Harnessing Expressed Single Nucleotide Variation and Single Cell RNA Sequencing To Define Immune Cell Chimerism in the Rejecting Kidney Transplant

Abstract

BackgroundIn solid organ transplantation, donor-derived immune cells are assumed to decline with time after surgery. Whether donor leukocytes persist within kidney transplants or play any role in rejection is unknown, however, in part because of limited techniques for distinguishing recipient from donor cells.MethodsWhole-exome sequencing of donor and recipient DNA and single-cell RNA sequencing (scRNA-seq) of five human kidney transplant biopsy cores distinguished immune cell contributions from both participants. DNA-sequence comparisons used single nucleotide variants (SNVs) identified in the exome sequences across all samples.ResultsAnalysis of expressed SNVs in the scRNA-seq data set distinguished recipient versus donor origin for all 81,139 cells examined. The leukocyte donor/recipient ratio varied with rejection status for macrophages and with time post-transplant for lymphocytes. Recipient macrophages displayed inflammatory activation whereas donor macrophages demonstrated antigen presentation and complement signaling. Recipient-origin T cells expressed cytotoxic and proinflammatory genes consistent with an effector cell phenotype, whereas donor-origin T cells appeared quiescent, expressing oxidative phosphorylation genes. Finally, both donor and recipient T cell clones within the rejecting kidney suggested lymphoid aggregation. The results indicate that donor-origin macrophages and T cells have distinct transcriptional profiles compared with their recipient counterparts, and that donor macrophages can persist for years post-transplantation.ConclusionsAnalysis of single nucleotide variants and their expression in single cells provides a powerful novel approach to accurately define leukocyte chimerism in a complex organ such as a transplanted kidney, coupled with the ability to examine transcriptional profiles at single-cell resolution.

Kidney resident macrophages in the rat have minimal turnover and replacement by blood monocytes

“…A recent study analyzed immune cell chimerism during kidney transplant rejection in humans (13). In these studies, the authors showed using single cell RNA sequencing that a population of macrophages expressing C1QC and CD81 was maintained in the donor kidney for several days/weeks post-transplant when the kidney was not undergoing rejection (13). In contrast, the authors also showed that the number of donor macrophages was significantly reduced as the kidneys underwent rejection.…”

Ferroptotic stress promotes the accumulation of pro-inflammatory proximal tubular cells in maladaptive renal repair

“…6D and Fig. S12A), (60). We found a cell population that is highly enriched for genes expressed in mouse damage-associated PT cells, including SOX9, VCAM1, and CDH6.…”

Single-Cell RNA Sequencing Reveals the Immunological Profiles of Renal Allograft Rejection in Mice

“…Among macrophage subtypes, infiltrating Ly6C hi Chil3 + macrophage and Ly6C lo patrolling macrophages are commonly found in normal kidneys (16,42), whereas IFNIC and Mrc1 + macrophages are more common to disease models like unilateral ureteric obstruction model (42). Study of kidney allograft biopsies from human recipients undergoing AR and CR also identified immune populations like those in mice, including monocytes, B cells, T cells, plasma cells (43)(44)(45), therefore independently supporting a role of these cells in kidney rejection in humans. However, due to the limited size of human biopsy samples and the large percentage of parenchymal cells, further analysis of immune cell subtypes is lacking.…”