Potential conflict of interest: Nothing to report.
Chronic hepatitis B virus (HBV) infection is characterized by a weak immune response to HBV. Regulatory T cells (Treg) can suppress the function of effector T cells and may thus be key players in this impaired immune response. Changes in the functionality or number of Treg could explain the decreased antiviral response in chronic HBV patients. To investigate the role of Treg in chronic HBV infection, we compared the proportional frequency and functionality of Treg in peripheral blood of 50 chronic HBV patients, 23 healthy controls, and 9 individuals with a resolved HBV infection. A higher percentage of Treg, defined as CD4, CD25, CD45RO, and cytotoxic T-lymphocyte–associated antigen 4–positive cells, was detected within the population of CD4+ cells in peripheral blood of chronic HBV patients compared with healthy controls and individuals with a resolved HBV infection. Accordingly, chronic HBV patients displayed a higher FoxP3 messenger RNA level than healthy controls. Depletion of CD25+ cells from peripheral blood mononuclear cells (PBMC) of chronic HBV patients resulted in an enhanced proliferation after stimulation with HBV core antigen. Reconstitution of these depleted PBMC with CD4+CD25+ Treg resulted in a dose-dependent reduction of both HBV-specific proliferation and interferon γ production. In conclusion, chronic HBV patients harbor an increased percentage of Treg in peripheral blood compared with controls. Treg have an immunosuppressive effect on HBV-specific T helper cells. The presence of HBV-specific Treg could contribute to an inadequate immune response against the virus, leading to chronic infection. (HEPATOLOGY 2005;41:771–778.)
Hepatitis B virus (HBV) is a common noncytopathic DNA virus. Infection with HBV in adults results frequently in a self-limiting, acute hepatitis, which confers protective immunity and causes no further disease. In 10% of infected adults, HBV leads to a chronic infection. Chronic HBV infection is an important risk factor for the development of cirrhosis and hepatocellular carcinoma. Worldwide, 350 million people suffer from chronic HBV infection, and approximately 1 million people die annually from HBV-related liver disease.1, 2
T helper 1 type cytokines such as interferon γ (IFN-γ) and interleukin 2 are involved in cell-mediated immunity and play a crucial role in the protection against intracellular pathogens, including HBV.3 In patients with an acute self-limiting HBV infection, a multispecific CD4+ and CD8+ T-cell response with a type 1 cytokine profile is important for control of the infection.4 These multispecific T-cell responses are maintained for decades after clinical recovery.5 In contrast, patients with a chronic HBV infection lack such a vigorous multispecific response. These patients have a weak or undetectable virus-specific T-cell response.4 The precise mechanism responsible for this T-cell hyporesponsiveness or tolerance is still unknown. One scenario that has not been explored in relation to chronic HBV infection is the potential role of host-mediated immunosuppressive mechanisms that might be activated in the face of persistent antigenic exposure.
Peripheral T cells contain an immunoregulatory subpopulation that expresses CD4, CD25 (the interleukin 2 receptor α chain), and CD45RO, as well as the cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4). These regulatory T cells (Treg) are capable of inhibiting the effector functions of CD4+, CD8+, and natural killer T cells.6–9 Treg express the forkhead/winged helix transcription factor gene (FoxP3). Retroviral gene transfer of FoxP3 converts naïve T cells into CD4+CD25+ Treg capable of suppressing the proliferative response of other CD4+ T cells.10 This indicates that FoxP3 is a key regulatory gene for the development of Treg. Several studies have shown that Treg play an important role in maintaining the peripheral immune tolerance.7, 11, 12 Furthermore, there is increasing evidence that CD4+CD25+ Treg contribute to the immunological hyporesponsiveness against several pathogens, resulting in chronic infections.13–16 It has been suggested that Treg can be induced through repetitive stimulation of T cells by high concentrations of antigen for extended periods.17
The aim of this study was to determine whether Treg are involved in the inadequate immune response leading to chronic HBV infection. We hypothesized that patients with a chronic HBV infection have a higher proportion of Treg compared with healthy controls and people who have resolved their HBV infection and that Treg may be responsible for the inability of patients to clear infection. For this purpose, we investigated the amount, phenotype, and function of Treg in the peripheral blood of chronic HBV patients, healthy controls, and individuals with a resolved HBV infection.
HBV, hepatitis B virus; Treg, regulatory T cell; HCV, hepatitis C virus; PBMC, peripheral blood mononuclear cell; IFN-γ, interferon γ; CTLA-4, cytotoxic T-lymphocyte–associated antigen 4; HBcAg, hepatitis B core antigen; mRNA, messenger RNA; HBeAg, hepatitis B e antigen; DC, dendritic cell.
