SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference

Function exhaustion of specific cytotoxic CD8+ T cell in chronic virus infection partly results from the low levels of CD4 help, but the mechanisms by which CD4 help T cell required to control hepatitis B virus infection are not well understood. In this study, we investigated the role of interleukin-21-producing CD4+ T cell response in viral control of hepatitis B virus infection. HBcAg-specific interleukin-21-producing CD4+ T cells in blood were detected in patients with hepatitis B virus infection. Patients with acute hepatitis B had greater HBcAg-specific interleukin-21-producing CD4+ T cells in blood compared with chronic hepatitis B patients, and there was no statistical significance between immune active chronic hepatitis B patients and inactive healthy carrier patients for these cells, whereas frequencies of these cells negatively correlated with HBV DNA levels but positively correlated with HBc18-27-specific IFN-γ-producing CD8+ T cells. Moreover, interleukin-21 sustained HBc18-27-specific CD8+ T cells in vitro, and interleukin-21 production by HBcAg-specific IL-21-producing CD4+ T cells of acute hepatitis B patients enhanced IFN-γ and perforin expression by CD8+ T cells from chronic hepatitis B patients. Our results demonstrate that HBcAg-specific interleukin-21-producing CD4+ T cell responses might contribute to viral control by sustaining CD8+ T cell antiviral function.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference

The quantity and quality of adaptive antiviral immune response influences clinical outcome of infection by the non-cytopathic, hepatotropic hepatitis B virus (HBV) [1]. The multispecific and vigorous CD4+ T cell and CD8+ T cell reactivity was present in acute HBV-infected patients who succeed in clearing HBV infection. However, in chronic HBV infection, the immune responses are weak and oligoclonal. The HBV-specific cytotoxic CD8+ T cells, which are believed to play a crucial role in viral clearance, show exhausted antiviral function characterized by an inability to produce cytokines such as IFN-γ and TNF-α, low cytotoxic activities or low proliferation in response to cognate antigen [2]. Studies in other persistent virus infection have shown that exhaustion of specific cytotoxic CD8+ T cell response mainly result from the high levels of virus antigen and low levels of CD4 help T cell[3]. Indeed, virus-specific CD4+ T cell responses are required for the efficient development of effector-specific cytotoxic CD8 T cell and B cell antibody production particularly during chronic HBV infection [4, 5]. A recent study showed that early activation of CD4+ T cells correlates with an influx of HBV-specific CD8+ T cells into the liver in a chimpanzee model of acute HBV infection, and animals depleted of CD4+ T cells become persistently infected when inoculated with a dose of HBV [6, 7]. These data indicate that virus-specific CD4+ T cell subsets play a critical role in determining immune responses to the virus and disease outcome. However, the mechanisms by which CD4 help T cell required to control HBV infection are not well understood.

Recently, several studies in animal model of LCMV infection demonstrate that interleukin-21 (IL-21), a common γ-chain cytokine, is essential for sustained specific CD8+ T cell response and control of viraemia in persistent viral infection [8-10]. IL-21, mainly produced by CD4+ T cells such as Th17 cells and T follicular helper (TFH) cells, is critical for the generation of plasma cells, IgG isotype switching and antibody production and promotes the expansion of Ag-specific CD8+ T effector cells and enhances their cytolytic potential [11-13]. In human virus infection, HIV-1-specific IL-21+ CD4+ T cell responses are shown to be induced in viraemic HIV infection and likely contribute to viral control by affecting CD8+ T cell maintenance [14, 15].

Until now, the role of IL-21 in patients with HBV chronic infection is not well understood. Recently, Ma et al. reported [16] that high serum IL-21 levels after 12 weeks of antiviral therapy predicted HBeAg seroconversion in patients with chronic hepatitis B (CHB). Furthermore, they demonstrated that circulating CXCR5+ CD4+ T cells, by producing IL-21, may have a significant role in facilitating HBeAg seroconversion [17]. The results show that IL-21 has an important role in the control of HBV replication by promoting anti-HBe-secreting B cell proliferation and HBeAg-IgG secretion in CHB patients. However, the role of IL-21-producing CD4+ T cells in function of HBV-specific CD8+ T cells in CHB patients is not fully defined yet. In this study, we examined IL-21-producing CD4+ T cell response induced by purified HBcAg in PBMCs from patients with acute HBV infection or chronic HBV infection. Furthermore, we explored the role of HBcAg-induced IL-21-producing CD4+ T cells in function of CD8+ T cells and in HBV infection control.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference

