaccine.32 This is important, as we showed here that HBeAg status is critical for successful immunostimulation in chronic HBV patients. The vast majority of HBeAg-negative patients treated with new analogs (entecavir and tenofovir) have undetectable HBV DNA, but have nearly no chance to achieve HBs antigen clearance. These patients, who need to be treated throughout their lives, would be the ideal target for the pDC-based immunotherapy in the future.

The advantage of pDCs over mDCs in eliciting immune responses was clearly demonstrated in our previous work,27, 28 where we directly compared the two cell types and their capacity to elicit immune responses to a variety of tumor and viral antigens. Furthermore, in contrast with autologous mDCs that required patients' cells, the pDC strategy could be directly applied to all HLA-A*0201+ patients. These settings have already been shown to be safe in chronic HBV patients.11, 32 Our previous work clearly demonstrates that the pDC strategy generates potent HLA-A0201-restricted antigen-specific cytotoxic T cells without cross-reactivity to different HLA alleles and without bystander alloreactivity.27, 28

Mutations within HBV antigens have been shown to occur during the progression of HBV infection.37 However, it appears that T cell escape mutants are not common in chronic HBV patients, as an intact core 18-27 epitope has been described in more than 92% of chronic HBV patients.38 In addition, CTLs specific for the wild-type HBc18-27 epitope could still recognize target cells presenting a mutated HBc18-27 epitope,37 therefore limiting the complete ineffectiveness of such an immunotherapy. As mutations occur in limited positions, mutated epitopes could also be used to load the pDCs to trigger CTLs toward the mutated epitopes.

We developed a new Hepato-HuPBL mouse model consisting in humanized mice engrafted with HBV-antigen expressing hepatocytes. Indeed, the existing chimeric and transgenic models are not suitable for testing such immunotherapies that required both the context of a human immune system and HBV antigen-expressing human hepatocytes. The human liver-uPA-SCID model further infected with HBV39 is devoid of immune cells, the HBV transgenic mouse40 is restricted to a murine context, and HLA-A2 transgenic mice13 allow epitope discovery but not therapeutic testing. Considering the huge number of PBMCs required to perform such in vivo experiments, PBMCs from chronic HBV-infected patients could not be used due to ethical limitations. Because we observed a similar amplification rate of HBV-specific immunity in vitro upon pDC stimulation between chronic HBV-infected patients and patients with resolved HBV infection, we used the latter group to establish our model. Using the Hepato-HuPBL mouse model, we clearly showed that in vivo, pDCs can elicit fully functional virus-specific T cells that are able to slow down the development of HBV-transfected hepatocytes and, importantly, reduce the viral load dramatically. This model appears to be helpful to perform preclinical in vivo studies of new immunotherapeutic approaches currently developed to fulfill HBV-specific cellular immune responses.

