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Keywords:

  • chronic hepatitis B;
  • hepatocellular carcinoma;
  • immune response;
  • regulatory T lymphocyte

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary.  Chronic hepatitis B (CHB) virus hepatitis B virus (HBV) infection is the key cause of hepatocellular carcinoma (HCC) in Asians. Recent studies have shown that levels of CD4+CD25+ regulatory T cells (Tregs) were increased and were linked to an impaired immune response in patients with CHB. Evaluating whether Tregs are involved in the progression of CHB to HCC will provide insight into the immunopathogenesis of HCC. In the present study, we showed that circulating and liver-residing Tregs increased in CHB (n = 15) and HCC (n = 49) patients, particularly in the peripheral blood of HCC patients with HBV infection (n = 29). The increased Tregs in CHB patients suppressed the specific immune response induced by not only HBV antigen, but also by HCC tumour antigen. When peripheral blood mononuclear cells (PBMC) were co-cultured with human hepatoma cell lines that are stably transfected with HBV (HepG2.2.15), CD4+CD25+ Treg populations increased and upregulated the expression of forkhead box P3 transcriptional regulator (FoxP3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor (TNF) receptor family gene (GITR). In contrast, PBMCs co-cultured with HepG2 cells (the parental cell line of HepG2.2.15) did not. CD4+CD25+ Tregs isolated from PBMCs that were co-cultured with HepG2.2.15 cells also had a greater suppressive ability with respect to the tumour antigen-specific immune response induced by NY-ESO-1 or MAGE-A3 compared with CD4+CD25+ Tregs isolated from PBMCs co-cultured with HepG2 cells. The results offer evidence that the expansion of CD4+CD25+ Tregs and the enhancement of the suppressor function of CD4+CD25+ Tregs induced by HBV infection-related factors could suppress the anti-tumour immune response to HCC tumour antigen and inhibit tumour immuno-surveillance against HCC, which may be involved in the immunopathogenesis from CHB to HCC.


Abbreviations:
CFSE

carboxyfluorescein succinimidylester

CHB

chronic hepatitis B

CTLA-4

cytotoxic T lymphocyte-associated antigen-4

CTL

cytotoxic T lymphocyte

DC

dendritic cell

ELISPOT

enzyme-linked immunospot

FITC

fluorescein isothiocyanate

FoxP3

forkhead box P3 transcriptional regulator

GITR

glucocorticoid-induced TNF receptor family gene

HBcAg

hepatitis B core antigen

HBeAg

hepatitis B e antigen

HBV

hepatitis B virus

HCC

hepatocellular carcinoma

IFN-γ

interferon-γ

LIL

liver-infiltrating lymphocyte

PBMC

peripheral blood mononuclear cell

PE

phycoerythrin

TNF

tumour necrosis factor

Treg

regulatory T cell

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Hepatitis B virus infection usually causes acute and chronic hepatitis. Approximately 360 million people worldwide are chronically infected. Chronic HBV infection is an important risk factor in the development of cirrhosis and hepatocellular carcinoma (HCC) [1–4]. During HBV infection, the pathogenesis of liver damage is immune-mediated and dependent on the balance between viral replication and the virus-specific CD8+ T-cell response [5–7]. Virus-specific CD8+ T cells are defective in patients with persistent HBV infection compared with patients who have resolved infection [8]. Recent studies have provided convincing evidence that the HBV-specific CD8+ T-cell response could be modulated by CD4+CD25+ regulatory T cells (Tregs) in patients with HBV infection [9–11]. CD4+CD25+ Tregs not only inhibited effectors of the immune response to HBV infection, but also influenced the disease prognosis in patients with hepatitis B [11].