Patients

Sixty-seven chronic hepatitis B (CHB, 33 are HLA-A2+) patients and 13 acute hepatitis B (AHB, 5 are HLA-A2+) patients attending a hepatitis clinic or admitted to hospitalization in our unit at xuzhou medical college hospital from March 2010 to August 2010 were recruited for study. CHB patients were divided into two groups: 30 patients confirmed to be inactive healthy carrier (IHC, 12 are HLA-A2+) with undetectable serum HBV DNA (<1000 copies/ml) and normal serum ALT levels (0–40 U/l) and 37 patients defined as immune active (IA, 21 are HLA-A2+) individuals with active HBV replication and significantly high levels of ALT. Patients with CHB or AHB were diagnosed according to the guidelines for hepatitis B diagnosis of the American Association for the Study of Liver Diseases (AASLD) [18]. Twenty age- and sex-matched healthy individuals (11 are HLA-A2+) were enrolled as controls. HLA-A2 typing was confirmed by flow cytometry. All patients were negative for HCV, HDV and HIV and had no histories of other liver diseases. No subject had received any antiviral or immunosuppressive medication within 6 months. Baseline clinical data of all these patients in this study are shown in Table 1. All subjects gave signed informed consent. The study was conducted in full compliance with the ethical principles of the Declaration of Helsinki and was consistent with Good Clinical Practice guidelines and applicable local regulatory requirements.

Table 1. Demographic and clinical characteristics of subjects
ParameterAHB (= 13)IA (= 37)IHC (= 30)
  1. AHB, acute hepatitis B; IA, immune active; IHC, inactive healthy carrier; ALT: alanine aminotransferase.

Age, years25.3 ± 5.931.7 ± 7.233.5 ± 6.7
Sex, M/F3/1011/269/21
HBsAg positive9 (69%)37 (100%)30 (100%)
HBsAb positive4 (31%)00
HBeAg positive4 (31%)37 (100%)0
HBeAb positive9 (69%)030 (100%)
HBcAb positive13 (100%)37 (100%)30 (100%)
HBV DNA level (log copies/ml)3.9 ± 2.26.1 ± 2.1Undetectable
Serum ALT level (U/l)792.6 ± 203.4183 ± 89.617.6 ± 6.8

PBMC preparation

Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized blood by Ficoll–Hypaque density gradient centrifugation according to the manufacture's recommendation, suspended and then cryopreserved in medium containing 90% foetal calf serum (FCS) and 10% dimethyl sulphoxide. A part of freshly isolated PBMCs were resuspended in RPMI 1640 supplemented with 10% heat-inactivated FCS, 100 U/ml of penicillin and 100 mg/ml of streptomycin.

Flow cytometric analysis

To determine antigen-specific IL-21-producing CD4+ T cells, fresh PBMCs at 1 × 106 cells per well were incubated with or without rHBcAg (10 μg/ml; Kitgen, Hangzhou, China) for 12 h in 10% FCS RPMI 1640 at 37 °C in humidified 5% CO2 atmosphere. Anti-CD28 and anti-CD49d Abs (each at 1 μg/ml) (Biolegend, San Diego, CA, USA) were added to the cultures for further 5 h. Brefeldin A (1 μg/ml; Sigma-Aldrich, St Louis, MO, USA) was added to the cultures in the last 4 h of the incubation period. After a wash with 2% FCS/PBS, cells were stained with PerCPcy5.5-conjugated anti-CD3, FITC-conjugated anti-CD4 (both from Biolegend, USA). The same isotype-matched antibodies were used as controls. Cells were then fixed and permeabilized using the Fix and Perm Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's procedure followed by staining the cells with PE-conjugated anti-IL-21 (Biolegend). Lastly cells were washed and resuspended with PBS and then acquired by flow cytometry (FACS Calibur Beckton/Dickinson USA), and data were analysed with CellQuest software. At least 2 × 105 events per run were acquired.

