Nearly 300 million people around the world have chronic hepatitis B infection (1), which might gradually develop into later-stage liver diseases like liver fibrosis, cirrhosis, and cancer. Therefore, CHB represents a key stage for medical interventions to prevent subsequent development of severe liver diseases. The Hepatitis B virus consists of the core protein (HBcAg, nucleocapsid), hepatitis B e antigen (HBeAg), hepatitis B surface antigens (HBsAg, including the large, medium, and small envelope proteins), and the X protein (HBx). Host DNA repair mechanisms convert incoming HBV double-stranded DNA into covalently closed cyclic DNA (cccDNA), which are viral persistence reservoirs and a key obstacle to the complete cure of chronic hepatitis B (2, 3). CHB infection is classified into four clinical phases according to serum HBV DNA load, alanine aminotransferase (ALT) levels, and HBeAg status: the HBeAg-positive immune tolerant (IT) and immune active (IA) phases, as well as the HBeAg-negative inactive carrier (IC) and hepatitis phases (ENEG) (4). Hepatitis B virus (HBV) replicates in hepatocytes, but it’s not cytopathic by itself. HBV associated liver damage is due to anti-HBV immune response in the liver (5). Persistent CHB infection is the consequence of interactions between HBV and the host’s innate and adaptive immune systems.

When HBV has entered the host cells, pattern recognition receptors (PPRs), like Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and NOD-like receptors (NLRs), recognize pathogen-associated molecular patterns (PAMPs) from HBV, resulting in activation of innate immune cells (e.g., NK cells, macrophages, dendritic cells, monocyte), stimulation of signaling pathway (e.g., Janus kinase-signal transducer and activator of the transcription signaling pathway, NF-κB signaling pathway), and production of chemokine, proinflammatory cytokines and interferons (IFNs), together to form innate defenses against HBV (6, 7). Innate immune response is vital for the early control of HBV infection, as well as for generating HBV-specific adaptive immune response (8). CD8⁺ T cells are the crucial cellular effectors contributing to HBV clearance from the liver with the help of NK cells (9–11). CD8⁺ T cells and B cells encounter HBV antigens presented by antigen-presenting cells (APCs), such as Kupffer cells (KCs), dendritic cells (DCs), and liver sinusoidal endothelial cells (LSECs), leading to the generation of HBV-specific CD8⁺ T cells and anti-HBV antibodies -producing B cells (12, 13). On the other hand, CD4⁺ T cells play a crucial role in facilitating CD8⁺ T cell activation and immune memory responses during HBV infection (14).

However, HBV usually escapes innate and adaptive immune surveillance and establishes persistent infection. HBV reduces the expression of Toll-like receptor 3 (TLR3), melanoma differentiation-associated protein 5 (MDA-5), and retinoic acid-inducible gene I (RIG-I) in DCs and hepatocytes, which reduces responsiveness to PAMPs from HBV and impairs IFN-I production (15). In addition, HBV infection led to reduced expression of TLR2 on hepatocytes in the liver of HBeAg-positive CHB patients, which is associated with a functional reduction in cytokine production (16). HBV also escapes immune recognition by RNA adenosine deaminase ADAR1-dependent viral RNA editing in hepatocytes (17). Furthermore, CHB infection causes functional exhaustion of T cells. For example, HBcAg increases PD-1 expression on CD4⁺ T cells and disturbs the function of CD4⁺ T cells through the ERK, JNK, and PI3K/AKT signaling pathways (18). CHB -associated CD8⁺ T cells are also exhausted and reduced secretion of IL-12,IFN-γ, and TNF-α, as well as cytotoxic activity (19). Moreover, HBV infection led to increased expression of the proapoptotic molecule Bcl-2-like protein 11 in HBV-specific CD8⁺ T cells, possibly leading to CD8⁺ T cell clonal depletion during CHB infection (20). Finally, the humoral immune response is impaired in CHB, as evidenced by fewer HBsAg-specific B cells in CHB patients (21). In addition, Kupffer cells in HBV -carrier mice also mediate humoral immune tolerance by IL-10 production (22). Together these studies demonstrated the compromised anti-HBV immune response in CHB.

NK cells are heterogeneous. In mouse, liver NK cells contain two distinct subsets, the CD49a^-CD49b⁺ conventional NK cells (cNK) and CD49a⁺CD49b^- liver resident NK cells (lrNK) (25). Each accounts for about half of total NK cells in the liver. cNK circulates in the blood, whereas lrNK mainly resides in the hepatic sinusoids (26). Compared with cNK, lrNK display distinct phenotypes, such as expression of CXCR6 and TRAIL. Besides, cNK and lrNK originate from distinct progenitors. cNK originate in the bone marrow, while lrNK are generated from Lin^-CD122⁺CD49a⁺ progenitors in the fetal liver (27). Similarly, human NK cells include the liver resident NK cell subset. However, their phenotypes and transcription factor requirements are different from those of mouse NK cells. In humans, lrNK could be identified by CXCR6⁺CD49E^- markers (28, 29).

