Single-Cell Characterization Of Hepatic CD8~+T Cells In A Murine Model Of Primary Biliary Cholangitis

Background:Primary biliary cholangitis（PBC）,a chronic and organ-specific autoimmune disease,is characterized by the injury of immune microenvironment to biliary epithelial cells（BECs）and intrahepatic small-median bile ducts,progressive cholestasis and the appearance of anti-mitochondrial antibodies（AMAs）,and with inflammatory cell infiltrates within the liver.Clinically,there are mainly manifestations like fatigue,pruritus,portal hypertension,osteoporosis and also accompanying with other autoimmune diseases such as sjogren’s syndrome（SS）,systemic sclerosis（SSc）and systemic lupus erythematosus（SLE）.The serological characteristics of PBC were highly expression of alkaline phosphatase（ALP）and γ-glutamyl transferase（GGT）.Up to now,the first-line treatment of PBC is still ursodeoxycholic acid（UDCA）,in some extent changing the natural history of the disease and improving the clinical manifestations and prognosis.However,approximately 40%patients cannot well response to or tolerate to this therapy,unfortunately finally resulting in progression of disease and even suffering liver transplantation.One of the most significant reasons attributes to the unclear pathogenesis.Although previous studies have demonstrated that CD8⁺T lymphocytes are the major pathogenic immune cells contributing to the pathogenesis of human PBC,they do not further clarify whether the diverse subsets of CD8⁺T cells participating in the disease progression or how much they contribute to the disease outcome.In present study,we aim to explore the differentiation and development of CD8⁺T cell subsets in a well-constructed PBC animal model（dnTGF-βRⅡ mice）and make sure the pathogenicity of these cells,and discussing their weight to PBC progression,which would be helpful to expand our understanding of the pathogenesis of PBC and may potentially lead to identifying novel therapeutic strategies.Methods:We use the well-acknowledged PBC animal model,the dominant negative TGF-β receptorⅡ transgenic（dnTGF-βRⅡ）mice for processing this project.Adoptive transfer experiment was performed for determine the pathogenicity of CD8⁺T cells and their different subsets.The high-throughputs single-cell RNA sequencing（scRNA-seq）technology and bioinformatics were applied to study the functional heterogeneity among hepatic CD8⁺T cells subsets in dnTGF-βRⅡ mice and C57BL/6 mice,confirming the differentiation and development trajectory of these cells in vivo,and exploring the specific subsets participating in the disease progression and ensuring their property.Flow cytometry experiment and multi-color immunohistochemistry confirmed the existence and function of CD8αα⁺T cells and CD8αβ⁺T cell in vivo by staining several markers and antibodies.RNA-seq was performed for determine the TCR repertoire of CD8αα⁺T cells and CD8αβ⁺T cells in the liver of dnTGF-βRⅡ mice.Results:1.Adoptive transfer experiments confirmed that CD8⁺T lymphocyte is the key pathogenic immune cell related to the PBC progression.Further respectively adoptive transfer experiments(different CD8⁺T cell subsets contained na?ve CD8⁺T cells,central memory CD8⁺T cells or effector memory CD8⁺T cells into Rag1^-/-mice)was performed and livers from these mce all showed the PBC-like pathology and the livers from Rag1^-/-mice transferred na?ve CD8⁺T cells showed more inflammatory infiltration compared with other transferred groups（no statistical differences）.2.scRNA-seq confirmed CD8⁺T lymphocyte is the most significant change of all immune cells in the hepatic pathogenic microenvironment of dnTGF-βRⅡ mice compared with C57BL/6 mice,and among the whole group of CD8⁺T cell,CD8⁺Tem cells play a more important pathogenic role with their special transcriptome characteristics compared with na?ve CD8⁺T cells and CD8⁺Tcm cells.3.Bioinformatics showed four different CD8⁺Tem subsets（terminally differentiated CD8αα⁺TCRαβ+Tem cells,active CD8αβ⁺Tem cells,chemotactic CD8αβ⁺Tem cells and terminally differentiated and proliferative CD8αβ⁺Tem cells）.Compared with other CD8αβ⁺Tem cells,CD8αα⁺TCRαβ+Tem cells exhibit the unique signature:①they are in the terminally differentiated state and their generation in the liver possibly attribute to the down-regulation of CD8β chain.②these terminally differentiated CD8αα⁺Tem cells acquire the higher cytokine production ability（like IFN-gamma）,higher cytotoxicity（high gene expression for Gzmb）.③ these CD8αα⁺TCRαβ+Tem cells showed the property of tissue resident memory T cells.On the other hand,the proliferative CD8αβ⁺Tem cells also showed a terminally differentiated state and specially these cells are in a sustained proliferative state with highly anergy consumption,which need more biochemical process about glycolysis,ATP synthesis and oxidative phosphorylation for keeping the whole CD8⁺T cell amount and in case response to antigen invasion or sustain the hepatic inflammatory environment.4.TCR repertoire in dnTGF-βRⅡ showed the different property between CD8αα⁺T cells and CD8αβ⁺T cells.TCR repertoire in CD8αα⁺T cells perform an oligoclonal state,while the repertoire of CD8αβ⁺T cells are in a polyclonal state.5.Adoptive transfer experiments about CD8αα⁺T cells or CD8αβ⁺T cells showed the unexpected different outcome.Livers from Rag1^-/-mice transferred CD8αα⁺T cells performed lower inflammatory infiltration compare with the group transferred CD8αβ⁺T cells,in detail expressed in lower infiltration of hepatic mononuclear cells and CD8⁺T cells.Conclusion:To sum up,under the natural history background of dnTGF-βRⅡ mice,we verified CD8⁺T lymphocytes are the key pathogenic immune cells resulting in autoimmune cholangitis and we also found a CD8⁺T cell subset named CD8αα⁺TCRαβ+Tem cells possessing a special differentiative and developmental trajectory,owing more specific cell phenotype and property with a potential of cytotoxicity.Further researches should be processed for clarifying the expect effect of CD8αα⁺TCRαβ+Tem cells on BECs or liver parenchymal cells,confirming the pathogenic contribution of this subset to PBC.