To determine the frequency of antigen-specific CD8+ T cells, fresh 1 × 106 PBMCs were stimulated with 10 μg/ml the HLA-A2-limited epitope peptide core 18-27(FLPSDFFPSV) (SBS Genetech Co. Ltd., Beijing, China) in the presence of IL-21 (Peprotech, Rocky Hill, NJ, USA) at 100 ng/ml or IL-2 at 50 U/ml or in medium alone and harvested at 5 days. HBcAg-specific CD8+ T cells were detected as previously reported [19]. Briefly, the harvested cells were incubated with HLA-A2-restricted epitope HBcAg 18-27 MHC/pentamer-PE (Proimmune LTD, Oxford, UK) at 4 °C in the dark for 20 min. Followed by discarding the supernatant and washing the cells, the resuspended cells were incubated with PerCPcy5.5-conjugated anti-CD3 and APC-conjugated anti-CD8 (Biolegend) at the dark for 20 min and were washed and then fixed using 1% paraformaldehyde. Gated on CD3+ T cells, the frequency of HBcAg 18-27 MHC-pentamer-PE/CD8-APC double-positive cells was analysed using FACSCalibur instrument (Becton Dickinson) and CellQuest software as described above.

ELISA for IL-21 concentrations

The cryopreserved PBMCs were thawed, washed and resuspended in RPMI 1640 and supplemented with 10% heat-inactivated FCS. The cell viability tested by 0.5% Trypan Blue, was always more than 95% and then used for assay. PBMCs at 1 × 106 cells per well in 200 μl complete medium were plated in a 96-well plate and stimulated with or without HBcAg (10 μg/ml) for 7 days. The cell-free supernatants were harvested and assessed for IL-21 by ELISA kit (Biolegend), according to the manufacturer's instructions.

ELISPOT

The thawed PBMCs were obtained from 31 HLA-A2-positive HBV-infected individuals. The frequencies of HBc 18-27-specific IFN-γ-producing CD8+ T cells were quantified by an ELISPOT assay using PBMC after 24-h period of stimulation with HBc 18-27 peptides according to the manufacturer's instructions (Dakewe Biotech Com., Shenzhen, China). Briefly, the 96-well plate was coated with 5 μg/ml mouse anti-human IFN-γ monoclonal antibody overnight at 4 °C, followed by six washes with sterile PBS, and freshly isolated PBMCs (2 × 105 cells) were added into the wells and incubated in 5% CO2 at 37 °C for 24 h in supplemented minimal essential medium with HBc 18-27 peptides (FLPSDFFPSV 10 μg/ml) or PMA/ionomycin (Alexis Biomol, San Diego, CA, USA) as a positive control. Cells in culture medium with HCV core 132–140 peptides (DLMGYIPLV) (SBS Genetech Co., Ltd.) were used as negative controls. Followed by removing the medium and cells and incubating with 200 μl deionized water on ice for 10 min, plates were washed ten times with PBS containing 0.05% Tween-20, and then, 100 μl biotinylated secondary anti-human IFN-γ monoclonal antibody was added into cells and incubated at 37 °C for 1 h. After washing, the plates were incubated with HRP-labelled streptavidin at 37 °C for 1 h. Plates were then washed again, and AEC solution (100 μl/well) was then added and incubated for 30 min at room temperature. The colour reaction was stopped by washing with distilled water. Plates were air-dried, and spots were counted with an automated ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH, USA). Each spot represented an IFN-γ-producing cell. The number of specific spot-forming cell (SFC) per 1 × 106 PBMC was determined as the mean number of spots in the presence of HBcAg 18-27 peptides minus the mean number of spots in the wells with medium only. ELISPOT response was defined as positive when the ratio of SFC with versus without antigen was higher than 2.5.