NK cells recognize “stressed” cells, such as infected or tumor cells, without major histocompatibility complex restriction or prior sensitization for cytotoxicity and cytokine secretion. NK cells can recognize and display cytolytic activity against virally infected cells by release of lytic granules, or by inducting apoptosis of infected cells by death receptors (30). In addition to cytolytic activity, NK cells are major producers of interferon-γ (IFN-γ) early in the innate immune response (31), as well as producers of tumor necrosis factor-α (TNF-α), interleukin (IL)-10, granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) (31). Furthermore, NK cells secrete chemokines, such as lymphotactin (XCL1) and C-X-C motif chemokine ligand 8 (CXCL8, IL-8) (32), which is key to their colocalization with other hematopoietic cells in areas of inflammation (33).

NK cells mediate their antiviral effects through the release of cytokines (e.g., IFN-γ, TNF-α), secretion of cytolytic granules for lysis of infected cells, and induction of target cell apoptosis through crosslinking of cell surface death receptors (e.g., FasL, TRAIL) (34). Among NK cell subsets, lrNK play an antiviral role in the early stage of host infection (35). lrNK produce less IFN-γ than cNK in human and in mice (36, 37), but lrNK produce IFN-γ more rapidly than cNK in response to murine cytomegalovirus (MCMV) infection (35). In addition, lrNK secrete more TNF-α and GM-CSF than cNK following in vitro stimulation (37). Murine lrNK expressed higher levels of TRAIL and FasL than cNK, but expressed lower levels of granzyme B (GzmB) and perforin (37). In addition to NK –intrinsic functions, NK cells can also cross-talk with dendritic cells (DC) by cytotoxicity against immature dendritic cells and by interacting with DCs via proinflammatory cytokines and chemokines (32, 33). Tissue-resident XCR1⁺ conventional dendritic cells (cDC1) can crosstalk with lrNK to limit early viral load (35).

NK cells express the germline-encoded inhibitory receptor, which recognizes “self” -expressing major histocompatibility complex (MHC) class І. NK cells also express activating receptors that recognize “stressed” cells, such as infected or tumor cells. In addition, NK cells express cytokine receptors to type I interferons (IFN-α and IFN-β), interleukin-15 (IL-15), IL-21, IL-18, and IL-12 for activation, as well as receptors to immunosuppressive factors, such as transforming growth factor-β (TGF-β) and IL-10.

The activation of NK cells is determined by the balance between activating signals and inhibitory signals input from cell surface receptors. NK cell activating receptors include natural cytotoxicity receptors (NCR, e.g., NKp30, NKp44, and NKp46), lectin-like receptors (e.g., NKG2D and NKG2C), IgG Fc receptors, and SLAM family receptors (e.g.,2B4, CRACC, and NTB-A) (38). NCRs are immunoglobulin-like type I transmembrane glycoproteins and mediated NK cell activation by the immunoreceptor-based activation motif (ITAM) motif. NKG2D is expressed on NK cells, γδ T cells, and CD8⁺ T cells. Human NKG2D interacts with DAP10 for NK cell activation (39). NK cells respond to “stressed” cells not only directly via activating receptors of stressed-induced ligands but also via the activating Fc receptor CD16, which recognizes a variety of targets containing antigens that IgG can bind. The initiation of NK-cell effector functions through CD16 depends on transmembrane signaling adaptors, such as FcϵRIγ (also known as FcRγ) and CD3ζ expressed by mature NK cells (40). SLAM-related receptors include 2B4, NTB-A, and CRACC. Src-family kinases phosphorylate SLAM family receptors in the immunoreceptor tyrosine-based switch motifs (ITSM) (41), which is required for further association with SAP-related adapters family. SAP is crucial for human NTB-A and 2B4-mediated NK cell activation (42). However, SLAM family receptors can also negatively regulate NK cell functions (42). In SAP-deficient human NK cells, 2B4 and, possibly, NTB-A turned into inhibitory receptors (43).

In addition to cell surface receptors, NK cell activation is also stimulated by cytokines such as type I IFN, IL-12, IL-15, and IL-18, which are usually produced in response to virus infection (44, 45).Moreover, the interaction with accessory cells also modulates NK cell functions. For example, DC -derived IL-12 promotes both IFN-γ; production and cytotoxicity of NK cells (46).

