Interleukin (IL)-35 is an inhibitory cytokine consisting of IL-12A and Epstein-Barr virus-induced gene 3 (Ebi3) and is required by regulatory T-cells (Tregs) for maximal activity. During chronic hepatitis B virus (HBV) infection, Tregs have immunosuppressive effects on HBV-specific T helper (Th) cells, yet little is known about the complex regulation of Tregs and their contribution to the inadequate immune system response to the virus. In the present study, we investigated whether IL-35 is involved in HBV-related cellular immune responses. Cluster of differentiation (CD)4+ T-cells from peripheral blood were derived from healthy volunteers, resolved HBV individuals and chronic active hepatitis B patients and stimulated with CD3/28-conjugated beads. We analysed mRNA and protein levels of IL-35 and assessed the inhibitory effect of IL-35 on HBV core antigen-specific cytotoxic T lymphocytes (CTLs), dendritic cells (DCs) and effector T-cells (Teffs). Correlation analyses between liver inflammation and HBV DNA load were conducted. Results show that chronic HBV patients harbour significantly higher levels of Ebi3 mRNA and protein in CD4+ T-cells compared with healthy volunteers and resolved HBV individuals. IL-35 suppressed the proliferation of HBV antigen-specific CTLs and interferon (IFN)-γ production in vitro. Ex vivo, IL-35 decreased the proliferation of CD4+CD45RA+ naïve T-cells, especially in CD4+CD25CD45RA+ naïve Teffs. IL-35 inhibited the expansion of CD11c+ DCs. Our data indicate that IL-35 is highly expressed in chronic HBV CD4+ T-cells and plays an important role in the inhibition of the cellular immune response in chronic HBV.

CLINICAL PERSPECTIVES

  • The newly characterized suppressive cytokine IL-35 plays a critical role in inhibiting various inflammation and immune responses. However, the role and mechanism of IL-35-mediated immune/inflammation suppression in regulating hepatitis B infection remained unknown.

  • We have shown that IL-35 is highly expressed in CD4+ T-cells from CHB patients and it plays an important role in the inhibition of cellular immune response in chronic HBV infection.

  • Elucidation of how IL-35 regulates the pathogenesis of HBV infection is important, which may identify a new target for future development of new therapeutics for hepatitis B infection and other inflammatory diseases.

INTRODUCTION

Hepatitis B virus (HBV) infection is widely present in Asian countries [1] and can lead to severe liver disease, including cirrhosis and hepatocellular carcinoma [2]. Cellular immune responses play an important role in viral clearance and disease pathogenesis [3,4]. In chronic hepatitis B (CHB) patients, one of the most important factors determining the chronicity of infection is the absence of a specific T-cell response [5,6].

The mechanism underlying the failure of the immune system during chronic HBV infection is still not understood. Previous studies have indicated that regulatory T-cells (Tregs) and dendritic cells (DCs) play important roles in the maintenance of peripheral immune tolerance [79]. Treg pathogenesis prevents the development of strong HBV-specific cytotoxic T lymphocyte (CTL) responses. Moreover, DC function is suppressed in patients with chronic HBV infections [10,11]. However, the roles of Tregs and DCs in persistent viral infections have not been well defined. Both Tregs and DCs can suppress the function of effector T-cells (Teffs) and may play a crucial role in impaired immune responses. Little is known about the suppressive mechanisms of these cells.

One factor that may play a role in the impaired immune response of HBV infection is interleukin-35 (IL-35). IL-35 is a heterodimeric cytokine composed of the Epstein-Barr virus-induced gene 3 (Ebi3) and IL-12A. Collison et al. [12] demonstrated that this novel Ebi3–IL-12A heterodimeric cytokine is preferentially secreted by Tregs and contributes to their suppressive activities. Both Ebi3 and IL-12A are required by Tregs for maximal regulatory activity in vivo and in vitro. Subsequent studies by Collison et al. [13] showed that human and mouse conventional T-cells (Tconvs) can be converted into a new population of T-cells with suppressive properties using IL-35 but not with IL-10 or transforming growth factor-β (TGF-β). These novel inducible Tregs are called IL-35-induced Tregs (iTR35 cells) and do not express Foxp3 (forkhead box P3).

Previous studies have shown that IL-35 can be detected in cluster of differentiation (CD)4+ T-cells from the peripheral blood of CHB patients, a result that provides evidence for the possible role of IL-35 in immune suppression during HBV infection [14]. Whether IL-35 or iTR35 cells are involved in the development of chronic HBV infection is unclear. The aim of the present study was to determine whether IL-35 contributed to HBV-related cellular immune responses and whether increased IL-35 in peripheral blood is associated with the viral load in patients with untreated chronic HBV infection. We characterized the expression of IL-35 in the peripheral blood CD4+ T-cells of CHB patients and the inhibitory effect of this cytokine on HBV-specific CTLs and DCs.