CD4+CD25+ Tregs comprise 5–10% of the human CD4+ T-cell population [12]. They express CD4, CD25, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) [12,13] and forkhead box P3 transcriptional regulator (FoxP3) [14,15], and they suppress the activation and proliferation of both CD4+ and CD8+ T cells [16,17]. CD4+CD25+ Tregs play a vital role in immunological self-tolerance, anti-tumour immune responses, antiviral immune responses and transplantation [18,19]. This cell type increases in patients with CHB [20] and HCC [21–24]. The removal of CD4+CD25+ Tregs can enhance the immune response to HBV or tumours in vitro [9,21]. The mechanisms that mediate the regulatory effect of CD4+CD25+ Tregs are controversial, with evidence supporting regulation through either suppressive cytokines or direct cell–cell contact [25]. Recent experiments showed that antigen-primed stimulation and recognition is necessary for the activation and suppression abilities of Tregs in vivo and in vitro [26]. Once CD4+CD25+ Tregs are activated, they can suppress the activation and proliferation of both CD4+ and CD8+ T cells in a non-antigen-specific manner [16,17,27]. It was noted that the depletion of CD4+CD25+ Tregs in CHB patients could expand the melanoma antigen-specific CD8+ T cells [9]. However, it is unclear whether CD4+CD25+ Tregs in CHB patients can suppress the HCC tumour antigen-specific immune response when HCC occurs due to chronic HBV infection. Here we propose that CD4+CD25+ Tregs in CHB patients can inhibit tumour immunosurveillance against HCC by suppressing the HCC tumour antigen-specific immune response and that they are involved in the immunopathogenesis from CHB to HCC. Evaluating the role of Tregs in the progression of CHB to HCC will provide insight into the immunopathogenesis of HCC.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Study participants

The study included 15 HLA-A2+ patients with CHB (who had no evidence of cirrhosis and no antiviral therapy during the 6 months before blood sampling) from Peking University Hepatology Institute (Peking, China), 49 HLA-A2+ HCC patients from Guilin Medical University (Guilin, China), and 25 healthy donors from the Beijing Red Cross Blood Center (Beijing, China). All participants gave informed consent before blood or tissue donation, and the Ethics Committee of Peking University People’s Hospital approved the study protocol. Patient and control characteristics are shown in Table 1.

Table 1.   Patient and control characteristics
 Hepatocellular carcinoma (HCC) patients (n = 49)Chronic hepatitis B (CHB) patients (n = 15)Healthy donors (n = 25)
Age (years)49.9 ± 12.945.7 ± 11.246.9 ± 14.5
Sex36 men/13 female9 men/6 female20 men/5 female
 HBsAg positive29150
 HBeAg positive13150
hepatitis B virus (HBV) DNA copies2.7 × 103–7.8 × 1071.4 × 107–2.2 × 109None
TNM(Tumor, Node, Metastasis) stage Not applicableNot applicable
 II8  
 III19  
 IV22  
Cirrhosis
 No181325
 Yes3120
ALT(Alanine aminotransferase)
 ≤40 U/L22025
 >40 U/L27150
Serum AFP (n-fetoprotein)level
 ≥20 ng/mL2200

Peptides and pentamers

The HLA-A2-restricted HBV core peptide (p18–27, FLPSDFFPSV), NY-ESO-1b peptide (p157–165, SLLMWITQC) and MAGE-A3 peptide (p271–279, FLWGPRALV) were synthesized by Sangon (Shanghai, China). The purity after high pressure liquid chromatography analysis was >95%. Pentamers of each peptide were purchased from Proimmune (Oxford, UK).

Isolation of peripheral blood mononuclear cells and liver-infiltrating lymphocytes

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation from either 100 mL heparinized blood or 40 mL leukapheresis-derived PBMC-enriched sample. Liver tissues were carefully washed with phosphate-buffered saline (PBS) solution containing 2% foetal calf serum (FCS) and 1% ethylenediaminetetraacetic acid (EDTA) to remove peripheral blood. The tissue was then whittled into small pieces, homogenized and pressed between two semifrosted microscope slides. The dissociated cell suspension was mixed on ice for 15 min. The upper part of the suspension was carefully recovered, passed through a 100 μm cell strainer (BD Labware, Bedford, MA, USA), and underlaid onto Ficoll-Hypaque separation solution. Liver-infiltrating lymphocytes (LIL) were then isolated by density gradient centrifugation. The viability of isolated cells was determined by trypan blue exclusive staining.

