The dual role of regulatory T cells in hepatitis B virus infection and related hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a major contributor to cancer‐related deaths worldwide. Hepatitis B virus (HBV) infection is a major etiologic factor leading to HCC. While there have been significant advancements in controlling HBV replication, achieving a complete cure for HBV‐related HCC (HBV‐HCC) remains an intricate challenge. HBV persistence is attributed to a myriad of mechanisms, encompassing both innate and adaptive immune responses. Regulatory T cells (Tregs) are pivotal in upholding immune tolerance and modulating excessive immune activation. During HBV infection, Tregs mediate specific T cell suppression, thereby contributing to both persistent infection and the mitigation of liver inflammatory responses. Studies have demonstrated an augmented expression of circulating and intrahepatic Tregs in HBV‐HCC, which correlates with impaired CD8+ T cell function. Consequently, Tregs play a dual role in the context of HBV infection and the progression of HBV‐HCC. In this comprehensive review, we discuss pertinent studies concerning Tregs in HBV infection, HBV‐related cirrhosis and HCC. Furthermore, we summarize Treg responses to antiviral therapy and provide Treg‐targeted therapies specific to HBV and HCC.


INTRODUCTION
Hepatitis B virus (HBV) is a member of the Hepadnaviridae family and specifically infects hepatocytes [1], leading to the development of acute or chronic hepatitis B infections.A staggering 250 million individuals worldwide are chronic carriers of this virus [2], experiencing either asymptomatic persistence or progression to severe outcomes, such as liver cirrhosis, liver failure and hepatocellular carcinoma (HCC) [3].Alarming global statistics report 799 000 annual deaths attributed to HBV infection, with a significant proportion of cirrhosis and liver cancer cases being HBV-associated [4].HBV is the most common cause of HCC, with an estimated prevalence of 44%-55% of HCC cases worldwide [5].Particularly in China, a region with high hepatitis B prevalence, over 80% of HCC cases are linked to HBV infection [6].HBV infection constitutes a highly intricate disease that involves intricate immune mechanisms, encompassing both innate and adaptive immune responses, with particular emphasis on the latter, which plays a pivotal role in controlling acute HBV infection.In contrast, chronic HBV infection is characterized by a weakened T-cell response to HBV antigens [7].It is evident that the host immune response assumes a critical function in the pathogenesis of liver inflammation, liver fibrosis and HCC [8].Collectively, HBV infection is accompanied by a compromised immune system function, leading to heightened viral replication and progression of liver disease, ultimately culminating in the development of HCC as a long-term complication [9].
Tregs represent a distinct subset of CD4 + T lymphocytes that serve as key guardians of immune tolerance and are essential in controlling excessive immune activation [10].Tregs can be classified into two subsets based on their origin: natural regulatory T cells (nTregs) and induced regulatory T cells (iTregs) [11].While nTregs arise in the thymus, iTregs are derived from naive CD4 + T lymphocytes under the influence of tolerogenic conditions and various factors, such as IL-10 and TGF-β [12].Initially, the surface marker CD25 (IL-2 receptor α chain) was identified as a hallmark of Tregs [13].Later, the transcription factor Foxp3 was recognized as a specific marker of Tregs in both rodents and humans [14][15][16].These CD4 + CD25 + Foxp3 + Tregs constitute approximately 5%-10% of the total CD4 + helper T (Th) cells [17].The expression of Foxp3 confers functional and phenotypic distinctions between Tregs and non-Tregs [18].
Notably, ectopic expression of Foxp3 in conventional T cells (Tconvs) bestows them with Treg-like suppressive function in vivo and in vitro, indicating that the FOXP3 gene is a master regulator of Treg inhibitory function [14,16].In human studies, Tregs have been identified through the use of cell surface markers CD127 and CD45RA (the naive T cell marker), in addition to CD25 and Foxp3 [19][20][21].Upon stimulation, naive Tregs (CD4 + CD45RA + Foxp3 low CD127 low CD25 low cells) can differentiate into effector Tregs (CD4 + CD45RA À Foxp3 high CD25 high cells), which exhibit heightened suppressive capabilities in vitro [10].Tregs' impairment has been implicated in various autoimmune diseases, including type 1 diabetes [22], arthritis [23] and thyroiditis [24].However, in HCC patients, both circulating and tumourinfiltrating FoxP3 + Tregs were found to be enriched [25], thus exerting suppressive effects on anti-tumour immunity.Given the dual role of Tregs in autoimmune diseases and HBV-related HCC (HBV-HCC), this review aims to elucidate the function and mechanism of Tregs and find possible Treg-targeted therapies in HBV-related liver diseases.

