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J Immunol. Author manuscript; available in PMC 2013 April 15.
Published in final edited form as:
PMCID: PMC3480715
EMSID: UKMS49765

IL-10 producing regulatory B cells in the pathogenesis of chronic HBV infection1,2

Abstract

A regulatory subset of B cells has been found to modulate immune responses in autoimmunity, infection and cancer but has not been investigated in the setting of human persistent viral infection. IL-10 is elevated in patients with chronic hepatitis B virus infection (CHB)3 but its cellular sources and impact on antiviral T cells have not been addressed. We investigated the role of IL-10 and regulatory B cells in the pathogenesis of CHB. Serum IL-10 levels were studied longitudinally in patients with CHB undergoing spontaneous disease flares. There was a close temporal correlation between IL-10 levels and fluctuations in viral load or liver inflammation. Blockade of IL-10 in vitro rescued polyfunctional virus-specific CD8 T cell responses. To investigate the potential contribution of regulatory B cells, their frequency was measured directly ex vivo and after exposure to stimuli relevant to HBV (CpG or HBV antigens). IL-10-producing B cells were enriched in patients, and their frequency correlated temporally with hepatic flares, both after stimulation and directly ex vivo. Phenotypically, these cells were predominantly immature (CD19+CD24hiCD38hi) ex vivo; sorted CD19+CD24hiCD38hi cells suppressed HBV-specific CD8 T cell responses in an IL-10-dependent manner. In summary, these data reveal a novel IL-10-producing subset of B cells able to regulate T cell immunity in CHB.

Introduction

B cells have been identified as potent regulators of T cell immune responses in studies of autoimmunity, infection and cancer (1-7). IL-10 is the primary mechanism by which B cells modulate other immune cells, however additional contributory mechanisms include engagement of co-stimulatory molecules (CD80/CD86), antigen presentation and induction of regulatory T cell populations (8). These ‘regulatory B cells’ are pathogenic in parasitic infections, subverting CD4 T cells towards a Th2 phenotype favorable to pathogen survival, and can reduce antigen-specific CD8 T cell responses through production of IL-10 in murine viral infection (9, 10). In contrast, IL-10 producing B cells are protective in the setting of autoimmune disease, chronic intestinal inflammation and allograft survival (2, 3, 11). For instance, adoptive transfer of these cells can ameliorate established disease in murine models of collagen induced arthritis and experimental autoimmune encephalomyelitis; absence of these cells is associated with increased Th1 and Th17 responses, reduced numbers of FoxP3+ regulatory T cells and exacerbated disease (12). B cell function is therefore pleiotropic, and not confined to antibody production.

In humans it has recently been demonstrated that analogous regulatory B cell subsets (Bregs) can suppress CD4 T cell proliferation and IFNγ and TNFα production by CD4+ T cells and regulate TNFα release by monocytes (13-15). The suppressive effects are mediated by IL-10 and are TGFβ independent. Phenotypically, B cells producing IL-10 have been found to be enriched within CD19+CD24hiCD38hi transitional B cells (13), CD19+CD24hiCD27+ B10 cells (15) or both CD27+ memory and CD38hi transitional B cell subsets together (14); whether these phenotypic distinctions are partially dependent on the mode of stimulation remains to be determined. Dysfunction within these cells has also been described; B cells from patients with systemic lupus erythematosus or multiple sclerosis have impaired capacity to produce IL-10; these cells are refractory to CD40 stimulation in the former disease and recover their IL-10 production upon treatment in multiple sclerosis (13, 16).

A role for regulatory B cells in human viral infection has not yet been reported. To investigate whether IL-10 and regulatory B cells contribute to the pathogenesis of chronic hepatitis B virus infection (CHB), we studied a cohort of patients undergoing spontaneous ‘flares of liver disease’. These rapid fluctuations in disease activity provide a unique opportunity to study the relationship between immune and clinical parameters of viral control and liver damage. Longitudinal analysis in these patients revealed that serum IL-10 levels and the frequency of IL-10 producing B cells were enriched in temporal correlation with the peak of viral load or liver inflammation. Ex vivo phenotypic characterization of IL-10 producing B cells revealed that these cells were predominantly contained within the immature B cell subset. Depletion of this B cell subset resulted in an expansion of functional HBV-specific CD8+ T cells in vitro. These data provide the first demonstration of the capacity of B regs to regulate antigen-specific CD8+ T cells in humans and implicate these cells in HBV pathogenesis.

