Circulating Donor Heart Exosome Profiling Enables Noninvasive Detection of Antibody-mediated Rejection

BACKGROUND. Endomyocardial biopsy remains the gold standard for distinguishing types of immunologic injury—acute versus antibody-mediated rejection (AMR). Exosomes are tissue-specific extracellular microvesicles released by many cell types, including transplanted heart. Circulating transplant heart exosomes express donor-specific human leukocyte antigen (HLA) I molecules. As AMR is mediated by antibodies to donor HLAs, we proposed that complement deposition that occurs with AMR at tissue level would also occur on circulating donor heart exosomes. METHODS. Plasma exosomes in 4 patients were isolated by column chromatography and ultracentrifugation. Donor heart exosomes were purified using anti-donor HLA I antibody beads and complement C4d protein expression was assessed in this subset as marker for AMR. RESULTS. Three patients had no rejection episodes. Circulating donor heart exosomes showed troponin protein and mRNA expression at all follow-up time points. One patient developed AMR on day 14 endomyocardial biopsy that was treated with rituximab, IVIG/plasmapheresis. Time-specific detection of C4d protein was seen in donor heart exosome subset in this patient, which resolved with treatment. C4d was not seen in other 3 patients’ donor exosomes. CONCLUSIONS. Anti-donor HLA I specificity enables characterization of circulating donor heart exosomes in the clinical setting. Further characterization may open the window to noninvasively diagnose rejection type, such as AMR.