NATURAL HISTORY OF HBV INFECTION
Patients infected with HBV can develop acute hepatitis, ranging from subclinical to icteric hepatitis with yellow staining in the skin mucosa and sclera as the main manifestation and even acute liver failure, which develops in approximately 1% of patients with acute hepatitis B(AHB) and jaundice [26].In most cases, acute HBV infection is self-limited, and the risk of chronicity in immunocompetent adults is not more than 5%.During recovery, HBV surface antigen (HBsAg) is cleared, followed by undetectable HBV DNA from serum [27].However, the risk of chronic hepatitis B(CHB) is remarkably increased in newborns (up to 90%), whose immune system is thought to be immature [28].The natural history of chronic HBV infection is mainly staged according to the characteristics of virology, biochemistry and histology [29,30].The clinical course of CHB can be classified into four stages, reflecting the dynamic relationship between the host immune system and viral replication.The immune-tolerant (IT) or hepatitis B envelope antigen (HBeAg)-positive chronic infection phase is the first phase of CHB, characterized by high HBV DNA, positive HBeAg, normal alanine aminotransferase (ALT) and minimal or no liver damage.The phase is more common and prolonged in perinatally infected subjects.Studies have revealed that events involving the initiation and promotion of HCC, such as HBV integration and clonal hepatocyte expansion, may start in this early phase of chronic infection [31].The second phase is named the 'immune active or clearance HBeAg positive phase'.During this phase, liver fibrosis can develop rapidly, and in some cases, this liver fibrosis can progress to cirrhosis or even liver failure.The second phase is characterized by high levels of HBV DNA.The third phase is the 'low replication phase', which is characterized by the absence of HBeAg and the presence of anti-HBeAg, low serum HBV DNA levels and normal ALT.This phase was termed the 'Inactive HBeAg negative phase'.The final phase is the 'Reactivation phase'.The low replication phase can persist lifelong, but some patients may subsequently develop HBV DNA replication either spontaneously or triggered by active immunosuppression, with or without e-antigen seroconversion, elevated HBV DNA load [7,9,30].Notably, the phases of chronic HBV infection are not necessarily sequential.The prevalence and natural history of hepatitis B virus in patients who did not meet the criteria of the traditional phases (i.e., indeterminate phase) is unclear.Patients in the indeterminate phase are characterized by increased ALT but low HBV DNA levels or increased HBV DNA but low ALT levels.In addition, there was no significant correlation between HBeAg and indeterminate phase (Figure 1a,b) [32].A recent study reported that adults who are HBsAg-negative/HBcAbpositive can still develop cirrhosis or HCC [33].

SUPPRESSIVE MECHANISMS OF TREGS
Tregs play a key role in maintaining peripheral tolerance, limiting the inflammatory response and preventing autoimmune diseases.However, they also restrict beneficial responses such as anti-tumour immunity [34] ligands (PD-L1 and PD-L2), blocking T-cell activation and function [36].(c) CD73 and CD39 are responsible for hydrolyzing extracellular ATP and ADP to AMP (by CD39) and converting AMP to adenosine (by CD73), which binds to the cell surface A2A receptor of effector cells [37].(d) Lymphocyte activation gene-3 (LAG-3) expressed on the surface of Tregs, with a high homology with CD4, binds to MHC II molecules on dendritic cells (DCs) with a higher affinity.This interaction suppresses DC maturation and function [38].T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) is highly expressed on Tregs.Research has demonstrated that the interaction of TIGIT with CD155 on human DCs can suppress DC function [39].T-cell immunoglobulin-3 (TIM-3), a negative regulator of type 1 T helper (Th1) immunity, is expressed by a significant number of tumour-infiltrating lymphocyte (TIL) Tregs.It has been suggested that Tim-3 + Tregs are related to more immunosuppressive activity than Tim-3 À Tregs in head and neck squamous cell carcinoma [40].However, the functional roles of Tim-3 + Tregs in HBV remain unclear.There remain many challenges about whether a few mechanisms are important or whether different mechanisms are required in different diseases.
Contact-independent mechanisms of suppression include Treg secretion of soluble mediators including IL-10, TGF-β and IL-35 [34].IL-2 receptor (IL-2R) is composed of three subunits: alpha chain (CD25, α), beta chain (CD122, β) and gamma chain (CD132, γ).All these subunits participate in high affinity binding through a rapid association rate initiated by the capture of ligand by IL-2Rα [41].Tregs constitutively express high levels of the IL-2Rα chain, thus having a higher affinity for IL-2 and compete for this growth factor with proliferating cells.In addition, Tregs only consume IL-2 without producing it, which induces cell apoptosis [42,43].Exosomes belonging to extracellular vesicles (EVs) are considered mediators of intercellular communication and contain a range of contents, such as lipids, proteins and nucleic acids [44].Yang et al. reported that HBV-positive exosomes transmit HBV to uninfected hepatoma cells and therefore, contribute to its spread [45].In contrast, Li et al. found that interferon-α (IFN-α) induced the transfer of resistance to HBV from liver nonparenchymal cells (LNPCs) to HBV-infected hepatocytes via exosomes [46].In addition, exosomes can transfer IFN-α-related miR-NAs from macrophages to HBV-infected hepatocytes and exhibit antiviral activities against HBV replication and expression [47].Tregs have been demonstrated to release immunosuppressive exosomes.However, an exploration of exosomes secreted by Tregs in the context of HBV has yet to be conducted.Prospective investigations are warranted.In addition, Tregs can release cytotoxic granules (perforin and granzymes), leading to apoptosis of the target cell.Other mediators also play a role in the inhibitory function of Tregs, such as neuropilin-1, amphiregulin and interleukin-34 (IL-34) (Figure 2) [48].immune response.An appropriate immune response can lead to viral clearance and recovery, an excess immune response can lead to liver failure, and an inadequate immune response will result in sustained HBV infection [49].During the acute phase of infection, the majority (>95%) of adult patients are able to clear HBV spontaneously.Understanding the underlying mechanisms of successful host immune responses leading to viral clearance and the underlying mechanism of immune failure in persistent infections is of great importance.Innate immunity is the host's first line of defense against HBV infection.After infection, HBV employs immune evasion strategies to induce little or no innate immune responses [50].Many studies demonstrated that adaptive immune cell responses significantly affect HBV infection clearance during AHB, especially HBV-specific effector CD8 + T cells [51].An early CD4 T-cell response to HBV infection may be necessary to induce the CD8 T-cell response needed to clear the infection [52].During acute resolving hepatitis B, there is a robust, coordinated adaptive immune response with virus-specific CD8 + T cells mediating clearance of infected cells, B cells secreting neutralizing antibodies against HBsAg that block further infection and CD4 + T cells supporting effective viral clearance [53].Statistically, more than 95% of HBV infections are self-limited in adults [54].However, Tregs have an immunosuppressive effect on the HBV-specific T-cell responses, which may result in persistent HBV infection [55].