Materials and methods

Patients and controls

Written informed consent was obtained from all patients. The study was approved by the local ethical committees (Royal Free Hospital, Royal London Hospital, Camden Primary Care Ethics Review Board). All patients recruited were negative for HIV and HCV antibodies. A total of 57 patients and 22 healthy donors took part in the study. All patients were untreated. Details of patient demographics are shown in table I. Serum samples were collected at multiple timepoints over the course of spontaneous flares of liver disease from 10 patients with HBV e antigen (eAg) negative CHB and five patients with eAg positive CHB. Viral load was measured either by an in house real-time polymerase chain reaction based assay or by the Bayer Versant HBV DNA 1.0 Assay. Liver biopsies (surplus to diagnostic requirements) were obtained from eight patients with CHB; paired peripheral blood samples were available from five of these patients (table II).

Table I
Patient characteristics1
Table II
Patient characteristics for liver samples

Quantification of IL-10 in serum and supernatant

Serum IL-10 was quantified using the Cytometric Bead Array (CBA) inflammation kit (BD biosciences) according to manufacturer’s instructions.

To detect IL-10 in supernatant, PBMC from patients and controls were stimulated with phorbol myristate acetate (PMA; 3ng/ml) and ionomycin (100ng/ml) (Sigma) for five hours, after which supernatant was collected and stored at −80°C. IL-10 in batch collected supernatant samples was then quantified with the Human IL-10 Elisa Kit (Diaclone) as per manufacturer’s instructions. The range of detection for this kit was between 1.56pg/ml and 50pg/ml. Alternatively, PBMC from healthy donors were stimulated with HBV core (HBcAg) (Rhein Biotech GmbH), surface (HBsAg) (Rhein Biotech GmbH) or e antigens (HBeAg) (Siemens Healthcare Diagnostics Products GmbH) for 96 hours, following which IL-10 was measured in supernatant.

Isolation of PBMC and intrahepatic lymphocytes

PBMCs were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation and frozen or immediately used. Intrahepatic lymphocytes were isolated as described previously (17).

IL-10 production by intracellular cytokine staining

PBMC were incubated with 1μM CpG-B ODN2006 (InVivoGen) for 96 hours at 37°C. PMA (3ng/mL) and ionomycin (100ng/mL) were added in the last four hours in the presence of 10μg/mL brefeldin A (Sigma). Alternatively, PBMC were stimulated with HBcAg (10μg/ml) for 16 hours (kindly provided by Rhein Biotech GmbH, Dusseldorf, Germany). Cells were then surface stained for markers CD19 PerCP (BD), CD24 PE and CD38 FITC, fixed and permeabilised and intracellularly stained with anti-IL-10 APC (BD Pharmingen). PBMC were acquired on a FACS calibur flow cytometer (Becton Dickinson). Flowjo software (Treestar) was used for analysis. For ex vivo analysis of IL-10 production, unstimulated PBMC were stained by the same protocol as above.

Fluorescence activated cell sorting of immature B cells

PBMC were isolated as described above and surface stained with anti-CD38 FITC (BD Pharmingen), anti-CD19 Pe-Cy7 (eBioscience) and anti-CD24 PE (BD Pharmingen). Immature B cells were depleted from PBMC on the basis of high expression of CD24 and CD38 by FACSAria (Becton Dickinson). Additionally, PBMC which had been stained with the same antibodies were passed through the machine untouched as a control.

Detection of HBV-specific CD8 T cell responses with IL-10/IL-10 Receptor blockade

PBMC were seeded in duplicate into a 96 well plate (0.25×106/well) in the presence of 1μM viral peptide and 50 U/mL IL-2, with or without anti-IL-10 (eBioscience) 5μg/mL and anti-IL-10 Receptor (BD Pharmingen) 10μg/ml. HLA-A2+ patients were stimulated with a panel of peptides representing immunodominant HLA-A2 restricted epitopes from HBV (envelope: FLLTRILTI, WLSLLVPFV, LLVPFVQWFV, GLSPTVWLSV; core: FLPSDFFPSV, and polymerase: GLSRYVARL, KLHLYSHPI) or CMV (pp65: NLVPMVATV) (Proimmune). HLA-A2-patients were stimulated with overlapping peptides (pool of 15mer peptides overlapping by 10 residues) spanning core of HBV genotype D or the pp65 protein of CMV. Medium was refreshed on day 4 with further addition of exogenous IL-2 (50U/mL), anti IL-10 (2.5μg/mL) and anti IL-10 receptor (5μg/mL). On day 10, PBMC were pulsed for a further 5 hours with 1μM peptide in the presence of Brefeldin A (10μg/ml) and then stained with anti-CD8 APC, anti-CD3 PerCPCy5.5 (BD biosciences) and intracellularly stained with anti-IFNγ FITC (R&D Systems). The frequency of IFN-γ positive CD3+CD8+ T cells represented the virus-specific CD8 T cell population.

