Sequencing of Physically Interacting Cells in Human Kidney Allograft Rejection to Infer Contact-dependent Immune Cell Transcription

Abstract

Background. Rejection requires cell–cell contact involving immune cells. Inferring the transcriptional programs of cell–cell interactions from single-cell RNA-sequencing (scRNA-seq) data is challenging as spatial information is lost. Methods. We combined a CD45pos enrichment strategy with Cellular Indexing of Transcriptomes and Epitopes by sequencing based quantification of leukocyte surface proteins to analyze cell–cell interactions in 11 human kidney transplant biopsies encompassing a spectrum of rejection diagnoses. scRNA-seq was performed using the 10X Genomics platform. We applied the sequencing physically interacting cells computational method to deconvolute the transcriptional profiles of heterotypic physically interacting cells. Results. The 11 human allograft biopsies generated 31 203 high-quality single-cell libraries. Clustering was further refined by combining Cellular Indexing of Transcriptomes and Epitopes by sequencing data from 6 different leukocyte-specific surface proteins. Three of 6 doublet clusters were identified as physically interacting cell complexes; macrophages or dendritic cells bound to B cells or plasma cells; natural killer (NK) or T cells bound to macrophages or dendritic cells and NK or T cells bound to endothelial cells. Myeloid-lymphocyte physically interacting cell complexes expressed activated and proinflammatory genes. Lymphocytes physically interacting with endothelial cells were enriched for NK and CD4 T cells. NK cell–endothelial cell contact caused increased expression of endothelial proinflammatory genes CXCL9 and CXCL10 and NK cell proinflammatory genes CCL3, CCL4, and GNLY. Conclusions. The transcriptional profiles of physically interacting cells from human kidney transplant biopsies can be inferred from scRNA-seq data using the sequencing physically interacting cells method. This approach complements previous methods that estimate cell–cell physical contact from scRNA-seq data.