TREGS IN ACUTE HBV INFECTION
Tregs in patients with HBV have been investigated over the past few years, but the function and frequency of Tregs in these patients remain controversial.The conflicting results may be due to different cell surface markers of Tregs, different stages of HBV infection and patient heterogeneity.Trehanpati et al. revealed that the frequencies of peripheral blood Tregs are higher in AHB patients than in CHB patients and healthy controls (HCs) [56].Peng et al. reported that the frequency of CD4 + CD25 + Tregs in AHB patients was comparable to that in healthy controls, while it was significantly increased in CHB patients [57].Xu et al. revealed that in AHB patients, circulating CD4 + CD25 + Treg frequency was initially low, and with time, the profile reversed to show an increased number of circulating Tregs during the convalescent phase and was restored to normal levels after resolution [58].As discussed above, changes in Tregs in AHB are not consistent.Further studies are needed to explore the role of Tregs in AHB.
Although helper T 17 (Th17) cells are also derived from CD4 + T cells, the function of Th17 cells is different from that of Tregs.Th17 cells play a critical role in host protection from extracellular pathogens [59], and Th17 cells can produce the proinflammatory cytokines IL-17 and IL-22, which are involved in liver damage and viral clearance after HBV infection [60].Another study revealed that the dynamic changes in the frequencies of Th17 and Tregs and the Th17/Treg ratio may be associated with the outcome of AHB patients.Compared with those in the control group, the acute stage group showed significant increases in the frequencies of Th17 and Tregs.Compared with those in the acute stage group, the early recovery group showed a significant reduction in the frequency of Th17 cells, a substantial increase in the frequency of Tregs and a significant decrease in the Th17/Treg ratio.Compared with those in the early recovery group, the full recovery group showed a slight increase in the frequency of Th17 cells, a significant reduction in the frequency of Tregs, which was significantly higher than that in the control group and a slight increase in the Th17/Treg ratio, which showed no significant difference between this group and the control group.In the acute stage of AHB, HBsAg and HBeAg levels were positively correlated with the Th17/Treg ratio [61].

TREGS IN CHRONIC HEPATITIS B VIRUS INFECTION
The role of Tregs in AHB seems different from that in CHB.Scientists have explored the Treg levels in HBVrelated liver disease (Table 1, Figure 1c).Bahabayi et al. showed that circulating Treg levels were elevated and positively correlated with serum HBV load and HBsAg levels in CHB patients [62].In most studies, elevated Tregs are relevant to persistent HBV infection, but the regulatory mechanism of Treg levels remains unclear.In addition, Patients with HBeAg-positive CHB harboured a higher percentage of peripheral blood and intrahepatic Tregs than those with HBeAg-negative CHB [63].HBeAg has been considered to promote chronicity, and this function might be partially accomplished by inducing Tregs.Compared with HCs, Tregs also accumulate in the liver tissues and peripheral blood of CHB patients and are associated with the severity of liver inflammation in CHB patients [64,65].A recent study found that human leukocyte antigen (HLA)-DQ + Tregs have more robust inhibitory functions than HLA-DQ À Tregs.Reducing HLA-DQ + Tregs can enhance the antiviral immune response to clear HBV [66].HLA-DQ, a class of MHC molecules, is constitutively expressed primarily by mature antigen-presenting cells (APCs) [67].It has been reported that a single-nucleotide polymorphism (SNP) of HLA-DQ can influence the clearance of HBV and the development of HCC [68,69].Similarly, Feng et al. found that Tregs increased significantly in the HBV group, but no relationships existed between Tregs and HBV-DNA load [70].
The exact mechanisms underlying Treg upregulation in CHB are still unclear.Recently, in vitro experiments suggested that Furin and transforming growth factor-β1 (TGFβ1) formed a positive feedback loop to activate Tregs.Furin or TGFβ1 knockdown in Tregs promoted Teff cell proliferation, stimulated IL-2 and IFN-γ secretion and inhibited HBV replication.Furthermore, furin or TGFβ1 depletion in Tregs can enhance the killing activity of cytotoxic T lymphocytes (CTLs) against HBV1.3P-HepG2 cells [71].A study showed that the significant source of TGF-β in the liver is hepatic stellate cells (HSCs), which are activated during chronic inflammation [72].Therefore, persistent infection with HBV might contribute to TGF-β production from HSCs and result in the differentiation of conventional CD4 + T cells into iTregs [73].Inhibitors of DNA-binding (Id) are natural negative regulators of E proteins that can initiate the transcription of many genes.Liu et al. reported that inhibitors of DNA-binding 3 (Id3) are favourable for Treg differentiation, which could further inhibit antiviral immunity to promote the chronicity of infection [74].Autophagy, an intracellular process, plays a role in maintaining cell homeostasis and survival [75].Enhanced autophagy was shown to favour Treg expansion and function [76].High-mobility group protein B1 (HMGB1) and its receptors are significantly upregulated in both the peripheral blood and intrahepatic tissue of CHB patients, and HMGB1-induced autophagy can maintain the survival and functional stability of CHB-Tregs [77].In addition, another study showed that Galectin-9 contributes to CD4 + CD25 + FOXP3 + PB HC < CHB 2020 [130] 2021 [80] CD4 + CD25 + FOXP3 + PB, IH HbeAg (À) CHB < HbeAg (+) CHB 2020 [63] CD4 + CD25 + FOXP3 + PB HC > LC 2019 [99] not described PB HC < CHB, LC 2019 [112] CD4 the expansion of Tregs through galectin-9/Tim-3 interaction [78].Additionally, plasmacytoid dendritic cells (pDCs) from HBV-infected patients induced the generation of a higher proportion of CD4 + CD25 + Tregs than those from healthy patients [79].It was recently shown that 5-aza-2 0 -deoxycytidine (5-aza-CdR), a general methylation inhibitor, can induce naive T-cell differentiation towards Tregs by mediating demethylation (Figure 3) [80].The mechanism by which Tregs are regulated still needs to be further explored.Another study documented that no obvious distinction was found in viral-specific Tregs between CHB and asymptomatic HBV carriers (ASCs).However, the Treg/ Th17 ratio was decreased in CHB patients compared with that in ASC patients [84].In contrast to Tregs, Th17 cells can produce the proinflammatory cytokine IL-17 and can participate in liver damage and viral clearance after HBV infection [60].A few studies reported that circulating Tregs and Th17 cells were increased in CHB and related ACLF patients compared with those in HCs.Furthermore, the highest frequency was observed in active CHB patients, while Th17 cells were most abundant in ACLF patients.These studies also found that the Treg/Th17 ratio gradually decreased with the progression of ACLF [70,85,86].Liang et al. found that Treg frequency increased gradually during HBV-ACLF.However, the Th17 frequency gradually increases during progression from CHB to ACLF but decreases markedly from the peak point to the recovery point.In addition, these studies reported that an increased Treg/Th17 ratio was associated with the survival of HBV-ACLF patients [87].Other studies have reported similar findings [88].However, Zhang et al. found that there was a decrease in Tregs with a concomitant increase in Th17 cells in the peripheral blood of patients in the remission stage of ACLF when compared with that of patients in the progression stage, CHB patients or normal controls [89].Although the role of Tregs in HBV-ACLF is not unclear, we can still speculate that Tregs may play a protective role in ACLF to control inflammation and antagonize the proinflammatory effect of Th17 cells.Compared to HCs, the increased Tregs in ACLF seem to be a consequence rather than a driver of the disease process in some studies.An imbalance of Th17 to Tregs may be used as a prognostic marker to predict disease progression.Therefore, more research and evidence are needed in the future.