Determination of CD8 T cell polyfunctionality

In six chronic individuals, polyfunctionality of virus-specific CD8+ T cell responses was analysed. PBMC were stimulated with viral peptides, and production of IFNγ, IL-2, TNFα, proliferation (CFSE-carboxyfluorescein diacetate, succinimidyl ester) and degranulation (CD107a) was determined after 10 days in vitro culture. The following panel of antibodies was used; anti-CD3 ECD (IOTest), anti-CD107a PE (BD Pharmingen), anti-IFN-γ V450 (BD), anti-TNFα APC (BD Biosciences), CFSE FITC (Serotec), LIVE/DEAD Near-IR Fluorescent Reactive Dye (Invitrogen). The above described protocol for detection of virus-specific CD8 T cell responses was used with the exception that PBMC were additionally stained with CFSE dye on day 0 and anti-CD107a antibody and monensin were added in addition to Brefeldin A upon restimulation with peptide on day 10. After gating on live CD3+ CD8+ T cells, the percentage frequencies of the 16 different combinations of IFNγ, TNFα, CFSE and CD107 responses were determined. Boolean gate arrays created in Flowjo were exported to PESTLE (version 1.7) for background subtraction (from medium alone samples) and graphical representations of polyfunctional CD8 T cell responses were generated using SPICE (Simplified Presentation of Incredibly Complex Evaluations, Version 5.1); software obtained from M. Roederer (National Institutes of Health, Bethesda, MD) (18).

Detection of HBV-specific CD8 T cell responses after depletion of immature B cells

PBMC or PBMC depleted of CD19+CD24hiCD38hi B cells isolated by FACSAria were stimulated with HBV peptides in the presence of IL-2 as described above. The frequency of virus-specific CD3+CD8+IFNγ+ T cells was determined on day 10.

Functional B cell assays

Sorted immature (CD19+CD24hiCD38hi) B cells (0.6×105) were stimulated with PMA/ionomycin for 2 hours, washed in RPMI and then co-cultured at a 1:4 ratio with PBMC (2.5×105) derived from the same HLA-A2+ patients in the presence of viral peptide and IL-2 as described above. The frequency of virus-specific CD8 T cells was determined at day 10 and compared with PBMC, which had been stimulated with HBV or CMV peptides in the absence of immature B cells.

Statistical analysis

Statistical significance was calculated by the non-parametric Mann-Whitney U test. A p value of <0.05 was deemed significant. All paired samples were analysed by the Wilcoxon matched pairs test.

Results

Temporal correlation between serum IL-10 levels and flares of liver disease

To determine the role for IL-10 in chronic HBV infection, serum levels were measured longitudinally in fifteen patients sampled repeatedly during disease fluctuations. Ten patients had eAg-negative CHB characterized by recurrent spontaneous hepatic flares; for comparison, we studied five patients with eAg-positive CHB. In all eAg-negative patients undergoing hepatic flares, IL-10 levels showed dynamic changes, with a close temporal correlation with disease activity. IL-10 peaked in tandem with the increase in viral load or liver inflammation or both, and decreased at the same time or shortly after the resolution of liver inflammation (Fig. 1). For comparison, we examined five patients with eAg positive disease in whom there was persistently elevated HBV DNA (>106 log at all timepoints). In 4 out of 5 patients with eAg+ CHB, IL-10 levels were raised above the low levels seen in healthy donors and in eAg-negative CHB between flares (Fig. 1 and (19)). However, unlike the close temporal correlation between IL-10 and recurrent hepatic flares in eAg-negative disease, it was not possible to detect any correlation between IL-10 levels and disease activity in these eAg+ patients.

