Immunoprofiling of Alcohol-Activated Hepatic Stellate Cells Reveals Mechanisms of Immune Evasion through NK/T Lymphocyte Checkpoint Signaling

Abstract

Chronic alcohol consumption induces the pathogenic activation of hepatic stellate cells (HSC) and their conversion into proliferative myofibroblasts (Myo), which together constitute a disease hub in alcohol-associated liver disease (AALD). While natural killer (NK) lymphocytes efficiently target early activated HSC and ameliorate liver fibrosis in mouse models of diet- and alcohol-induced liver disease, late-activated HSC evade immune surveillance. To gain insight into evasive resistance mechanisms, we profiled the expression of immunoregulatory ligands by HSC and showed that HSC dynamically express CD80, a B7-family ligand that suppresses NK and T cell responses. Using a mouse model of acute-on-chronic alcohol consumption, we show that combined blockade of the CTLA-4//TIGIT/PD-1 inhibitory checkpoints overcomes this resistance mechanism, promoting the selective elimination of activated HSC (aHSC)/Myo, yet fails to diminish fibrosis or ameliorate liver function. Single-cell transcriptome profiling of liver non-parenchymal cells revealed that checkpoint blockade promotes hepatic infiltration of pro-fibrotic Th1 and Th17 T cell subpopulations, while decreasing immunosuppressive Treg. Strikingly, antibody-directed engagement of the PD-1 and TIGIT checkpoints also fails to reduce fibrosis or improve liver function. Thus, selective targeting of aHSC/Myo may be necessary to achieve significant therapeutic benefit.

Keywords

AALD / Immunotherapy / Checkpoint / Hepatic stellate cell

Acknowledgments

We thank Qihong Yang, Stephanie Pan, and the Southern California Research Center for ALPD and Cirrhosis for excellent technical assistance with animal experiments and cell isolation.

Author Contributions

Conceptualization, A.K. and D.E.F.; Methodology, A.K., P.H. and D.E.F.; Statistical Analysis, Y.C.; Writing—Original Draft Preparation, A.K. and D.E.F; Writing, S.B. and D.E.F.; Visualization, S.B. and D.E.F.

Ethics Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Animal Care and Use Committee of the University of Southern California (Protocol #21365, approved 28 January 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

Single-cell RNA-seq data, including unfiltered matrices, and combined and sublibrary reports, have been deposited to Mendeley Data (doi:10.17632/5b9vd5fdtn.1).

Funding

This research was funded by grant 1R21AA029741-01 from the National Institutes of HealthH/National Intitute on Alcohol Abuse and Alcoholism, the Robert E. and May R. Wright Foundation Trust, and the James H. Zumberge Faculty Research and Innovation Fund to D.E.F.

Declaration of Competing Interests

The authors declare no competing interests.

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