Transwell co-incubation assay

The fresh PBMCs from AHB patients were CD8+ T cell-deleted by magnetic cell sorting (MACS) (CD8+ T cell isolation kits, Miltenyi Biotec). At the same time, CD8+ T cells and CD4+ T cells were deleted from partial PBMCs by MACS (CD4+ T cell and CD8+ T cell isolation kits, Miltenyi Biotec). The CD8 T cell-deleted PBMCs or CD4-CD8 T cell-deleted PBMCs were rested or stimulated with rHBcAg (2 μg/ml; Kitgen) for 5 h at 37 °C. After washed twice with PBS, 1 × 106 cells were plated in the bottom chambers of transwell plates. CD8+ T cells from PBMCs of IA patients were isolated using microbeads according to the manufacturer's instructions (Miltenyi Biotech). 3 × 105 CD8+ T cells were placed in the upper chambers. Unpulsed CD8 T cell-depleted PBMCs in the bottom chamber with isolated CD8+ T cells in the upper chamber served as a negative control. Cells were cocultured with medium alone or anti-IL-21 neutralizing antibodies (10 μg/ml, ReliaTech, Germany, CA 102-P236) or IL-21 (10 ng/ml; Peprotech) for 12 h at 37 °C, 5% CO2. The CD8+ T cells were collected for the detection of perforin and IFN-γ expression.

RNA extraction and real-time reverse-transcriptase polymerase chain reaction (RT-PCR)

Total RNA was extracted from harvested CD8+ T cells using TRIzol (Invitrogen) according to the manufacturer's instructions, followed by reverse transcription using oligo (dT) primers at 42 °C for 30 min and at 95 °C for 5 min. The cDNA was used as a template for real-time PCR amplification. The real-time PCR was performed using the following conditions: 95 °C for 3 min, and 95 °C for 30 s, 60 °C 30 s, 72 °C 1 min for 40 cycles and then 72 °C 10 min. The expression level of GAPDH mRNA was measured as an internal control, and relative expression was determined using the △△Ct calculation method. Relative perforin or IFN-γ expression between control and experimental groups was calculated using the 2−△△Ct formula. The primer sequences were as follows: perforin (forward) 5′-CATGTAACCAGGGCCAAAGTC-3′ and (reverse) 5′-ATGAAGTGGGTGCCGTAGTTG-3′; IFN-γ (forward) 5′ CTAATTATTCGGTAACTGACTTGA-3′ and (reverse) 5′ ACAGTTCAGCCATCACTTGGA. Human GAPDH was amplified as an internal control using the forward primer (5′-ACCCACTCCTCCACCTTTGA-3′) and the reverse primer (5′-TGGTGGTCCAGGGGTCTTAC-3′). Real-time PCR was performed on an ABI 7500 Real-Time PCR System using the SYBR Green qPCR SuperMix UDG Kit (Invitrogen).

HBsAg, HBeAg and HBV DNA detection

Serum HBsAg, HBsAb, HBeAg, anti-HBe and HBcAb were determined quantitatively using an electrochemiluminescence immunoassay (ECLIA) on the Roche Elecsys 2010 immunoassay analyser (Roche, Basel, Switzerland). Serum levels of HBV DNA were quantified with a high-sensitivity fluorescent real-time polymerase chain reaction kit (DaAn Gene Co., Guangzhou, China) and amplified in a PE5700 fluorescence PCR apparatus (Perkin-Elmer, Boston, MA, USA). The results were expressed as HBV DNA copies per millilitre of serum, and the detection sensitivity of the PCR assay was 1 × 103 copies/ml.

Statistical methods

Data were expressed as mean ± standard deviation. The Mann–Whitney U-test was used to perform nonparametric statistical analysis between two independent groups of patients with the SPSS 13.0 for Windows (SPSS, Chicago, IL, USA). Spearman's correlation or linear regression was used for correlation analysis. A P-value of <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference

Induction of HBV-specific IL-21-producing CD4+ T cells in PBMCs from HBV-infected patients

Because HBcAg of HBV is known to have strong immunogenicity for eliciting antigen-specific CD4+ T cell and humoral response, we stimulated PBMCs of HBV-infected patients with rHBcAg and examined for antigen-specific IL-21-producing CD4+ T cells by intracellular cytokine flow cytometry. As shown in Fig. 1A, although HBcAg-specific IL-21+ CD4+ T cells were undetectable in healthy controls, HBcAg-specific IL-21-producing CD4+ T cells can be detected in HBV-infected individuals. The frequencies of HBcAg-specific IL-21-producing CD4+ T cells in AHB patients were significantly higher than that in patients with chronic HBV infection, regardless of disease stage. Interestingly, there was no statistical significance between IA group and IHC group for the frequencies of HBcAg-specific IL-21-producing CD4+ T cells (Fig. 1B), although the frequencies of HBcAg-specific IL-21-producing CD4+ T cells were slight higher in IA group than that in IHC group. The findings were also verified by IL-21 ELISA, in which PBMCs from 5 AHB patients produced greater production of IL-21 in response to HBcAg in culture, compared with that from 8 IHC patients or 14 IA patients (Fig. 2).

