*Purpose: Cardiac allograft tolerance in non-human primates (NHPs) has been achieved by our group via hematopoietic mixed chimerism consisting of an allogeneic bone marrow (BM) and kidney co-transplantation from the same donor after non-myeloablative conditioning. In this setting, T cell tolerance is believed to rely on presentation of donor MHC molecules in the host’s primary and secondary lymphoid organs. In this study, we investigated whether donor MHC chimerism could be achieved using donor bone marrow derived extracellular vesicles (exosomes) instead of cells.

*Methods: Exosomes were isolated through serial ultracentrifugation from supernatants of donor BM cells cultured for 48 hours (expressing H38 MHC class I epitope). One H38^– cynomolgus macaque recipient received conditioning consisting of ATGAM and thymic irradiation before a heart and kidney co-transplantation. The NHP underwent a short course of immunosuppression with a calcineurin inhibitor and co-stimulation blockade with anti-CD154 mAb. A single dose of H38⁺ donor bone marrow derived exosomes was infused into the H38^– recipient on post-operative day 2. In addition to graft survival, image flow cytometry was used to detect the presence of recipient cells displaying donor MHC class I on their surface (cross-dressed) in the blood, thymus, and spleen.

*Results: Infusion of donor bone marrow derived exosomes resulted in the presence of recipient peripheral blood leukocytes displaying donor MHC class I molecules in the recipient. Flow cytometric analysis showed that MHC class I chimerism was restricted to the myeloid cell compartment. Protocol biopsy on post-operative day 75 showed kidney allograft rejection without cardiac allograft rejection. The cardiac allograft had good contraction until post-operative day 89. H38⁺ cross-dressed cells were present in the thymus and spleen at time of necropsy.

*Conclusions: Donor MHC chimerism in the form of MHC cross-dressing can be achieved in a NHP model using donor bone marrow derived exosomes. These findings suggest that bone marrow derived exosomes could be used in place of whole bone marrow in chimerism-induced allograft tolerance protocols. The use of exosomes could allow reduction of recipient conditioning and eliminate the risk of GVHD making this approach safer than traditional BMT when translated to human patients.

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