Title Donor tissue specific extracellular vesicle characterization for monitoring rejection in lung transplantation Project Summary (Abstract) Lung transplantation remains the only curative therapy in patients with end stage respiratory failure. However, morbidity and mortality in lung transplant patients remains the highest amongst all solid organ transplants, and is directly associated with transplant lung rejection and complications of immunosuppression. Yet to this date, there is no reliable biomarker platform to monitor the transplant lung status. An accurate, non- invasive, time-sensitive biomarker platform would have significant translational impact on patient survival and quality of life as it would enable early intervention and immunosuppression titration. Extracellular vesicles (EVs), including exosomes, are released by many tissues into peripheral circulation. EVs represent stable and tissue-specific proteomic and RNA signature profiles that reflect the conditional state of their tissue of origin. Tissue specific EV profiles and their RNA signatures are distinct in conditions of health versus injury/ pathology. Therefore, understanding tissue specific EV profiles from bodily fluids has promise to serve as a ?liquid biopsy? of the status of their tissue of origin. But no group has studied tissue specific EV profiles in transplantation. From work done over the past three years in our laboratory, we found that transplanted tissues/ organs release a stable and detectable pool of donor-specific EVs into the recipient blood/ bodily fluids. Further, immune rejection of the transplanted tissue leads to distinct changes in the donor EV quantity and in their proteomic and microRNA profiles. These findings have now been validated in the clinical setting. In this mentored research project we propose to investigate the role of donor lung specific EV proteomic and RNA signatures from recipient plasma as a novel biomarker platform in the field of lung transplantation diagnostics. Since no study to date has characterized transplant lung specific and recipient immune cell EV pools, we propose to investigate this novel concept in two in vivo transplant models: (1) established mouse model of orthotopic lung transplantation under controlled settings of acute rejection versus acceptance, and; (2) human lung transplantation. In both settings we will characterize transplant lung specific and recipient T cell specific EV pools, their proteomic and RNA signatures in conditions of health versus rejection to discover distinct profiles associated with transplant rejection. Successful completion of this project proposal would lay the foundation for future development of a non-invasive, EV-based novel biomarker assay to monitor lung transplant rejection.