image

Figure 1. HBcAg-specific IL-21-producing CD4+ T cells are detected in peripheral blood of patients with HBV infection. (A) Representative flow cytometric analysis of intracellular staining for IL-21 production by HBV-specific CD4 T cells from a HC subject (top panel), an AHB patient, a IA patient, and a IHC patient, in which in vitro PBMCs were stimulated 12 h with HBcAg and then stained with anti-CD3/anti-CD4/anti-IL-21. The values in dotplots represent the percentage of HBcAg-specific IL-21-producing CD4+ T cells. (B) Summary data of IL-21-producing CD4 T cells in response to HBcAg. The χ-axis represents clinical group. The y-axis represents % CD4+ T cells producing IL-21. P-values are shown. IHC, inactive healthy carrier; IA, immune activated; AHB, acute hepatitis B.

Download figure to PowerPoint

image

Figure 2. IL-21 production of PBMCs stimulated with HBcAg. PBMCs from 5 AHB patients, 8 IHC and 14 IA CHB patients were cultured in the presence of HBcAg(10 ug/ml) in the complete medium, and supernatants were assessed for IL-21 by ELISA at 7 days. P-values are shown. IHC, inactive healthy carrier; IA, immune activated; AHB, acute hepatitis B.

Download figure to PowerPoint

HBV-specific IL-21+ CD4+ T cells were associated to HBV DNA levels in patients with IA CHB

Chronic hepatitis B patients at inactive stage had plasma virus <1000 copies/ml, and IA CHB patients often had higher viral load. In this study, we found there was a significant negative correlation between HBV DNA levels and IL-21-producing CD4+ T cell response to HBcAg in CHB patients at IA stage (R2 = 0.410, P = 0.001, Fig. 3A). In contrast, the frequency of IL-21-producing CD4+ T cells to HBcAg was not correlated with the levels of ALT (R2 = 0.023, = 0.474) as shown in Fig. 3B.

image

Figure 3. Correlation between IL-21-producing CD4+ T cells in response to HBcAg and HBV DNA levels, ALT levels or frequency of HBcAg-specific IFN-γ-producing CD8+ T cells in the peripheral blood of IA patients. IA, immune activated; R2, the correlative coefficient and P-values are shown.

Download figure to PowerPoint

HBV-specific IL-21+ CD4+ T cells correlate with HBc 18-27-specific IFN-γ-producing CD8+ T cells

Given the above association between IL-21 production by HBcAg-specific CD4+ T cells and HBV virus load in IA patients, we next evaluated whether HBV-specific IL-21+ CD4+ T cells might correlate with HBV-specific CD8+ T cell response. Following HLA-A2 genotype screening, we detected IFN-γ-producing CD8+ T cells of PBMCs stimulated with HBc 18-27 peptide for 24 h by ELISPOT in 14 IA CHB patients. The data showed that HBV-specific IL-21+ CD4+ T cells positively correlate with HBc 18-27-specific IFN-γ-producing CD8+ T cells in IA patients (Fig. 3C).

IL-21 sustains HBc 18-27-specific CD8+ T cells and improves antiviral function of CD8+ T cells

To determine whether IL-21 could affect the frequency of HBc 18-27-specific CD8+ T cells from CHB patients, we compared the frequency of HBc 18-27-specific CD8+ T cells in PBMCs with or without IL-21 stimulation. The data showed that ex vivo HBc 18-27-specific CD8+ T cells from CHB patients could be easily sustained and survived if cocultured with IL-21, and the frequency of HBc 18-27-specific CD8+ T cells was similar to that with IL-2 stimulation (Fig. 4A).

