As professional antigen-presenting cells (APCs), B cells use their receptors (BCRs) to specifically recognize HBV antigens on the surface of Kupffer cells or dendritic cells (DCs). Following antigen recognition, the BCR-antigen complexes are internalized into endosomes via receptor-mediated endocytosis, where B cells subsequently process these antigens and present them on MHC-I and MHC-II molecules within lysosomes. These molecules then bind to processed HBcAg or HBsAg antigens, forming HBcAg-MHC-II and HBsAg/HBcAg-MHC-I complexes, respectively. Subsequently, these complexes are transported to the plasma membrane surface (Cai and Yin, 2020). Simultaneously, under the activation signals from multiple pairs of co-stimulatory molecules, including B7 and CD28, CD40 with CD40L, naïve CD4⁺ T cells are activated by B cells presenting the HBcAg-MHC-II complex. This activation initiates a CD4⁺ T cell immune response. However, CD8⁺ T cells are activated by B cells presenting the HBcAg/HBsAg-MHC-I complex, collectively inducing a Cytotoxic T-lymphocytes (CTLs) cytotoxic response (Barnaba et al., 1990; Gehring et al., 2013; McIntosh et al., 2014; Cai and Yin, 2020; Sajjad et al., 2021). In addition, follicular dendritic cells (FDCs) may participate in specialized antigen presentation, a pathway linked to the formation of memory B cells and plasma cells(PCs). FDCs are a class of cells that line the antigen-exposed surfaces of B-cell follicles in lymph nodes and spleens. These cells engage in close interactions with B cells, express Fc receptors, and possess the unique ability to preserve antigen-antibody complexes for an extended duration. This preservation is achieved through the formation of immune complex-encapsulated vesicles on their dendrites. FDCs may participate in the immune memory response of B cells, which may take up antigens from FDCs and process and present them to T cells (MacLennan, 1994; Heath et al., 2019). However, low expression of the co-stimulatory molecule CD40 on B cells of patients with chronic HBV infection may affect the co-stimulation between CD40 and CD40L, thus suppressing the growth and activation of B cells (Xing et al., 2013). Furthermore, the surface of B cells in patients with CHB also lacks the expression of the co-stimulatory molecule CD80 (Xu et al., 2015). As a key type of APCs, B cells may also be implicated in T cell damage during chronic HBV infection. The research by Barnaba et al. showed that HBsAg-specific B cells cross-present HBsAg fragments to CTLs via the MHC-I molecule. Nevertheless, the induction of cytotoxicity in CTLs during this process might lead to the death of HBsAg-specific B cells (Barnaba et al., 1990; Keller et al., 2009), as shown in Figure 2 .

B cells form a germinal center by recruiting specific CD4⁺ T cell helpers. This process stimulates the proliferation and differentiation of B cells following induction by antigenic signals, involving the identification of HBV peptide-MHC-class II molecular complexes on the surface of B cells by Th cells. Co-stimulatory signals, facilitated by the interaction of CD40 and B7 on the B cell surface with various co-stimulatory molecules on the Th cell surface (including CD40L and CD28), further contribute to this immune response. The CD4⁺ T cell receptor (TCR) on T cell surfaces binds to HBV antigenic peptide-MHC-class II molecular complexes. Non-specific co-stimulatory signals, facilitated by co-stimulatory molecules like B7 and CD40, activate CD4⁺ T cells, leading them into a proliferative and differentiated state. Notably, the co-stimulatory molecule B7 is essential for T cell activation and differentiation, with interactions between B7 and CD28 playing a key role in delivering essential stimulatory signals to T cells (Azuma et al., 1993; Freeman et al., 1993; Janakiram et al., 2017). Anti-HBs are secreted by B cells and act as a protective antibody to directly neutralize pathogens and block HBV replication and its entry into hepatocytes (Lu et al., 2018). Additionally, anti-HBs also fulfill other multi-effect functions during chronic HBV infection. Immunohistological studies have shown a significant correlation between immune-mediated hepatitis injury during chronic HBV infection and hepatocyte membrane-associated HBsAg. It is suggested that membrane HBsAg could potentially act as a target antigen for T cell-mediated immune lysis of hepatocytes (Ray et al., 1976; Huang and Neurath, 1979). The finding of cytoplasmic/membrane HBsAg on infected hepatocytes showed that anti-HBs IgG may bind HBsAg, triggering the release of perforin/granzyme in NK cells. This activation results in antibody-dependent cytotoxicity (ADCC), leading to the exhaustion of HBV-infected hepatocytes (Chu and Liaw, 1987; Ackerman et al., 2016; Lu et al., 2018). Theoretically, anti-HBs also bind to HBsAg and induce Kupffer cells to engage in antibody-dependent cellular phagocytosis (ADCP) and contribute to the immune clearance process of HBV (Lu et al., 2018). Beyond the induction of NK cells for ADCC-mediated lysis of hepatocytes infected with HBV, driven by membrane-associated HBsAg and HBcAg, contributes to hepatocyte injury through complement system activation. In patients with chronic HBV infection, elevated levels of anti-HBc IgG in the liver may bind to HBcAg, leading to extensive formation of antigen-antibody complexes on the surfaces of hepatocytes and Kupffer cells. This further activates the classical complement pathway and complement system through a series of zymogenic cascade reactions. Ultimately, this culminates in the assembly of membrane attack complexes (MACs) to lyse virus-infected hepatocytes and cause hepatocyte necrosis (Farci et al., 2010; Mutti et al., 2018).

