Clinical implementation of single-cell RNA sequencing using liver fine needle aspirate tissue sampling and centralized processing captures compartment specific immuno-diversity

Abstract

Blood samples are frequently collected in human studies of the immune system but poorly represent tissue-resident immunity. Understanding the immunopathogenesis of tissue-restricted diseases, such as chronic hepatitis B, necessitates direct investigation of local immune responses. We developed a workflow that enables frequent, minimally invasive collection of liver fine-needle aspirates in multi-site international studies and centralized single-cell RNA sequencing data generation using the Seq-Well S3 picowell-based technology. All immunological cell types were captured, including liver macrophages, and showed distinct compartmentalization and transcriptional profiles, providing a systematic assessment of the capabilities and limitations of peripheral blood samples when investigating tissue-restricted diseases. The ability to electively sample the liver of chronic viral hepatitis patients and generate high-resolution data will enable multi-site clinical studies to power fundamental and therapeutic discovery.

Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra-hepatic environment

“…2) were only detectable in the liver, thereby making intra-hepatic studies indispensable for the comprehensive identification of DACS. Recent studies also underscore the importance of interrogating the liver environment to identify distinct DACS specific to the liver environment that may otherwise be missed by exclusively analyzing PBMCs (16, 38, 40–42). Second, we identified a shift in the immunological profile of the liver from an immune-suppressed environment found in CHB patients to a pro-inflammatory, immune-active environment in FC patients.…”

“…These studies allude to a potential function for innate immune cells in the resolution of CHB (11)(12)(13). Even though recent studies have explored the dynamics of the liver environment in different phases of CHB using multiplexed imaging and bulk RNA sequencing (14,15) in biopsies, and fine-needle aspirates (FNA)-based single cell RNA-sequencing (scRNA-seq) (16), the interrogation of DACS in functionally cured patients has remained elusive. This presents a clear unmet need for a comprehensive, high-resolution investigation of key cell types or cell-states and regulatory cell-cell interactions that may modulate underlying mechanisms involved in sustenance of FC.…”

Immunological biomarker discovery in cure regimens for chronic hepatitis B virus infection

“…Phenotyping of lymphocytes from liver biopsies Specific immune subsets are enriched within the liver, and may re-circulate through the peripheral blood, but to what extent the functional or transcriptional phenotypes of these subsets are different in the liver compared to the blood is not yet clear 50,51 (Genshaft et al is unpublished data). We may miss an essential piece of the HBV-specific immune response using only blood for analysis.…”

“…However, due to the nature of collection, peripheral blood contamination can be an issue if the needle penetrates a large vessel, requiring methods to control for contamination. Methods to quantify and assess the level of blood contamination that allow for standardisation of serial FNAs are being developed (e.g OPPT-FNA [optimising practical and processing techniques for FNA]) 51,53 (Genshaft et al is unpublished data). Furthermore, cryopreservation may be possible, but protocols are not yet standardised.…”

Unraveling the Complexity of Liver Disease One Cell at a Time

“… 37 38 39 40 41 42 43 ScRNA-seq combined with serial sampling of the liver via fine-needle aspiration biopsies has been recently employed to longitudinally study hepatic immune diversity during the course of HBV infection. 36 Several studies in humans and mice have also employed transcriptional and epigenetic approaches to examine the cellular programs and effector functions of virus-specific CD8 + T-cells before and after viral antigen clearance. 37 38 39 40 42 Furthermore, the impact of direct acting antiviral therapy for chronic HCV infection has been mapped transcriptionally, revealing treatment-induced alterations in innate immune and interferon signalling.…”