Figure 1
Temporal correlation between serum IL-10 and chronic flares of liver disease. IL-10 levels were measured in serum samples collected before, during and after chronic flares of liver disease in ten eAg negative and five eAg positive patients with chronic ...

Role for IL-10 in suppression of HBV-specific CD8 T cell responses

We next investigated a potential role for IL-10 in modulation of the anti-viral immune response. HBV-specific CD8 T cell responses were quantified following in vitro stimulation with a pool of HBV peptides with or without blockade of IL-10 and its receptor (Fig. 2A). There was little response above background in patients with high viral load; this is in keeping with previous data that have characterized profound qualitative and quantitative defects in the HBV-specific CD8 T cell response in high level carriers (20, 21). Eight out of fifteen chronic HBV infected patients showed recovery of HBV-specific responses upon IL-10 blockade (Fig. 2B). In five individuals, blockade of IL-10 revealed new HBV-specific CD8 T cell responses which were previously undetectable. There was no relationship between response to blockade and viral load, liver inflammation or eAg status in this small group of individuals studied. To determine whether IL-10 blockade could recover additional T cell effector functions, we investigated the polyfunctionality of CD8 T cell responses (defined as ability to produce IFNγ, IL-2 or TNFα, proliferate (CFSE) or degranulate (CD107)) after stimulation with HBV or CMV peptides in 6 patients. Contour plots depict representative effector responses of HBV-specific CD8 T cells after IL-10 blockade (Fig. 2C). No IL-2 production above background was detected in any of the patients studied. As shown in Fig. 2D, we observed recovery of cytolytic potential in three out of six patients with CHB. Analysis of CD8 T cell polyfunctionality was perfomed utilising software (SPICE (Simplified Presentation of Incredibly Complex Evalulations), obtained from M. Roederer NIH), grouping responding CD8 T cells by their ability to produce either one, two, or three or more effector functions. By this method, the contributions of each individual response profile to the total virus-specific CD8 T cell response before and after IL-10 blockade was analysed (Fig. 2E). From this analysis, we observed that in five out of six patients, IL-10 blockade uncovered novel polyfunctionality, with an increased proportion of the total CD8 T cell response represented by dual and polyfunctional cells (Fig. 2F). One patient, who already had an unusually polyfunctional response to HBV, did not show any enhancement of this upon IL-10 blockade (Fig. 2F). Polyfunctionality of CMV-specific CD8 T cell responses from patients with CHB was also enhanced upon IL-10 blockade (Supplemental Fig. 1), suggesting that IL-10 may have a global suppressive effect. This is in line with our previous finding that CD8 T cells are functionally impaired in CHB, regardless of their antigen-specificity (22). These data imply that different effector functions of virus-specific CD8 T cells are susceptible to suppression by IL-10. IL-10 blockade was not effective in all patients studied, in line with findings showing that multiple mechanisms are known to contribute to the exhaustion of the T cell response in CHB (17, 20, 23).

Figure 2
Blockade of IL-10 rescues polyfunctionality of virus-specific CD8 T cell responses. PBMC were stimulated with a pool of HBV peptides with or without blockade of IL-10 and its receptor. A, Representative contour plots to show the effect of IL-10/R blockade ...

IL-10 producing B cells are enriched in chronic HBV and correlate temporally with disease flares

IL-10 producing B cells have recently been identified in humans as potent regulators of T cell proliferation and cytokine production in healthy donors (14) and in autoimmune disease (13, 15). However, their role in human infection is unknown. To first assess the role of circulating rather than intrahepatic mononuclear cells to IL-10 release in CHB, we quantified levels in supernatant after stimulation with mitogens (PMA and ionomycin). As shown in Fig. 3A, PBMC from patients with CHB produced more IL-10 than healthy donors (p<0.05). We next investigated the contribution of IL-10 producing B cells to this enhanced capacity for IL-10 production within circulating leukocytes. PBMC were stimulated with CpG, a TLR-9 agonist, relevant in the context of the DNA virus HBV, which has been shown previously to potently induce B cell IL-10 release (14, 24, 25). In response to this stimulus, we observed a mean five-fold increase in the frequency of B cells producing IL-10 in patients (n=25) compared to controls (n=14; p<0.0001) (Fig. 3B; left panel). Additionally, the frequency of IL-10 producing B cells was enriched during flares of disease activity (Fig. 3B lower panel). As shown in Fig. 3C, there was no difference in the frequency of total CD19+ B cells between healthy donors and patients, suggesting that a subset of IL-10 producing B cells may arise from the existing pool during active flares of disease.