Tregs in HBV mouse models
Characteristics of the intrahepatic specific T-cell response against HBV in AHB patients have not been studied due The article reported that male mice were treated with a neutralizing anti-CD25 antibody to deplete Tregs.As expected, serum HBsAg levels were significantly decreased after Treg depletion, showing that Treg depletion enhanced HBsAg clearance [92].One study, using an HBV-persistent B6J mouse model, showed that HBsAg-specific Tregs mediate follicular helper T (Tfh)-cell-dependent anti-HBs dysregulation and HBV persistence.In addition, this study also found that HBsAg seroconversion in these mice could be delayed by the adoptive transfer of Tregs [93].In addition, Tregs have been shown to have an anti-cirrhosis effect in a mouse model [94].These findings suggest that the immunomodulatory role of Tregs during HBV infection is a double-edged sword.Tregs prevent severe inflammation and immunopathology but also interfere with viral clearance.

Treg response to antiviral therapy
CHB infection is associated with a compromised antiviral immune response.Tregs were positively associated with serum HBV DNA and HBsAg levels in CHB patients.Currently, antiviral drugs mainly include nucleoside/ nucleotide analogues (NAs) and pegylated interferon (PEG-IFN) [95].Previous studies revealed that IL-2, IFNγ, TNF-α and IL-4 increased in CHB patients after NA treatment, and the numbers of Tregs were reduced [96,97].Nevertheless, Zhang et al. contended that the levels of Tregs identified as CD4 + CD25 + were reduced in the cohort displaying a positive response to lamivudine treatment compared to the non-responsive group.No significant change in Tregs was observed in the entecavir treatment group [98], possibly due to the difference in the specific markers selected for Tregs.Th17 cells mediate nonspecific inflammation, and the Th17/Treg imbalance is associated with disease progression in patients with CHB infection [99].Liu et al. reported that the Th17/Treg (CD4 + CD25 + CD127 dim ) ratio was reduced in the therapeutic response (TR) group that responded to NA therapy, and no differences were found between the TR group and HCs [100].In addition, the frequency of CD25 + CD4 + T cells was reduced following Peg-IFN-α-2b therapy [101].Pan et al. conducted a 96 week follow-up of the HBV-infected patients undergoing antiviral therapy and found that patients with a high HBV viral load had higher levels of Tregs before antiviral therapy than after antiviral therapy [102].Other studies also showed that timely and effective antiviral therapy can continuously inhibit viral replication and antigen production, resulting in a reduction in Tregs and a resumption of the immune response [103,104].In summary, these findings suggest that Tregs inhibit the HBV-specific T cell responses in patients with CHB.Meanwhile, antiviral therapy can downregulate express levels of Tregs and enhance the immune response, which is more conducive to virus clearance.

TREGS IN THE PROGRESSION OF HBV-INDUCED LIVER FIBROSIS
The global prevalence of HBV infection in patients with liver cirrhosis (LC) is 42% [105].Cirrhosis imposes a substantial health burden on many countries and has increased globally level since 1990, partly due to population growth and ageing [4].Many inflammatory factors are expressed during liver cirrhosis.Tregs suppress the activation of T and other immune cells and thus inhibit inflammation [94].Studies have substantiated a strong correlation between elevated Tregs, impairment of CD8+ T cells and diminished survival outcomes in HCC [106].However, the role of Tregs in the development of liver fibrosis (LF) remains controversial.Some studies have reported that Tregs can activate HSCs to promote LF.In turn, activated HSCs upregulate the levels of Tregs via the PGE2/EP2 and EP4 pathways.TGF-β produced by Tregs may also worsen fibrosis by activating HSCs.However, Tregs are likely to be only a minor source of TGF-β in the liver [107,108].In addition, Additionally, many studies have suggested that the Treg/ Th17 ratio is negatively related to the severity of liver fibrosis [110][111][112].Currently, there is limited research on the role of Tregs in HBV-LF.In the future, more studies are necessary to elucidate the exact function of Tregs, especially in the liver microenvironment.