Flow cytometric analysis

To determine the frequency and phenotype of Tregs, multicolour flow cytometric analysis was performed. Cells were washed once in PBS containing 1% FCS and then they were stained with fluorescently labelled anti-CD4, anti-CD25, anti-CD152 (BD PharMingen, Heidelberg, Germany), phycoerythrin (PE)-conjugated anti-FoxP3 (Biolegend, San Diego, CA, USA) and anti-glucocorticoid-induced tumour necrosis factor (TNF) receptor family gene (GITR) (R&D Systems, Weisbaden, Germany). Either three- or four-colour flow cytometry was done using a Becton Dickinson(San Diego, CA, USA) FACS Calibur and FlowJo 7.0 software. Isotype-matched antibodies were used as controls for all of the samples.

Immunofluorescence of CD4+ and FoxP3+ T cells in liver tissue

Tissues were stained with mouse antihuman CD4 (BD PharMingen) and goat antihuman FoxP3 IgG (Ab2481, 1:100 dilution; Novus Biologicals, Littleton, CO, USA), followed by Alexa Fluor 488-conjugated goat antimouse IgG1 and Alexa Fluor 488-conjugated donkey antigoat IgG (all 2 μg/mL, Molecular Probes, Leiden, The Netherlands). Positive cells were quantified by ImagePro Plus software (Media Cybernetics) and were expressed as mean ± SD in 10 high power fields (h.p.f.) using fluorescence microscopy.

Cell culture and co-culture experiments

The human hepatoma cell line HepG2.2.15 (a HBV stably transfected cell line, kindly provided by Professor J. T. Guo, Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Philadelphia, PA, USA) and its parental cell line HepG2 (purchased from ATCC, American Type Culture Collection, Manassas, USA) were maintained in DMEM(Dulbecco’s Modified Eagle Media) supplemented with 100 mL/L foetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were incubated at 37 °C in an atmosphere containing 5% CO2. Cells were seeded onto six-well plates at 2 × 105 cells per well. After cells had attached to the dishes, they were co-cultured with 2 × 106 PBMCs from HLA-A2+ healthy donors for 3 days in a transwell culture system. PBMCs were cultured with interleukin (IL)-2 (10 units/mL) as a control.

Cell isolation and sorting

Peripheral blood mononuclear cells were isolated from heparinized blood of CHB patients and were co-cultured with human hepatoma cell lines. To isolate CD4+CD25+ Tregs and CD4+CD25 T cells, PBMCs were further separated using a regulatory T-cell isolation kit (StemCell, Vancouver, Canada), according to the manufacturer’s instructions. CD8+ T cells were isolated using a CD8+ T-cell enrichment kit (StemCell). Enriched cells were determined by flow cytometry, and the purity after sorting was >95%.

Proliferation and suppression assays

CD4+CD25 and CD4+CD25+ cells were isolated and purified as described above. The CD4+CD25+ Tregs were isolated from co-culture systems. The CD4+CD25 T cells as the response cells were stimulated with anti-CD3/anti-CD28 Dynabeads (Invitrogen Dynal AS, Oslo, Norway) for 3 days. Then, CD4+CD25 T cells were labelled with carboxyfluorescein succinimidylester (CFSE) before co-culture by incubation with 5 μmol/L CFSE for 10 min at 37 °C in PBS, followed by addition of ice-cold complete medium and PBS washes. After 3 days of co-culture with CD4+CD25+ Tregs and CD4+CD25 T cells at a ratio of 1:5, the proliferation of T cells was measured using CFSE labelling and flow cytometry.