T follicular helper (Tfh) cells play a role in regulating regulatory B (Breg) cells HBV-associated humoral immune response (Hu et al., 2014). IL-21 is a cytokine primarily released by activated CD4⁺ T cells (primarily Tfh cells and Th17 cells). It plays a crucial role in sustaining the effector response of CD8⁺ T cells during chronic viral infections (Zander et al., 2022). HBV antigenic peptides are presented to CD4⁺ T cells via MHC-II, and these interactions stimulate IL-21 production by CD4⁺ T cells, especially Tfh (Asao, 2021). IL-21 can induce the differentiation of naive and memory B cells into PCs, leading to substantial production of IgG antibodies. Moreover, IL-21 plays a role in regulating the differentiation, proliferation, and activation of T cells, B cells, and NK cells (Ettinger et al., 2005; Shbeer and Ahmed Robadi, 2023). IL-21 produced by Tfh cells also elevates the number of IgG-expressing B cells and stimulates the diversification of HBV-specific T-cell responses. This includes an enhancement in the production of IFN-γ (Publicover et al., 2011). Beyond its involvement in promoting the proliferation, differentiation, and activation of B and CD4⁺ T cells, IL-21 is also capable of augmenting the cytotoxicity of CD8+ T and NK cells, along with increasing IFN-γ production (Yi et al., 2010). The hallmark of T cell senescence in humans is the absence of CD28 expression. IL-21 exhibits the capability to enhance the growth of CD28(+) CD8⁺ T cells by preventing the downregulation of CD28 (Alves et al., 2005; Nguyen and Weng, 2010; Wang et al., 2020). Furthermore, IL-21 can directly act on CD8⁺ T cells to maintain CD8⁺ T cell responsiveness in chronic viral infections (Yi et al., 2010). Also, IL-21 inhibits Foxp3+ regulatory CD4⁺ T cells, resulting in a reduction in the number of suppressor regulatory T cells (Tregs), which indirectly promotes the production of antigen-specific CD8⁺ cytotoxic T lymphocytes (Li and Yee, 2008). Recent research indicated the capacity of IL-21 to promote the generation of HBV-specific CD8⁺ T cells while downregulating the expression of PD-1 and TIM-3. This observation highlights that IL-21 could contribute to the clearance of HBV (Tang et al., 2019). These prior outcomes indicated that the pleiotropic effects of IL-21 exert a positive immune influence during viral infection.

