Single-Cell Transcriptome Analysis Reveals The Role And Mechanism Of Birc5 In Acute Allograft Rejection After Murine Heterotopic Heart Transplantation

Objective:1.To describe dynamical changes of immune cell clusters in acute allograft rejection after heart transplantation at the single-cell level.2.To study the cellular and molecular mechanism of Birc5 knockout in T cells to protect mouse heart transplant rejection,and explore potential therapeutic targets for acute transplant rejection.Methods:1.Construction of murine heterotopic heart transplantation model,performed Single-cell transcriptome sequencing（sc RNA-seq）,after data filtration,batch effect correction,unsupervised clustering,dimensionality reduction,visualization and cell type annotation.Unsupervised clustering,dimensionality reduction and visualization analysis and more detailed cell type annotations were performed in conjunction with literature and databases.Subsequently,differentially expressed genes（DEGs）analysis,pathway enrichment analysis and pseudotime trajectories analysis were performed in the interest cell subsets.2.After obtain the dataset of human endocardial biopsy（EMB）expression profile from public database,analyze the gene sets related to rejection,and combine sc RNA-seq results in this experiment to establish a gene screening system to obtain candidate gene sets related to rejection.3.Construction of murine heterotopic heart transplantation model,the expression level of survivin（a Birc5 encoded protein）and positive proportions of annexin V（early apoptosis）was analyzed by FCM（flow cytometry）in CD4⁺T cells and CD8⁺T cells isolated from cardiac grafts of isograft group and allograft group.The survival curves of cardiac grafts in the isograft group,WT group,and Birc5^-/-group were observed.On day 6 after surgery,H&E staining of cardiac graft slices was used.4.In WT and Birc5^-/-group,CD4⁺T cells were sorted from spleen,and activated in vitro for 48 h.Next,the number of CD4⁺T cells and the positive proportion of Ki67（proliferation marker）and Annexin V were analyzed by FCM.During activation in vitro,Birc5^-/-CD4⁺T cells were treated with Ac-DEVD-CHO,an inhibitor of caspase-3.The expression level of Cleaved Caspase-3 and Cleaved PARP in each group was detected by Western blot.5.Construction of mouse heart transplant model and treated with YM155（the small molecule inhibitor of survivin）,survival curves and graft H&E slices was observed.PBMC（Peripheral blood mononuclear cells）of healthy people was activated in vitro,following treatment with YM155.The positive proportion of Annexin V was measured by FCM.Results:1.After filtered and batch effect correction,sc RNA-seq data of the four groups named as Ctrl-SP,Allo-SP,Ctrl-HT and Allo-HT were grouped together.18,678 cells in heart grafts and spleens were divided into 18 cell clusters by unsupervised clustering.Next,all cell clusters were classified as 6 major cell types,including T cells,NK cells,B cells,myeloid cells,neutrophils and fibroblasts.the main myeloid cells in Allo-HT changed from cardiac-resident macrophages in Ctrl-HT to ISG^hi macrophages.The marker genes of ISG^himacrophages were mainly enriched in pathways associated with migration,chemotactic and response to interferons.Ccl5^hi CTL（cytotoxic T lymphocytes）and Mki67^hi CTL were accounted for nearly half of Allo-HT,with strong proliferation capacity and cell killing ability.2.Birc5 is highly expressed in T cells undergoing transplant rejection.Birc5^-/-can attenuate mouse heart transplant rejection.Birc5^-/-T cell apoptosis is partially mediated by caspase-3.Treatment with YM155 can attenuate mouse heart transplant rejection and increase T cell apoptosis of human PBMC in vitro.Conclusions:1.At single-cell level,the dynamic change of the proportion of immune cell cluster in acute allograft rejection after heart transplantation was revealed.The functional markers and differentiation trajectories of various cell cluster in myeloid cells,B cells and T cells were identified,including ISG^hi macrophages,Alloreactive B cells,Mki67^hi CTL and Ccl5^hi CTL.2.The expression of survivin in heart grafts infiltrated T cells was upregulated during acute rejection,and the proportion of apoptosis was increased.Birc5 knockout in T cells attenuate murine heart transplant rejection.The apoptosis induced by Birc5 knockout in T cells was partly dependent on caspase-3 activation.The small molecule inhibitor YM155protects mouse heart transplant rejection.Birc5 may be a potential therapeutic target for acute transplant rejection.