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Primary biliary cholangitis (PBC), an organ-specific autoimmune disease, is characterized by injury to small bile ducts, inflammatory cell infiltrates within the liver, progressive cholestasis, and in some cases, cirrhosis with unclear pathogenesis. We aimed to clarify the importance role of hepatic immunce cells in the pathogenesis of human and experimental PBC.The dominant-negative TGFβ receptor type II transgenic (dnTGFβRII) mice, a well-studied and established murine model of PBC were used to identify changes of immune cells, especially the pathogenic CD8+ T cells. The high-throughput single-cell RNA sequencing technology were applied and found functional heterogeneity among the hepatic CD8+ T cells subsets in dnTGFβRII mice. CD8+ T cells were confirmed the key cells leading to the pathogenesis of PBC in dnTGFβRII mice, and identified the terminally differentiated CD8αα T cells and CD8αβ T cell subsets in the liver of dnTGFβRII mice. While terminally differentiated CD8αα T cells have higher cytokine production ability and cytotoxicity, the terminally differentiated CD8αβ T cells retain their proliferative profile. Our work suggests that there are developmental and differentiated trajectories of pathogenic CD8+ T cell subsets in the pathogenesis of PBC. A further clarification of their roles would be helpful to our understanding of the pathogenesis of PBC and may potentially lead to identifying novel therapeutic modalities.

Regulatory B cells (Breg) are considered as immunosuppressive cells. Different subsets of Breg cells have been identified both in human beings and in mice. However, there is a lack of unique markers to identify Breg cells, and the heterogeneity of Breg cells in different organs needs to be further illuminated. In this study, we performed high-throughput single-cell RNA sequencing (scRNA-seq) and single-cell B-cell receptor sequencing (scBCR-seq) of B cells from the murine spleen, liver, mesenteric lymph nodes, bone marrow, and peritoneal cavity to better define the phenotype of these cells. Breg cells were identified based on the expression of immunosuppressive genes and IL-10-producing B (B10) cell-related genes, to define B10 and non-B10 subsets in Breg cells based on the score of the B10 gene signatures. Moreover, we characterized 19 common genes significantly expressed in Breg cells, including Fcrl5, Zbtb20, Ccdc28b, Cd9, and Ptpn22, and further analyzed the transcription factor activity in defined Breg cells. Last, a BCR analysis was used to determine the clonally expanded clusters and the relationship of Breg cells across different organs. We demonstrated that Atf3 may potentially modulate the function of Breg cells as a transcription factor and that seven organ-specific subsets of Breg cells are found. Depending on gene expression and functional modules, non-B10 Breg cells exhibited activated the TGF-β pathway, thus suggesting that non-B10 Breg cells have specific immunosuppressive properties different from conventional B10 cells. In conclusion, our work provides new insights into Breg cells and illustrates their transcriptional profiles and BCR repertoire in different organs under physiological conditions.