PATIENTS AND METHODS

Study subjects

The whole protocol was performed with 82 treatment-naive chronic active hepatitis B patients, who were recruited from the First Affiliated Hospital of the Zhejiang University School of Medicine. The detailed information of those patients for each experiment (including mRNA and protein levels of Ebi3 and IL-12A and IL-35 concentrations in CD4+ T-cells from the peripheral blood, as well as the effect roles of IL-35 on HBV-specific CTLs, DCs and Teffs) is shown in the Figure 1. Age- and sex-matched healthy volunteers (n=42) and resolved HBV individuals (n=21) served as controls. The diagnostic criteria for chronic hepatitis B and resolved HBV individuals referred to American Association for the Study of Liver Diseases (AASLD) Practice Guidelines [15]. The clinical characteristics of all study subjects are presented in Table 1. Patients co-infected with HIV, hepatitis A virus, hepatitis C virus or hepatitis D virus were excluded from the present study. Patients and controls that were immunocompromised, pregnant or had received antiviral or immunomodulatory HBV treatment during the 6 months prior to blood sampling were also excluded. All patients and healthy volunteers gave informed consent to participate in the present study. The study protocol conforms to the guidelines of the Declaration of Helsinki and was approved by the Ethics Review Committee of the First Affiliated Hospital of the Zhejiang University School of Medicine.

Flowchart of patients for each experiment in the present study

Figure 1
Flowchart of patients for each experiment in the present study

* and **, among those patients, five of them were used for both IL-35 concentration detection and the pentamer experiment.

Figure 1
Flowchart of patients for each experiment in the present study

* and **, among those patients, five of them were used for both IL-35 concentration detection and the pentamer experiment.

Table 1
Baseline characteristics of subjects enrolled in the present study

Abbreviations: n.a., not applicable; Neg, negative.

Chronic HBV patients (n=82)Resolved HBV individuals (n=21)Healthy volunteers (n=42)
Male, n (%) 64 (78.0%) 13 (61.9%) 28 (66.7%) 
Age (years)* 32.6±0.7 40.3±2.7 32.7±1.6 
Serum ALT (units/l)* 292.6±34.5 24.1±1.7 19.3±1.5 
HBV DNA (log10 copies/ml)* 6.6±1.7 Neg Neg 
HBeAg-positive 61 (74.4%) n.a. 
Chronic HBV patients (n=82)Resolved HBV individuals (n=21)Healthy volunteers (n=42)
Male, n (%) 64 (78.0%) 13 (61.9%) 28 (66.7%) 
Age (years)* 32.6±0.7 40.3±2.7 32.7±1.6 
Serum ALT (units/l)* 292.6±34.5 24.1±1.7 19.3±1.5 
HBV DNA (log10 copies/ml)* 6.6±1.7 Neg Neg 
HBeAg-positive 61 (74.4%) n.a. 

*mean ± S.E.M.

Preparation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were isolated by Hypaque–Ficoll density centrifugation (Amersham Pharmacia) and cultured in 1640 medium (Invitrogen Life Technologies) supplemented with 10% heat-inactivated fetal calf serum (GIBCO), 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 10 mM HEPES, adjusted to a density of approximately 1×106/ml and cultured at 37°C with 5% CO2.

Isolation and stimulation of primary CD4+ T-cells

CD4+ T-cells were enriched from PBMCs by positive selection using magnetic-activated cell-sorting columns (Miltenyi Biotec), adjusted to a cell density of ~1×106/ml and stimulated for 6 days with CD3/28-coated Dynabeads® (Invitrogen; beads–cells=3:1) and IL-2 (40 units/ml). Cells were then collected for flow cytometry and real-time PCR analysis. For IL-35 ELISA and flow cytometry experiments, the cell stimulation cocktails (1:500, eBioscience) were added in the last 5 h. The CD4+ T-cell-depleted PBMCs (CD4-cells) were cryopreserved and used for enzyme-linked immunosorbent spot (ELISPOT) tests.

Flow cytometric analysis

To detect IL-35, CD4+ T-cells were incubated with anti-CD4-FITC (BD Pharmingen) and anti-CD25-PerCP-cy5.5 (BD Pharmingen) at room temperature. After fixation and permeabilization, cells were incubated with allophycocyanin (APC)-conjugated Ebi3 antibody (R&D Systems) and phycoerythrin (PE)-conjugated IL-12A (R&D Systems) or an IgG1 control (eBioscience) at room temperature. Cells were then immediately analysed using a FACScan system (Becton Dickinson).

