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Potential conflict of interest: Dr. Leroy consults for, is on speakers' bureau of, and received grants from Bristol-Myers Squibb, Gilead, and Roche.
Supported by the French Blood Service and the French National Agency for Research on AIDS and Viral Hepatitis.
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)
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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.
ALT, alanine aminotransferase; CFSE, carboxyfluorescein succinimidyl ester; CTL, cytotoxic T lymphocyte; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen, HBV, hepatitis B virus; HLA-A, human leukocyte antigen A; IFN, interferon; LIL, liver-infiltrating lymphocyte; mDC, myeloid dendritic cell; PBMC, peripheral blood mononuclear cell; pDC, plasmacytoid dendritic cell; TCR, T cell receptor.
Patients and Methods
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
Liver Biopsies (n = 6)
Inactive Carriers (n = 17)
Untreated Chronic HBV Patients
Treated HBV Patients
HBeAg− (n = 24)
HBeAg+ (n = 5)
HBeAg− (n = 42)
HBeAg+ (n = 6)
Data are presented as the mean ± SD unless indicated otherwise.
Abbreviations: ALT, alanine aminotransferase; F, female; HBeAg, hepatitis B e antigen; HBV, hepatitis B virus; M, male.
45 ± 9
43 ± 13
31 ± 10
55 ± 13
49 ± 21
52 ± 14
Sex, %, M/F
ALT level, IU/L
22 ± 15
44 ± 31
217 ± 400
35 ± 22
49 ± 23
27 ± 7
Viral load, log IU/mL
1.51 ± 1.83
6.66 ± 7.35
7.86 ± 8.03
2.86 ± 3.66
3.17 ± 3.54
3.79 ± 4.07
HBsAg, log IU/mL
2.83 ± 1.06
3.78 ± 0.61
4.19 ± 0.38
3.23 ± 0.71
3.68 ± 0.3
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).
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).
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 analyses were performed using a Mann-Whitney nonparametric U test, Wilcoxon matched pairs test, and unpaired t test using Prism software.
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.
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.
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.
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.
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.
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.
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.
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.