Expansion of peptide-specific CD8+ T cells

CD8+ T cells were isolated from PBMCs of CHB patients and PBMCs of healthy donors with a >95% purity. As described previously [28,29], CD8+ T cells were seeded onto 48-well plates at a concentration of 5 × 105 cells per well in 10% human AB serum-RPMI 1640 medium. For antigen presentation, autologous dendritic cells (DCs) were incubated with 2.5 μg/mL beta-2 microglobulin (Sigma, St Louis, MO, USA) and 10 μg/mL peptide for 2 h, and then they were irradiated at 30 Gy. After washing, DCs were added to the plates at a ratio to CD8+ T cells of 1:5. Autologous Tregs isolated from PBMCs of CHB patients or PBMCs of co-culture systems were also added to the plates at a ratio to DCs and CD8+ T cells of 1:1:5 (that is, 1 DC : 1 Treg : 5 CD8+ T cell). Then, IL-2 (10 units/mL; R&D System, Minneapolis, MN, USA) was added. The CD8+ T cells were restimulated at Day 8 and in some samples at Day 15, with peptide-loaded, irradiated autologous DCs.

Enzyme-linked immunospot assays

Interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assays were performed on the peptide-specific CD8+ T cells mentioned above. Immobilon-P membrane microtitre 96-well plates (Millipore, Bedford, MA, USA) were coated with 100 μL of 5 μg/mL anti-IFN-γ monoclonal antibody, and human IFN-γ ELISPOTs were performed according to the manufacturer’s instructions (ELISpot kit; DIACLONE, Besancon Cedex, France). The peptide pulsed T2 cells (5 × 104 cells per well) were added together with CD8+ effector T cells (5 × 104 cells per well). After incubation for 18–20 h at 37 °C, the cells were removed and the plates were developed with a second (biotinylated) antibody to human IFN-γ and streptavidin-alkaline phosphatase. Plates were developed for 10 min at room temperature in the dark, and the reaction was stopped by rinsing with distilled water. The membranes were air-dried. The spots were counted using an ELISpot reader system (ImmunoSpot Image Analyzer 3B, Cellular Technology Ltd., Cleveland, OH, USA). The number of specific spot-forming cells was determined as the mean number of spots in the presence of an antigen minus the mean number of spots in the wells containing medium only, and was expressed per 5 × 104 cells.

Staining with HLA-pentameric complexes

The pentamers were used to stain short-term lines produced from the patients’ CD8+ effector T cells as described previously [28,29]. T cell lines were incubated for 10 min at room temperature with 10 μL of PE-labelled tetrameric complex. The cells were washed in PBS and incubated at 4 °C for 20 min with saturating concentrations of directly conjugated anti-CD8 fluorescein isothiocyanate (FITC) monoclonal antibody (Becton Dickinson.). After further washing, the cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry (FACS Calibur; Becton Dickinson) using FlowJo 7.0 software.

Statistical analysis

Continuous variables were expressed as mean ± SD. Differences of continuous parameters between the groups were analyzed, and a one-way analysis of variance followed by the Scheffe’s post-hoc test was used. Statistical analysis was done using the Student’s t-test to assess differences between the study groups. A value of P < 0.05 was considered significant. The data were analyzed using spss software version 11.0 (SPSS, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Circulating and liver-residing CD4+CD25+ Tregs in CHB and HCC patients

We determined the frequency of circulating CD4+CD25+ Tregs by measuring the numbers of CD4+CD25+FoxP3+ T cells (Fig. 1a). The study revealed that the frequency of circulating Tregs was significantly higher in HCC patients than in healthy controls (4.26 ± 4.18% and 1.67 ± 0.73% in HCC patients and healthy controls, respectively; P 0.01). We also found that CHB patients had a higher percentage of circulating CD4+CD25+ Tregs than healthy controls (4.39 ± 1.45% and 1.67 ± 0.73% in CHB patients and healthy controls, respectively; P < 0.01). The frequency of circulating CD4+CD25+ Tregs in CHB patients was not significantly different from that in HCC patients.

