Uncovering Salient Cellular Heterogeneity in Human Lung Transplantation Using Single-Cell RNA Sequencing

Abstract

Lung transplant (LT) recipients experience a low survival rate compared with other solid organ transplant recipients mainly due to development of chronic lung allograft dysfunction (CLAD). Acute rejection (AR) is a major risk factor for CLAD. We have applied single-cell RNA sequencing (scRNAseq) to allograft-derived cells during quiescence, AR, and CLAD, in order to determine key cellular elements in dysfunctional lung allografts. Fresh bronchoalveolar lavage (BAL) cells from one LT patient (4792 cells) with stable lung function and one with AR (1791) as well as enzymatically and mechanically dissociated CLAD lung tissue (9797 cells) were subjected to barcoding and library construction using 10X Genomics 3' expression V2 chemistry. Library constructs were then sent for scRNAseq. Data were analysed using the Seurat package in R. scRNAseq analysis of BAL cells demonstrated at least three distinct subsets of macrophages including a cluster with a clear inflammatory phenotype that was highly present in AR (cluster 5, Fig 1A). Another macrophage subset with a regenerative phenotype was represented mostly in the stable patient sample (cluster 11, Fig 1A). The AR sample also contained a distinct cytotoxic T cell cluster (cluster 14, Fig 1A), absent in the stable patient sample (Fig 1A, B, and C). Comparison of CLAD lung tissue to the AR BAL sample revealed co-clustering of cytotoxic T cells (cluster 1, Fig1D), suggesting further expansion of these cells as allograft dysfunction progresses. In addition, unique cell subsets of T, B, and NK cells were seen in CLAD lung tissue but not in BAL (Fig 1D, E, and F). Using scRNAseq, we offer insights into the cellular composition of stable and AR BAL and created a map of tissue-resident cells in a CLAD lung. Further application of this powerful tool is underway and will deepen our understanding of the cellular complexity of LT immunobiology and its role in allograft injury and dysfunction.