IL-35 detection by ELISA

CD4+ T-cells (stimulated for 6 days) were collected and lysed by lysis buffer (CST, Cell Signaling Technology). Then the cells were centrifuged and the supernatants were used for IL-35 detection by human IL-35 ELISA kit (Biolegend). All procedures were in accordance with the manufacturer's instructions.

IL-35 treatment in vitro

Purified CD4+ T-cells were stimulated for 24 h at 37°C with HBV core antigen (1 μg/ml; Meridian, BioDesign)+PBS (control; GIBCO), HBV core antigen+IL-35 (Enzo Life Sciences International;10 ng/ml), HBV core antigen+IL-35 (40 ng/ml), HBV core antigen+IL-35 (100 ng/ml), CD3/28-coated beads (beads–cells=1:1)+PBS (control), CD3/28+IL-35 (10 ng/ml), CD3/28+IL-35 (40 ng/ml) or CD3/28+IL-35 (100 ng/ml). Subsequently, the cell culture supernatants were collected and stored at −80°C for further analysis using ELISAs. Additionally, cells were collected for flow cytometry. PBMCs were also stimulated for 24 h with HBV core antigen (1 μg/ml)+PBS, HBV core antigen+IL-35 (100 ng/ml) and subsequently collected for flow cytometry to detect surface markers of DCs.

HBV core 18–27 pentamer staining

For this pentamer staining, patients were all human leucocyte antigen (HLA)-A*0201-positive. PBMCs were stimulated with HBV core antigen peptide 18–27 (10 μg/ml; ANASPEC) for 11 days at 37°C in the presence of 100 ng/ml IL-35 or PBS (control). HCV (hepatitis C virus) NS3 peptide 1073–1081 (10 μg/ml; Proimmune) was also used as control. The cells were then collected for pentamer staining by flow cytometry as previously described [16].

Preparation and stimulation of adherent cells

PBMCs were cultured in six-well plates at 37°C. Suspended cells were then removed and plates were washed with PBS. Remaining adherent cells (ACs) were stimulated with HBV core antigen (1 μg/ml) and either 100 ng/ml IL-35 or PBS at 37°C. Subsequently, the cell culture supernatants were collected and stored at −80°C for ELISA analysis.

Analysis of cytokine secretion with ELISA

Sandwich ELISA technology was used to measure the concentrations of human IL-10, IL-6 and IL-12p70 in CD4+ T-cell or AC culture supernatants. IL-6 and IL-10 Quantikine ELISA kits (R&D systems) and IL-12p70 Quantikine ELISA kit (Biolegend) were used according to manufacturer's instructions.

Determination of interferon-γ-producing cells by ELISPOT

CD4 cells (2×105) were cultured in 96-well culture plates coated with interferon-γ (IFN-γ) antibody (ELISPOT Plus, Mabtech). The cells were incubated with HBV core antigen (1 μg/ml)+PBS (control), HBV core antigen+IL-35 (10 ng/ml), HBV core antigen+IL-35 (40 ng/ml) or HBV core antigen+IL-35 (100 ng/ml) for 48 h at 37°C. The group stimulated by HCV core antigen (abcam, 1 μg/ml) with or without IL-35 (100 ng/ml) treatment was also performed. Cells without HBV core antigen stimulation were used as control. The remaining procedures were performed in accordance with Mabtech instructions. The number of specific IFN-γ-secreting cells was calculated by subtracting the value for the unstimulated control from the value for the stimulated sample.

RNA extraction and real-time PCR analysis

CD4+ T-cell RNA was isolated by TRIzol extraction followed by reverse transcription using the Toyobo cDNA kit (FSQ-101). The IL-35 (Ebi3 and IL-12A) mRNA levels were assessed by quantitative real-time PCR using POWER SYBR® Green PCR Master Mix (Applied Biosystems). β-Actin was used as the reference gene. The following Ebi3 primers were used: 5′-ATGGCTCCCTACGTGCTCAAT-3′ (sense) and 5′-CGCAGCTCCCTGACGCTTGTA-3′ (anti-sense). The following IL-12A primers were used: 5′-AATG-TTCCCATGCCTTCACCA-3′ (sense) and 5′-CTAAGGC-ACAGGGCCATCATA-3′ (anti-sense). The following β-actin primers were used: 5′-CACGAAACTACCTTCAACTCC-3′ (sense) and 5′-CATACTCCTGCTTGCTGATC-3′ (anti-sense). PCR conditions: 95°C for 10 min; 40 cycles of 95°C for 15 s, 60°C for 30 s and 72°C for 30 s.

