A total of 61 patients with chronic HBV infection, including 37 patients with CHB and 24 asymptomatic HBV carriers (ASC), were enrolled in this study. All patients were hospitalized or followed up in The Second Hospital Jilin University from March 2014 to June 2015. The diagnoses of CHB and ASC were made in accordance with diagnostic standard of Chinese Guideline of Prevention and Treatment for CHB (2010 Version). No patients received antiviral or immunomodulatory therapy before baseline sampling. Patients who were co-infected with HIV or other hepatitis viruses, or afflicted with immune disorder or end-stage liver diseases were excluded from the study. All CHB patients received entecavir (ETV) therapy (0.5 mg 1/daily) after baseline sampling, and blood samples were also collected 48 weeks post-therapy. For normal controls, 20 of healthy individuals with matched age and sex ratio were also enrolled. The baseline characteristics of all enrolled subjects were shown in Table 1. The study conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The protocol was approved by the Ethics Committee of The Second Hospital of Jilin University, and written informed consent was obtained from each participant.

Hepatitis B virus (HBV) DNA was quantified by a commercial real-time Polymerase Chain Reaction (PCR)-Fluorescence Quantitative Detection Kit for HBV DNA (DaAn Gene, Guangzhou, Guangdong Province, China) with the detection limit of 2 log10 IU/mL. Serum biochemical assessments were tested by Hitachi 7500 automatic analyzer (Hitachi, Tokyo, Japan).

Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Hypaque (Sigma-Aldrich, St Louis, MO, USA). CD4⁺CD25⁺CD127^dim/− Tregs were purified using CD4⁺CD25⁺CD127^dim/− regulatory T cell isolation kit II (Miltenyi, Bergisch Gladbach, Germany). CD8⁺ T cells were purified using human CD8⁺ T cell isolation kit (Miltenyi). The purity of enrich cells was more than 90% by flow cytometry determination.

HepG2.2.15 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/L, Tiangen, Beijing, China), and streptomycin (0.1 mg/mL). Moreover, G148 (final concentration, 6.5 mg/mL) was added to the culture medium to maintain HepG2.2.15 cells. Cells were incubated at 37°C under 5% CO₂ condition. CD8⁺ T cells purified from chronic HBV-infected patients who were positive for HLA-A2 were stimulated with recombinant human IL-35 (final concentration 1 ng/mL; Peprotech, Rocky Hill, NJ, USA) for 6 h. Cells were washed twice, and were co-cultured in direct contact and in parallel in indirect contact (effector and target cells were separated by a 0.4 μm membrane, which allowed the passage of soluble factors only) with target HepG2.2.15 cells (ratio of effector cells to target cells = 1: 5) in the presence of HBV core 18-27 epitope (HBc 18-27, sequence: FLPSDFFPSV, final concentration 10 μg/mL) for 48 h, as described previously (Phillips et al., 2010). The supernatants and target cells were harvested for further studies. CD4⁺CD25⁺CD127^dim/− Tregs were stimulated with recombinant human IL-35 (final concentration 1 ng/mL; Peprotech) for 6 h. Cells were washed twice, and were co-cultured in direct contact with autologus CD4⁺CD25⁻ cells at the ratio of 1: 4 in the presence of anti-CD3/anti-CD28 (final concentration, 1 μg/mL, eBioscience, San Diego, CA, USA) or recombinant HBV surface antigen (HBsAg, final concentration 10 μg/mL; AbD Serotec, Oxford, United Kingdom) for 48 h. The supernatants and cultured cells were harvested for further experiments.

Concentration of IL-35 was measured by commercial ELISA kits (CUSABIO, Wuhan, Hubei Province, China) according to instructions from the manufacturer.

The following cytokines levels in the cultured supernatants, including interferon (IFN)-γ, IL-1β, IL-10, IL-12p70, IL-6, IL-8, and tumor necrosis factor (TNF)-α, were tested by Human Proinflammation 7-Plex Base Kit (Meso Scale Discovery, Rockvillie, MD, USA) using SECTOR Imager (Meso Scale Discovery) following manufacturer's instructions.