Figure 3
IL-10 producing B cells are enriched during active flares of disease. A, Cross-sectional comparison of IL-10 levels in supernatant between patients with CHB (n=19) and controls (n=9) following 5 hour stimulation of PBMC with mitogens (PMA and ionomycin). ...

We next investigated whether B cells could produce IL-10 in response to HBV derived antigens. HBV core antigen potently induced IL-10 production from healthy donor PBMC (Fig. 4A). Stimulation with HBsAg and HBeAg did not induce IL-10, suggesting that the effect of HBcAg was not mediated by a non-specific contaminant in the antigen preparations. The effect of HBcAg could be due to its unique ability to activate B cells, regardless of their antigen-specificity, by cross-linking surface receptors (26). Similarly, there was an enrichment of B cells producing IL-10 in response to HBV core antigen stimulation that mirrored the spontaneous flares of disease observed in two patients (Fig. 4B).

Figure 4
B cells produce IL-10 in response to HBV core antigen. A, Graph shows IL-10 production in supernatant in response to stimulation of healthy donor PBMC (n=4) with HBcAg, HBsAg or HBeAg (10μg/ml). B, Longitudinal analysis to show the frequency of ...

Immature CD19+CD24hiCD38hi B cells producing IL-10 are enriched during the peak of disease flares

To investigate the phenotype of IL-10 producing B cells, peripheral blood leucocytes were surface stained for the markers CD19, CD24 and CD38 post-stimulation, which differentiate B cells into mature (CD24intCD38int), memory (CD24hiCD38neg) and immature (CD24hiCD38hi) subsets. Upon CpG stimulation, all B cell subsets could produce IL-10 (Fig. 5A-representative plot). We next studied whether the contribution of each B cell subset to total IL-10 production varied according to disease activity. During the peak of liver inflammation, immature B cells showed a 5-7 fold increase in their capacity to produce IL-10 (Fig. 5B; upper panels). In an eAg negative patient undergoing a flare, almost a third of total IL-10 production (28 percent) was produced by immature B cells during this peak, despite this subset representing the minority of total B cells. IL-10 production by immature cells did not show analogous large fluctuations in a patient with consistently high levels of viraemia and liver inflammation (eAg-positive, high DNA, high ALT). Mature B cells produced IL-10 at constitutively higher levels than immature or memory subsets in all individuals. However, the proportion of mature or memory subsets able to produce IL-10 did not fluctuate over the course of a flare (Fig. 5B lower panels).

Figure 5
Immature B cells are enriched in CHB and have increased capacity for IL-10 production at the peak of liver disease. PBMC were stimulated with the TLR ligand CpG (96hrs) and PMA/ionomycin (4 hours), following which the phenotype of IL-10 producing B cells ...

Immature B cells produce IL-10 directly ex vivo in patients with active HBV infection

In vitro stimulation with CpG may alter the phenotype of regulatory B cells. To identify the putative regulatory B cell subset without the influence of in vitro stimulation, we analysed the phenotype of IL-10 positive B cells directly ex vivo at serial timepoints before, during and after a hepatic flare of disease. As shown in Fig. 6A, IL-10+ B cells were detectable at low frequencies in the absence of any stimulation. IL-10 producing B cells were enriched and clustered together, predominantly within the CD19+CD24hiCD38hi immature cells at the peak of viral load. There was no comparable clustering of IL-10-producing CD24hiCD38hiB cells on examination of four healthy donors ex vivo (Fig. 6A lower panel). Total immature B cell frequency correlated temporally with peak viral load or liver inflammation in two patients undergoing flares but not in an eAg-positive CHB patient with a persistently elevated viral load and liver inflammation consistently four times greater than the upper limit of normal (>200IU/L) (Fig. 6B; upper panels). In this case, immature B cells were persistently elevated at twice the mean frequency observed in patients with high viral load (range 5-8% compared to mean of 4%, Das et al. unpublished observations). Temporal fluctuations in mature, memory and total B cell frequencies did not correlate with either viral load or liver inflammation (Fig. 6B; lower panels). Thus, not only do immature cells have an increased capacity for IL-10 production during the peak of liver inflammation relative to other B cell subsets, they are also enriched in number in patients with CHB ex vivo.