image

Figure 1.  Frequency of circulating and liver-residing CD4+CD25+ Tregs in CHB and hepatocellular carcinoma (HCC) patients. (a) Representative prevalence of CD4+CD25+FoxP3+ Tregs of peripheral blood mononuclear cell from HCC patient (HCC), CHB patients (CHB) and healthy control (NC). (b) The percentage of CD4+CD25+ Treg cells in the peripheral blood of HCC patients (n = 49), healthy donors (n = 25) and CHB patients (n = 15). Individual cell frequencies in each patient are shown. CD4+CD25+FoxP3+ T cells are presented as a percentage of the total CD4+ cell population. The prevalence of CD4+CD25+FoxP3+ Tregs in HCC and CHB patients was higher than the prevalence in healthy controls (P < 0.01). (c) The percentage of CD4+CD25+ Tregs in the liver of CHB patients and HCC patients. liver-infiltrating lymphocyte (LIL) were isolated from the liver tissue of six CHB patients, nine HCC patients and four healthy donors. HCC patients and CHB patients had a dramatic increase in the number of LIL compared with healthy donors. (d) Immunofluorescence staining for CD4 and FoxP3 within liver tissue cryosections of healthy controls, CHB patients and HCC patients. h.p.f., ×400; l.d.f., ×100. Positive cells were stained green.

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As shown in Fig. 1b, the frequency of circulating CD4+CD25+ Tregs in HBsAg-positive HCC patients was higher than the frequency of circulating CD4+CD25+ Tregs in HBsAg-negative HCC patients (5.58 ± 4.90% and 2.35 ± 1.46% for HBsAg positive and HBsAg negative, respectively; P < 0.01). However, there was no difference in the circulating CD4+CD25+ Tregs frequency between HBsAg-negative HCC patients and healthy controls (Fig. 1b).

To investigate the accumulation of CD4+CD25+ Tregs in the liver of CHB patients and HCC patients, LIL were isolated from the liver tissue of six CHB patients, nine HCC patients and four healthy donors. HCC patients and CHB patients had a dramatic increase in the number of LIL compared with healthy donors. According to flow cytometric analysis, the frequencies of CD4+CD25+ Tregs in CHB patients and HCC patients were significantly higher than in healthy controls (CHB patients vs healthy controls, 4.95 ± 1.33%vs 1.43 ± 0.66%; HCC patients vs healthy controls, 5.99 ±2.09%vs 1.43 ± 0.66%) (Fig. 1c). Immunofluorescence staining was performed to visualize the severity of inflammation and the distribution of FoxP3+ Tregs. As shown in Fig. 1d, HCC patients and CHB patients showed a dramatically increased infiltration of CD4+ and FoxP3+ LIL compared with healthy controls (HCC patients FoxP3+ LIL 26.7 ± 14.2/h.p.f.; CHB patients FoxP3+ LIL 21.5 ± 12.6/h.p.f.; healthy controls FoxP3+ LIL 1.9 ± 1.5/h.p.f.).

Frequency and phenotype of CD4+CD25+ Tregs from peripheral blood mononuclear cells co-cultured with HepG2 or HepG2.2.15 cells

To evaluate the effect of HBV-infected liver cells on CD4+CD25+ Treg frequency and phenotype, human hepatoma cell lines HepG2.2.15 (with HBV stably transfected) and its parental cell line HepG2 (without HBV transfected) were co-cultured with PBMCs from HLA-A2+ healthy donors. After 3 days of co-culture, CD4+CD25+ Treg frequency and phenotype were measured using flow cytometry. In the co-cultures, human hepatoma cell lines (HepG2 and HepG2.2.15) increased the expansion of CD4+CD25+ Tregs, but HepG2.2.15 cells strongly increased the frequency of CD4+CD25+ Tregs compared to HepG2 (21.68 ± 1.07%vs 14.86 ± 1.04%) (Fig. 2a).

image

Figure 2.  Frequency and phenotype of CD4+CD25+ Tregs from peripheral blood mononuclear cell (PBMCs) co-cultured with HepG2 or HepG2.2.15 cells. (a) Representative expression of FoxP3 in CD4+CD25+ T cells of PBMCs co-cultured with HepG2 or HepG2.2.15 cells (cells gated from CD4+ T cells). (b) The percentage of CD4+CD25+ Tregs in the PBMCs co-cultured with HepG2 or HepG2.2.15 cells. (c) The expression of FoxP3, CTLA-4 and GITR on Tregs from PBMCs, PBMCs co-cultured with HepG2 and PBMCs co-cultured with HepG2.2.15 cells. Asterisks (*) represent significant statistical difference compared with the group of PBMCs co-cultured with HepG2 (P < 0.05).