TREGS IN HBV-HCC The interaction of Tregs with adaptive immune cells in HBV-HCC
As immunosuppressive cells, Tregs and their associated factors, such as metabolites and secreted cytokines, mediate the immune tolerance of the tumour microenvironment.A recent study reported that the HBV-HCC microenvironment is more immunosuppressive and exhausted than the nonviral-HCC microenvironment [113].It is well-documented that HCC patients with a high lymphocyte infiltration level in the tumours have a lower risk of recurrence and a better prognosis [114].The proportion of Tregs is elevated in HBV-HCC.This upregulation of Tregs may impede the function of CD8 + T cells, thereby correlating with cancer progression and poor survival outcomes in HBV-HCC [115].In addition, an elevated level of Tregs is accompanied by reduced infiltration of CD8 + T cells in tumour regions compared with nontumour regions.CD8 + T cells in HCC tumour tissues can differentiate into CTLs, which then produce cytokines (IFN-γ, TNF-α) and cytotoxic enzymes (perforin and granulosin B) to clear cancer cells [116].However, the expression of granzyme A, granzyme B and perforin was decreased significantly in tumour-infiltrating CD8 + T cells [115].The exhausted CD8 + T cells eventually weaken the tumour surveillance of the adaptive immune system and lead to the immune escape of tumour cells.
The proportion of Tregs in the HBV-HCC microenvironment was significantly higher than that of non-virus-associated HCC, and the expression levels of inhibitory receptors PD-1 and LAG-3 were higher, showing a more potent immunosuppressive capability [113].Qiu et al. demonstrated that Furin or TGFβ1 knockdown in Tregs promoted Teff proliferation and enhanced the killing activity of CTLs against HCC cells and HBV in vitro [71].The chemokine receptor CCR4 is expressed on Tregs and other T helper cells, binds to its ligands CCL22 and CCL17, and is involved in lymphocyte trafficking [117].Subsequent to HBV infection and during ensuing pathological processes, there is an elevation in the expression of the chemokine CCL22 within the liver microenvironment, facilitating the recruitment of Tregs.Nishida et al. reported that CCR4 + Tregs were the principal type of Tregs in HBV-associated HCC, and were related to sorafenib resistance and HBV load titers.In addition, CCR4 + Tregs can express more IL-10 and IL-35 than CCR4 À Tregs, which enhances their ability to suppress CD8 + T cells [118].The HBV-encoded gene HBx induces substantial IL8 by production through activating MEK-ERK signalling.The activity of IL8 is mainly determined by binding to its receptors CXCR1 and CXCR2.The IL8-CXCR1 axis on the hepatic vascular endothelium (liver sinusoidal endothelial cells, LESCs) promotes the growth and dissemination of HCC by recruiting Tregs in the liver microenvironment [119].Notably, the study also discovered that the expression of CXCR1 was higher and that the IL8-CXCR1 axis increased the percentage of CTLA-4 + Tregs in HBV-positive HCC tissues compared with that in HBV-negative HCC tissues, so the combined targeting of IL8 and CTLA-4 may provide more clinical benefits for HBV-associated HCC.Another study indicated that the CD25, CTLA-4, CXCR3, 4-1BB and OX40 expression levels in Tregs were significantly different among the HBV-, hepatitis C virus (HCV)-and nonalcoholic steatohepatitis (NASH)-related HCC groups.The frequency of Tregs was higher in the HCV-related HCC than in the other two groups, and the CXCR3 expression level in Tregs was lowest in HBV-HCC [120].Exploring the Treg fate in different etiologies of HCC can enhance our understanding of tumour immunity in cancers and will help guide the design of novel immunotherapeutics.In general, Tregs play a vital role in HBVrelated diseases.Thus, an in-depth investigation of factors that regulate Treg functions is crucial for improving the treatment of HBV-HCC and CHB.

The interaction of Tregs with innate immune cells in HBV-HCC
In the liver, a pivotal site for HBV replication, natural killer (NK) cells play a critical role in innate immunity [121].Recent research has revealed a direct regulatory mechanism where CD4 + CD25 + Tregs suppress NK cell-mediated hepatocytotoxicity.This suppression occurs through the involvement of membrane-bound TGF-β (mTGF-β) and interactions between OX40 and OX40L in the context of HBV-associated liver disease [122].Liu et al. demonstrated that MicroRNA-15a/16-1 serves as a key player in preventing Kupffer cells (KCs) from overproducing CCL22.This is achieved by inhibiting nuclear factor-κB, a factor responsible for CCL22 transcription.MicroRNA-15a/16-1 has demonstrated the capacity to impede the function of Tregs by diminishing the binding of CCL22 to C-C chemokine receptor type 4 [123].Consequently, it disrupts the communication between KCs and Tregs, presenting novel ideas for HCC treatment.In a hypoxic microenvironment, tumour tissues exhibit an abundance of triggering receptors expressed on myeloid cell-1 positive (TREM-1+) tumour-associated macrophages (TAMs).Notably, CCR6 has been identified as the sole receptor for CCL20.The TAMs are actively involved in enhancing CCL20 production within the tumour microenvironment.Their presence leads to the recruitment of Tregs that characterized by high CCR6 expression [124].Additionally, Wu et al. reported that at advanced stages of HCC, TREM-1 + TAMs indirectly impair the cytotoxic function of CD8 + T cells, induce CD8 + T-cells apoptosis and promote the recruitment of CCR6 + Foxp3 + Tregs into tumour tissues.This recruitment is achieved by the secretion of the chemokine CCL20, ultimately promoting the progression of HCC [124].
The myeloid-derived suppressor cells (MDSC) constitute a regulatory immune cell population localized within the liver.In the context of HBV infection, MDSCs play a pivotal role by promoting the secretion of immunosuppressive factors such as TGF-β and IL-10.This, in turn, triggers the formation of Tregs.APCs play a crucial role in activating T cell-mediated, antigen-specific adaptive immune responses.Within this context, a member of the B7 family member, B7-H4, is known to exert negative regulation on T cell responses [125].A study conducted by Kryczek et.al shed light on the complex interplay of these immune components.Tregs have been shown to induce APCs to produce high levels of IL-10, which subsequently leads to the upregulation of B7-H4 expression in APCs.This mechanism highlights how APCs can effectively suppress T cell activation through the induction of B7-H4 [126].Additionally, TIGIT, a receptor found on regulatory, memory and activated T cells, comes into focus.Poliovirus receptor (PVR, also called CD155), highly expressed on DCs, bound TIGIT with high affinity.The TIGIT-CD155 interaction enhances the production of IL-10 by DCs, effectively impeding CD4 + T-cell proliferation and function [127].In acute HBV infection, NK cells have been shown to be critical for HBV clearance [128].NK cell dysfunction is associated with impaired CD8 + T cell responses in CHB [129].The research from Ma et al. highlights the impact of HBeAg on immune regulation.HBeAg induces the production of IL-10 in Tregs, subsequently upregulating the expression of NKG2A on NK cells.This event leads to lead to NK cell exhaustion, which, in turn, suppresses the antitumour immunity of organisms and allows evasion of immune surveillance in HCC (Figure 4) [130,131].

