Lijie Ma,Xuehua Sun,Xiaoni Kong,Yueqiu Gao

Institute of Clinical Immunology,Department of Liver Diseases,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai,China

ABSTRACT Chronic hepatitis B(CHB)remains a global health problem.The persistence of hepatitis B surface antigen(HBsAg)in the blood for longer than 6 months after the initial infection is a sign of CHB.The therapeutic goal for the functional cure of CHB is the generation of antibodies against HBsAg.However,the adaptive immune response of patients with CHB cannot generate an efficient antiviral response.Many previous studies have evaluated T cell function and T cell therapy specifically designed to counter hepatitis B virus(HBV)infection.As one of the major components of adaptive immunity,B cells also display dysfunctions in anti-HBsAg antibody (HBsAb) production and antigen presentation.Patients with CHB have amplification of CD19+CD10-CD27-CD21-atypical memory B cell subsets and CD19+CD24hiCD38hi regulatory B cells.Currently,no reviews have summarized specific B cell responses during CHB infection.Thus,in this study,we summarized B cell dysfunction during CHB progression and the potential mechanisms behind these dysfunctions to further our understanding of the mechanisms of adaptive immune response of B cells in the process of CHB development and help provide new methods and ideas for the treatment of CHB.

Keywords:Chronic hepatitis B virus infection B cell dysfunction Hepatitis B surface antibody Atypical memory B cells (atMBCs)Antigen-presenting cells (APCs)Regulatory B cells (Bregs)

1.Introduction

Hepatitis B virus (HBV) is a noncytopathic hepatotropic enveloped DNA virus that contains a partially double-stranded relaxed circular DNA genome and causes a serious global health problem.More than 250 million people worldwide are estimated to have chronic hepatitis B (CHB),and over 887,000 deaths are caused by cirrhosis and hepatocellular carcinoma annually.1,2Studies have shown that both the persistent replication and immune clearance of HBV are involved in the pathogenesis of CHB.The host immune response,particularly T cell-mediated immune response,is considered the principal factor in regulating the outcome of HBV infection.However,B cell-mediated humoral immune response may also play important roles in patients with CHB.3In fact,B cells have been increasingly recognized to contribute to the continuous control of HBV.4,5Although considerable progress has been made in identifying the targets for T cell exhaustion under sustained HBV stimulation,6－9analogous defects in B cell responses have been less commonly studied.4In this review,we summarized the currently available information about B cell dysfunctions in CHB.

2.Anti-hepatitis B surface antigen antibodies(HBsAbs)and B cells

HBV-specific memory B cells can differentiate into plasma cells and are capable of producing polyclonal antibodies to hepatitis B surface antigen (HBsAg),hepatitis B e antigen (HBeAg),and hepatitis B core antigen (HBcAg) during CHB.Circulating HBsAbs subsequently clear the virus and prevent HBV re-infection.10The production of HBsAbs is the hallmark of the functional cure of CHB.HBsAbs are considered important for both the prevention of reinfection and maintenance of ongoing immune control.HBsAbs block the entry of HBV into hepatocytes and remove circulating antigens and infectious HBV particles.11Thus,there is growing recognition of the importance of humoral immune responses in the production of HBsAbs during the control of HBV processes.12,13More than 30 years ago,the inherent dysfunctions of B cell synthesis in HBsAbs were considered a key feature of CHB.14,15However,studies have not yet clarified what inhibits the production of HBsAbs by B cells in CHB.

First,an immune evasion mechanism has been postulated,in which HBsAgs are released in several rounds by subviral particles rather than by the whole virus,which suppresses the innate and adaptive immune responses directly,including HBsAg-specific B cell differentiation.16This inhibits HBsAb production (Fig.1a) and the formation of immune complexes in circulation,17,18which eventually induces specific immune tolerance and prevents host eradication of the HBV infection.The long-term suppression of HBsAgs is thought to promote HBsAg-specific B cell responses.19－21

Second,atypical memory B cells(atMBCs)exhibit dysfunction in generating HBsAbs (Fig.1b),and their accumulation as antigenspecific B cell responses is likely inhibited in patients with CHB.In addition to inhibitory receptors expressed by atMBCs,high doses of antigens and deficiencies of T helper cells (Th cells) lead to inadequate HBsAb production.22Future research analyzing HBsAgspecific B cells and Th cells may help better determine the effect of defective T cells on B cell-mediated HBsAb production.