Antibodies

The isotype controls were all purchased from eBioscience. FITC anti-human CD11c, PE/Cy7 anti-human CD86, PE anti-human CD25, APC anti-human CD45RA, APC anti-human CD45RO and APC anti-human HLA-DR were purchased from Biolegend. Pro5® Recombinant MHC Pentamer was purchased from ProImmune.

Statistical analysis

The data were analysed using GraphPad Prism and shown as the mean ± S.E.M. The Mann–Whitney U-test was used to compare the HBV group with the healthy control group. The Wilcoxon signed rank test was used to analyse the differences between the IL-35-untreated and IL-35-treated groups. Spearman's non-parametric correlation test was used for the correlation analysis. P<0.05 was considered significant.

RESULTS

Peripheral blood CD4+ T-cell expression of IL-35 after CD3/28-coated bead stimulation

CD4+ T-cells were stimulated with CD3/28-coated beads in vitro for 6 days. Then, a comparative analysis of IL-35 (both Ebi3 and IL-12A) was performed using all CD4+ T-cells and CD4+CD25+ Treg cells. The Ebi3 and IL-12A mRNA levels in the peripheral blood of CHB and healthy individuals were determined. The ratio of Ebi3 mRNA to actin mRNA was significantly higher in chronically infected patients than in healthy individuals (P=0.023; Figure 2A). No difference was found in IL-12A mRNA expression (P=0.78; Figure 2B). We also detected Ebi3 and IL-12A protein levels by flow cytometry and the results were in accordance with the mRNA levels. Higher expression levels of Ebi3 were detected in both total CD4+ T-cells and CD4+CD25+ Treg cells from individuals with CHB than in cells from healthy individuals (P=0.016 and P=0.004 respectively). However, no difference was detected in IL-12A protein expression level between the CHB group and the control group (P=0.107 and P=0.121 respectively; Figures 2C–2E). Similarly, IL-35 concentration was detected by ELISA, finding a significant increase in CD4+ T-cells from CHB patients, compared with healthy controls and resolved individuals (both P<0.001). No difference was found between resolved group and healthy group (P=0.121; Figure 2F). These results suggested that IL-35 is highly expressed in the CD4+ T-cells of CHB patients and returned to the normal levels when the HBV infection was controlled or the virus was cleared.

IL-35 levels in CD4+ T-cells in CHB and healthy individuals after CD3/28-bead stimulation

Figure 2
IL-35 levels in CD4+ T-cells in CHB and healthy individuals after CD3/28-bead stimulation

(A) Ebi3 mRNA and (B) IL-12A mRNA in CD4+ T-cells from CHB and healthy individuals. (C) Histogram of Ebi3 (left, black unfilled), IL-12A (right, black unfilled) and isotypes (grey filled) in CD4+ T-cells from CHB patients. (D) Mean fluorescence intensity (MFI) for Ebi3 protein in CD4+ T-cells and CD4+CD25+ Tregs from CHB and healthy individuals. (E) MFI of IL-12A protein in CD4+ T-cells and CD4+CD25+ Tregs in CHB compared with healthy individuals. (F) IL-35 concentrations in CD4+ T-cells from CHB patients, healthy volunteers and resolved individuals.

Figure 2
IL-35 levels in CD4+ T-cells in CHB and healthy individuals after CD3/28-bead stimulation

(A) Ebi3 mRNA and (B) IL-12A mRNA in CD4+ T-cells from CHB and healthy individuals. (C) Histogram of Ebi3 (left, black unfilled), IL-12A (right, black unfilled) and isotypes (grey filled) in CD4+ T-cells from CHB patients. (D) Mean fluorescence intensity (MFI) for Ebi3 protein in CD4+ T-cells and CD4+CD25+ Tregs from CHB and healthy individuals. (E) MFI of IL-12A protein in CD4+ T-cells and CD4+CD25+ Tregs in CHB compared with healthy individuals. (F) IL-35 concentrations in CD4+ T-cells from CHB patients, healthy volunteers and resolved individuals.

IL-35 suppresses the HBV antigen-specific CTL response in vitro

Our results showed that IL-35 is highly expressed in chronically infected HBV patients. To determine whether this cytokine is involved in suppression of the HBV antigen-specific immune response, PBMCs were treated with recombinant IL-35 in vitro. PBMCs from CHB patients were stimulated with HBV core antigen peptide 18–27 for 11 days. HBV antigen-specific CTLs were then analysed with pentamer staining. The proportion of HBV-specific CTLs among CD8+ T-cells was significantly lower in the IL-35-treated group than that in the IL-35-untreated group (P=0.003; Figures 3A–C). PBMCs from CHB patients were stimulated with HCV NS3 peptide for 11 days (same as HBV peptide18–27 stimulation) and the PENTAMER+CD8+ T-cells were negative (result not shown). It demonstrated that the Pentamer+CD8+ T-cells were specific for HBV peptide.