image

Figure 4. CD8+ T cell response to IL-21 stimulation. (A) PBMCs from 10 IA patients were stimulated with HBc 18-27 peptides in the presence of IL-21 or IL-2 or in medium alone for 5 days and then detected for frequency of HBc 18-27-specific CD8+ T cells by flow cytometry as described in Materials and Methods. (B) Following stimulated with HBcAg for 5 h, the CD8+ T cell-deleted or CD4+ CD8+ T cell-deleted PBMCs from 7 AHB patients were placed in the bottom chamber of a transwell plate and co-incultured for 12 h with the isolated CD8+ T cell from PBMCs of IA patient in the upper chamber. For blockage of IL-21 signal, the neutralizing anti-IL-21 was added to the CD8+ T cell in the upper chamber. After the incubation, CD8+ T cells were harvested and relative levels of interferon-γ (IFN-γ) mRNA or perforin mRNA were detected by qPCR. P-values are shown. IA, immune activated; AHB, acute hepatitis B.

Download figure to PowerPoint

Next, to determine whether IL-21 secretion by HBV-specific CD4+ T cells could directly improve the antiviral function of CD8+ T cells through IL-21 signal, we depleted CD8+ T cells of PBMCs from 7 AHB patients with strong IL-21 responses and then stimulate the CD8+ T cell-deleted PBMCs with HBcAg for 1 h. After complete removal of the remaining antigen, we added the HBcAg-stimulated CD8+ T cell-deleted PBMCs from each individual in the bottom chamber of a transwell plate. The isolated CD8+ T cell from PBMCs of IA patient was placed in the upper chamber. After co-incultured for 12 h, it was similar to additional rIL-21-induced IFN-γ mRNA and perforin mRNA expression of CD8+ T cells, which the HBcAg-pulsed CD8-deleted PBMCs of AHB patients induced markedly increased IFN-γ mRNA and perforin mRNA expression in the CD8+ T cells (Fig. 4B), although the levels of IFN-γ mRNA and perforin mRNA expression of CD8+ T cells were lower in HBcAg-pulsed CD8 deleted PBMCs than in CD4-CD8 T cell-deleted PBMCs plus rIL-21. And the effect can be significantly abrogated by the addition of a neutralizing antibody to IL-21(Fig. 4B).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference

Available data indicate that the induction of efficient antiviral CD8+ cytotoxic T lymphocyte (CTL) response for viral clearance depends on the early CD4+ T cell priming to HBV infection [1]. However, the mechanisms by which CD4 T help cells required to control HBV infection has yet to be elucidated. In this study, we investigated HBcAg-specific IL-21 producing CD4+ T cell responses in patients with HBV infection. We found a significantly higher frequency of HBcAg-specific IL-21+ CD4+ T cells in AHB patients than that in patients with chronic HBV infection, suggesting a role for IL-21 production of HBcAg-specific CD4+ T cells in inducing an effective immune response for viral clearance in patients with HBV infection. Because all of the patients with AHB enrolled in this study completely cleared the virus in the end, we have not yet been able to demonstrate a role for IL-21 in converting a self-limited HBV infection to chronic infection. In CHB patients, however, the frequency of HBcAg-specific IL-21+ CD4 T cells did not change significantly between IA patients and IHC individuals. This is different from recent findings where HBV-specific CD4+ T cells producing IL-21 were significantly higher in IHC versus HBeAg-positive IA CHB patients [16]. The cause of this difference may be related to patients' selection. Although IL-21 is induced only in the presence of large amounts of Ag [15], it is well known that there are lower circulating HBV-specific CD4+ T cells or CD8+ T cells in IA CHB patients with too high levels of serum HBV DNA (especially more than 10copies/ml), compared with relative low HBV DNA levels. This means that too high viral loads or viral antigen may sharply suppress HBV-specific CD4+ T cell response in CHB patients. The study by Ma et al. [16] was focused on CHB patients with median 8.5 log10 copies/ml levels of serum HBV DNA. However, the HBV DNA levels of IA CHB patients were moderate (6.1 log10 copies/ml) in our study. So, circulating HBV-specific CD4+ T cells producing IL-21 in our study may be relative high. This may explain the discrepancy of findings between the two studies.

