Introduction: Using donor splenocytes fixed with cross-linker ethylcarbodiimide (ECDI-SPs), our lab has induced tolerance in a variety of murine and non-human primate models when one dose of ECDI-SPs is administered before transplant and one dose after. We aimed to induce tolerance post-transplant in a murine model by depleting donor macrophages prior to transplant. Once successful, we investigated the roles of extracellular vesicles (EVs) released by donor macrophages in alloimmunity.

Methods: We transplanted BALB/c pancreatic islets under the kidney capsule of C57BL/6J mice made diabetic with streptozotocin. Prior to islet harvest, donors were treated with either anti-CSF1R antibody to deplete macrophages or an isotype control. Recipients received two doses of ECDI-SPs following transplant. Graft rejection was defined as hyperglycemia for two consecutive days. For in vitro studies, EVs were generated by stimulation of RAW 264.7 macrophages of BALB/c origin. These EVs were fed to C57BL/6J dendritic cells, which were then cultured with 4C or TCR75 T cells, specific to direct and indirect presentation of BALB/c MHC respectively. T cells were analyzed for proliferation via CFSE dilution on flow cytometry.

Results: Depletion of donor macrophages followed by treating the recipient with two doses of ECDI-SPs resulted in indefinite graft survival in 90% of recipients, while non-depleted donors resulted in only 30% graft survival. We found that in vitro, C57BL/6J dendritic cells fed EVs released by BALB/c macrophages were able to stimulate 4C and TCR75 T cells to proliferate. This demonstrates the ability of DCs to present MHC from allogeneic EVs both semi-directly and indirectly. DCs fed BALB/c lysate stimulated TCR75, but not 4C, T cells, suggesting that intact EVs are needed for this semi-direct presentation. T cells did not respond when cultured with EVs alone, demonstrating the necessity for dendritic cells.

Conclusions: Depleting donor macrophages permitted tolerization of islet grafts with post-transplant treatments. In vitro results suggest that EVs released by donor macrophages when stimulated are capable of contributing to both semi-direct and indirect stimulation of recipient T cells. Dendritic cells play a crucial role in this phenomenon, likely contributing costimulation signals for T cell proliferation.