Effect of IL-35 on HBV core antigen-specific CTL responses

Figure 3
Effect of IL-35 on HBV core antigen-specific CTL responses

Representative plots of Pentc18-27+CD8+ T-cells in PBMCs after stimulation with HBV core peptide 18–27 (A) without IL-35 and (B) with IL-35. Graphs show events after gating of CD8+ T-cells. (C) Stimulation with 100 ng/ml recombinant IL-35 significantly decreases the proportion of HBV core antigen peptide 18-27+ CTLs. (D) Comparison of HBV-specific IFN-γ production after 48 h of incubation with antigens with or without IL-35.

Figure 3
Effect of IL-35 on HBV core antigen-specific CTL responses

Representative plots of Pentc18-27+CD8+ T-cells in PBMCs after stimulation with HBV core peptide 18–27 (A) without IL-35 and (B) with IL-35. Graphs show events after gating of CD8+ T-cells. (C) Stimulation with 100 ng/ml recombinant IL-35 significantly decreases the proportion of HBV core antigen peptide 18-27+ CTLs. (D) Comparison of HBV-specific IFN-γ production after 48 h of incubation with antigens with or without IL-35.

IL-35 inhibits HBV antigen-specific IFN-γ-producing CTLs in vitro

CD4 cells from chronically infected patients were stimulated with HBV core antigen for 48 h with or without IL-35 at increasing concentrations. Then, IFN-γ-producing cells were detected by ELISPOT. The cell counts were as follows: 237±28 [Antigen (Ag) only], 216±35 (Ag+10 ng/ml IL-35), 185±36 (Ag+40 ng/ml IL-35) and 170±36 (Ag+100 ng/ml IL-35). The number of IFN-γ-producing cells was slightly decreased by 10 ng/ml IL-35 (P=0.37). At higher concentrations however, IL-35 dramatically reduced the number of IFN-γ producing cells (40 ng/ml IL-35: P=0.01; 100 ng/ml IL-35: P=0.007). At higher concentrations of IL-35, fewer spots were detected. The reduction caused by increasing the IL-35 concentration was statistically significant (P=0.029 and P=0.004 compared with the 10 ng/ml IL-35 group; P=0.05 compared with the 40 ng/ml IL-35 group; Figure 3D). Similarly, the result of the ELISPOT experiment showed that IL-35 (100 ng/ml) treatment did not suppress HCV antigen-specific IFN-γ secretion significantly (result not shown).

IL-35 suppresses CD4+CD45RA+ naïve T-cells in vitro

CD4+ T-cells were cultured with an antigen-specific (HBV core antigen) or non-antigen-specific stimulus (CD3/28-coated beads) with or without different concentrations of IL-35. CD4+T-cell surface markers including CD45RA, CD45RO and CD25 were analysed. The proportions of CD45RA+ cells among CD4+ T-cells were 36.09±5.93% (Ag only), 32.75±6.24% (Ag+10 ng/ml IL-35) 33.50±5.41% (Ag+40 ng/ml IL-35) and 32.23±5.43% (Ag+100 ng/ml IL-35). IL-35 at 10 ng/ml was sufficient to significantly reduce the antigen-specific expression of CD45RA on CD4+ T-cells. Higher concentrations of IL-35 were associated with lower CD45RA expression levels (Figures 4A and 4B). We then analysed the proportions of CD25+CD45RA+ and CD25CD45RA+ cells among CD4+ T-cells and found that the proportion of CD4+CD25CD45RA+ naïve Teffs was significantly decreased by IL-35 at 40 ng/ml and 100 ng/ml (10 ng/ml, 40 ng/ml, 100 ng/ml IL-35 treatment compared with IL-35 untreated, P=0.063, P=0.031 and P=0.031 respectively). However, the change in the proportion of CD4+CD25+CD45RA+ naïve Tregs among CD4+ T-cells was not significant (Figure 4B). When the effect of IL-35 on the non-antigen-specific expression of CD45RA on CD4+ T-cells was analysed, no significant difference was found. In addition, no significant changes in CD25 or CD45RO expression were observed after IL-35 treatment (result not shown).

