Single cell RNA sequencing reveals naïve T cells ready for effector function in livers of Primary Sclerosing Cholangitis

Question:

Although genetic associations point towards immune dysregulation as part of the pathogenesis of Primary Sclerosing Cholangitis (PSC), the role of specific immune cell populations within the liver of patients with PSC remains elusive. Here, we report on a comprehensive phenotypic and functional investigation of the liver infiltrating T cells of patients with PSC.

Methods:

Liver infiltrating and circulatory T cells were isolated from patients with PSC (n = 16). As controls we used T cells isolated from liver tissue and blood from patients undergoing transplantation for alcoholic liver disease (ALD, n = 16) and from blood of healthy donors (n = 10). Liver infiltrating T cells were sorted and processed for single cell RNA sequencing (scRNA-Seq) and cellular indexing of transcriptome and epitopes by sequencing (CITE-Seq). T cells were stained for measurements of surface markers by flow cytometry. Finally, naïve T cells were analyzed for proliferation capacity, cytokine production and differentiation capacity in vitro.

Results:

By combining scRNA-Seq, CITE-Seq and multi-parameter flow cytometry, we observed a unique immunophenotype of liver infiltrating and peripheral T cells in PSC, when compared to ALD and healthy controls. A subset of liver infiltrating naïve-like CD4+ T cells was identified in PSC patients with end-stage liver disease. Using flow cytometry, we confirmed the presence of phenotypically naïve CD4+ T cells in PSC livers and found they were increased in frequency compared with ALD. Transcriptome analysis using scRNA-Seq revealed high expression of markers such as STAT3 suggesting a possible predisposition to acquire a Th17 phenotype. In vitro, PSC derived naive CD4+ T cells showed higher rates of proliferation and production of cytokines associated with Th17 cells compared to healthy controls.

Conclusion:

We here provide the first comprehensive atlas of intrahepatic T cells in PSC. Naïve-like CD4+ T cells ready to differentiate into Th17 cells were identified within the liver and their abundance was increased in liver and blood of PSC compared to ALD. We propose these cells as potential contributors to disease pathogenesis.