Human lung allografts experience persistent fibrogenic shift following acute cellular rejection
1Cincinnati Children's Hospital Medical Center, 2Baylor College of Medicine, 3University of Cincinnati College of Medicine, *Corresponding author
Description
Single-cell RNA sequencing of freshly collected transbronchial lung biopsies from 11 pediatric and adult lung transplant recipients. 134,167 cells over 26 biopsies and the authors' own 60 cell states, grouped into 5 compartments: epithelial, endothelial, stromal, myeloid and lymphoid. Biopsies carry one of four conditions: acute cellular rejection at ISHLT grades A1 to A4 (22,742 cells over 6 biopsies), resolved acute cellular rejection after high-dose corticosteroid treatment (48,551 cells over 7), surveillance biopsies that never showed rejection (34,674 cells over 10), and three further biopsies showing fungal infection, aspiration or diffuse alveolar damage (28,200 cells). The viewers contrast rejection against never-rejected, and resolved against never-rejected, within each cell state.
RATIONALE: Acute cellular rejection (ACR) remains a significant challenge in lung transplantation, with incomplete understanding of its molecular mechanisms and pathways linking ACR to chronic lung allograft dysfunction (CLAD). OBJECTIVES: To characterize the cellular and molecular mechanisms underlying ACR in lung allografts using single cell genomics and identify potential therapeutic targets for CLAD. METHODS: Single cell RNA-sequencing of freshly collected lung tissue was performed across 8 pediatric and adult patients with ACR, Resolved ACR, and surveillance biopsies without ACR. Validation included gene microarray analysis, immunofluorescence, and single cell ATAC-seq. MEASUREMENTS AND MAIN RESULTS: Gene set enrichment analysis revealed persistent TGF-β signaling and PI3K/AKT/mTOR pathway activation in both ACR and Resolved samples, validated by immunofluorescence showing sustained elevation of mTOR activation marker phosphorylated-S6 ribosomal protein and COL3A1. Fibrogenic cells exhibited myofibroblast gene signatures via mesenchymal state transitions rather than epithelial- or endothelial-to-mesenchymal transition. Cell communication analysis showed increased Type II Interferon signaling, with Jak/Stat pathway activation in endothelial and basal cells, and reduced VE-Cadherin staining in ACR. Compositional analysis revealed increased cytotoxic, memory T cells and dendritic cells, with persistent reduction of natural killer cells in ACR and Resolved. Donor/recipient analysis revealed predominantly recipient-derived immune cells in ACR. CONCLUSIONS: Persistent TGF-β and mTOR pathway activation following histologic ACR resolution provides molecular insight into ACR-CLAD linkage and suggests mTOR inhibition and TGF-β blockade as potential therapeutic mechanisms to prevent CLAD.
Dataset ID
LMEX0000090007
Assay Type
Single-cell RNA-seq
Organism
Human
Stages
Adolescent | Adult
Cell Count
134167
Technology
10x Genomics Chromium Single Cell Gene Expression
Reference
Potter et al. (2026)
External databases
GEO: GSE274199
| File | Description | Size |
|---|---|---|
| LTx_ACR_analysis.h5ad | Analysis object for this study, HDF5 AnnData, gzip compressed. 134,167 cells x 35,519 genes over the authors' 60 cell states and 26 biopsies. X holds log2 counts per ten thousand, converted exactly from the authors' natural-log counts-per-ten-thousand matrix; obs carries the cell state, the compartment, the biopsy, the donor, the ISHLT grade and the rejection status; obsm carries the authors' UMAP. The source object holds no raw integer counts, so none is offered here. The sequencing data is at GEO GSE274199. | 2391.7 MB |
| LTx_ACR_library_metadata.tsv | Library metadata for the 26 transbronchial biopsies, one row each. Carries the biopsy, its donor, the ISHLT rejection grade, the rejection status used by the viewers, the 10X chemistry, the transplant indication, the donor sex, age and reported ethnicity, and the number of cells the biopsy contributes. The Samples tab shows the donor level and the fourteen columns the site defines for a human sample, so this file carries the biopsy level and the rest. | 2.4 KB |
| LungMAP ID | Alt ID | Species | Sex | Race | Age | Age Group | Health Status |
|---|---|---|---|---|---|---|---|
| LMSPLTX0000001 | Patient 01 | Homo sapiens | Female | White | 17 years | Adolescent | ACR |
| LMSPLTX0000002 | Patient 02 | Homo sapiens | Female | White | 13 years | Adolescent | ACR, Resolved |
| LMSPLTX0000003 | Patient 03 | Homo sapiens | Male | White | 27 years | Adult | Never |
| LMSPLTX0000004 | Patient 04 | Homo sapiens | Female | Arabic | 15 years | Adolescent | Never |
| LMSPLTX0000005 | Patient 05 | Homo sapiens | Female | White | 15 years | Adolescent | Never |
| LMSPLTX0000006 | Patient 06 | Homo sapiens | Male | European | 15 years | Adolescent | Never |
| LMSPLTX0000007 | Patient 07 | Homo sapiens | Male | Black | 13 years | Adolescent | ACR, Resolved |
| LMSPLTX0000008 | Patient 08 | Homo sapiens | Female | Hispanic | 41 years | Adult | ACR |
| LMSPLTX0000009 | Patient 09 | Homo sapiens | Male | Hispanic | 15 years | Adolescent | ACR, Never, Other, Resolved |
| LMSPLTX0000010 | Patient 10 | Homo sapiens | Female | White | 17 years | Adolescent | Other |
| LMSPLTX0000011 | Patient 11 | Homo sapiens | Male | White | 13 years | Adolescent | Other |