Intrahepatic T cell analysis reveals Th17 and MAIT17 enrichment in primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is an immune-mediated liver disease where the exact contribution of T cells is unclear. To deeply characterize intrahepatic T cells in PSC, we combined single-cell RNA sequencing (scRNA-seq) with T cell receptor sequencing (TCR-seq) and functional confirmation, using primary biliary cholangitis (PBC) and alcohol-related liver disease (ALD) as disease controls.

METHODS

Intrahepatic T cells from 12 PSC, nine PBC and three ALD explants were enriched by negative selection, viability-sorted and analyzed by scRNA-seq for total gene and TCR expression. T helper 17 (Th17) and IL-17+ mucosal-associated invariant T cell (MAIT17) proportions were analyzed by flow cytometry and liver immunohistochemistry. Liver TCRs in blood were evaluated in an independent PSC, PBC and healthy cohort previously analyzed by TCR β-chain sequencing. Antigenicity of MAIT TCR-transduced reporters was assessed using MAIT-specific tetramers (5-OP-RU-MR1) and MR1+H69 cholangiocyte co-cultures.

RESULTS

ScRNA-seq of 40,897 intrahepatic T cells revealed increased Th17/MAIT17 proportions in PSC livers compared with PBC (1.9-fold) and ALD (3.5-fold). Liver Th17 and MAIT17 expressed tissue-resident markers (CD69, CXCR6, ITGAE) and relative abundances measured by scRNA-seq correlated with proportions detected by flow cytometry. Immunohistochemistry liver staining of a validation cohort (n = 37) showed higher fractions of RORγt+ cells in PSC and greater cumulative frequencies of liver Th17 and MAIT17 TCR clonotypes in PSC blood compared with PBC. Seven of ten TCR reporters representing the most-common and expanded MAIT17 clonotypes in PSC livers bound 5-OP-RU-MR1 tetramer and were activated by 5-OP-RU-loaded MR1+H69 cholangiocytes.

CONCLUSIONS

Multimodal analysis of intrahepatic T cells in PSC revealed a novel enrichment of Th17 and MAIT17 which may reflect increased IL-17 polarization of T cells within the PSC microenvironment.

IMPACT AND IMPLICATIONS: PSC is a progressive cholestatic biliary disease featuring liver T cell infiltration of unknown significance. To better define the role of T cells in PSC, we phenotyped intrahepatic T cells by scRNA-seq and detected increased proportions of tissue-resident Th17 and MAIT17 cells in PSC relative to disease controls. TCR sequencing also revealed that liver Th17 and MAIT17 TCR clonotypes were more frequent in blood from individuals with PSC compared with PBC. Collectively our results suggest that PSC is associated with a greater expansion of liver Th17 and MAIT17 which may contribute to the pathogenesis of PSC.