Peripheral blood mononuclear cells (PBMCs) or HepG2.2.15 cells were trypsinized, and were resuspened in 500 μL of Annexin V binding buffer. 5 μL of Annexin V-FITC (Beyotime Biotech, Wuhan, Hubei Province, China) and 5 μL of propidium iodide (PI, Beyotime Biotech) were added for 10 min incubation at room temperature in the dark. Cell apoptosis was analyzed with FACS Calibur analyzer (BD Biosciences), and were analyzed using FlowJo software version 8.6.2 (Tree Star, Ashland, OR, USA).

Cellular proliferation was determined by Cell Counting Kit-8 (CCK-8, Beyotime Biotech) according to instructions from the manufacturer.

Western blot analysis was performed as previously described (Liu et al., 2017). Briefly, total proteins were loaded and separated on SDS-PAGE gels, and were electroblotted onto PVDF membrane. The membrane was soaked for 2 h in a blocking solution (PBS containing 5% non-fat milk and 0.05% Tween 20) and incubated overnight in the presence of rabbit polyclonal antibodies targeting phosphorylated signal transducers and activators of transcription 1 (STAT1, phospho Y701, ab30645), STAT1 (ab31369), or mouse monoclonal anti-GAPDH (Abcam, 1:2,000 dilution). Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibody IgG (Abcam, 1: 2,000 dilution) was added to the membrane and incubated for an additional 2 h. Antigen-antibody complexes were observed using enhanced chemiluminescence (Western Blotting Luminol Reagent).

The cytotoxicity of HepG2.2.15 cells was assessed by measuring the lactate dehydrogenase (LDH) expression in the supernatants at the end of each incubation period using LDH Cytotoxicity Assay Kit (Beyotime) according to the manufacturer's instructions.

Statistical significance was determined by SNK-q test or paired t-test using SPSS version 19.0 for Windows (SPSS, Chicago, IL, USA). Values of P < 0.05 were considered as significant differences.

We firstly investigated IL-35 expression in the serum from all enrolled subjects, including 20 of normal controls (NC), 37 of CHB, and 24 of ASC. Serum concentration of IL-35 was significantly elevated in CHB (37.33 ± 12.72 pg/mL) and ASC (33.65 ± 13.64 pg/mL) in comparison with NC (24.17 ± 4.99 pg/mL; P < 0.0001 and P = 0.0053; Figure 1A). However, there was no remarkable difference of IL-35 level between CHB and ASC (P = 0.287; Figure 1A). Moreover, IL-35 expression was positively correlated with HBV DNA level in patients with chronic HBV infections (r = 0.316, P = 0.013; Figure 1B). However, there was no significant correlation between IL-35 concentration and serum ALT levels (r = 0.162, P = 0.615). Moreover, serum IL-35 levels were also measured in CHB patients receiving 48-week ETV therapy. ETV treatment led to significant down-regulation of HBV DNA levels, only one patient did not reach virological response at 48 weeks of therapy. Inhibition of viral replication resulted in remarkable reduction in IL-35 concentration in CHB patients (19.88 ± 11.40 pg/mL; P < 0.0001; Figure 1C).

2 × 10⁵ of PBMCs isolated from 10 patients with CHB, which were randomly selected from the Figure 1A, were stimulated with HBsAg (10 μg/mL) in presence or absence of IL-35 (1 ng/mL) for 24 h. Cells and supernatants were harvested for further analyses. The results of CCK-8 assay showed that the growth of IL-35-treated PBMCs [cell counting, (7.43 ± 1.14) × 10⁵] were slower than those with HBsAg stimulation only [cell counting, (8.56 ± 1.12) × 10⁵; P = 0.015; Figure 2A]. The proinflammatory cytokine secretions in the cultured supernatants were also measured. The concentrations of IFN-γ, IL-1β, IL-6, and, IL-8, which were produced by PBMCs, were notably reduced in response to IL-35 stimulation (Table 2). In contrast, IL-10 level was remarkably elevated in HBsAg and IL-35 co-stimulated PBMCs in comparison with HBsAg stimulation only (Table 2). Reduction of proinflammatory cytokine secretion in HBsAg and IL-35 co-stimulated PBMCs was accompanied by the decreased phosphorylation of STAT1 in comparison of HBsAg stimulation only (Figure 2B). Furthermore, flow cytometry was also performed to assess the apoptotic cells. Representative Annexin V/PI stained PBMCs for apoptosis analysis were shown in Figure 2C. Annexin V⁺PI⁻ cells represented early stage apoptotic cells, while Annexin V⁺PI⁺ cells represented late stage apoptotic cells. IL-35 stimulation led to significant elevation in both early and late stage apoptotic PBMCs (P = 0.0064 and P = 0.038, respectively, Figures 2D,E).

