Abstract

Hepatitis B virus (HBV) infection is restricted to the liver where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage have relied almost solely on animal models and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome practical obstacles of liver sampling using fine-needle aspiration (FNA) and develop an optimized workflow to comprehensively compare the blood and liver compartments within chronic hepatitis B (CHB) patients using single-cell RNA sequencing (scRNAseq). We developed a workflow that enabled multi-site international studies and centralized scRNAseq. Blood and liver FNAs were collected, and cellular and molecular capture were compared between the Seq-Well S³ picowell-based and the 10x Chromium reverse-emulsion droplet-based scRNAseq technologies. Both technologies captured the cellular diversity of the liver but Seq-Well S³ effectively captured neutrophils, which were absent in the 10x dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver FNAs captured a heterogeneous liver macrophage population. Comparison between untreated CHB patients and patients treated with nucleoside analogues showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.