Chronic hepatitis B virus (HBV) infection remains a major global health burden, largely because current antiviral therapies rarely achieve a functional cure defined by sustained hepatitis B surface antigen (HBsAg) loss [
1]. While extensive efforts have focused on restoring HBV-specific T cell immunity, innate immune components, particularly natural killer (NK) cells, have received comparatively less attention. NK cells are enriched in the liver and represent a critical first line of antiviral defense, yet their precise functional contribution to HBV control in the context of antigen-specific, antibody-mediated responses, especially in the chronic phase, has remained elusive. Elucidating how HBV escapes immunity and understanding dysfunctional immune responses is crucial for advancing effective therapeutic strategies. In this context, the study by Tang et al. [
2] offers timely and important insights by interrogating antibody-mediated NK cell responses and uncovering previously unappreciated heterogeneity within the CD16
hi NK compartment that is associated with HBV outcomes.
NK cells exert antiviral effects through direct cytotoxicity, cytokine secretion, and antibody-dependent cellular cytotoxicity (ADCC), the latter mediated by FcγRIIIa (CD16) [
3]. CD16 enables NK cells to engage the Fc portion of antibodies bound to infected cells, triggering ADCC, granule release, and inflammatory mediator production to eliminate target cells. In acute viral infections, antibody-mediated NK activation can synergize with humoral immunity to accelerate viral clearance. However, in chronic HBV infection, NK cells are widely regarded as functionally altered, exhibiting reduced cytokine production alongside with high expression of inhibitory immune checkpoint molecules [
3-
5]. Most prior studies have relied on cytokine-mediated
in vitro stimulation and less detailed phenotypic analyses, leaving the status of “HBV-specific” antibody-dependent NK responses largely unexplored.
Tang et al. [
2] address this gap through an elegant approach: repurposing a T cell receptor-like antibody specific for the HBV core 18-27 peptide (cTCRL-Ab). Originally developed to visualize peptide-major histocompatibility complexes (MHCs) complexes and deliver interferon (IFN)-α selectively to infected hepatocytes, this antibody also retains an intact Fc domain capable of engaging CD16 on NK cells. By leveraging this tool, the authors establish a system that directly probes HBV-infected cells through recognition of peptide-MHCs and subsequently triggers NK effector functions through CD16 binding, namely cytolytic activity and IFN-γ production. This methodological approach represents a conceptual advance, enabling functional assessment of NK responses in an antigen-specific and Fc-dependent manner. In other words, this tool offers a controlled, antigen-restricted model of Fc-dependent NK engagement that captures key element of antibody-mediated immunity and helps to investigate immune interactions in HBV infection.
Using the cTCRL-Ab system and hepatitis B core (HBc) 18−27 pulsed target cells, the study demonstrates that antibody- mediated NK cell responses are indeed compromised in chronic HBV infection [
2]. Compared with healthy controls, NK cells from chronically infected patients exhibit diminished IFN-γ production in response to cTCRL-Ab engagement, while retaining degranulation capacity. This pattern echoes the well-described “functional dichotomy” of NK cells in chronic HBV, reinforcing the notion that cytokine production is particularly vulnerable to chronic antigenic stimulation and inhibitory signaling. Importantly, these impairments are not static. The authors show that pegylated IFN-α (Peg-IFNα) therapy, long known to modulate NK cell activity [
6], selectively suppresses CD16-dependent, antibody-mediated NK responses. Mechanistically, this suppression is linked to downregulation of CD16 expression, which was demonstrated both
in vivo and
in vitro. At first glance, these findings appear counterintuitive, given the established role of IFN-α in activating NK cells and its association with favorable virological outcomes. However, the work by Tang et al. [
2] underscores a crucial principle: IFN-α does not uniformly “activate” NK cells, but rather reshapes their functional landscape in a receptor- and subset-specific manner.
One of the important contributions of the study from Tang et al. [
2] lies in the use of single-cell RNA sequencing to dissect NK cell heterogeneity. Traditional dichotomization of NK cells into CD56
dimCD16
hi (mostly cytotoxic) and CD56
brightCD16
lo (mostly cytokine producers) subsets appears insufficient to capture their full complexity in chronic HBV infection. Tang et al. [
2] reveal that within the CD16
hi compartment itself, there exist at least two functionally divergent populations with distinct transcriptional and phenotypic signatures. On one hand, the authors identify a CD16
hi-dysfunctional subset characterized by exhaustion-associated features. This population preferentially undergoes CD16 downregulation in response to Peg-IFNα, correlating with reduced antibody-mediated IFN-γ production. On the other hand, they describe a functionally activated CD16
hi subset defined by expression of CX3C motif chemokine receptor 1 (
CX3CR1) and absence of killer cell lectin like receptor C2 (
KLRC2), which is encoding NKG2C: the CX3CR1⁺KLRC2⁻ CD16
hi NK cells. This subset displays high cytotoxic potential, is enriched in the blood of patients with favorable treatment responses, and shows signs of functional restoration in the liver of individuals who achieve HBsAg loss.