This study demonstrates the potential of pDCs in triggering functional virus-specific T cells from HBeAg-negative chronic HBV patients. It contributes to the identification of critical factors for successful restoration of antiviral immunity and establishes a preclinical model to test anti-HBV immunotherapeutic strategies. Following antiviral treatments, the elimination of persistently infected hepatocytes remains a major therapeutic goal to cure chronic HBV infection. Our strategy, which restores functional anti-HBV effectors critical for the control and clearance of the virus, could be the basis for a potential novel immunotherapeutic approach to treat chronic HBV infection.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank C. Morand, I. Michaud, and F. Bernard from EFS Rhone-Alpes for providing blood samples; F. Blanquet, R. Balouzat, and S. Kamche for expert animal care; F. Herodin for animal irradiation; P. Morand for allowing virological analysis; and A. Marlu for providing clinical data. We thank Abbott Laboratories for providing reagents to perform the Architect HBsAg QT assays. We are grateful to M. K. Maini for helpful discussions. Finally, we thank the patients who consented to participate in this study.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Deny P, Zoulim F. Hepatitis B virus: from diagnosis to treatment. Pathol Biol (Paris) 2010; 58: 245253.
  • 2
    Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 2009; 137: 15931608.
  • 3
    Jung MC, Pape GR. Immunology of hepatitis B infection. Lancet Infect Dis 2002; 2: 4350.
  • 4
    Guidotti LG, Chisari FV. Immunobiology and pathogenesis of viral hepatitis. Annu Rev Pathol 2006; 1: 2361.
  • 5
    Maini MK, Boni C, Lee CK, Larrubia JR, Reignat S, Ogg GS, et al. The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection. J Exp Med 2000; 191: 12691280.
  • 6
    Webster GJ, Reignat S, Brown D, Ogg GS, Jones L, Seneviratne SL, et al. Longitudinal analysis of CD8+ T-cells specific for structural and nonstructural hepatitis B virus proteins in patients with chronic hepatitis B: implications for immunotherapy. J Virol 2004; 78: 57075719.
  • 7
    Rehermann B, Lau D, Hoofnagle JH, Chisari FV. Cytotoxic T lymphocyte responsiveness after resolution of chronic hepatitis B virus infection. J Clin Invest 1996; 97: 16551665.
  • 8
    Maini MK, Boni C, Ogg GS, King AS, Reignat S, Lee CK, et al. Direct ex vivo analysis of hepatitis B virus-specific CD8(+) T-cells associated with the control of infection. Gastroenterology 1999; 117: 13861396.
  • 9
    Shimada N, Yamamoto K, Kuroda MJ, Terada R, Hakoda T, Shimomura H, et al. HBcAg-specific CD8 T-cells play an important role in virus suppression, and acute flare-up is associated with the expansion of activated memory T-cells. J Clin Immunol 2003; 23: 223232.
  • 10
    Yang PL, Althage A, Chung J, Maier H, Wieland S, Isogawa M, et al. Immune effectors required for hepatitis B virus clearance. Proc Natl Acad Sci U S A 2010; 107: 798802.
  • 11
    Lau GK, Suri D, Liang R, Rigopoulou EI, Thomas MG, Mullerova I, et al. Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen. Gastroenterology 2002; 122: 614624.
  • 12
    Fisicaro P, Valdatta C, Massari M, Loggi E, Biasini E, Sacchelli L, et al. Antiviral intrahepatic T-cell responses can be restored by blocking programmed death-1 pathway in chronic hepatitis B. Gastroenterology 2010; 138: 682693.
  • 13
    Loirat D, Lemonnier FA, Michel ML. Multiepitopic HLA-A*0201-restricted immune response against hepatitis B surface antigen after DNA-based immunization. J Immunol 2000; 165: 47484755.
  • 14
    Depla E, Van der Aa A, Livingston BD, Crimi C, Allosery K, De Brabandere V, et al. Rational design of a multiepitope vaccine encoding T-lymphocyte epitopes for treatment of chronic hepatitis B virus infections. J Virol 2008; 82: 435450.
  • 15
    Livingston BD, Crimi C, Grey H, Ishioka G, Chisari FV, Fikes J, et al. The hepatitis B virus-specific CTL responses induced in humans by lipopeptide vaccination are comparable to those elicited by acute viral infection. J Immunol 1997; 159: 13831392.
  • 16
    Chen W, Shi M, Shi F, Mao Y, Tang Z, Zhang B, et al. HBcAg-pulsed dendritic cell vaccine induces Th1 polarization and production of hepatitis B virus-specific cytotoxic T lymphocytes. Hepatol Res 2009; 39: 355365.
  • 17
    Gehring AJ, Xue SA, Ho ZZ, Teoh D, Ruedl C, Chia A, et al. Engineering virus-specific T-cells that target HBV infected hepatocytes and hepatocellular carcinoma cell lines. J Hepatol 2010; 55: 103110.
  • 18