Finally,not all antibodies against HBsAg have an equal ability to clear HBV.The antigen epitope is a key factor in determining the efficacy of HBsAbs.Only antibodies that recognize the unique sA epitope (HBsAg-aa119－125 as the core motif) can effectively mediate HBsAg clearance.23－25In some situations,HBsAbs may clear HBsAg from the peripheral blood,but they do not eliminate viral particles from the liver.26Therefore,although the B cellmediated immune response is an integral part of the host’s defense against HBV infection and contributes to HBV suppression,clearance,and protection,it is not enough to clear HBV infection alone.

3.AtMBCs and the host immune response in CHB

In CHB,a substantial proportion of HBsAg-specific B cells display the atMBC phenotype,which is dysfunctional in generating an HBsAb response.27,28AtMBCs interfere with the signal transduction,survival,cytokine production,and differentiation of plasma cells in host immune responses.

RNA sequencing has revealed that multiple inhibitory receptors,including Fc receptor-like (FcRL) family members,are upregulated and antigen presentation genes are downregulated in the B cells of patients with CHB.29,30HBV has been reported to directly cause the induction of the inhibitory receptors FcRL4,FcRL5,and programmed death(PD)-1 on the surface of B cells;furthermore,FcRL5 has been identified as the key factor in the generation of atMBCs.31－33An expansion of the atypical memory CD19+CD10-CD27-CD21-subset of B cells with high levels of FcRL5 expression has been consistently reported in patients with CHB.34FcRL4 is expressed in an HBVdependent manner.In addition,FcRL4 expression by atMBCs was shown to be significantly higher than that by MBCs during chronic viral infections.29,31Furthermore,atMBCs are associated with the abnormal expansion of a subset of T follicular helper cells(Tfh)with high CD40 ligand (CD40L) expression (Fig.1c).Activated CD40L expression in Tfh cells is correlated with altered B cell differentiation and the accumulation of a higher number of atMBCs in patients with CHB.31,35

Although atMBCs can be present in peripheral global B cell compartments and in non-HBV-infected livers,they are commonly found in HBV-infected liver cells (Fig.1d) with high levels of PD-1 expression,36－41which suggests that the liver microenvironment may facilitate the development of the atMBC phenotype.At the same time,the liver microenvironment and HBV antigen have a combined effect on driving the atMBC and PD-1hiphenotypes,which has been verified by thein vitrodetection of B cells isolated from a large number of human livers.4

4.Antigen-presenting B cells in CHB

The key to inducing T cell-mediated immunity is efficient antigen presentation by antigen-presenting cells(APCs).In addition to playing an essential role in the humoral immune response,B cells also have an important function in antigen presentation.42－44Major histocompatibility complex class I and class II molecules are upregulated in CD40L-activated B cells,which induce the expression of CD80 and CD86 and internalize and present antigens to T cells (Fig.1e).45－47Therefore,the depletion of B cells by rituximab or obinutuzumab may result in defective T cell responses to HBV and reactivation of the virus.However,the incidence of reactivation after obinutuzumab- or rituximab-containing immunochemotherapy varies substantially among studies and thus remains largely unknown.48－51

B cells could serve as APCs and present the core peptides of HBV to CD8+T cells;52in fact,the HBcAg antigenic component has a unique ability to bind to and activate a high frequency of naive human and murine B cells.53In vivoexperiments have shown that the ability of HBcAg-specific B cells to internalize,process,and present HBcAgs to naive Th cells is 105times more efficient than that of classical APCs.54However,HBsAgs are preferentially internalized and presented by classical APCs such as dendritic cells and macrophages.55,56A mouse study has reported that HBsAgs do not induce costimulatory molecules on atMBCs (Fig.1f).54

Fig.1.B cells are defective in CHB.(a) HBV inhibits HBsAb production.(b) AtMBCs are dysfunctional in generating HBsAbs.(c,d) Tfhs are correlated with the accumulation of atMBCs in HBV-infected liver cells.(e)B cells may efficiently internalize and present antigens to CD8+T cells.(f)AtMBCs are correlated with the dysfunctional antigen presentation of B cells.(g) Bregs inhibit proinflammatory cytokine production in CD4+ T cells,(h) convert CD4+CD25- T cells into Tregs,(i) and suppress HBV-specific CD8+ T cell responses.Abbreviations:AtMBC,atypical memory B cell; Breg,regulatory B cell; CHB,chronic hepatitis B; CK,cytokines; HBV,hepatitis B virus; HBsAb,anti-hepatitis B surface antigen antibody;HBeAb,anti-hepatitis B e antigen antibody;HBcAb,anti-hepatitis B core antigen antibody;IL-10,interleukin 10;Treg,regulatory T cell;Tfh,T follicular helper cell;TGF-β,transforming growth factor beta.

