Single-cell transcriptomic profiling of the intrahepatic immune compartment in chronic hepatitis B infection

Abstract

The intrahepatic immune environment is the primary determinant of disease trajectory in chronic hepatitis B virus infection, yet the cellular and molecular events that govern progression to cirrhosis, hepatocellular carcinoma (HCC), or functional cure remain incompletely understood. Current clinical phase classification relies on peripheral serum biomarkers reflecting both host and viral biology: viral load, alanine aminotransferase, hepatitis B envelope antigen (HBeAg), and hepatitis B surface antigen (HBsAg). Though these biomarkers have clinical utility, they provide limited resolution into the immunological events occurring within the liver. For most chronically infected patients, functional cure is rarely achieved spontaneously and remains elusive even with antiviral therapy, underscoring a fundamental gap between viral suppression and immune-mediated viral control. Closing this gap requires a deeper understanding of the intrahepatic immune states associated with each phase of infection and the specific conditions that distinguish patients who have achieved functional cure from those who have not. Motivated by this, the present dissertation aims to advance the field’s understanding of the functional programming and systems-level coordination of intrahepatic immune populations across the full natural history of chronic hepatitis B. To address this, single-cell RNA sequencing of intrahepatic immune, parenchymal, and stromal cells obtained by liver fine-needle aspiration from patients spanning all clinically defined disease phases was performed and 22 major cell types and 112 distinct transcriptional programs were identified. Chapter two describes the extensive heterogeneity of cell states and programs observed across phases of disease within each broad cell group, underscoring the complexity of the intrahepatic immune landscape relative to what serum biomarkers alone can resolve about the immune response to hepatitis B virus infection. Chapters three systematically examines differential program usage across four clinical axes aligning with the biomarkers used to define clinical phase and demonstrate that each variable independently modulates distinct aspects of intrahepatic immune activity in ways not captured by phase classification alone. High viral load in the absence of hepatic injury is associated with a coordinated, multi-compartment remodeling of innate and adaptive functions: myeloid cells mount broad interferon-stimulated gene expression programs, conventional dendritic cell pro-inflammatory and antigen presentation programs are suppressed, CD8+ effector T cells and NK cells exhibit activation-associated, non-terminal dysfunction, and B cells skew toward a chronically stimulated, atypical transcriptional phenotype. Active hepatitis is instead associated with transcriptional reprogramming rather than compositional change, characterized by loss of NK cell cytolytic capacity despite activation marker expression, tolerogenic myeloid states, and simultaneous engagement of pro-inflammatory and regulatory T cell programs. Loss of HBeAg in the context of active hepatitis is associated with broad myeloid de-escalation of interferon-stimulated programming toward patrolling and surveilling phenotypes. Loss of HBsAg representing the transition to functional cure in patients with maintained viral suppression, is associated with a transcriptionally distinct immune state characterized by redistribution of innate-like sentinel programming from tissue-resident to central memory CD8+ T cells, persistent myeloid and granulocytic signaling activity, and an anti-correlated relationship between adaptive surveillance and innate inflammatory program modules. Chapter four addresses the relationships between these immune programs and the liver-resident stromal and parenchymal populations within the dataset. Using 8,955 hepatocytes, cholangiocytes, liver sinusoidal endothelial cells, vascular endothelial cells, erythrocytes, and platelets from which 26 transcriptomic programs were identified, and significant associations were identified between cholangiocyte senescence programs and HBsAg, and between cholangiocyte viral sensing programs and viral load. Cellular connectivity analysis revealed endothelial and cholangiocyte recruitment of NK cell subsets in high viremia. Cellular connectivity analysis across the HBsAg transition revealed a shift from complement and damage-associated signaling in HBsAg-positive patients toward endothelial fibrinogen and integrin-mediated T cell tissue retention in functionally cured patients. Combined, the work presented in this thesis provides a high-resolution transcriptional map of the intrahepatic niche across the natural history of chronic hepatitis B and identifies program-level immune features with potential implications for patient stratification, treatment timing, and rational design of immunotherapeutic strategies aimed at achieving functional cure.