THERAPIES TARGETING TREGS IN HBV OR HBV-HCC
Tregs are central to maintaining peripheral tolerance and have attracted considerable attention as a potential therapeutic avenue for averting autoimmunity and promoting transplantation tolerance [132].However, in the context of HCC, immune suppression can facilitate the immune escape of tumour cells.Over the past years, Tregs have been implicated in the suppression of virus-specific immune responses, thereby providing a mechanism for the persistence of HBV.As discussed above, HBV and HBV-HCC are the two different stages of HBV infection.If not treated, the patients infected with HBV can progress to HBV-HCC.Treg plays a dual role in different periods, and the intervention method and timing are very important.Therefore, our primary focus involved an extensive discussion on the potential roles of Tregs at various stages of the disease.This deliberation aimed to ascertain whether Tregs function as allies, adversaries or bystanders in the pathological processes.For instance, Tregs behave differently during acute and chronic HBV infection.The depletion of Tregs during AHB has been shown to impede its transition to a chronic state [18].However, down-regulated Tregs in chronic hepatitis may progress to liver failure.Moreover, Tregs and associated factors contribute to forming an immunosuppressive tumour microenvironment of HCC, thus highlighting the potential of targeting Tregs to inhibit HCC progression.Immune checkpoint inhibitors (ICIs) are designed to counteract immune checkpoints that restrain immune cell activity, exhibiting anti-tumour effects.Several immune checkpoints, such as TIM-3, TIGIT and LAG-3 are expressed in Tregs, offering promising avenues for future research in HCC treatment aimed at inhibiting Treg function [133,134].Furthermore, it is worth noting that non-virally associated HCC generally exhibits higher levels of IFN-γ, IL-17, Granzyme B and TNFα, while virally-associated tumours display increased PD-1 expression on T cells.This heightened PD-1 expression is indicative of a more suppressive environment fostered by HBV infection [113].
Research indicated that Tregs induce immune suppression via the secretion of immunosuppressive cytokines such as IL-35, IL-10 and TGF-β.IL-35 can restrain the proliferation of HBV-specific CTL cells and IFN-γ secretion in vitro.Importantly, IL-35 has been associated with HBV-related hepatic complications, including CHB, cirrhosis and HCC [135].Elevated levels of IL-10 have shown connections to HBV serum titers and the degree of liver inflammation [136].Recent research indicates that TGFβ1 knockdown in Tregs can effectively inhibit HBV replication in vitro [71].Henceforth, endeavours aimed at inhibiting the expression of IL-10, IL-35 and TGFβ1 present a promising avenue for reinstating the compromised functionality of Teffs.Furthermore, a recent study highlighted that CCR4 + Tregs represent the predominant Tregs subtype recruited to HBV-HCC.Their presence is associated with sorafenib resistance and HBV load titers.Targeting intratumoural CCR4 + Tregs has demonstrated promise in overcoming sorafenib resistance and sensitizing tumours to immune checkpoint blockade in mouse models of liver cancer [118].This approach exhibits the potential for targeted depletion of intratumoural Tregs, mitigating the risk of pronounced autoimmunity.
Emerging research has revealed the impact of metabolic pathways on Treg generation and function.Recent studies demonstrated that blocking glycolysis can promote the generation of Tregs through the hypoxiainducible factor 1α (HIF1α).Notably, the concentration of amino acids has been found to affect the differentiation and function of Tregs.The findings provide novel avenues for potential therapeutic approaches during viral infection [137,138].In the context of CHB patients with type 2 diabetes mellitus, IL-15 was observed to be downregulated.IL-15 can suppress Treg function and inhibit the expression of immune checkpoint molecules in Tregs [139].Moreover, Huang et al. have reported that exosomal circGSE1 originating from HCC cells contributes to the progression of HCC by inducing the expansion of Tregs.This occurs through the regulation of the miR-324-5p/TGFBR1/Smad3 axis [140].The discovery of exosomal circGSE1 potential as a biomarker holds promise for future HCC immunotherapy.Other findings may open up new avenues for the development of therapeutic strategies aimed at enhancing specific anti-HBV immunity by modulating Treg autophagy [77].
In conclusion, Tregs have emerged as key players in the maintenance of HBV infection, HBV-related cirrhosis and HCC.This underscores the urgency of investigating strategies targeting Tregs molecules and the modulation of Treg activity during specific phases of HBV infection and HCC.Based on the above discussion, restoring the T-cell immune response is key to treating hepatitis B virus.Fisicaro et al. reported that combined blockade of PD-1 and activation of CD137 (CD137L) increased responses of intrahepatic HBV-specific T cells.PD-1 blocking could reduce Treg suppressive activity, and an anti-CD137 agonist antibody can induce a decrease in the Treg number [141].A phase II study revealed the promising therapeutic potential of the toll-like receptor 8 (TLR8) agonist GS-9668 for HBV infection.In vitro treatment of peripheral blood mononuclear cells isolated from patients with CHB with GS-9688 induced a decrease in Tregs frequency (NCT03615066) [142].TLRs are crucial for immune response induced by some viral infections, including respiratory syncytial virus and hepatitis C virus [143].TLR8 is important for inducing TNF-α production that corresponds to recovery [144].Boni et al. proposed a phase II study through a combination of GS-4774 and tenofovir for the treatment of adults with CHB (NCT02174276).The yeast vector-based GS-4774 vaccine contains HBV antigens.Both in vivo and in vitro experiments showed enhanced specific CD8 + T cell responses after vaccination with GS-4774 [145].Whilst, the yeast component promotes the differentiation of natural T cells to Th17 cells, but not Tregs, resulting in a decrease in Treg frequency [146].Therefore, delving into the regulatory mechanisms governing Tregs holds significant promise as a prospective avenue of investigation in CHB.Such exploration contributes to the advancement of immunotherapeutic strategies aimed at targeting Tregs.Novel ICIs targeting molecules such as anti-TIM-3, anti-TIGIT or anti-LAG-3 have demonstrated the ability to restore the functionality of tumour-infiltrating T cells in vitro settings [147] and are currently undergoing evaluation in early-stage clinical trials.Furthermore, depleting Tregs in patients with solid tumours via administration of an anti-CCR4 monoclonal antibody (mAb) is being explored in a phase IB clinical trial(NCT01929486), revealing the potential to enhance anti-tumour immunity in select patients [148].Cyclophosphamide has shown promise in reducing Treg frequencies.A phase III clinical trial assessing Tregs reduction through cyclophosphamide administration in advanced HCC is currently underway (NCT00396682) [149].Another candidate is OX40, a costimulatory molecule of the tumour necrosis factor receptor superfamily that is constitutionally expressed by Tregs, is being investigated in an ongoing phase I clinical trial.This trial demonstrates that an agonistic anti-OX40 monoclonal antibody mediates anti-tumour effects by mitigating Tregs-mediated immune suppression(NCT02315066) [150].The glucocorticoid-induced tumour necrosis factor receptor-related protein (GITR), a costimulatory molecule expressed at low levels by resting CD4 + and T A B L E 2 Potential therapy targeting Tregs in the management of CHB and HCC.