Patients and Methods
Patients and Healthy Controls.
Heparinized peripheral blood samples were obtained from 50 chronic HBV patients, 23 healthy controls, and 9 individuals with resolved HBV infections (Table 1). Patients coinfected with HIV, hepatitis A virus, hepatitis C virus (HCV), or hepatitis D virus and patients with a resolved viral hepatitis (other than HBV) were excluded from this study. Patients and controls who were immunocompromised or pregnant and patients that received antiviral or immunomodulatory HBV treatment during the last 6 months before blood sampling were also excluded. All participants gave informed consent before blood donation.
Table 1. Patient and Control Characteristics
Chronic HBV Patients n = 50
Controls n = 23
Resolved HBV Patients n = 9
Abbreviations: ALT, alanine transaminase; n.a., not applicable; neg, negative
Isolation of Peripheral Blood Mononuclear Cells and Flow Cytometric Analysis.
Peripheral blood mononuclear cells (PBMC) from chronic HBV patients and controls were obtained via ficoll separation (Ficoll-Paque plus; Amersham Biosciences, Buckinghamshire, UK). The PBMC were immediately frozen in medium containing 10% dimethyl sulfoxide and stored at −135°C until further use. Flow cytometric analysis was performed on the stored samples using fluorochrome-conjugated antibodies specific for the surface markers CD4, CD45RO, and CD25 diluted in phosphate-buffered saline/0.3% bovine serum albumin. The cells were fixed by incubation with intraprep reagent 1 and permeabilized by incubation with intraprep reagent 2 (Beckman-Coulter, Marseille, France). Anti–CTLA-4 antibody was added during permeabilization. The following antibodies were used: anti-CD4-PerCP-Cy5.5 (SK3) (Pharmingen, San Diego, CA); anti-CD45RO-APC and anti-CD45RO-FITC (UCHL1) (Becton Dickinson, San Jose, CA); anti-CD25-FITC and anti-CD25-APC (2A3) (Becton Dickinson); anti-CTLA-4-PE (BNI3) (Immunotech, Marseille, France); anti-CD62L-FITC (FMC46) (Serotec, Oxford, United Kingdom); and anti-GITR-FITC (110416) (R&D Systems, Oxon, United Kingdom). For the CD45RO, CD25, CTLA-4, and GITR antibodies, isotype-matched control antibodies were used to determine the level of background staining. After staining, the cells were analyzed with a four-color cytometer (FACScalibur; CELLQuest Pro software, Beckton Dickinson).
CD4+CD25+ T-Cell Isolation.
Fresh PBMC were used for CD4+CD25+ isolation. CD4+ T cells were isolated from PBMC by negative selection using the untouched CD4+ T-cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD25+ T cells were isolated from CD4+ T cells using anti CD25-microbeads (Miltenyi Biotec). The isolations were performed according to the manufacturer's instructions. The CD4− and CD4+CD25− fraction were pooled and used as CD25 (Treg)–depleted responder cells. Purity of the cell fractions was determined by flow cytometry analysis with antibodies against CD3, CD4, and CD25. The following antibodies were used: anti-CD3-FITC (UCHT1) (Immunotech); anti-CD4-PerCP-Cy5.5 (SK3) (Becton Dickinson); and anti-CD25-PE (M-A251) (Pharmingen). An isotype-matched control antibody was used to determine the level of background staining for the CD25-PE antibody. The CD4+CD25+ Treg purification method resulted in a Treg fraction containing more than 90% pure CD4+CD25+ Treg and a Treg–depleted cell fraction containing all other cell types present in PBMC.
