DESCRIPTION (provided by applicant): Lung transplantation (LTx) is a viable treatment option for end-stage lung diseases. Although short-term survival has improved, acute and chronic rejection remains as hurdles for long-term function of the organ. Diagnosis of both acute and chronic rejection following human LTx remains an important challenge. Transbronchial lung biopsy has been the gold standard for the diagnosis of acute rejection. However, there is significant interobserver variability, and up to 40% of biopsies are insufficient for evaluation of rejection. Diagnostic features for Ab mediated rejection also varies widely. Lastly, chronic rejection after LTx is characterized histologically by obliterative bronchiolitis (OB), a fibroproliferative lesion involving terminal and respiratory bronchioles. Transbronchial lung biopsy is an insensitive method for the detection of OB. Therefore, chronic lung allograft rejection is diagnosed and staged according to decrements in spirometry measurements, a downstream event that induces airway fibrosis and obliteration. Therefore, further insights into the mechanisms of rejection and identifying specific biomarkers for rejection after LTx is critical for long term function of the transplanted lungs. Using both human LTxR and a murine model of chronic rejection, obliterataive airway disease (OAD), we demonstrated that: 1) In human LTxR, circulating exosomes are detectable during acute cellular rejection (A1 and A2). 2) Exosomes can be detected in the sera earlier to acute cellular rejection. 3) De novo development of DSA is accompanied by exosomes in the bronchoalveolar lavage fluid (BAL). 4) Exosomes contained Collagen V (Col-V) which has been shown to be selectively expressed in the lung parenchyma. 5) Exosomes are present in the local site (BAL) much before clinical evidences of OAD lesions, and 6) Sera from LTxR diagnosed with BOS also have Col-V containing exosomes which are detectable in the sera much before BOS. Based on these findings, we propose to determine; 1) The mechanisms by which exosome induction following donor specific immune responses lead to lung allograft rejection; and 2) Determine the kinetics of exosome development with the goal to employ exosome detection in the sera as a potential biomarker for rejection and treatment of rejection. Towards this, we will determine the kinetics of exosomes present in the BAL and serum following LTx using ultracentrifugation and protein isolation methods and correlate the findings with acute cellular, acute Ab mediated, and chronic rejection. Subsequent experiments will; a) define the composition of exosomes; b) determine the cells contributing to the exosome; and c) determine whether HLA specific messenger RNA (mRNA) present in the exosomes will transfer the donor HLA to recipient epithelial and endothelial cells. This is based on our premise that the exosomes will contain several microRNAs involved in immune regulation and donor HLA mRNA. Further, mRNA for HLA in the exosomes may transfer the message to the recipient's epithelial or endothelial cells leading to continued immune responses against the donor HLA resulting in chronic rejection following human LTx.

PUBLIC HEALTH RELEVANCE: This proposal is towards determining the potential use of exosomes in the circulation and body fluids, induced following donor specific immune responses, as a novel and non-invasive biomarker for lung allograft rejection. Further, in depth characterization of exosomes will provide new insights into the mechanisms by which exosomes perpetuate lung allograft rejection especially chronic rejection following human lung transplantation.