Background
Reconstructive transplantation has become an enthusiastically employed means for reconstruction of devastating tissue defects However, conventional immunosuppression required to maintain allograft survival hinders its widespread application The treatment concept presented here is designed to induce immunosuppression-free allograft survival Methods: Murine skin, heart, and hindlimb transplants were performed across a full MHC mismatch barrier Recipients were treated with PTCy (ie non-myeloablative TBI, T-cell depletion and a single dose of post-transplant cyclophosphamide) with and without donor BM and splenocytes (DBM) Mixed- and regulatory T cell chimerism as well as Vβ-TCRstaining was performed Donor-specific unresponsiveness was tested via mixed lymphocyte reactions (MLR) and by secondary skin and heart transplant challenge.
Results
Untreated animals rejected allogeneic skin grafts, hearts and hindlimbs acutely within 14 ± 1 days, 9 ±2 days, and 8 ± 1 days, respectively The PTCy extended skin and heart graft survival (32 ± 8; 65 ± 4 days, respectively) Additional DBM augmentation lead to allograft survival of >150 days in skin and heart Indefinite graft survival of >250 days was observed in all animals receiving the induction regimen and a VCA ± DBM In groups receiving a VCA with and without DBM, donor chimerism was detected at 3017% ± 872% and 2251% ± 596%, respectively Regulatory T cell chimerism showed recipient-derived Tregs predominantly contributing to the Treg pool (926+/− 42%) in the early phase after transplantation (POD 14–30) whereas at later time points (POD 60-100) donor-derived Foxp3+ cells contributed equally (462 +/− 113%). Gradual depletion of developing T cell clones by donor-derived cells, shown by progressive reduction in circulating Vβ5/11+ T cells, indicates actuation of central mechanism for tolerance induction All long-term survivors showed donor-specific T cell unresponsiveness in-vitro (MLR) while demonstrated proliferation against 3rd party stimulators In-vivo, tolerant animals rejected 3rd party skin while donor-matched tissues were accepted long-term (skin and heart).
Conclusion
Robust tolerance and immunosuppression-free long-term allograft survival can be induced combining DBM transplantation with PTCy in a fully MHC-mismatched murine models of skin, heart, and VCA The intragraft vascularized bone marrow component of a hind limb provides sufficient amounts of DBM to induce stable multi-lineage mixed chimerism and immune tolerance.