Interestingly, we found a significantly negative correlation between HBV DNA levels and IL-21-producing CD4+ T cell response to HBcAg in IA CHB patients. The immune state between IHC and IA stage in patients with CHB is different. There is a kind of balance between antiviral response and low HBV replication in IHC CHB patients. However,it is fluctuant between antiviral response and HBV replication in IA CHB patients. HBV replication would be suppressed if the antiviral response was strong. Studies in murine models with human hepatitis B have shown that IL-21-producing CD4+ T cells are necessary for HBV antigen clearance [20]. Recently, Li et al. [17] reported that circulating CXCR5+ CD4+ T cells, by producing IL-21, may have a significant role in facilitating HBeAg seroconversion and viral control in patients with chronic HBV infection. Furthermore, IL-21 also counteracts regulatory T cell-mediated immune suppression [21]. So, high circulating HBV-specific IL-21+ CD4+ T cells in present study may contribute to the suppression of HBV replication in IA patients with CHB.

Previous studies have demonstrated that CD4+ T help cells probably contribute indirectly to the control of HBV infection by facilitating the induction and maintenance of the virus-specific B cell and CD8 T cell response [2]. We find in this study that the frequency of HBcAg-specific IL-21+ CD4+ T cells positively correlate with HBc 18-27-specific IFN-γ-producing CD8+ T cells, which were crucial for non-cytopathic inhibition of HBV replication in hepatocytes. In addition, we observed the effect of IL-21 on the frequency of HBc 18-27-specific CD8+ T cells in vitro by flow cytometry in IA CHB patients. These data suggest that IL-21 might maintain survival and function of HBV-specific CD8+ T cells, but also support their amplification in chronic HBV infection.

The HBV-specific CD8+ T cell responses play a crucial role in viral clearance through the production of antiviral cytokines such as IFN-γ and granzyme/perforin-mediated cytotoxicity [7]. To further investigate the effect of HBcAg-specific IL-21+ CD4+ T cell response on the function of CD8+ T cells, we next used transwells to coculture the HBcAg-stimulated CD8+ T cell-deleted PBMCs from AHB individual with isolated CD8+ T cell from PBMCs of IA patient. The mRNA expression of perforin and IFN-γ was significantly upregulated in the isolated CD8+ T cells placed in the upper chamber, and the upregulation can be counteracted in the presence of anti-IL-21 antibody. These data indicate that HBcAg-specific IL-21+ CD4+ T cell response could directly promote antiviral activity of CD8+ T cells through IL-21 signalling. Our findings were consistent with some previous reports, demonstrating that HIV-1-IL-21-producing CD4+ T cell response contribute to viral control by the modulation of CD8+ T cell function in patients with HIV infection [15].

A recent report by Hu et al. [22] demonstrated that frequency of IL-21-secreting CD4+ T cells increased in both hepatitis B-related acute-on-chronic liver failure and severe chronic hepatitis B and was associated with the disease severity. However, in the present study, we could not find the relationship between frequencies of HBcAg-specific IL-21-secreting CD4+ T cells and liver damage in IA CHB patients. The possible explanation is that IL-21 might be produced by active different CD4+ T cell subsets and NKT cells [23]. In addition to T follicular help (TFH) cells, interleukin-17-producing CD4+ T cells (Th17) also secrete IL-21 [24, 25]. The highly increased frequency of Th17 cells in PBMCs has been observed in CHB patients with severe liver damage [25, 26]. So the increased IL-21-secreting CD4 T cells might not be mainly derived from TFH cells, but also from Th17 cells in hepatitis B-related acute-on-chronic liver failure and severe chronic hepatitis B as Hu's report demonstrating IL-21 mainly derived from IL-17A+ IL-21+ CD4+ T cells [22]. In addition, the frequency of HBV-specific IL-21-secreting CD4+ T cells did not be detected in the Hu's study, which could not directly be involved in liver damage in HBV infection.

In summary, the study presented here demonstrates that HBc-specific IL-21-producing CD4+ T cell response is decreased in patients with CHB than AHB. These data support the hypothesis that decreased IL-21 secreted from HBV-specific CD4+ T cells partly contributes to the exhaustion of specific cytotoxic CD8+ T cell response in chronic HBV infection. These findings provide clues for rational design of new therapeutic strategy against chronic HBV infection.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference

This work was supported by the National Grand Program on Key Infectious Disease of China (Grant no. 2012ZX10002007) and Specialized Research Fund for the Doctoral Program Construction of Higher Education in China (No 53410903).

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Reference

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. Reference