Ethylenecarbodiimide-Fixed Donor Splenocyte Infusions Differentially Target Direct and Indirect Pathways of Allorecognition for Induction of Transplant Tolerance

Strategic exposure to donor Ags prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pretransplant infusion of donor splenocytes treated with the chemical cross-linker ethylenecarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allospecificities. To target indirect allospecific T cells, ECDI-SPs induce upregulation of negative, but not positive, costimulatory molecules on recipient splenic CD11c(+) dendritic cells phagocytosing the injected ECDI-SPs. Indirect allospecific T cells activated by such CD11c(+) dendritic cells undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allospecific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4(+)CD25(+)Foxp3(+) T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SP infusions target host allogeneic responses via a multitude of mechanisms, including clonal depletion, anergy, and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.