Gut microbiota (GM) modifies the intrahepatic immune microenvironment, but the underlying mechanisms remain poorly understood. Liver fibrosis-associated imprinting is predicted to be reflected in GM. This study investigated the link between GM and the intrahepatic T cell receptor (TCR) immune repertoire (IR), and whether GM modulates the intrahepatic immune microenvironment via TCR IR during liver fibrosis. We analyzed the correlation between GM and TCR IR during liver fibrogenesis. Accordingly, 16S rRNA gene sequencing (16S-seq) and bulk immune repertoire sequencing (IR-seq) were performed to characterize GM and intrahepatic TCR IR. Fecal microbial transplant (FMT) and TCRβ knockout (TcrbKO) mouse models were employed to determine the biological link between GM and TCR IR in liver fibrosis. We found that GM and intrahepatic TCR IR are highly correlated, with both showing reduced diversity and centralized distribution during liver fibrosis. The restoration of normal intestinal microbiota may reshape intrahepatic TCR IR and delay liver fibrosis. Interestingly, TCR IR ablation abrogated the impact of GM on liver fibrogenesis. Furthermore, GM modulated hepatic stellate cell (HSC) activation via TCR IR-mediated intrahepatic immune milieu. Our study demonstrates that GM, which exhibits cross-talk with the intrahepatic TCR IR, influences the intrahepatic immune microenvironment and liver fibrosis progression.

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Aberrant immune cell infiltrates and microcircumstances represent characteristic features of liver fibrosis. In this study, we profiled the transcriptomes of intrahepatic CD45+ immune cells, from mice, using single-cell RNA sequencing (scRNA-seq) technology to understand the landscape of intrahepatic immune cells during the pathogenesis of fibrosis. Analysis of approximately 10,000 single-cell transcriptomes revealed an increase in dendritic cells (DCs), macrophages, and neutrophils and a decrease in T and natural killer T (NKT) cells. In addition, we report changes in the transcriptomes of diverse immune cell types, implying a deteriorating intrahepatic immune microcircumstance. Furthermore, we uncovered a novel fibrosis-associated CD8 T (Ccl5+, Ccl4+) and CD4 T (mt-Co1+) cell subpopulation, which infiltrates fibrotic liver and is characterized by abnormal activation or inactivation as well as a TCR decline. The results from scRNA-seq and bulk immune repertoire sequencing (IR-seq) revealed an obvious decline in T cell receptor (TCR) clonotypes combined with shrinking VJ and VDJ segment usage, as well as lower complementarity-determining region 3 (CDR3) amino acid (AA) diversity from fibrotic liver. Interestingly, a deficiency of TCR IR (TcrbKO mice) led to a deterioration of liver fibrosis, coupled with activation of hepatic stellate cells (HSCs) induced by the upregulation of macrophage and γδ T cell distribution in fibrotic TcrbKO livers. Our findings reveal the landscape and dynamics of single immune cells in liver fibrosis, and clarify the protective role of TCR IR in response to chronic liver injury.