    Bohne F, Chmielewski M, Ebert G, Wiegmann K, Kurschner T, Schulze A, et al. T-cells redirected against hepatitis B virus surface proteins eliminate infected hepatocytes. Gastroenterology 2008; 134: 239247.
  • 19
    Zoulim F, Berthillon P, Guerhier FL, Seigneres B, Germon S, Pichoud C, et al. Animal models for the study of HBV infection and the evaluation of new anti-HBV strategies. J Gastroenterol Hepatol 2002; 17(Suppl): S460-S463.
  • 20
    Barchet W, Cella M, Colonna M. Plasmacytoid dendritic cells—virus experts of innate immunity. Semin Immunol 2005; 17: 253261.
  • 21
    Colonna M, Trinchieri G, Liu YJ. Plasmacytoid dendritic cells in immunity. Nat Immunol 2004; 5: 12191226.
  • 22
    Villadangos JA, Young L. Antigen-presentation properties of plasmacytoid dendritic cells. Immunity 2008; 29: 352361.
  • 23
    Lui G, Manches O, Angel J, Molens JP, Chaperot L, Plumas J. Plasmacytoid dendritic cells capture and cross-present viral antigens from influenza-virus exposed cells. PLoS One 2009; 4: e7111.
  • 24
    Fonteneau JF, Gilliet M, Larsson M, Dasilva I, Munz C, Liu YJ, et al. Activation of influenza virus-specific CD4+ and CD8+ T-cells: a new role for plasmacytoid dendritic cells in adaptive immunity. Blood 2003; 101: 35203526.
  • 25
    Schlecht G, Garcia S, Escriou N, Freitas AA, Leclerc C, Dadaglio G. Murine plasmacytoid dendritic cells induce effector/memory CD8+ T-cell responses in vivo after viral stimulation. Blood 2004; 104: 18081815.
  • 26
    Chaperot L, Blum A, Manches O, Lui G, Angel J, Molens JP, et al. Virus or TLR agonists induce TRAIL-mediated cytotoxic activity of plasmacytoid dendritic cells. J Immunol 2006; 176: 248255.
  • 27
    Aspord C, Charles J, Leccia MT, Laurin D, Richard MJ, Chaperot L, et al. A novel cancer vaccine strategy based on HLA-A*0201 matched allogeneic plasmacytoid dendritic cells. PLoS One 2010; 5: e10458.
  • 28
    Aspord C, Laurin D, Richard MJ, Vie H, Chaperot L, Plumas J. Induction of antiviral cytotoxic t-cells by plasmacytoid dendritic cells for adoptive immunotherapy of posttransplant diseases. Am J Transplant 2011; 11: 26132626.
  • 29
    Chen MT, Billaud JN, Sallberg M, Guidotti LG, Chisari FV, Jones J, et al. A function of the hepatitis B virus precore protein is to regulate the immune response to the core antigen. Proc Natl Acad Sci U S A 2004; 101: 1491314918.
  • 30
    Milich DR, Chen MK, Hughes JL, Jones JE. The secreted hepatitis B precore antigen can modulate the immune response to the nucleocapsid: a mechanism for persistence. J Immunol 1998; 160: 20132021.
  • 31
    Milich D, Liang TJ. Exploring the biological basis of hepatitis B e antigen in hepatitis B virus infection. HEPATOLOGY 2003; 38: 10751086.
  • 32
    Luo J, Li J, Chen RL, Nie L, Huang J, Liu ZW, et al. Autologus dendritic cell vaccine for chronic hepatitis B carriers: a pilot, open label, clinical trial in human volunteers. Vaccine 2010; 28: 24972504.
  • 33
    Zhang Z, Zhang H, Chen D, Yao J, Fu J, Jin L, et al. Response to interferon-alpha treatment correlates with recovery of blood plasmacytoid dendritic cells in children with chronic hepatitis Br J Hepatol 2007; 47: 751759.
  • 34
    Gehring AJ, Sun D, Kennedy PT, Nolte-'t Hoen E, Lim SG, Wasser S, et al. The level of viral antigen presented by hepatocytes influences CD8 T-cell function. J Virol 2007; 81: 29402949.
  • 35
    Boni C, Penna A, Bertoletti A, Lamonaca V, Rapti I, Missale G, et al. Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B. J Hepatol 2003; 39: 595605.
  • 36
    Lok AS, McMahon BJ. Chronic hepatitis B. HEPATOLOGY 2007; 45: 507539.
  • 37
    Bertoletti A, Costanzo A, Chisari FV, Levrero M, Artini M, Sette A, et al. Cytotoxic T lymphocyte response to a wild type hepatitis B virus epitope in patients chronically infected by variant viruses carrying substitutions within the epitope. J Exp Med 1994; 180: 933943.
  • 38
    Rehermann B, Pasquinelli C, Mosier SM, Chisari FV. Hepatitis B virus (HBV) sequence variation of cytotoxic T lymphocyte epitopes is not common in patients with chronic HBV infection. J Clin Invest 1995; 96: 15271534.
  • 39
    Meuleman P, Leroux-Roels G. The human liver-uPA-SCID mouse: a model for the evaluation of antiviral compounds against HBV and HCV. Antiviral Res 2008; 80: 231238.
  • 40
    Chisari FV. Hepatitis B virus transgenic mice: models of viral immunobiology and pathogenesis. Curr Top Microbiol Immunol 1996; 206: 149173.

Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
HEP_25879_sm_SuppFig1.tif757KSupporting Information Figure 1
HEP_25879_sm_SuppFig2.tif2555KSupporting Information Figure 2

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.