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As shown in Figs 2a,c, FoxP3, CTLA-4 and GITR were upregulated after being co-cultured with HepG2 and HepG2.2.15 cells. In comparison with HepG2, HepG2.2.15 cells showed a significantly greater ability to induce the expression of FoxP3, CTLA-4 and GITR.

Increase of CD4+CD25+ Tregs in the circulation from CHB patients had a suppressive impact on tumour antigen-specific CD8+ responsiveness via tumour-specific antigen induction

We analyzed whether circulating CD4+CD25+ Tregs in CHB patients have an altered suppressive capacity for specific CD8+ responsiveness via tumour-specific antigen (NY-ESO-1 and MAGE-A3 peptides). Of eight HLA-A2+ CHB patients, six were found to have a specific CD8+ T-cell response to NY-ESO-1 and MAGE-A3 in their peripheral blood after antigen-driven DC cell expansion in vitro. Pentamer assays and IFN-γ release ELISPOT assays were performed. When the autologous CD4+CD25+ Tregs were added to the co-culture at a ratio of 1 DC:1 Treg:5 CD8+ T cells, the frequencies of HBVcore18–27-specific CD8+ T cell and specific IFN-γ-producing cells were decreased (Figs 3a,b). As shown in Figs 3c,d, the frequency of NY-ESO-1 pentamer+ CD8+ T cells with autologous Tregs added was also lower than NY-ESO-1 pentamer+ CD8+ T cells without autologous Tregs added (0.15 ± 0.10%vs 0.58 ± 0.35%). The frequency of MAGE-A3 pentamer+ CD8+ T cells with autologous Tregs added was lower than that in the group without autologous Tregs added (0.12 ± 0.10%vs 0.30 ± 0.16%). The average frequency of specific IFN-γ-producing cells counted by ELISPOT assay was similar in the results detected by pentamer assay.

image

Figure 3.  Circulating CD4+CD25+ Tregs in CHB patients have an altered suppressive capacity to specific CD8+ responsiveness. (a) The frequency of HBVcore18–27 pentamer+ CD8+ T cells with and without autologous Tregs added. (b) The average frequency of specific Interferon (IFN)-γ-producing cells with and without autologous Tregs added. (c) The frequency of NY-ESO-1157–165 pentamer+ CD8+ T cells with and without autologous Tregs added. (d) The frequency of MAGE-A3271–279 pentamer+ CD8+ T cells with and without autologous Tregs added. Treg– represents the expansion of antigen-specific CD8+ T cells without autologous CD4+CD25+ Tregs added; Treg+ represents the expansion of antigen-specific CD8+ T cells with autologous CD4+CD25+ Tregs added. SFC, spot-forming cells.

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CD4+CD25+ Tregs co-cultured with HepG2.2.15 cells had higher suppressive function on tumour antigen-specific CD8+ responsiveness than Tregs co-cultured with HepG2 cells

To evaluate the effect of HBV-infected liver cells on CD4+CD25+ Treg suppressive function, HepG2.2.15 cells and HepG2 cells were co-cultured with PBMCs from HLA-A2+ healthy donors. The CD4+CD25+ Tregs were then isolated from co-culture systems. The CD4+CD25 T cells as the response cells were stimulated by anti-CD3/anti-CD28 Dynabeads for 3 days. After 3 days of co-culture with CD4+CD25+ Tregs and CD4+CD25 T cells, the proliferation of T cells was measured using CFSE labelling and flow cytometry. The CD4+CD25+ Tregs isolated from HepG2.2.15 co-culture systems strongly inhibited the proliferation of anti-CD3/anti-CD28 Dynabead-stimulated T cells compared with CD4+CD25+ Tregs isolated from HepG2 co-culture systems (Fig. 4a).

image

Figure 4.  The suppressive function of CD4+CD25+ T cells isolated from co-culturing with HepG2.2.15 cells and HepG2 cells. (a) The suppression of CD4+CD25 T-cell proliferation by CD4+CD25+ T cells. The CD4+CD25+ Tregs isolated from HepG2.2.15 co-culture systems strongly inhibited the proliferation of anti-CD3/anti-CD28 Dynabead-stimulated T cells compared with CD4+CD25+ Tregs isolated from HepG2 co-culture systems. (b) The frequency of NY-ESO-1157–165 pentamer+ CD8+ T cells with and without autologous Tregs added. (c) The frequency of MAGE-A3271–279 pentamer+ CD8+ T cells with and without autologous Tregs added.