Freshly isolated PBMC and the pooled CD25-depleted cells were cultured in triplicate in a concentration of 1 × 105 cells per well in 100 μL RPMI 1640 (Bio Whittaker, Verviers, Belgium) containing 10% pooled human serum (Department of Immunohematology and Bloodbank, Leiden University Medical Center, Leiden, The Netherlands) and penicillin/streptomycin (Gibco, Paisley, UK). The cells were stimulated with 1 μg/mL HBV core antigen (HBcAg) (a kind gift from M. van Roosmalen, Biomerieux, Boxtel, The Netherlands), 5 μg/mL Phytohemagglutin (Murex, Paris, France), 1.5 limits of flocculation/mL purified tetanus toxin (SVM, Bilthoven, The Netherlands) or were not stimulated and cultured for 6 days. Inhibition of proliferation by CD4+CD25+ Treg was tested by adding 10% (11,000 cells), 20% (25,000 cells), or 30% (43,000 cells) Treg to 1 × 105 CD25- depleted cells in 100 μL RPMI 1640 containing 10% human serum and penicillin/streptomycin. The cells were stimulated with 1 μg/mL HBcAg and cultured for 6 days. After 5 days of incubation, 50 μL of the supernatant was harvested for ELISA and replaced with 50 μL identical medium, and the cells were pulsed with 0.25 μCi/well of [3H]thymidine (Radiochemical Centre, Amersham, Little Chalfont, UK). The cells were harvested 16 hours later. Proliferation for both assays was determined via liquid scintillation counting of the harvested cells. IFN-γ production was determined by measuring the IFN-γ concentration in the harvested supernatant using a cytokine ELISA kit from U-CyTech (Utrecht, The Netherlands) according to the manufacturer's instructions.
FoxP3 RNA Quantification.
RNA was isolated from thawed PBMC samples of a subgroup of HBV patients (n = 25) and healthy controls (n = 11) using TRIzol (Invitrogen, Breda, The Netherlands). Residual DNA was removed from the RNA sample using the DNA-FREE RNA kit (Zymo, Orange, CA). The amount of RNA was quantified using a Ribogreen quantification kit (Molecular Probes, Leiden, The Netherlands). Copy DNA was produced in a reverse-transcriptase reaction with a random primer hexamer (Promega, Leiden, The Netherlands) using 750 ng of the RNA. The FoxP3 messenger RNA levels were determined in a real-time polymerase chain reaction reaction with the TaqMan (Abi Prism 7700 sequence detector, Applied Biosystems, Foster City, CA), using GAPDH as an internal control. The FoxP3 polymerase chain reaction primers and probe were obtained from Applied Biosystems assay on demand, and the GAPDH primers and probe were obtained from Biosource (Camarillo, CA). The relative copy number FoxP3 was calculated as recommended by the manufacturer. The relative amount of messenger RNA per CD4+ cell was determined using the following formula: (copies messenger RNA/RNA concentration used in the reverse-transcriptase reaction)/percentage of CD4+ cells.
Flow cytometry and FoxP3 data were analyzed using the Mann Whitney U test. Analysis of the depletion assay data was performed using the Wilcoxon matched pairs signed rank sum test. SPSS version 11.5 for Windows (SPSS, Chicago, IL) was used for the analyses. Statistical analysis on the reconstitution experiments was performed by analyzing the logarithmic transformation of the dependent variable with random intercept and random slope using PROC Mixed in SAS version 8.2 (SAS Institute, Cary, NC).
Patients With Chronic HBV Infection Have an Increased Proportion of Treg in Peripheral Blood Compared With Healthy Controls.
To determine whether CD4+CD25+ Treg play a role in the persistence of a chronic HBV infection, we first compared the percentage of Treg present in the peripheral blood of chronic HBV patients (n = 50), healthy controls (n = 23), and individuals with a resolved HBV infection (n = 9) (see Table 1). The percentage of Treg was determined by flow cytometry. Several different markers known to be expressed on CD4+CD25+ Treg were investigated. The expression of CTLA-4, CD45RO, CD62L, and GITR on the CD4+CD25+ cells was compared for PBMC from chronic HBV-infected patients, healthy controls, and individuals with a resolved HBV infection. The CD4+CD25+ cells with high expression of CD25 all expressed CD45RO and CTLA-4; however, this was not the case for GITR and CD62L (data not shown). Based on a previous study, which showed that the CD4+CD25+ cells with high expression of CD25 are Treg,6 CD45RO and CTLA-4 were used as additional markers in our flow cytometry experiments. Typical dot plots obtained by flow cytometry for PBMC from a representative chronic HBV patient are shown in Fig. 1. The percentage of Treg was defined as the percentage of cells that stained positive for CD4, CD25, CD45RO, and CTLA-4 divided by the percentage of cells that stained positive for CD4. Patients with a chronic HBV infection showed a significantly higher percentage of Treg within their population of CD4+ T cells in peripheral blood compared with healthy controls (5.40% ± 0.54% vs. 3.23% ± 0.66% [mean ± SEM]; P = .003) and individuals who had resolved their HBV infection (5.40% ± 0.54% vs. 2.96% ± 0.61% [mean ± SEM]; P < .035). There was no significant difference in the percentage of Treg between healthy controls and individuals with a resolved HBV infection (Fig. 2).