Effect of IL-35 on surface marker expression and cytokine secretion on CD4+ T-cells and ACs

Figure 4
Effect of IL-35 on surface marker expression and cytokine secretion on CD4+ T-cells and ACs

(A) Representative contour plots of CD45RA expression on CD4+ T-cells after HBV antigen stimulation with or without IL-35. (B) Comparison of CD45RA expression on total CD4+ T-cells, CD4+CD25+ Tregs and CD4+CD25 Teffs after incubation with antigens with or without IL-35. (C) IL-10 secretion from CD4+ T-cells after stimulation with CD3/28-coated beads (left) or HBV antigen (right) with or without IL-35. (D) The proportion of CD11c+ cells among PBMCs decreases significantly in response to IL-35. (E) IL-35 significantly inhibits IL-6 secretion from ACs.

Figure 4
Effect of IL-35 on surface marker expression and cytokine secretion on CD4+ T-cells and ACs

(A) Representative contour plots of CD45RA expression on CD4+ T-cells after HBV antigen stimulation with or without IL-35. (B) Comparison of CD45RA expression on total CD4+ T-cells, CD4+CD25+ Tregs and CD4+CD25 Teffs after incubation with antigens with or without IL-35. (C) IL-10 secretion from CD4+ T-cells after stimulation with CD3/28-coated beads (left) or HBV antigen (right) with or without IL-35. (D) The proportion of CD11c+ cells among PBMCs decreases significantly in response to IL-35. (E) IL-35 significantly inhibits IL-6 secretion from ACs.

IL-35 induces IL-10 secretion from CD4+ T-cells in vitro

We used ELISA to detect cytokine levels in the culture supernatant of CD4+ T-cells incubated with or without IL-35 in vitro. The IL-10 concentrations in the four groups stimulated by CD3/28-coated beads were as follows: 128.3±36.5 ng/l (CD3/28 only), 143.1±36.08 ng/l (CD3/28+10 ng/ml IL-35), 155.5±36.14 ng/l (CD3/28+40 ng/ml IL-35) and 160.8±32.75 ng/l (CD3/28+100 ng/ml IL-35). Low concentrations of IL-35 (10 ng/ml) were sufficient to increase non-antigen-specific IL-10 secretion. At higher concentrations of IL-35, more significant increases were observed (P=0.008, P=0.002 and P=0.002 compared with the IL-35-untreated group; P=0.049 and P=0.037 compared with the 10 ng/ml IL-35-treated group). The IL-10 levels in the four groups stimulated with HBV core antigen were as follows: 71.32±6.10 ng/l, 39.56±6.26 ng/l, 57.36±3.95 ng/l and 61.60±5.48 ng/l. IL-35 at 10 ng/ml slightly decreased antigen-specific IL-10 secretion when compared with the IL-35-untreated group (P=0.063). However, when IL-35 was added at 40 ng/ml or 100 ng/ml, the decrease in secretion was reversed (P=0.125 and P=0.063 compared with the 10 ng/ml IL-35-treated group; Figure 4C). This result indicates that IL-35 can promote non-antigen-specific IL-10 secretion from CD4+ T-cells in vitro. Other cytokines, including IL-6 and IL-12p70, were also analysed in the same manner. No change in IL-6 was found with CD3/28 stimulation or antigen stimulation and the level of IL-12p70 was too low to be analysed (result not shown).

IL-35 inhibits DC expansion in vitro

DC surface markers, including CD11c, CD80, CD86 and HLA-DR, were analysed. The proportion of CD11c+ cells among PBMCs stimulated with HBV core antigen in vitro was significantly decreased by IL-35 treatment (IL-35-untreated group: 10.10±3.65%; IL-35 treatment group: 8.96±3.40%; P=0.02; Figure 4D). The expression levels of CD86 on CD11c+ cells were slightly increased by IL-35 treatment, but this change was not statistically significant (P=0.078). No effect on HLA-DR due to IL-35 was observed (result not shown).

Decreased IL-6 secretion from ACs after IL-35 treatment in vitro

ELISAs were used to detect the levels of cytokines in the culture supernatant of ACs stimulated with HBV core antigen in the presence or absence of IL-35. The IL-6 concentrations were 375.9±101.9 ng/l (Ag alone) and 313.0±99.23 ng/l (Ag+100 ng/ml IL-35). This result shows that IL-35 is antigen specific and can significantly increase the release of IL-6 from ACs in peripheral blood in vitro (P=0.002; Figure 4E). The secretion of IL-12p70 by ACs stimulated with HBV core antigen was undetectable (result not shown).

