Longitudinal liver sampling in patients with chronic hepatitis B starting antiviral therapy reveals hepatotoxic CD8+ T cells.

Accumulation of activated immune cells results in nonspecific hepatocyte killing in chronic hepatitis B (CHB), leading to fibrosis and cirrhosis. This study aims to understand the underlying mechanisms in humans and to define whether these are driven by widespread activation or a subpopulation of immune cells. We enrolled CHB patients with active liver damage to receive antiviral therapy and performed longitudinal liver sampling using fine-needle aspiration to investigate mechanisms of CHB pathogenesis in the human liver. Single-cell sequencing of total liver cells revealed a distinct liver-resident, polyclonal CD8+ T cell population that was enriched at baseline and displayed a highly activated immune signature during liver damage. Cytokine combinations, identified by in silico prediction of ligand-receptor interaction, induced the activated phenotype in healthy liver CD8+ T cells, resulting in nonspecific Fas ligand-mediated killing of target cells. These results define a CD8+ T cell population in the human liver that can drive pathogenesis and a key pathway involved in their function in CHB patients.

HepG2-NTCP Subclones Exhibiting High Susceptibility to Hepatitis B Virus Infection.

HepG2 cells reconstituted with Hepatitis B virus (HBV) entry receptor sodium taurocholate co-transporting polypeptide (NTCP) are widely used as a convenient in vitro cell culture infection model for HBV replication studies. As such, it is pertinent that HBV infectivity is maintained at steady-state levels for an accurate interpretation of in vitro data. However, variations in the HBV infection efficiency due to imbalanced NTCP expression levels in the HepG2 cell line may affect experimental results. In this study, we performed single cell-cloning of HepG2-NTCP-A3 parental cells via limiting dilution and obtained multiple subclones with increased permissiveness to HBV. Specifically, one subclone (HepG2-NTCP-A3/C2) yielded more than four-fold higher HBV infection compared to the HepG2-NTCP-A3 parental clone. In addition, though HBV infectivity was universally reduced in the absence of polyethylene glycol (PEG), subclone C2 maintained relatively greater permissiveness under PEG-free conditions, suggesting the functional heterogeneity within parental HepG2-NTCP-A3 may be exploitable in developing a PEG-free HBV infection model. The increased viral production correlated with increased intracellular viral antigen expression as evidenced through HBcAg immunofluorescence staining. Further, these subclones were found to express different levels of NTCP, albeit with no remarkable morphology or cell growth differences. In conclusion, we isolated the subclones of HepG2-NTCP-A3 which support efficient HBV production and thus provide an improved in vitro HBV infection model.

The Human Male Liver Is Predisposed to Inflammation Via Enhanced Myeloid Responses to Inflammatory Triggers.

Background & Aim: Men have a higher prevalence of liver disease. Liver myeloid cells can regulate tissue inflammation, which drives progression of liver disease. We hypothesized that sex alters the responsiveness of liver myeloid cells, predisposing men to severe liver inflammation. Methods: Luminex was done on plasma from Hepatitis B Virus infected patients undergoing nucleoside analogue cessation in 45 male and female patients. We collected immune cells from the sinusoids of uninfected livers of 53 male and female donors. Multiparametric flow cytometry was used to phenotype and characterize immune composition. Isolated monocytes were stimulated with TLR ligands to measure the inflammatory potential and the expression of regulators of TLR signaling. Results: We confirmed that men experienced more frequent and severe liver damage upon Hepatitis B Virus reactivation, which was associated with inflammatory markers of myeloid activation. No differences were observed in the frequency or phenotype of sinusoidal myeloid cells between male and female livers. However, monocytes from male livers produced more inflammatory cytokines and chemokines in response to TLR stimulation than female monocytes. We investigated negative regulators of TLR signaling and found that TOLLIP was elevated in female liver-derived monocytes. Conclusions: Our data show that enhanced responsiveness of myeloid cells from the male liver predisposes men to inflammation, which was associated with altered expression of negative regulators of TLR signaling.

Immunomodulation and RNA interference alter hepatitis B virus-specific CD8 T-cell recognition of infected HepG2-NTCP.

BACKGROUND AND AIMS: CD8 T cells are essential in controlling HBV infection. Viral control is dependent on efficient recognition of HBV-infected hepatocytes by CD8 T cells, which can induce direct lysis of infected hepatocytes. In addition, CD8 T cells produce interferon (IFN)-γ, which mediates noncytopathic viral clearance. Innate immunomodulators and HBV-targeted RNA interference (RNAi) are being developed to treat chronic hepatitis B (CHB), but may modify HBV antigen presentation and impact CD8 T-cell recognition, in addition to their primary mechanisms of action. APPROACH AND RESULTS: HBV-infected HepG2-NTCP cells were treated with tenofovir disoproxil fumarate (TDF), Toll-like receptor (TLR) 7/8 agonists, TLR7/8 conditioned media (CM) collected from immune cells, or RNAi using short interfering RNAs. The effect of these treatments on antigen presentation was measured through coculture with CD8 T cells recognizing human leukocyte antigen-A0201 restricted epitopes, HBc18-27 or HBs183-191. Cytokine profiles of TLR7/8 CM were measured using a cytometric bead array. TDF reduced viral replication, but not CD8 T-cell recognition, of infected cells. Direct exposure of infected HepG2-NTCP to TLR7/8 agonists had no impact on T-cell recognition. Exposure of infected HepG2-NTCP to TLR7/8 CM enhanced HBV-specific CD8 T-cell recognition through type 1 interferon (IFN) and IFN-γ-dependent mechanisms. RNAi rapidly suppressed HBV-DNA, HBcAg, and HBsAg expression, impairing recognition by HBV-specific CD8 T cells. CONCLUSIONS: Immunomodulation and RNAi, but not nucleos(t)ide analogues, alter the recognition of infected HepG2-NTCP by HBV-specific CD8 T cells. Understanding these changes will inform combination treatments for CHB.

Mechanisms of HBV immune evasion.

The concept of immune evasion is a longstanding topic of debate during chronic Hepatitis B Virus infection. The 292 million individuals chronically infected by HBV are clear evidence that the virus avoids elimination by the immune system. The exact mechanisms of immune evasion remain undefined and are distinct, but likely interconnected, between innate and adaptive immunity. There is a significant body of evidence that supports peripheral tolerance and exhaustion of adaptive immunity but our understanding of the role that central tolerance plays is still developing. Innate immunity instructs the adaptive immune response and subversion of its functionality will impact both T and B cell responses. However, literature around the interaction of HBV with innate immunity is inconsistent, with reports suggesting that HBV avoids innate recognition, suppresses innate recognition, or activates innate immunity. This complexity has led to confusion and controversy. This review will discuss the mechanisms of central and peripheral tolerance/exhaustion of adaptive immunity in the context of chronic HBV infection. We also cover the interaction of HBV with cells of the innate immune system and propose concepts for the heterogeneity of responses in chronically infected patients.