NK cells represent a major immune cell type in the liver, making up to 50% of hepatic lymphocytes and to an even higher frequency in the livers of patients with CHB infection (47–49). NK cells are essential for controlling CHB infection not only by direct antiviral functions but also by orchestration of adaptive immunity.

Although NK cells possess anti-HBV potentials, CHB infection induces NK cells into an exhaustion-like status with reduced NK cell percentages in the peripheral blood (58), with dominating expression of inhibitory cell surface receptors, and with decreased effector functions, underlying the dysfunctional host immune responses to HBV in these patients (24, 59).

NK cells not only control virus replication in the liver, but also could cause liver injury when NK cells become over activated in some contexts (5, 71, 72). Activated NK cells aggravate hepatic inflammation and stimulate hepatocyte death by secretion of inflammatory cytokines and by TRAIL-mediated apoptosis of hepatocytes in chronic hepatitis B patients (73, 74). NK cells also produced IFN-γ and TNF-α to exacerbate liver injury (74), and increased Fas ligand (FasL) or NKG2D ligand expression on hepatocytes to promote apoptosis (70, 75). Therefore, although NK cells contribute to virus control, they might also be pathogenic in CHB depending on the context.

The activity of NK cells is overall inhibited during CHB infection (101). Immune-checkpoint inhibitors, whose therapeutic value has been revealed in the field of cancer therapy, could potentially relieve suppression of NK cells’ activity also in CHB by preventing inhibitory signaling through inhibitory checkpoint receptors. Blockades of inhibitory receptors like NKG2A, Tim-3, and Siglec-9 have been shown to promote NK cell function in CHB infection (53, 62, 102) ( Table 2 ). For example, NKG2A⁺ NK cell is dysfunctional in HBeAg-positive CHB patients (64). Blocking NKG2A interaction with HLA-E increased in vitro cytolytic activity of peripheral NK cells from patients with active CHB (53). Similarly, Tim-3 expression is remarkably increased in circulating NK cells from CHB patients (62). In vitro cytotoxicity by PBMCs or NK cells from CHB patients was enhanced in the presence of Tim-3 blockers (62). Moreover, inhibitory receptor Siglec-9 is commonly expressed on CD3⁻CD56^dim NK cells. Blocking Siglec-9 on NK cells among PBMCs from HBsAg-positive CHB patients significantly improved the production of IFN-γ, TNF-α, and CD107a of NK cells (102), indicating that blocking Siglec-9 may restore the dysregulation of NK cells in HBsAg-positive CHB patients. Therefore, the blockade of immune checkpoint signaling may relieve the negative regulation of NK cells and revive the antiviral function of NK cells for CHB control.

Immunosuppressive cytokines contribute to NK cell receptor imbalance and NK cell dysfunction during CHB infection. TGF-β1 reduced the expression level of NKG2D/DAP10 and 2B4/SAP in patients in the immune tolerance (IT) phase of CHB infection, whose NK cells displayed impaired function (61). Neutralization of TGF-β1 restored the level of NKG2D and 2B4 on NK cells in vitro (61). Neutralizing IL-10 restored IFN-γ production by CD56^bright and CD56^dim NK cells in patients with active CHB infection (68), whereas in CHB patients without IL-10 elevation or noticeable liver inflammation, only blockade of both IL-10 and TGF-β restored NK function (68). Modulation of the cytokine milieu has shown potentials in cancer treatment (103), which might also be applied for treating CHB. Therefore, targeting immunosuppressive cytokines represents an intervention strategy to boost NK cell-based immunotherapy for CHB infection.

Protection from potential liver injury is also important for designing anti-HBV NK -based immunotherapy. Blocking chemokines responsive to γ–2/IFN-γ inducible protein 10/C-X-C motif chemokine ligand 10(CRG-2/IP-10/CXCL10) and monokine induced by interferon-γ/C-X-C motif chemokine ligand 9 (Mig/CXCL9) in vivo relieves liver injury while preserving the antiviral potential of HBV–specific cytotoxic T lymphocytes (96), suggesting that these are targets for liver protection in immunotherapy for CHB. Furthermore, combing NK-based immunotherapy with liver -protective drugs might prevent NK cell -mediated liver injury while preserving its antiviral functions. Vitamin C consumption attenuated liver injury of CCl4-treated mice by normalizing metabolism and relieving inflammatory responses in the liver (104), while Vitamin C is also an antioxidant that may stimulate or at least maintain the activation of NK cells (105, 106). Vitamin B is a conventional liver-protective drug, and it has been used clinically for the treatment of chronic hepatitis B (107), which, like vitamin C, could also promote the activation of NK cells (108).