*Purpose: To precisely define endothelial cell responses in AMR we performed comprehensive single cell RNA-seq on a human kidney biopsy with AMR.

*Methods: Single cell libraries were generated from an allograft core biopsy with a histologic diagnosis of AMR. Libraries were sequenced by NovaSeq to a depth of ~50k reads/cell. Gene-cell matrices were obtained from CellRanger and the downstream analysis were done using R and Seurat. This study had IRB approval. Receptor ligand analysis was performed by comparing known ligands or receptors highly expressed in endothelial cells with their cognate receptors in the dataset.

*Results: 11,592 cells (detecting 1,841 unique genes/cell) were analyzed, revealing most major kidney cell types including two endothelial cell subclusters (figure 1). A detailed cell-specific receptor-ligand analysis was performed across cell types. We focused on endothelium, and report that the ligands JAG1, JAG2 and DLL4 were highly expressed in endothelial cells and their cognate receptor NOTCH3 was highly expressed in pericytes (figure 2). Endothelial cells also highly expressed NOTCH4. The ligands JAG1 and THBS2 were also highly expressed in pericytes and fibroblasts, respectively.

*Conclusions: Single cell RNA-seq of an AMR biopsy has revealed novel intercellular communication pathways, including upregulated Notch signaling between endothelial cells, pericytes and fibrocytes. Given known roles for activated endothelium in precipitating AMR, these studies suggest a new role for Notch pathway activation in response to alloantibody binding.

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