Ebi3 mRNA expression in CD4+ T-cells positively correlates with the HBV DNA load

Ebi3 was highly expressed in CD4+ T-cells of CHB patients relative to the level of expression in the control group. We analysed the association between the Ebi3 mRNA expression in CD4+ T-cells from CHB patients and the patients’ serum alanine aminotransferase (ALT) and HBV DNA levels. A positive correlation was observed between Ebi3 mRNA expression and the serum HBV DNA level (r=0.405, P=0.027; Figure 5A). However, no association was found between the Ebi3 mRNA level and the serum ALT level (r=−0.055, P=0.401; Figure 5B).

Ebi3 mRNA levels in CD4+ T-cells from CHB patients are correlated with HBV DNA load

Figure 5
Ebi3 mRNA levels in CD4+ T-cells from CHB patients are correlated with HBV DNA load

Correlation of the Ebi3 mRNA levels in CD4+ T-cells with (A) the HBV DNA load and (B) the serum ALT level.

Figure 5
Ebi3 mRNA levels in CD4+ T-cells from CHB patients are correlated with HBV DNA load

Correlation of the Ebi3 mRNA levels in CD4+ T-cells with (A) the HBV DNA load and (B) the serum ALT level.

DISCUSSION

IL-35 is a new member of the IL-12 cytokine family and it is composed of an α chain (IL-12A) and a β chain (Ebi3) [12]. Although IL-35 is constitutively expressed by mouse Tregs it is controversial as to whether IL-35 is constitutively expressed by human Tregs [13,17]. IL-35 can convert Tconvs into a newly discovered T-cell subset, Treg-iTR35 cells, in mice and in humans [13]. IL-35 has also been shown to suppress the proliferation of CD4+CD25 effector cells and to inhibit the differentiation of T helper (Th)17 cells in vitro [18]. Liu et al. [14] found that IL-35 expression can be detected in CD4+ T-cells from the peripheral blood of CHB patients. The results of our study demonstrate that both Ebi3 protein and mRNA are highly expressed in the CD4+ T-cells of CHB patients relative to the levels in cells of healthy individuals. Although no significant difference was found in the expression of the α chain IL-12A, our flow cytometry results showed that IL-12A was expressed in almost all CD4+ T cells. Expression of IL-12A in combination with elevated expression of Ebi3 in CD4+ T cells suggests that the IL-35 heterodimer is highly expressed in CHB patients. It also indicated that IL-35 concentrations in CD4+ T-cells from peripheral blood increased in treatment-naive chronic active hepatitis B patients and returned to the normal level after the virus was cleared or infection was controlled.

When an individual is exposed to HBV, the strength of the immune response determines whether chronic infection will occur. The immune response, especially the adaptive immune response, has been reported to be impaired in CHB patients: the HBV-specific CTL response is low and CD4+CD25+Foxp3+ Tregs increase and promote infection tolerance [5,6,10,19].

To determine whether the high production of IL-35 by human peripheral blood CD4+ T-cells from CHB patients is related to immune tolerance in the context of chronic infection, we analysed functional changes of various cells, including CD8+ T-cells, CD4+ T-cells and ACs, in response to IL-35 treatment in vitro. Our results show that IL-35 suppresses the expansion of HBV core antigen peptide 18–27-specific pentamer+ CTLs and inhibits antigen-specific IFN-γ secretion by CTLs. It has been suggested that IL-35 plays an important role in the suppression of HBV antigen-specific CTL responses. This role is proposed to be independent of traditional CD4+CD25+Foxp3+ Tregs or other inhibitory cytokines, such as IL-10 and TGF-β. However, our results show that only a high concentration of IL-35 (≥ 40 ng/ml) can promote a suppressive effect and that high concentration of IL-35 didn't suppress HCV antigen-specific IFN-γ secretion significantly. To our knowledge, this study is the first to determine the effect of IL-35 on antigen-specific CTL responses.

Niedbala et al. [18] found that IL-35 can directly suppress the proliferation of mouse CD4+CD25 effector cells under more ‘physiological’ conditions; however, little or no effect on CD4+CD25+ Treg cells was found under these conditions. When we analysed the effect of IL-35 on CD4+ T-cells, we used two methods to stimulate CD4+ T-cells: CD3/28-coated beads (non-antigen specific) and HBV core antigen (antigen specific). The results show that IL-35 significantly decreases the antigen-specific expression of CD45RA in total CD4+ T-cells and CD4+CD25 Teffs. CD45RA expression on CD4+CD25+ Tregs was also slightly reduced by IL-35, but this difference was not statistically significant. IL-35 treatment in vitro is hypothesized to suppress the antigen-specific proliferation of naïve CD4+ T-cells, especially naïve CD4+CD25 Teffs. In addition, this suppression of antigen-specific proliferation can be achieved using low IL-35 concentrations (10 ng/ml). We did not observe any obvious changes in CD25 expression in CD4+ T-cells. One possible explanation for this result is that 24 h may not be a sufficient amount of time for stimulation.