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To evaluate the specific suppressive function of CD4+CD25+ Tregs, we prepared autologous tumour antigen-specific CD8+ T cells. Of six HLA-A2+ healthy donors, three were found to have a specific CD8+ T-cell response to NY-ESO-1 and MAGE-A3 in their peripheral blood after antigen-driven DC cell expansion in vitro. The CD4+CD25+ Tregs isolated from the HepG2.2.15 co-culture system strongly suppressed the expansion of NY-ESO-1- and MAGE-A3-specific CD8+ T cells compared with Tregs isolated from the HepG2 co-culture system (Figs 4b,c).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

To date, the factors that determine chronicity and progression of HBV infection and the reason for the absence of a specific T-cell response have not been clear. Low specific CD8+ T-cell response, negative selection, peripheral anergy and imbalance in lymphokine production all appear to contribute to maintaining the immunotolerant state [6]. In HCC, the absence of a specific T-cell response to a tumour also contributes to the development and progression of the tumour. The CD4+CD25+ Tregs in tumour patients suppress tumour-specific CD8+ T-cell cytotoxicity and contribute to tumourigenesis [27]. The tumourigenesis of HCC is associated with chronic HBV infection. Whether immunosuppressive agents (such as Tregs) caused by chronic HBV infection contribute to immune escape of tumour cells in early tumourigenesis is the hypothesis that we want to validate. The present study demonstrates the importance of Tregs in chronic HBV infection and tumour immunopathogenesis. We showed that circulating and liver-residing Tregs increased in CHB and HCC patients, particularly in the peripheral blood of HCC patients with HBV infection. Increased Tregs in CHB patients could suppress the specific anti-tumour immune response. In addition, the HBV stably transfected hepatoma cell lines could increase the frequency of CD4+CD25+ Tregs and induce the higher suppressive function on tumour antigen-specific CD8+ responsiveness compared with Tregs co-cultured with hepatoma cell lines that were not HBV transfected.

In our study, we found that circulating and liver-residing CD4+CD25+ Tregs increased in CHB patients compared with healthy controls. This result is similar to that of Janssen’s group [20], but it differs from the data published by Xu’s group and Bertoletti’s group [7,11]. The discrepancies might be caused by the selection of CHB patients. In our study, all of the CHB patients were HBeAg positive, and the serum HBV DNA levels were higher than 107 copies/mL. The data from Xu’s group also suggest the CHB patients with high levels of serum HBV DNA had a significant increase of circulating CD4+CD25+ Tregs compared with healthy controls [11]. In HCC patients, circulating CD4+CD25+ Tregs increased significantly compared with healthy controls, but it is noted that the HCC patients with HBsAg-positive serum had an increased percentage of Tregs compared with the HCC patients with HBsAg-negative serum. We also observed that CHB patients and HCC patients had a significant increase of liver-infiltrating CD4+CD25+ Tregs compared with healthy controls. Immunohistochemical analysis also verified a dramatic increase of CD4+ and FoxP3+ cells infiltrating the liver of CHB and HCC patients. These data imply that circulating and liver-residing Tregs increased in CHB and HCC patients, particularly in the peripheral blood of HCC patients with HBV infection.