CTLs also kill HBV-infected target cells by secreting cytokines, including IFN-γ and TNF-α. IFN-γ has a pleiotropic effect. IFN-γ possesses immunomodulatory properties, activating the functions of NK and T cells, along with macrophages. This activation helps regulate the immune microenvironment, applying immune pressure on infected hepatocytes. Simultaneously, IFN-γ impedes HBV replication through a non-cytolytic mechanism by inhibiting HBV transcription (Nosaka et al., 2020; Chen et al., 2021). However, T-cell exhaustion often occurs during persistent HBV infection, and cytokines (e.g., IL-2 and IL-12) can antagonize T-cell exhaustion. IL-2, primarily produced by activated T cells, functions as an autocrine cytokine that facilitates self-activation and proliferation. It is often referred to as the T cell growth factor. The signaling of IL-2 is closely linked to both antigen presence and co-stimulation, and the interaction of these two factors propels T-cell proliferation, sustains cell survival, and guides effector differentiation. Additionally, IL-2 is critically involved in the formation of long-lived CD8⁺ T cell memory (Abbas et al., 2018; Kalia and Sarkar, 2018). By receiving IL-2, virus-specific CD8⁺ T cells that were previously impaired regain their capacity for growth, differentiation, and IFN-γ production (Bénéchet et al., 2019). Furthermore, IL-12, a cytokine produced by activated APCs(e.g., dendritic cells, macrophages, etc.), serves as a potent inducer of IFN-γ production by T and NK cells. The activation of CD8⁺T cells requires a variety of signals. In addition to the antigen signal generated by TCR recognizing antigen peptide -MHC-I molecular complex and the co-stimulation signal generated by several pairs of co-stimulatory molecules (mainly CD28 and B7), pro-inflammatory cytokines IL-12 and/or IFN-α could also serve as the third signal of T cell activation to jointly promote the proliferation and differentiation of CD8⁺T cells. The activation of CD8⁺T cells requires a variety of signals. In addition to the antigen signal generated by TCR recognizing antigen peptide -MHC-I molecular complex and co-stimulation signal generated by several pairs of co-stimulatory molecules (mainly CD28 and B7), researchers gradually realize that pro-inflammatory cytokines IL-12 and/or IFN-α can be used as the third signal for T cell activation. This third signal collaborates to enhance the proliferation and differentiation of CD8⁺T cells (Curtsinger and Mescher, 2010). IL-12 is capable of down-regulating the expression of PD-1 on HBV-specific T cells, thereby restoring a diverse and multifunctional CD8⁺ T cell response in order to enhance cytotoxicity (Schurich et al., 2013). Moreover, Schurich’s team found that IL-12 can correct mitochondrial metabolic disorders in dysfunctional CD8⁺ T cells, enhance their mitochondrial potential, and reduce their reliance on glycolysis, thereby restoring HBV-specific T cell effector function (Schurich et al., 2016).

In addition to producing antibodies, B lymphocytes contribute to immune system activation through cytokine production and antigen presentation. B cells can be sorted into subpopulations as per their functional characteristics, including effector B (Beff) cells and Breg cells (Matsushita, 2019). Breg cells exhibit a dual function by exerting both negative regulatory immune responses and protective immune responses via the production of anti-inflammatory cytokines, including IL-10, IL-35, and transforming growth factor-β (TGF-β) (Matsushita, 2019). In contrast, Beff cells secrete cytokines, including IL-6, IFN-γ, and tumor necrosis factor-α (TNF-α), to exert pro-inflammatory immune responses and enhance responses of effector cells and memory CD4⁺ T cells (Shen and Fillatreau, 2015; Matsushita, 2019). These pro-inflammatory cytokines may induce cccDNA degradation, inhibit HBV replication and cccDNA transcription, thereby reducing the level of HBV cccDNA in hepatocytes and exerting a non-cytolytic antiviral effect on infected hepatocytes (Hösel et al., 2009; Palumbo et al., 2015; Xia et al., 2016). IL-6 has the capability to hinder the entry of HBV into hepatocytes by suppressing the expression of NTCP. Downregulated local NTCP in the hepatocyte membrane confers neonatal hepatocytes resistance to HBV reinfection, which may accelerate virus clearance during immune-mediated cell death and compensatory proliferation of living hepatocytes (Yan et al., 2012; Bouezzedine et al., 2015; Yan et al., 2019).

