EVLP Reprograms Donor Lung Transcriptomics and Attenuates Donor-Specific Immune Signatures

Background: Ex vivo lung perfusion (EVLP) is widely used to assess and recondition donor lungs; however, whether it fundamentally reshapes graft biology or primarily reflects pre-existing donor-derived programs remains unclear. We hypothesized that EVLP actively reprograms lung transcriptomics and modulates donor-specific molecular signatures.
Methods: In a prospective paired intra-donor study, lungs from human donors (DBD and controlled DCD) were subjected to either cold storage (ACS) or EVLP (AEx). Lung biopsies were obtained at baseline and after preservation. RNA sequencing was performed, followed by differential expression and pathway enrichment analyses (GO, KEGG, Reactome).
Results: EVLP induced extensive transcriptomic reprogramming, with over 2,000 differentially expressed genes compared to cold storage. Functional enrichment analyses revealed a coordinated activation of key inflammatory and immune signaling pathways, including cytokine–cytokine receptor interaction, TNF signaling, NF-κB signaling, and IL-17 pathways, together with processes related to leukocyte recruitment, chemokine activity, and cell adhesion.
In parallel, EVLP was associated with activation of pathways reflecting tissue stress and adaptation, including oxidative stress response (NFE2L2-related pathways), response to chemical stress, and extracellular matrix organization and remodeling, indicating a combined inflammatory and repair-oriented transcriptional program.
Donor type strongly influenced baseline lung biology, with DCD and DBD lungs exhibiting distinct immune-related transcriptional profiles. Under cold storage, these differences were amplified, with enrichment of pathways associated with response to bacterial-derived molecules (including LPS), humoral immune response, and complement and coagulation cascades, alongside sustained activation of cytokine signaling networks.
Following EVLP, the transcriptional landscape was substantially reorganized. While donor-specific differences persisted, they shifted toward a more structured and coordinated immune phenotype, characterized by enrichment of T-cell receptor signaling, ZAP-70 activation, CD28 co-stimulation, and immune checkpoint pathways (including PD-1 signaling), as well as interferon-mediated responses.
Importantly, EVLP attenuated the magnitude of transcriptomic differences between DCD and DBD lungs, suggesting a convergence toward a more uniform immune signaling profile across grafts.
Conclusions: EVLP is not a passive preservation strategy but an active biological modulator that reprograms donor lung transcriptomics. Rather than simply normalizing injury, EVLP reshapes the molecular landscape and attenuates donor-related differences, supporting a model in which early graft biology is dynamically redefined during ex vivo perfusion.