Figure 6
Ex vivo characterization of IL-10 producing B cells. A, Dot plots show the phenotypic characteristics of IL-10 producing B cells (unstimuated, ex vivo) over multiple timepoints in an eAg negative patient undergoing a flare (upper panel) or in four healthy ...

Little is known about the presence of different B cells within the human liver, the site of HBV replication. By extracting lymphocytes from surplus tissue available from liver biopsies from patients with CHB, we determined that the percent of total B cells and their subset composition was not significantly different from that seen in the periphery of the same patients. Immature B cells (CD19+CD24hiCD38hi), which have not been previously identified in the human liver, were present at a frequency of 3-7.3%, suggesting that they may also participate in IL-10-mediated regulation in this setting (Fig. 6C). In support of this, a small population of intrahepatic B cells from patients with CHB showed the capacity to produce IL-10 in response to CpG stimulation (Supplemental Fig. 2).

Functional suppression of HBV-specific CD8 T cell responses by immature B cells

To investigate whether B regs play a role in the immune tolerance characteristic of this disease, CD19+CD24hiCD38hi B cells were depleted from total peripheral blood by fluorescence activated cell sorting and HBV-specific CD8 T cell responses were determined after 10 days in vitro stimulation with pooled HBV peptides. We observed a 2 to 4 fold increase in HBV specific responses in six out of nine patients following selective depletion of immature B cells only, despite this subset accounting for less than 1% of total peripheral blood leucocytes (Fig. 7A). We observed a trend towards a greater fold change in HBV compared to CMV specific responses from individuals in the same cohort (Fig. 7B).

Figure 7
Suppression of HBV-specific CD8 T cell responses by immature B cells. PBMC from HLA-A2 positive donors were depleted of CD19+CD24hiCD38hi immature B cells by cell sorting. A, Summary data show the effect of CD19+CD24hiCD38hi B cell depletion on the HBV-specific ...

To further probe the potential of immature B cells to actively suppress antiviral responses, sorted highly purified CD19+CD24hiCD38hi B cells were added back to in vitro culture in a one to four ratio with peripheral blood leucocytes. In all three cases there was suppression of HBV-specific CD8 T cell responses compared to PBMC depleted of immature B cells (Supplemental Fig. 3; representative example Fig. 7C).

Human immature B cells have recently been shown to mediate their suppressive activity in healthy controls and those with autoimmune disease through IL-10. In order to define the role for IL-10 in the suppressive activity of immature B cells in the setting of a persistent viral infection, we added them into a short-term expansion of HBV-specific CD8 T cell responses with or without IL-10 blockade. The co-administration of antibodies to block IL-10 and the IL-10 receptor almost completely abrogated the suppression of HBV-specific CD8 T cell responses mediated by CD19+CD24hiCD38hi B cells (Fig. 7C). Immature B cells therefore show regulatory potential, and can suppress HBV-specific CD8 T cell responses partially through production of IL-10.

Discussion

In this study we showed that serum IL-10 levels correlated with spontaneous flares of liver disease and IL-10 blockade in vitro could restore HBV-specific CD8 T cell polyfunctionality. Regulatory B cells contributed to IL-10 release and were enriched in CHB, in particular during flares. Unstimulated IL-10 producing B cells were of immature phenotype ex vivo; depletion of this small subset was able to restore HBV-specific CD8 T cell responses. All B cell subsets were also found to be present in the liver, suggesting that they may be able to exert regulatory effects on HBV-specific CD8 T cell responses both in the circulation and the liver. These data point towards a pathogenic role for B cells and IL-10 in CHB.

We had access to a unique cohort of patients undergoing spontaneous flares of liver disease in whom we could correlate serum IL-10 levels with disease activity over time. In all individuals with eAg-negative CHB, IL-10 was raised at the time of the peak in viral load or liver inflammation. It was not possible to infer whether IL-10 was more closely linked to either viral load or liver inflammation due to their close association during the peak of flares, unlike in acute HBV infection in which IL-10 can start to increase in tandem with viral load, sometimes weeks before the onset of liver inflammation (27). However, previous cross-sectional data showed elevated serum IL-10 in patients with liver inflammation and increased IL-10 during flares induced by treatment withdrawal (19, 28). Thus it is likely that in the setting of spontaneous flares of CHB, IL-10 constitutes a physiological mechanism to dampen liver inflammation.