In chronic HBV infection, most Breg cells are capable of expressing the anti-inflammatory cytokine IL-10. This expression enhances the cellular function of Tregs while concurrently suppressing the immune function of effector T cells to maintain immune tolerance (Mauri and Bosma, 2012; Liu et al., 2016). In CHB patients, B cells that produce IL-10 primarily display immature/transitional phenotypes characterized by CD19 ⁺ CD24hi CD38hi expression. IL-10 secreted by Breg cells inhibits HBV-specific CD8⁺ T cell responses. Notably, the suppression can be restored after the exhaustion of Breg cells (Das et al., 2012). Bregs also have an impact on the activated CD4⁺ T cells. A study reveals that IL-10 secreted by Bregs exerts a negative regulatory effect on the T cell-dependent inflammatory response. This secretion inhibits both the proliferation of effector CD4⁺ T cells and the activation of antigen-specific CD8⁺ T cells (Catalán et al., 2021). Similarly, another experiment on IL-10-deficient mice shows the same conclusion. Compared with the new IL-10 transcription reporter mice, the B-cell-specific IL-10-deficient mice exhibit an increased specific CD8⁺ T cell response to murine cytomegalovirus and an elevation in the number of PCs (Madan et al., 2009).

It is crucial to emphasize that IL-35, another immunosuppressive factor secreted by Tregs and Breg cells, has the capacity to inhibit T-cell proliferation and effector functions (Xiang and Xie, 2015). Interestingly, IL-35, which exhibits elevated expression levels in CD4⁺ T cells from patients with chronic HBV infection, demonstrates the ability to suppress HBV-specific CTL cell proliferation and IFN-γ production in vitro (Li et al., 2015). In addition, it was found that IL-35 played a key function in various diseases, including inflammatory bowel disease (Li et al., 2014), autoimmune diabetes (Bettini et al., 2012), T-cell-dependent colitis (Wirtz et al., 2011), human prostate tumors (Olson et al., 2012), colorectal cancer (Zeng et al., 2013), and systemic lupus erythematosus (Okamoto et al., 2011). IL-35 can modulate T-cell-mediated specific immune responses (Collison et al., 2007; Okamoto et al., 2011; Wirtz et al., 2011; Bettini et al., 2012; Olson et al., 2012).

B cells are critically involved in the immune control of HBV through their antibody secretory function and immunomodulatory effects, etc. During chronic HBV infection, varying degrees of B cell immunodeficiency were observed, a phenomenon that impedes HBV clearance. A lack of HBsAg-specific B cells and significantly lower plasma anti-HBs levels in CHB patients could be partially restored in HBsAg seroconversion responders, which confirms the important roles of HBsAg-specific B cells and anti-HBs during HBV clearance (Xu et al., 2015). The acquired data suggest that the functional impairment observed in HBsAg-specific B cells, particularly in their ability to secrete anti-HBs antibodies, may contribute to the persistence of HBV infection. In addition, Breg cells and their secreted IL-10 and IL-35 also play a role in immunomodulating and maintaining immune tolerance by enhancing the function of Treg cells and inhibiting the immune function of effector T cells. While research on Breg cell-derived IL-35 in HBV infection is limited, accessed data from previous research indicated the immunosuppressive role of IL-35 in immune-related diseases. Based on this, we speculated that IL-35 played a vital role in the immune-mediated hepatic injury and immune tolerance observed throughout chronic HBV infection. Specifically, it is likely involved in suppressing the proliferation and differentiation of effector T cells. However, this may not be helpful for investigating HBV-associated immunopathogenesis due to the absence of activation and specific marker features on the surface of Breg cells. In pathogen immunity, memory B cells (MBCs) differentiate into antibody-secreting PCs. However, during HBV infection, there are significant alterations observed in peripheral and hepatic B-cell compartments, with HBsAg-specific B cells localizing to the infected liver and atypical memory B cells (atMBCs) preferentially accumulating and up-regulating PD-1 (Burton et al., 2018). The atMBCs present in the overall population of B cells and HBsAg-specific B cells exhibit enrichment in the liver and express a variety of IRs. The atMBC derived from CHB patients shows an attenuated BCR signaling response and reduced ability to secrete important antiviral cytokines such as IL-6 or TNF-α (Burton et al., 2018). Although most studies have focused on the high expression of IRs such as PD-1 on T cells, it is crucial to note that PD-1 is capable of inhibiting B-cell signaling, survival, and activation functions (Okazaki et al., 2001; Good-Jacobson et al., 2010; Titanji et al., 2010; Kardava et al., 2011; Thibult et al., 2013). The binding of PD-1 to BCR recruits tyrosine phosphatase 2 (SHP-2) to the C-terminal phosphotyrosine of PD-1. The subsequently phosphorylated SHP-2 dephosphorylates a key signal transducer of BCR signaling, leading to the inhibition of BCR signaling (Okazaki et al., 2001). Moreover, the collective evidence of reduced production of B-cell protective antibodies, compromised immune responses generated by Bregs, and impaired presentation of HBsAg peptides by B cells due to downregulated expression of co-stimulatory molecules suggests the presence of B-cell functional impairment during chronic HBV infection.