5 × 10⁴ of HepG2.2.15 cells (seeded in five independent wells) were stimulated with HBsAg (10 μg/mL) in presence or absence of IL-35 (1 ng/mL) for 24 h. Cells and supernatants were harvested for further analyses. Cell proliferation did not change significantly in HBsAg-stimulated HepG2.2.15 cells in response to IL-35 treatment [(7.22 ± 2.62) × 10⁵ vs. (6.96 ± 2.93) × 10⁵; P = 0.576; Figure 3A]. Neither IL-10 nor IL-12p70 could be detected in the supernatants from HepG2.2.15 cells, and the production of other five cytokines also did not reveal significant differences in response to IL-35 treatment (Table 3). Phosphorylated STAT1 expression in HBsAg-stimulated HepG2.2.15 cells did not change significantly in the presence or absence of IL-35 (Figure 3B). Furthermore, there were no remarkable differences in percentage of apoptotic HepG2.2.15 cells between HBsAg and HBsAg+IL-35 stimulation (P > 0.05, Figures 3C,D).

A total of 2.5 × 10⁴ purified CD4⁺CD25⁺CD127^dim/− Tregs from 14 CHB patients, which were also randomly selected from Figure 1A, were stimulated with IL-35 for 6 h, and cells were washed twice with DMEM to remove recombinant IL-35. Stimulated CD4⁺CD25⁺CD127^dim/− Tregs were co-cultured with autologous CD4⁺CD25⁻ T cells at ratio of 1: 4 in either a non-specific (anti-CD3/CD28 stimulation) or HBV antigen specific (HBsAg stimulation) manner. Cells and medium supernatants were harvested after another 48 h of culture. It was mainly CD4⁺ T cells which produced the cytokines since the co-culture of CD4⁺CD25⁺CD127^dim/− Tregs and CD4⁺CD25⁻ T cells. There were no remarkable differences in the suppressive capacities of purified Tregs between anti-CD3/CD28 and HBsAg stimulation in the absence of IL-35 stimulation (P = 0.160, Figure 4A). IL-35 treatment notably increased the inhibitory activity of Tregs in both groups, which manifested as down-regulation of cellular proliferation (P = 0.0003 and P = 0.0006, respectively, Figure 4A). The enhancement effect represented similar potent in co-cultures between the two groups (P = 0.348, Figure 4A). Moreover, the levels of IL-35, IL-10, IFN-γ, and TNF-α were measured in the supernatants of co-cultured cells. IL-35 treatment enhanced the production of IL-35 and IL-10 in both non-specific and HBV antigen-specific cultures (Figures 4B,C). In contrast, IFN-γ and TNF-α secretions was reduced in response to IL-35 treatment in co-cultures subjected to either anti-CD3/CD28 or HBsAg stimulation (Figures 4D,E).

2 × 10⁵ of purified CD8⁺ T cells from HLA-A2 restricted CHB patients (n = 9) were stimulated with IL-35 for 6 h, and were co-cultured in direct contact or in indirect contact with 10⁶ of HepG2.2.15 cells in the presence of HBc 18-27 peptide. As controls, HepG2.2.15 cells were cultured alone, and CD8⁺ T cells were cultured with HBc 18-27 peptide stimulation only. The supernatants were harvested 48 h post co-culture for further analysis. IFN-γ and TNF-α productions in the supernatants were significantly increased in the direct and indirect coculture system (Figures 5A,B). As expected, IL-35 treatment down-regulated both cytokines secretion in the direct and indirect coculture system (P < 0.05, Figures 5A,B). Moreover, the cytotoxicity of target HepG2.2.15 cells were assessed after direct and indirect contact with CD8⁺ T cells, and percentage of cell death was measured by LDH release. A maximum of nearly 50% cell death was reach in direct contact coculture system, and IL-35 stimulation reduced percentage of cell death to approximate 30% (P = 0.034, Figure 5C). However, no cytotoxicity was observed in the indirect system with or without IL-35 stimulation, as the proportion of cell death was similar to cultured HepG2.2.15 cells (Figure 5C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.