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

Significance Statement The combination of exome sequencing with single-cell RNA sequencing can reveal the recipient versus donor origin of each immune cell within human kidney allografts. This approach greatly improves upon previous techniques used to identify and describe leukocyte chimerism within a complex organ, such as Y chromosome identification for sex-mismatched transplants. Exome sequencing and single-cell RNA sequencing of single nucleotide variants indicated that donor-origin macrophages may contribute to the alloimmune response through antigen presentation and signaling, whereas donor-origin T cells remain quiescent. Therefore, teaming these techniques can paint a portrait of the chimerism that may lie behind rejection of a donor kidney. Background In 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. Methods Whole-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. Results Analysis 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. Conclusions Analysis 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. Podcast This article contains a podcast at https://www.asn-online.org/media/podcast/JASN/2020_08_07_JASN2020030326.mp3