This nuanced view reframes the apparent paradox of IFN-α therapy. It seems that rather than globally suppressing antibody-dependent NK function, IFN-α selectively constrains exhausted NK populations while permitting, or even promoting, the expansion and activity of a cytotoxic, treatment-responsive subset. Thus, changes observed at the bulk level, such as reduced CD16 expression or diminished ADCC readouts, may obscure beneficial reprogramming occurring within specific NK cell subsets.
The identification of CX3CR1⁺KLRC2⁻CD16
hi NK cells as a correlate of favorable HBV outcomes is particularly interesting. CX3CR1, the receptor for fractalkine (CX3CL1), has been implicated in the trafficking and survival of cytotoxic lymphocytes. In HBV infection, dendritic cells can secrete CX3CL1 upon HBV exposure [
7], and CX3CR1⁺CD8⁺ T cells have been linked to HBV clearance [
8]. CX3CR1⁺KLRC2⁻ CD16
hi NK cells may preferentially migrate toward CX3CL1-secreting dendritic cells (DCs), thereby favoring DC-NK crosstalk and enhancing NK functions. Thus, the enrichment of CX3CR1⁺ NK cells in treatment responders, together with prior observations, suggests that the CX3CL1/CX3CR1 chemokine axis plays a central role in the immune cell crosstalk, positioning and functional programming during HBV infection.
Noteworthy is also the absence of
KLRC2 (NKG2C) in this subset. Adaptive or “memory-like” NKG2C⁺ NK cells have been extensively studied in viral infections, but their role in HBV remains less clear [
9]. The CX3CR1⁺KLRC2⁻ phenotype identified here points to an alternative pathway of NK cell activation, distinct from classical adaptive or “memory-like” NK differentiation.
Notably, the study extends beyond peripheral blood to intrahepatic NK cells, albeit in a limited cohort. The liver is the primary site of HBV replication and immune-mediated pathology, and intrahepatic NK cells often differ substantially from their circulating counterparts. Tang et al. [
2] report that CX3CR1⁺KLRC2⁻CD16
hi NK cells within the liver tend toward functional restoration in individuals who achieve HBsAg loss. Although longitudinal intrahepatic sampling remains challenging, this observation strengthens the biological relevance of the identified subset and suggests that peripheral signatures, at least in part, reflects events occurring within the hepatic microenvironment.
Collectively, the findings of Tang et al. [
2] have important implications for the design of next-generation HBV therapies. First, they underscore the need to consider antibody-mediated NK functions, not merely natural cytotoxicity, when evaluating immune competence in chronic HBV. Second, they suggest that the effects of IFN-α on NK cells may be more nuanced, emphasizing its role in shaping of specific NK cell subsets. Third, they identify CX3CR1⁺ KLRC2⁻CD16
hi NK cells as a potential biomarker of treatment response and a candidate target for immunomodulatory strategies.
The cTCRL-Ab itself may have translational potential. Beyond serving as a mechanistic probe, it could be adapted to monitor immune restoration during therapy or to screen Fc-engineered molecules designed to enhance NK cell-mediated antiviral activity. In an era increasingly focused on combination therapies, pairing antivirals with immune modulators [
10,
11], such tools may be very promising. The use of cTCRL-Ab could additionally favor NK trafficking and retention towards HBV-infected cells, fostering NK-mediated killing of infected cells and driving favorable outcomes. Moreover, there is increasing evidence for a role of cell metabolism in orienting NK cell function during viral infections [
12]. CD16 engagement has been shown to enhance both glycolytic and oxidative pathways of NK cells through a metabolic rewiring sustained by upregulation of nutrient transporters, whose activity is required for IFN-γ production [
13]. Accordingly, cTCRL-Ab could also promote NK cell function through metabolic reprogramming. Thus, such a multifaced tool offers incredible potential.
Finally, the study by Tang et al. [
2] represents a significant advance in our understanding of NK cell biology in chronic HBV infection. By illuminating the antibody-mediated dimension of NK cell function and uncovering functionally distinct CD16
hi subsets, it challenges prevailing notions of uniform NK cell impairment and highlights the importance of cellular heterogeneity. The identification of a cytotoxic CX3CR1⁺KLRC2⁻CD16
hi NK subset linked to favorable outcomes not only enriches our conceptual framework of innate immunity in HBV, but also opens possible new avenues for therapeutic intervention to restore HBV-associated immune exhaustion. As the field moves toward immunebased cures for HBV, such refined insights into NK cell regulation will be essential for translating immunological knowledge into clinical success.
FOOTNOTES
-
Authors’ contribution
Zuzana Macek Jilkova: Conceptualization, writing of the manuscript, critical revision of the manuscript. Caroline Aspord: Writing of the manuscript, critical revision of the manuscript.
-
Acknowledgements
This work was supported by ANRS-MIE and EFS.
-
Conflicts of Interest
The authors have no conflicts to disclose.
Abbreviations
antibody-dependent cellular cytotoxicity
T cell receptor-like antibody specific for the HBV core 18-27 peptide
CX3C motif chemokine receptor 1
hepatitis B surface antigen
killer cell lectin like receptor C2
major histocompatibility complexes
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