Chronic HBV patients had a similar increase in the percentage of CD4+CD25+CD45RO+CTLA-4+ cells in the total lymphocyte population compared with healthy controls and resolved HBV subjects. In addition, we did not observe a difference between patients and controls for the percentage of CD4+CD25+ cells within the subset of CD4+ T cells (data not shown).
FoxP3 RNA Expression Is Increased in Patients With Chronic HBV Infection.
The relative FoxP3 messenger RNA (mRNA) levels of the peripheral blood samples from a subgroup of 25 chronic HBV patients and 11 healthy controls was determined via real-time reverse transcriptase polymerase chain reaction. FoxP3 mRNA levels per CD4+ cells in patients with chronic HBV infection were significantly higher compared with healthy controls (204 ± 21.5 vs. 96 ± 10.9 [mean ± SEM]; P = .001), respectively (Fig. 3).
To assess whether the relative FoxP3 expression was specific for CD4+CD25+ Treg, FoxP3 mRNA levels were determined in isolated CD4+CD25− and CD4+CD25+ cells. CD4+CD25+ cells contained a 193-fold higher FoxP3 mRNA level compared with CD4+CD25− cells (2047 ± 721.8 vs. 10 ± 4.9 [mean ± SEM]).
Treg Inhibit the HBcAg–Specific Proliferation in a Dose-Dependent Manner.
Next, we determined whether Treg present in peripheral blood of chronic HBV patients can functionally suppress HBV-specific T-cell responses. Depletion of CD4+CD25+ Treg from PBMC of 17 chronic HBV patients resulted in a significantly stronger proliferation upon stimulation with HBcAg compared with PBMC (P = .001) (Fig. 4A). For 11 patients, the proliferation of Treg-depleted T cells was compared with PBMC after stimulation with purified tetanus toxin. As shown in Fig. 4B, there was no increase in proliferation upon stimulation with tetanus toxin after depletion of CD4+CD25+ cells. PBMC from healthy controls did not proliferate upon stimulation with HBcAg (data not shown). The isolated CD4+CD25+ T cells were hyporesponsive, because they did not proliferate upon stimulation with the mitogen phytohemagglutin (data not shown).
To confirm the suppression of the HBcAg response by Treg, 10% (11,000), 20% (25,000), or 30% (43,000) CD4+CD25+ T cells were reconstituted to 1 × 105 CD25 depleted cells and the proliferation and IFN-γ production was determined. CD4+CD25+ T cells inhibited the HBcAg-specific proliferation in a dose-dependent manner (Fig. 5A). The IFN-γ concentration in the pooled supernatant of the proliferation assay was determined by ELISA. IFN-γ production was also inhibited in a dose-dependent manner by CD4+CD25+ T cells (Fig. 5B).
Treg in Relation to Clinical Parameters.
Among the 50 chronic HBV patients, 24 were HBV e antigen (HBeAg)-positive and 26 were HBeAg-negative. For analysis of Treg in relation to HBeAg, we excluded 5 patients with a precore mutant, because these patients are not able to produce HBeAg irrespective of HBV replication status. HBeAg-positive patients exhibited a higher percentage of Treg in peripheral blood compared with HBeAg-negative patients (P = .045) (Fig. 6). No correlation was observed between the viral load or hepatic inflammation (serum alanine aminotransferase level) and the percentage of peripheral blood Treg.
The factors that determine chronicity of HBV infection and the reason for the absence of a specific T-cell response are not clear. Negative selection, peripheral anergy, and imbalances in lymphokine production all appear to contribute to maintaining the immunotolerant state.2 Recently, much attention has focussed on Treg, because they have a prominent role in immunoregulation and tolerance.6–9 The present study demonstrates the importance of Treg in chronic HBV infection. We showed that patients with chronic HBV infection contained a higher percentage of Treg in their peripheral blood compared with healthy controls and individuals with a resolved HBV infection. In addition, the relative FoxP3 mRNA levels were higher in PBMC of patients with a chronic HBV infection. FoxP3 is a transcription factor specifically expressed by CD4+CD25+ Treg.10, 18–20 The higher relative level of FoxP3 mRNA per CD4+ cell indicates that chronic HBV patients have indeed a higher percentage of Treg within their population of CD4+ cells in peripheral blood.
