*Purpose: Exosomes are tissue specific, bilayered, membrane-bound nanoparticles released by many types of cells into bodily fluids including blood and urine. Exosomes carry protein and RNA within their cargo along with cell-specific protein markers on their surface. Cargo profiles are dynamic and reflect the conditional status of the tissue of origin. We propose that donor tissue-specific exosomes are released by the transplanted heart into recipient circulation and may be altered during immunologic rejection, which could serve as an accurate biomarker platform. We studied this concept in a mouse model of full major histocompatibility mismatch heterotopic heart transplantation.

*Methods: A full major histocompatibility mismatch (BALB/c [H2-Kd] into C57BL/6 [H2-Kb]) heterotopic heart transplantation was performed (n=15). Immunocompetent recipients fully rejected the donor heart, whereas immunosuppressed recipients accepted the allograft. Exosomes were isolated using size exclusion chromatography and ultracentrifugation. Donor heart specific exosomes were purified using H-2Kd antibody conjugated beads. The bead bound exosome fractions were utilized for protein and RNA cargo analysis.

*Results: Exosomes isolated from the recipient mouse plasma demonstrated presence of exosome specific surface markers (Fig.1). Bead bound exosomes showed expression of Troponin I, a cardiomyocyte marker, both at the protein and mRNA level. Troponin I was detected as early as 4 hours post-transplant (Fig.1b/c). The presence of tissue-restricted self-antigens cardiac Myosin and Vimentin was detected in recipient plasma exosomes. mRNA array analysis of donor specific exosomes showed differences in exosomal mRNA cargoes of rejected transplant groups at time points 4 hours, day 1, and day 2. Next generation sequencing demonstrated distinct changes in donor heart specific exosome microRNA cargoes at 24 versus 48 hour time points.

*Conclusions: The donor heart releases exosomes into recipient circulation within the first 4 hours after transplantation. Donor heart exosomes carry cardiac proteins and mRNAs as part of their intra-exosomal cargoes, which show differential expression before clinical onset of acute rejection. Collectively, these results demonstrate that cargo profiling of donor heart exosomes may serve as a novel biomarker for noninvasive monitoring of immunologic rejection in heart transplantation, and provide insights into their immunogenic potential.

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