5.Regulatory B cells (Bregs) in CHB

Recently,the role of Bregs in infectious and autoimmune diseases has drawn attention.57,58The phenotypes of human Bregs are unique in different diseases and must be further confirmed in patients with CHB.Different B cell populations,such as CD5+B-1a cells,59CD19hiCD1dhiCD5+B cells,60CD19+CD24hiCD38hitransitional B cells,61and CD19+CD24hiCD27+memory B cells,have been reported to play regulatory functions.A previous study showed that Bregs in patients with CHB were immature CD19+CD24hiCD38hiB cells.62An elevated Breg population can promote HBV replication and the progression of liver fibrosis but can also inhibit alanine aminotransferase activity and liver inflammation.63,64

Bregs exert regulatory functions mainly via the production of cytokines such as interleukin 10 (IL-10) and transforming growth factor beta,65－67which inhibit pro-inflammatory cytokine production in CD4+T cells (Fig.1g),convert CD4+CD25-T cells into Tregs(Fig.1h),suppress HBV-specific CD8+Tcell responses(Fig.1i),and increase the production of inhibitory cytokines.68Increased Breg populations in patients with CHB have been reported to produce large amounts of IL-10,65,68thereby inhibiting the antiviral ability of HBV-specific CD8+T cells,reducing immune-mediated liver injury,and increasing the HBV viral load.This is also consistent with the clinical characteristics of patients with immune tolerance.When IL-10 is blocked,the regulatory effects of Bregs on CD4+T cells and the conversion of CD4+CD25-T cells into Tregs are inhibited and the polyfunctionality of HBV-specific CD8+T cell responses are rescued.69In addition,inhibiting Bregs requires direct contact between Bregs and CD4+T cells.

6.Others

B cells are known to internalize B cell receptor (BCR)-linked antigens and deliver them to autophagosomes where they colocalize with toll-like receptor (TLR)-9 or TLR-7.70,71The crosstalk between TLR7/9 and the BCR signaling pathway may modulate the response of B cells to foreign or endogenous antigens.72,73The direct activation of TLR7 in B cells can also contribute to the development of effective antiviral antibody responses.74,75The intrinsic signaling pathway of TLR9 in B cells can increase immunoglobulin G production.76,77HBV-specific B cells,which function as the primary innate immune sensor,require the upregulation of myeloid differentiation primary response 88 (MyD88) and type I interferon genes.78HBcAgs can activate the expression of innate immune genes in B cells.Some scholars have found that the frequencies of HBcAg-specific B cells were temporarily increased,which indicates a possible link between HBcAg-specific B cells and liver damage in patients with CHB.79It is likely that HBcAgs are released from dying hepatocytes during hepatic flares,which leads to the temporary extensive formation of antigen-antibody complexes on the surface of hepatocytes and Kupffer cells and results in the activation of the classical complement pathway and massive liver necrosis.80However,whether anti-HBcAg antibodies(HBcAbs)are the initial factor for the host’s immune attack against HBV,which results in inflammatory and immunopathological processes in patients with CHB,requires further investigation.HBcAg- and HBsAg-specific B cells in patients with CHB differ in frequency,phenotype,and function.That is,HBcAg-specific B cell populations are larger than HBsAg-specific B cell populations;HBsAg-specific B cells are enriched for atMBC phenotypes;and a functional defect of maturation in antibody-producing B cells affects only HBsAgspecific B cells.

7.Conclusions

Although the above evidence indicates the importance of B cells in the control of HBV,there are still many complexities regarding B cell responses during CHB infection that remain unknown.

A detailed understanding of the constraints on pathogenspecific B cell responses will help identify new therapeutic targets for harnessing humoral immune responses.Vaccines that provide therapeutic antibodies may help induce humoral responses.This strategy will hopefully overcome immune tolerance in patients with CHB and allow the clinical efficacy of“therapeutic vaccination” to cure HBV infection.

Authors’ contributions

L.Ma wrote the manuscript.X.Sun designed the figure.X.Kong revised the manuscript.Y.Gao designed the manuscript.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81874436 to Y.Gao,81673935 to X.Sun).