CD137L
Activation of CD137 induces a significant decrease in Treg number. [141] GS-9688 GS-9688 induces a decrease in Tregs frequency.[142] GS-4774 GS-4774 promotes the differentiation of natural T cells to Th17 cells, resulting in a decrease in Treg frequency.
[145] HCC CCR4 CCR4 antagonist can block Treg accumulation in HCC and enhance therapeutic efficacy to PD-1 antibody and sorafenib. [148] Cyclophosphamide Cyclophosphamide decreases the frequency and suppressive function of circulating Tregs.
CD8 + T cells and at high levels by Tregs, is another promising candidate.Activating GITR signalling with an agonistic anti-GITR mAb or GITR ligands can inhibit the suppressive activity of Tregs.A phase IB study is evaluating the safety and efficacy of GITR agonist antibody, both as a monotherapy and in combination with Gemcitabine, Pembrolizumab or Nivolumab in patients with advanced solid tumours (NCT02628574) [151].
Another study also showed that Treg-mediated inhibition of IFN-γ production and cytotoxicity of CD8 + T cells can be partially reduced by anti-PD-1 and anti-PD-L1 antibodies in HCC (Table 2) [152].In the future, illustrating the underlying mechanisms of Tregs in HCC pathogenesis is important to find new therapeutic targets.