Several studies have demonstrated that IL-35 can play a suppressive role without input from other inhibitory cytokines, such as IL-10 and TGF-β. In addition, researchers have found that IL-35 can elevate IL-10 secretion levels from CD4+ T-cells [12,13,17,20,21]. In our research, we found a significant increase in IL-10 secretion from CD4+ T-cells after IL-35 treatment in vitro; however, this elevation was not HBV antigen-specific. IL-35 may play two different inhibitory roles: (1) directly suppressing the immune response through IL-35/IL-35 receptor signals and (2) indirectly promoting the secretion of the inhibitory cytokine IL-10.

We investigated the effects of IL-35 on the innate immune response and analysed the expression of various surface markers, including CD11c, CD86 and HLA-DR. The proportion of CD11c+ cells among PBMCs was significantly decreased by IL-35, but the expression of CD86 on CD11c+ cells increased slightly with IL-35 stimulation. CD11c is a DC marker, whereas CD86 is an activation marker. IL-35 may not only decrease the number of DCs in peripheral blood but also simultaneously activate DCs.

In the present study, we observed an association between the serum HBV DNA level and increased Ebi3 expression in the CD4+ T-cells from the peripheral blood of CHB patients. This suggests IL-35 may be involved in immune suppression during HBV infection. The concrete mechanism of IL-35/IL-35 receptor warrants further study. Although our results showed IL-35 could suppress HBV specific CTL immune response, emerging evidence has suggested that IL-35 is also important in other diseases, such as autoimmune disease and cancer [18,22,23]. In addition, Langhans et al. [24] found that Treg-cells inhibit HCV reporter T-cells via secretion of IL-10 and IL-35 rather than cell-contact-dependent mechanisms. It is demonstrated that IL-35 also played an inhibitory role in HCV infection. So in our opinion, just like cytokine IL-10, IL-35 may play a suppressive role in various diseases, including the HBV infection.

CONCLUSIONS

Both mRNA and protein levels of Ebi3 are significantly higher in CD4+ T-cells from CHB patients than in cells from healthy individuals. In addition, elevated IL-35 is associated with the inhibition of the cellular immune response in CHB patients, which contributes to the development and progression of hepatitis B.

AUTHOR CONTRIBUTION

Yu Chen and Lanjuan Li substantial contributions to conception and design and involved in editing the manuscript. Xuefen Li and Li Tian drafted the manuscript, analysis and interpretation of data. Yuejiao Dong, Yiyin Wang and Qin Ni acquisition of data and followed-up the patients and clinical works. Yuejiao Dong, Qiaoyun Zhu, Wenzheng Han and Xia Liu carried out cell biology and molecular biology experiments and provided analytical tools. All authors participated in interpretation of the findings and final approval of the version to be published.

We would like to thank all patients for participating, Dr Kezhou Liu for expert patient care and Ms Bo Ye for helpfully taking peripheral blood.

FUNDING

This work was supported by the National Natural Science Foundation of China [grant number 81171565]; the Major national S&T Projects for infectious diseases [grant number 2012ZX10002005]; and the Zhejiang Provincial Natural Science Foundation of China [grant number LY14H030001].

Abbreviations

     
  • AC

    adherent cell

  •  
  • ALT

    alanine aminotransferase

  •  
  • APC

    allophycocyanin

  •  
  • CD

    cluster of differentiation

  •  
  • CHB

    chronic hepatitis B

  •  
  • CTL

    cytotoxic T lymphocyte

  •  
  • DC

    dendritic cell

  •  
  • Ebi3

    Epstein-Barr virus-induced gene 3

  •  
  • ELISPOT

    enzyme-linked immunosorbent spot

  •  
  • Foxp3

    forkhead box P3

  •  
  • HBV

    hepatitis B virus

  •  
  • HCV

    hepatitis C virus

  •  
  • HLA

    human leucocyte antigen

  •  
  • IFN-γ

    interferon-γ

  •  
  • IL

    interleukin

  •  
  • iTR35

    IL-35-induced Treg

  •  
  • PBMC

    peripheral blood mononuclear cell

  •  
  • PE

    phycoerythrin

  •  
  • Tconv

    conventional T cell

  •  
  • Teff

    effector T-cell

  •  
  • TGF-β

    transforming growth factor-β

  •  
  • Th

    T helper

  •  
  • Treg

    regulatory T-cell

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Author notes

1

These authors contributed equally to this article.