Recent studies have demonstrated that the cellular immune response to HBV may be suppressed by the increased presence of Tregs found in patients with the virus. Franzese’s group reported that the depletion of CD4+CD25+ Tregs in CHB patients could expand melanoma antigen-specific CD8+T cells [9]. Indeed, we showed that the increased levels of Tregs in CHB patients could not only suppress the HBcAg-specific T-cell response but also suppress the specific immune response induced by HCC tumour antigen (NY-ESO-1 and MAGE-A3 peptides) in six patients. These results suggest that the suppressive effect of Tregs in CHB patients is non-HBV specific, which was supported by the suppressive mechanism of Tregs. CD4+CD25+ Tregs can be activated and expanded by antigen stimulation. Tregs can then suppress the activation and proliferation of both CD4+ and CD8+ T cells in a non-antigen-specific manner through either suppressive cytokines or direct cell–cell contact [16–18,27,34]. As shown in the results, when the autologous CD4+CD25+ Tregs isolated from CHB patients were added, the frequencies of NY-ESO-1/MAGE-A3-specific CD8+ T cells and specific IFN-γ-producing cells were decreased. As previously described, we found that NY-ESO-1 and MAGE-A3 as tumour-specific antigens can elicit specific cellular and humoral immune responses against tumour cells in HCC patients. The spontaneous CD8+ T-cell response to NY-ESO-1 and MAGE-A3 antigen in HCC patients is involved in the immune attack and killing of tumour cells [28–30]. These results imply that increased presence of Tregs in CHB patients may be involved in suppressing specific anti-tumour immune responses in the early tumourigenesis of CHB patients.

In the experimental setting, it is difficult to acquire enough liver-residing CD4+CD25+ Tregs from CHB patients to dissect the suppressive effect on specific immune responses. To evaluate the effect of HBV-infected liver cells on CD4+CD25+ Treg suppressive function, we co-cultured human hepatoma cell line HepG2.2.15 (stably transfected with HBV) and its parental cell line HepG2 with healthy donor PBMCs. The experiments, performed in a transwell culture system, showed that HepG2.2.15 cells strongly increased the frequency of CD4+CD25+ Tregs compared with the frequency of CD4+CD25+ T cells co-cultured with HepG2 cells (untransfected with HBV). The expression of FoxP3, CTLA-4 and GITR was unregulated in the group co-cultured with HepG2.2.15. Recent data showed that carcinoma cell supernatants could expand CD4+CD25+ Tregs and enhance their suppressor ability via the high level secretion of transforming growth factor (TGF)-β [31,32]. HCC cell supernatants could increase the expansion and function of CD4+CD25+ Tregs [33]. In our previous work, we found that the secretion of TGF-β in HepG2.2.15 cell supernatants was higher than that in HepG2 cell supernatants. In the present study, whether the increased expansion and function of CD4+CD25+ Tregs is due to the high level secretion of TGF-β in HepG2.2.15 cells after being stably transfected with HBV remains to be determined.

As to the suppression of Tregs isolated from PBMCs co-cultured with HepG2.2.15 or HepG2, it was similar to the phenomenon that increased Treg levels in CHB patients suppressed specific immune responses induced by HCC tumour antigen. The CD4+CD25+ Tregs isolated from HepG2.2.15 strongly suppressed the proliferation of autologous CD4+CD25 T cells and expansion of NY-ESO-1- and MAGE-A3-specific CD8+ T cells. These data indicate that the suppressive effect of increased Tregs expanded by HepG2.2.15 cells could suppress the effector T cells in a non-antigen-specific manner.

In conclusion, the results offer evidence that HBV infection-related factors not only induce and expand CD4+CD25+ Treg cells but also enhance their suppressor ability toward anti-tumour immune responses to HCC tumour antigen. These findings suggest that CD4+CD25+ Tregs in CHB patients can inhibit tumour immunosurveillance against HCC by suppressing the HCC tumour antigen-specific immune response and that they are involved in the immunopathogenesis from CHB to HCC.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the National Natural Science Foundation of China (grants no. 30640091 and no. 30772499), the National Key Sci-Tech Special Project of China (grant no. 2008ZX10002-019), the Program for New Century Excellent Talents (985-2-099-113), the National Basic Research Program (grants no. 2005CB522902 and no. 2007CB512906) and National 863 plans projects (2007AA021103).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References