HBV-related vaccines offer potential benefits for addressing B cell dysfunction, with different types serving distinct purposes: preventive vaccines help induce protective antibodies and establish immune memory, while therapeutic vaccines aim to enhance HBV-specific immune responses (Jansen et al., 2021; Mahmood et al., 2023). In a phase 1b clinical trial, the therapeutic vaccine TG1050 demonstrated a favorable safety profile in patients treated for NUC, it was found to elicit HBV-specific cellular immune responses effectively (Zoulim et al., 2020). GS-4774, a novel therapeutic vaccine, encodes three Hepatitis B Virus (HBV) proteins - HBsAg, HBcAg, and HBx. It is designed to stimulate strong and specific T-cell responses against HBV. Current trials indicate that it is both safe and well-tolerated (Gaggar et al., 2014; Lok et al., 2016). Consequently, the suppressive effect of GS-4774 on the virus remains undetermined. A phase 2 clinical trial using GS-4774 showed that though GS-4774 did not reduce the HBsAg levels in chronic HBV-infected patients, it produced a strong immune stimulation effect on the CD8⁺ T cells of vaccinated patients (Boni et al., 2019). The Phase III clinical trial involving a therapeutic vaccine that comprises HBsAg and HBcAg (NASVAC) demonstrates that NASVAC more effectively reduces the viral load to an undetectable level in Chronic Hepatitis B patients when compared to Peg-IFN treatment (Al Mahtab et al., 2018). However, the clinical efficacy and safety of HBV vaccines are influenced by various immunological and clinical factors, and therapeutic vaccines continue to face significant challenges in restoring robust HBV-specific immunity. Consequently, future research will focus on the development of next-generation HBV vaccines.

TCR-T cell therapy is a form of adoptive immunotherapy that uses genetic engineering to modify patient T cells with tumor antigen-specific TCRs, enabling precise recognition and targeting of tumor cells. This approach is particularly suited for patients lacking tumor-specific T cells. In recent years, TCR-T therapy has become a research focus and is regarded as one of the most promising immunotherapeutic strategies. The application of HBV-specific TCR-redirected T (HBV-TCR-T) cellular immunotherapy has been seen in clinical practice, specifically in preventing the reemergence of HCC following liver transplantation (Qasim et al., 2015; Hafezi et al., 2021). Furthermore, over-transfer of mRNA HBV-specific TCR-electroporated T cells to HBV-infected human hepatic chimeric mice prompts a gradual decrease in viraemia. This process, however, simultaneously induces limited, transient hepatic inflammation and cellular damage (Kah et al., 2017). Similarly, adoptive transfer of engineered T cells expressing HBsAg or HBcAg-specific TCR into humanized mice infected with HBV, in combination with the viral entry inhibitor myrcludex B, ensures long-term control of HBV infection (Wisskirchen et al., 2019). In a phase I pilot study involving eight patients with HBV-associated advanced HCC, the safety and tolerability of HBV-TCR-T cellular immunotherapy have been primarily observed. This was particularly evident in patients with advanced HBV-HCC but without prior liver transplantation. Of the eight patients, seven demonstrated decreased or stable serum HBsAg levels, and three showed tumor shrinkage. This antitumor activity and antiviral efficacy may be attributed to HBV-TCR-T cells specifically targeting HBV-expressing metastatic tumor cells (Meng et al., 2021). However, this immunotherapy has significant limitations: in chronic HBV-infected patients with HCC, both normal HBV-infected hepatocytes and HBV-DNA-integrated HCC cells express HBV antigens. Targeting these normal infected hepatocytes may lead to severe liver injury, thereby increasing the risk of extrahepatic inflammatory events (Bertoletti et al., 2015; Tan and Schreiber, 2020). Moreover, longer follow-ups are needed in a larger sample size to observe the potential risk of HBV-TCR-T cell immunotherapy on tumor metastasis and/or bleeding as well as its safety and efficacy. In parallel, CAR-T therapy represents another innovative targeted cancer treatment that utilizes genetic engineering to equip T cells with chimeric antigen receptors (CARs), allowing precise recognition and attack of tumor cells. T cells expressing HBV envelope protein-specific CARs localize and act within the liver, effectively and rapidly inhibiting HBV replication. Animal studies indicate that this process induces only transient liver injury (Krebs et al., 2013); however, these results may not fully translate to humans. In clinical applications, the primary adverse effects of CAR-T cell therapy are cytokine release syndrome (CRS) and neurotoxicity. CRS is triggered by extensive CAR-T cell activation of the immune system and subsequent release of inflammatory cytokines, posing life-threatening risks if not promptly managed. Therefore, close monitoring is essential following CAR-T cell infusion. Therefore, in clinical practice, it is essential to account for the potential risk of acute or chronic hepatitis induced by adoptive T-cell therapy.