Whether or not IL-10 impairs HBV-specific CD8 T cell responses in this setting was next investigated. As shown previously, IL-10 is known to have a dual role during infection (29). It can both protect the host against Th1 mediated immunopathology during malaria and Toxoplasma gondii infections, yet conversely tolerise local and systemic anti-viral responses and favour viral persistence in murine chronic infection (19, 30-32). Blockade of IL-10 in these studies improved vaccine efficacy and disease resolution (33-35). In this study, we showed that IL-10 blockade in vitro could recover polyfunctional virus-specific CD8 T cell responses. Based on this, we hypothesize that raised IL-10 during hepatic flares may serve a physiological role in attenuating inflammation, but as a bystander effect may inadvertently dampen adaptive immune responses. This is supported by studies of Leishmania Major, in which IL-10 producing T cells suppressed immunopathology in subjects with high antigen load, however additionally rendered Th1 responses ineffective, resulting in prevention of lesion healing (36).

The source of IL-10 in CHB is likely to be multi-cellular, as noted in HIV infection (37). Within the liver, Toll-like receptor activated Kuppfer cells (38), LSEC primed CD4 T cells (39), CD4+CD25hiFoxp3+(40) and antigen induced CD8 T cells are known to produce IL-10 (41); IL-10 can suppress T cell proliferative responses and NK cell function (19, 42). In this study, we showed that mitogen-activated peripheral blood mononuclear cells were additionally capable of IL-10 production ex vivo, and upon expansion with CpG, B cells were potent IL-10 producers. Recent findings suggest IL-10-producing B cells may play an apical role in the induction and maintenance of other regulatory populations such as Tregs (8); it will be interesting to investigate whether their suppressive effects can be amplified by other cell types in CHB.

B cells have classically been associated with antibody production and humoral immunity; anti-envelope antibodies have been associated temporally with clearance of HBV and persisting memory B cells maintain ability to mount a rapid humoral response upon antigen re-encounter, even in patients who have clinicially sub-therapeutic antibody responses (43, 44). B cells are more activated in CHB compared to healthy donors (45), and can uniquely interact with viral proteins; the core antigen of HBV can cross-link surface receptors of B cells (BCRs) irrespective of their antigen specificity, and is postulated to be presented mainly by B cells, not dendritic cells (45, 46). The unique capacity of HBV core antigen to cross-link the B cell receptor in a non-antigen specific manner suggests that it may be an alternative stimulus in addition to activation through HBV-specific BCRs. This is supported by our observation that B cell IL-10 responsiveness to HBcAg correlated with disease activity.

The potential importance of B cells in ongoing control of HBV has been highlighted recently by cases of HBV reactivation triggered by the B cell depleting drug Rituximab (47). An immunoregulatory role is attributed to B cells in another liver disease, primary biliary cirrhosis. Rituximab treatment in this setting exacerbates cholangitis, associated with a large T cell inflammatory infiltrate into the liver and pro-inflammatory cytokine production (48). In concordance with these observations, recent studies show an emerging role for non-antibody dependent B cell functions; primarily modulation of T cell responses through production of soluble cytokines, antigen-presentation and interaction with co-stimulatory molecules (8). Regulatory B cells suppress inflammation and auto-immunity in mice (2, 3), and can suppress T cell pro-inflammatory cytokine production in healthy human donors (13); this is mediated primarily by IL-10 production. Importantly, the absence of endogenous B cell derived IL-10 alone has been shown to favour a pro-inflammatory environment in murine arthritis with preferential generation of Th1 and Th17 cells over regulatory T cells. B cell-derived IL-10 has similarly been shown to suppress pathogenic T cell responses in experimental autoimmune encephalomyelitis (7) and Salmonella infection (49). These data highlight that a relatively small fraction of total B cells have capacity to potently modulate T cell responses in vivo (12).

Naive B cells express TLR 9 and TLR 10, which are upregulated upon activation, in contrast to TLR-4 which lacks significant expression on human B cells (50, 51). Stimulation via toll like receptors has been shown to induce regulatory function and IL-10 production in B cells by a ‘two-step model’; TLR stimulation first primes B cells towards IL-10 production which are then further potentiated towards a regulatory phenotype upon BCR and CD40 ligation (52). In this study, we observed that stimulation with CpG, an agonist of TLR-9, could induce IL-10 production by B cells, and that IL-10 B cells were more enriched in CHB than in healthy donors.