In our study, we observed an association between the presence of serum HBeAg and an increased percentage of Treg, suggesting that HBeAg might be involved in the induction of Treg. This is supported by the observation that HBeAg can elicit T-cell tolerance in murine experimental studies.21, 22 No correlation was found between HBV DNA levels or alanine aminotransferase and the percentage of Treg. One could argue whether peripheral blood is the most appropriate compartment in HBV infections to assess accurately the presence of Treg and the relation to viral load and liver inflammation. Other studies have shown that Treg accumulate and expand locally at the site of infection, where they exert their suppressive activity.23–26 Therefore, we are currently assessing whether the relation between percentage of intrahepatic Treg and the viral load or liver inflammation is more prominent.
Several studies have demonstrated that the cellular immune response to HBcAg is generally low to undetectable in chronic HBV patients.27–31 This immune response may be suppressed by the increased presence of Treg found in these patients. Indeed, we showed that depletion of CD4+CD25+ cells clearly increased the HBcAg-specific T-cell response in 17 patients. The effector cells suppressed by Treg are mainly CD4+ T cells, because HBcAg has been described to induce predominantly a CD4+ T-cell response.27 Furthermore, when the isolated Treg were reconstituted to the Treg-depleted fraction, the proliferation and IFN-γ production were suppressed in a dose-dependent manner.
In the present study, depletion of CD4+CD25+ cells did not affect the tetanus toxin response, suggesting that the suppressive effect of Treg was HBV-specific. Whether CD4+CD25+ cells inhibit the response to the recall antigen tetanus toxin is still a matter of debate. In chronic Helicobacter pylori infection, CD25 depletion resulted in an increased proliferation against membrane proteins from the bacterium, while the tetanus response was unaffected.15 In two other studies, CD4+CD25+ cells inhibited the response against tetanus toxin.16, 32
In our experimental setting, it is difficult to dissect the mechanism of immune suppression by Treg. In most in vitro models, suppression is caused by a cell–cell contact-dependent mechanism, yet the precise mechanism of suppression in vivo is not known.6, 7, 33 It was recently shown that Treg from patients with chronic HCV infection produce interleukin 10 and transforming growth factor β upon stimulation. The suppressive effect of the Treg was neutralized through the addition of an anti–transforming growth factor β antibody, and experiments in a transwell culture system showed that the suppression was contact-dependent.16 Further experiments will be necessary to determine the mechanism responsible for the suppression in an HBV-specific experimental setting.
Although we cannot exclude that the increased percentage of Treg was preexistent, predisposing the host to a chronic infection, the high number of Treg able to suppress HBV-specific T-cell responses is most likely caused by the viral infection itself. It has been suggested that Treg can be induced through repetitive stimulation of T cells by high concentrations of antigen for long periods of time.17 Chronic HBV-infected patients are known to have large amounts of HBV antigens in their peripheral blood. Results from studies of other chronic viral infections, such as cytomegalovirus, HIV, and HCV have previously shown a role for Treg in the persistence of these infections.13, 14 Depletion of CD4+CD25+ T cells from peripheral blood enhances T-cell responses to cytomegalovirus and HIV antigens.13 In patients with chronic HCV infection, a higher percentage of Treg was detected in peripheral blood compared with healthy controls and compared with individuals with a resolved HCV infection. The CD4+CD25+ Treg could directly suppress HCV-specific CD8+ T-cell IFN-γ production.14
Treg can be induced by immature dendritic cells (DC).34 Recently our group showed that myeloid DC isolated from chronic HBV-infected patients were impaired in their maturation and less capable of stimulating T cells compared with DC isolated from healthy individuals.35 Increased numbers of Treg are capable of inhibiting the maturation of DC and other antigen-presenting cells, resulting in a decreased T-cell stimulatory capacity.33, 36 This process could lead to the generation of more Treg and immature DC through a self-maintaining regulatory loop, which propagates HBV tolerance and chronicity of disease. An additional factor that may contribute to the increased percentage of Treg is the tolerogenic environment of the liver,37 the primary site of HBV replication.
In conclusion, patients with chronic HBV infection have a higher percentage of Treg in their peripheral blood compared with healthy controls and individuals with a resolved HBV infection. These Treg are capable of inhibiting the HBV-specific immune response, which could contribute to persistence of HBV infection.
The authors thank J. Kwekkeboom for helpful discussion, M. van Roosmalen for providing recombinant HBcAg, and W. Mol for technical assistance with the FoxP3 polymerase chain reaction. We also want to thank D. Sprengers, R.A. de Man, W.F. Leemans, J. Sarneel, A. Keizerwaard, L.A. van Santen-van der Stel, and C. van de Ent-van Rij for their help with obtaining peripheral blood samples.