Numerous studies have demonstrated the efficacy of blocking PD-1 (Fisicaro et al., 2010), CD244 (Raziorrouh et al., 2010), CTLA-4 (Schurich et al., 2011), and Tim (Sakuishi et al., 2010) in restoring virus-specific CD8⁺ T-cell immune responses. However, PD-1 pathway blockade therapy dominated in terms of the expression level of IRs and the effect of blockade response (Bengsch et al., 2014). A phase Ib study utilizing PD-1 inhibitors has confirmed the safety and efficacy of nivolumab monotherapy. Among the twelve patients under monotherapy with nivolumab, 11 CHB patients presented a reduction in HBsAg levels, with one even displaying sustained loss of HBsAg (Gane et al., 2019). Blockage of the PD-1 and PD-L pathway could potentially revive the exhausted subpopulation of CD8⁺ T cells selectively. The majority of CD8⁺ T cell subsets that respond well to PD-1 blocking therapy are those at a lesser degree of differentiation, indicating a proliferation explosion. However, the response from more differentiated and exhausted subsets of CD8⁺ T cells remains unsatisfactory (Blackburn et al., 2008; Im et al., 2016). In conclusion, the combined blockade therapy of multiple IR pathways may be more beneficial for rescuing the function of exhausted T cells. Designing strategies to rescue the subgroups of exhausted CD8⁺ T cells that are intolerant to PD-1 blockade is an important target for the future.

Agonists that target innate immune receptors like TLR7, TLR8, and TLR9 can potentiate type I interferon responses, thereby improving the compromised virus-specific immune functions associated with chronic HBV infection. Stimulation in vitro using the TLR8 agonist GS-9688 (Selgantolimod) prompted the expression of IFN-γ and TNF-α by NK cells, and elevated the proportion of IFN-γ-expressing HBV-specific CD8⁺ T cells in approximately half the CHB patients (Amin et al., 2021). The TLR7 agonist (GS-9620) was used to stimulate human PBMCs. GS-9620 achieved a long-term inhibition of HBV in human hepatocytes through a Type I interferon-dependent mechanism, and it also enhanced the antigen presentation of HBV (Niu et al., 2018). Another clinical trial corroborated the influence of GS-9620 on the reactivity of HBV-specific T cells and NK cells in patients with chronic HBV infection (Boni et al., 2018). Agonists of TLR7/8/9, novel immunomodulators in the oral delivery form, are under preclinical or clinical development. However, limited studies have yet to observe these drugs’ efficacy in reducing serum HBsAg levels.

Dysfunction of B cells can be addressed through targeted therapies to restore their virus-specific responses. Salimzadeh et al. showed that in CD40L-expressing feeder layer cells, B-cell maturation cytokines, like IL-2 and IL-21, can partially restore HBsAg-specific B-cell maturation. Furthermore, they observed that the addition of a PD-1 blocker further promotes the functional recovery of dysfunctional HBsAg-specific B cells and their ability to secrete anti-HBs (Salimzadeh et al., 2018). Although the PD-1/PD-Ls complex is an inhibitor of the B-cell activation cascade, deregulation of immunosuppression by Toll-like receptor 9 (TLR9) agonists blocking the PD-1/PD-Ls pathway enhances B-cell activation, proliferation, and inflammatory cytokine production (Thibult et al., 2013). The resulting data on virus-associated B-cell exhaustion provide potential targets for ameliorating impaired B-cell responses during persistent viral infection.