IL-10 B cells have previously been assigned distinct phenotypic profiles including CD19+CD24hiCD38hi transitional or CD27+CD24hi B10 cells (13, 15). However, we observed that all B cell subsets had capacity for IL-10 production, similar to that reported by Bouaziz et al. who showed a heterogenous spread of IL-10 B cells amongst immature and memory B cell compartments (14). Discrepancies in phenotype between studies is partially due to an imperfect panel of markers to characterise this subset at this time, and different methods of stimulating these cells including TLR ligands, CD40 and stimulation through the B cell receptor, all of which could impact on their phenotype. To remove any potential bias of stimulation, we phenotyped IL-10 positive B cells directly ex vivo, in the absence of stimulation. In this setting, we observed that IL-10 producing B cells were predominantly of immature phenotype, and immature B cells were the only B cell subset to correlate with hepatic fares ex vivo. This is consistent with a phenotypic subset of human transitional regulatory B cells described by Blair et al. and is in line with studies of HIV and HCV infection, in which there is a peripheral enrichment of this immature subset (53, 54).

On this pretext, we chose to study immature B cells as the putative regulatory subset in CHB. Depletion of this small subset by cell sorting recovered virus-specific CD8 T cell responses in vitro. These observations are similar to those seen in murine arthritis, in which an analogous transitional 2 marginal zone precursor B cell subset could suppress antigen specific T cell responses and ameliorated established disease (1). Immature B cells are generated in the bone marrow and traffic to the spleen, where they can mature after encounter with antigen and CD4 T cell help. Re-circulation to non-splenic secondary lymphoid organs and sites of inflammation has been reported, however immature cells actively downregulate integrin-mediated homing to lymph nodes (55). In this study, we observed that all B cell subsets including immature B cells were present in the liver, at a similar frequency to that observed in paired peripheral blood. This was despite absence of significant liver inflammation at the timepoint that liver biopsies were taken from patients. This first demonstration of immature B cells present in the human liver sheds new light on the unexpected trafficking potential of this subset. Whilst the intrahepatic localisation of this subset remains to be defined, a recent study has shown that B cells have the potential to infiltrate hepatic parenchyma and form lymphoid aggregrates (56). These data allude to an additional site where B cells may encounter and modulate T cells apart from the peripheral blood and spleen, the principle sites at which human immature B cells have been identified (57).

Thus, B cells have the capacity to suppress CD8 T cell responses, which could further constrain anti-viral responses already dysregulated by co-inhibitory signals and pro-apoptotic pathways (17, 20, 23). The capacity of regulatory B cells to suppress virus-specific CD8 T cell responses implicates a pathogenic role for regulatory B cells and IL-10 in CHB. An in vivo role for B regs is supported by the mirroring between their expansion and contraction and disease activity. We hypothesize that IL-10 may be triggered in this setting as a feedback mechanism to dampen liver inflammation, but results in suppression of HBV-specific CD8 T cell responses as an inadvertent bystander effect. Our findings highlight the capacity of B regs to not only curtail pro-inflammatory adaptive responses, but also to downregulate antigen-specific T cell responses. Taken together our data provide the first evidence for a role of B regs in human infection, revealing that they can potently modulate the immune response to HBV.

Supplementary Material

Acknowledgments

We thank all the patient and staff who provided blood and liver samples.

Footnotes

Authorship Contributions

Study concept and design AD, CM, PB, MKM; acquisition of data AD, GE, CP, ARL, PK, DP, AC; analysis and interpretation of data; AD, GE, CP, ARL, PK, DP, AC, CM, MKM; drafting of the manuscript AD, CM, MKM; critical revision of the manuscript; AD, GD, UG, PTK, MB, PL, CM, MKM; technical or material support; PK, GD, UG, PTK, MB, PL.

Conflict of Interest Disclosures

None

1This work was funded by Medical Research Council Awards G108515 and G0801213 to MKM and by an MRC PhD studentship to AD.

2Corresponding author address; Mala K. Maini, Division of Infection and Immunity, Rayne Institute, 5 University Street, UCL, London, WC1E 6JF.

3Chronic Hepatitis B Virus (CHB), Hepatitis B Virus (HBV), regulatory B cells (Breg), HBV core antigen (HBcAg), HBV surface antigen (HBsAg), HBV e antigen (HBeAg), Toll like receptor (TLR), Intrahepatic lymphocytes (IHL).

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