CONCLUSION
Collectively, the role of Tregs in hepatitis viral infections appears to exhibit a dual nature.On one hand, Tregs mediate targeted suppression of T cells in the context of HBV infection, potentially contributing to viral persistence, while simultaneously safeguarding against excessive liver damage.However, it remains uncertain whether these divergent roles can be ascribed to the same Treg populations or if they involve distinct Treg subpopulations.Moreover, the underlying mechanisms governing these distinct mechanisms mediate these disparate functions and require further investigation.A promising avenue for enhancing HBVrelated disease management involves the specific targeting of Tregs and a refined understanding of the optimal time frame for such intervention.In addition, Tregs have the ability to shape a tumour-prone immune microenvironment for HCC formation by weakening the immune surveillance function of the innate and adaptive immune systems.Consequently, strategies aimed at inhibiting Treg function and promoting an enhanced immune state within the tumour microenvironment hold considerable promise for the treatment of HCC.Exploring the modulation of Treg activity and its potential impact on HCC treatment merits further research and consideration.
. Therefore, defining the mechanisms of Treg function is clearly of crucial importance.The mechanisms by which Tregs are involved in the immune response can be divided into two main categories: contact-dependent and contact-independent.The former includes the following: (a) Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is expressed on Tregs and effector CD4 + and CD8 + T cells and is a coinhibitory molecule.Its interaction with ligands affects the function and activation of effector T cells (Teffs, CD4 + CD25 À T cell) [35].(b) Programmed death-1 (PD-1) delivers a negative signal when interacting with its negative/anti-HBeAg positive F I G U R E 1 Nature history of hepatitis B virus infection with Treg levels at different stages.(a) Self-limited acute hepatitis B virus (HBV) infection.A robust response of CD4 + T cells and CD8 + T cells is elicited to eliminate HBV.(b) Chronic HBV infection.Four stages are delineated, including the 'immune tolerant' phase marked by a high HBV DNA and low-inflammation, the 'immune active' phase characterized by HBV-specific CD8 + T cell response and antibody production resulting in chronic liver inflammations, the 'immune inactive' phase featuring diminished HBV DNA and limited inflammation, and the 'immune reactive' phase wherein chronic hepatitis progressed to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC).(c) Treg levels in acute hepatitis B (AHB), chronic hepatitis B (CHB), acute-on-chronic liver failure (ACLF), liver cirrhosis (LC) and HCC.Both increased and decreased Treg levels are observed in AHB.Tregs are upregulated in CHB and HCC.Tregs are predominantly downregulated in LC and ACLF.
HBV does not directly cause liver cell injury.The outcomes after infection are closely related to the hostF I G U R E 2Mechanism of immune function of Tregs.In the realm of contactdependent mechanisms, Tregs suppress DC function through CTLA4, LAG3 and TIGIT.PD-1 attenuates T-cell activation and expansion after binding to PD-L1.CD39 and CD73 on the Treg surface mediate the conversion of proinflammatory extracellular ATP into immunosuppressive extracellular adenosine.Galectin-9 contributes to the expansion of Tregs through galectin-9/ Tim-3 interaction.Contact-independent mechanisms involve molecules such as CD25, IL-10, TGF-β and IL-35.Extracellular vesicles (Evs) are recognized as pivotal mediators of intercellular communication between Tregs and Teffs.Tregs can release cytotoxic granules (perforin and granzymes), leading to apoptosis of the target cells.
T A B L E 1 Changes in Tregs in HBV-related liver disease in human.Markers (Tregs) Position Frequency Citation CD4 + CD25 + PB ACLF < HC, CHB 2021 [83]CD4 + CD25 + FOXP3 + PB HC < ACLF < CHB 2021[85] Treg activity in acute on chronic liver failure CHB can progress to acute-on-chronic liver failure (ACLF).ACLF is characterized by acute deterioration of liver function, coagulopathy and subsequent multiple organ failure with 28-day mortality in the Asia-Pacific and African regions[3,81,82].A recent study revealed that HBV-related acute-on-chronic liver failure (HBV-ACLF) patients had significantly decreased Tregs compared with those of HCs and CHB patients.Furthermore, the study suggests that the model for end-stage liver disease (MELD) score was negatively correlated with CD4 + CD25 + Tregs[83].This characteristic may help to predict HBV-ACLF severity and indicate a prognosis response to guide the treatment of HBV-ACLF.

F I G U R E 3
Mechanism of Tregs elevation in chronic hepatitis B. High mobility group box 1 protein (HMGB1) initiates autophagic processes to sustain Treg activity; Elevated levels of Furin, TGFβ1, Galectin-9, Id3, and 5-aza-CdR enhance the immunosuppressive function of Tregs.Plasmacytoid dendritic cells (pDCs) contribute to the expansion of Tregs.to the risk of complications associated with liver biopsies.Therefore, research using animal models is needed.Stross et al. observed a rapid escalation in the abundance of Tregs within the liver subsequent to the initial phase of HBV replication in a mouse model.Tregs alleviated immune-mediated liver damage by attenuating the antiviral activity of effector T cells but did not affect the development of HBV-specific CD8 + T cells[90].Kosinska et al. reported that male mice showed functionally suppressed woodchuck hepatitis virus-specific CD8 + T-cell responses in the liver and notably higher numbers of intrahepatic Tregs than females.Subsequently, they found that functional blockade of Tregs by anti-CD25 antibody during transient HBV infection contributed to increased numbers of CD8 + T cells.This increase in CD8 + T cells was related to a significant reduction in woodchuck hepatitis virus (HBV/WHV) viral loads.Given the role of Tregs in the immune response, some studies have focused on efforts to deplete Tregs to improve the immune response in mice.For instance, highly effective depletion of Tregs in depletion of regulatory T cells (DEREG) mice has been shown to improve HBV-specific T-cell responses and accelerate virus clearance, although at the expense of increased liver damage[91].A similar result was found in another study.

IL- 10
produced by Tregs has an antifibrotic effect by inhibiting the activation and proliferation of HSCs [109].Instead, Deng et al. reported that Tregs had an antifibrotic function by increasing the anti-cirrhosis activity of human amniotic mesenchymal stem cells (hAMSCs) [94].The different Treg identification, species (human/mouse) and disease models might partially explain the different roles of Tregs in LF.As discussed above, Tregs and Th17 cells have a common origin and belong to CD4 + T-cell subsets.Tregs and Th17 cells have antagonistic effects on each other during inflammation.A study revealed that the frequencies of CD4 + CD25 + Tregs and Th17 cells significantly increased.Elevated Tregs are induced to antagonize Th17 cells.A further study found that Tregs directly downregulated the pro-fibrotic features of HSCs [64].Lan et al. found that circulating Tregs were reduced in LC patients compared with those in HCs, and Tregs and Th17 cells were negatively correlated [99].

F I G U R E 4
Interaction between Tregs and other cells in HBV-related liver disease.Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) play a pivotal role in the anti-tumour immune response.Their efficacy is hindered by Tregs.Liver Sinusoidal Endothelial Cells (LSECs), Hepatic Stellate Cells (HSCs) and Myeloid-Derived Suppressor Cells (MDSCs) actively participate in fostering the expansion of Tregs.Within the liver tumour microenvironment, the recruitment of Tregs is facilitated through the secretion of chemokines, specifically CCL22 and CCL17.