The severity of lung injury that develops within the first several days after lung transplantation (LT) is a key indicator of which LT recipients are at greatest risk of death or early development of chronic lung allograft dysfunction. The fact that 30% of lungs deemed suitable for LT rapidly develop severe lung injury suggests that there may be unrecognized subclinical injury already present in donor lungs that renders the lung allograft susceptible to further injury at the time of LT. A critical unmet need is improved ability to detect and interpret the consequences of subclinical donor lung injury that may drive poor clinical outcomes after LT. In this Katz R01 proposal, an accomplished physician scientist will develop a new area of research investigating how subclinical donor lung injury triggers a cascade of events that ultimately results in CLAD. Recent studies have identified donor-derived cell-free DNA (cfDNA) as a biomarker of lung allograft injury, with increased cfDNA detected prior to clinical recognition of acute rejection. However, it is unknown which specific cells and injury mechanisms cause cfDNA release from the donor lung. cfDNA can also mechanistically exacerbate lung inflammation. In models of non-LT lung injury, cfDNA detection in bronchoalveolar lavage or plasma activates the stimulator of interferon genes (STING). STING then promotes ongoing dysregulated inflammation by activating inflammatory pathways previously implicated in lung injury after LT, including NF-B, NLRP3, and MKLK. In this Katz R01 proposal, an ESI physician scientist with clinical expertise in LT and scientific expertise in animal models of ARDS proposes an integrated approach to define mechanisms of donor lung injury that drive cfDNA release and poor outcomes after LT. Using single-cell genomics and CyTOF-based immune cell profiling of serial samples from human donor lungs coupled with a new multi-hit murine model of subclinical donor lung injury, we will determine the specific cellular and molecular mechanisms through which donor lung injury affects early allograft dysfunction. We hypothesize that subclinical donor lung injury drives severe allograft injury through release of donor-derived cfDNA into the allograft airspace, triggering a feed-forward cycle of inflammation and ongoing cellular injury that results in poor clinical outcomes. The Specific Aims are: (1) to test whether subclinical donor lung injury is associated with release of donor-derived cfDNA and poor clinical outcomes in humans, using single-cell RNA sequencing and mass cytometry on donor lung biopsies collected before and after LT and (2) to use a novel animal model of sequential subclinical lung injury prior to ischemia reperfusion to determine how subclinical donor lung injury releases cfDNA to prime the donor lung to develop excessive STING-dependent inflammation after ischemia-reperfusion injury. Together, the combination of longitudinal human observational data and mechanistic mur...