PD-1+CD8+ T Cell-Mediated Hepatocyte Pyroptosis Promotes Progression of Murine Autoimmune Liver Disease

The specific mechanisms underlying effector pathways in autoimmune liver disease remain enigmatic and therefore constructing appropriate murine models to investigate disease pathogenesis becomes critical. A spontaneous severe murine model of autoimmune liver disease has been previously established in dnTGFßRII Aire-/- mice, exhibiting disease phenotypes that resemble both human primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH). The data suggest that auto-reactive liver-specific CD8+ T cells are the primary pathogenic cells in liver injury. In this study, these data are advanced through the use of both single-cell sequencing and extensive in vitro analysis. The results identify a specific expanded pathogenic subset of PD-1+CD8+ T cells in the liver, exhibiting strong functional activity and cytotoxicity against target cells. Depletion of PD-1+CD8+ T cells using CAR-T cells effectively alleviates the disease. GSDMD-mediated pyroptosis is found to be aberrantly activated in the livers of model mice, and treatment with a GSDMD-specific inhibitor significantly inhibits disease progression. In vitro experiments reveal that PD-1+CD8+ T cells can induce the pyroptosis of hepatocytes through elevated production of granzyme B and perforin-1. These results provide a novel explanation for the cytotoxic activity of pathogenic liver PD-1+CD8+ T cells in autoimmune liver diseases and offer potential therapeutic targets. Overall design: CD45+ cells were sorted using the FACS Aria II cell sorter and dispensed into individual microwell plates. The transcriptome of CD45+ lymphocytes from dnTGFßRII, Aire-/-, dnTGFßRII Aire+/- and dnTGFßRII Aire-/- mice (each sample mixed with hepatic cells from 2-3 mice) was split to construct the single cell TCR library and the targeted single cell mRNA library, which was amplified by multiplexed polymerase chain reaction and sequenced using an Illumina NovaSeq platform. To explore the function state of hepatic cells in different groups, we extracted the total RNA of liver tissues or 5×10^5 hepatic CD8+ T cells from dnTGFßRII Aire-/- mice and their control littermates, then we build the cDNA library and performed the bulk RNA sequencing. To explore the function state of hepatic cells in different groups, we extracted the total RNA of liver tissues from dnTGFßRII Aire-/- mice with/without PD-1 targeting CAR-T cells and wild type mice, then we build the cDNA library and performed the bulk RNA sequencing.