Foxp3+ Treg Cells Resident within Donor Bone Marrow are Essential for Costimulation Blockade-Induced Long-Term Survival of Murine Limb Transplants

Introduction Vascularized composite allotransplantation (VCA) would likely be more widely performed if maintenance immunosuppression was not essential for graft acceptance. VCA procedures that include donor bone marrow cells are of particular interest given concerns these cells may promote graft immunogenicity or provoke graft vs. host disease. As a result, depletion of donor bone marrow cells might be expected to enhance VCA survival. However, our experimental data points to the contrary. Materials and Methods We used murine models of heterotopic and orthotopic limb transplantation (BALB/c->C57BL/6) to assess the efficacy of peri-transplant therapy in achieving long-term VCA survival. Results Injection of CD40L mAb (MR-1) and donor splenocyte transfusion (5x106 cells), plus 30 days of rapamycin (RPM, 2 mg/kg/d) induced long-term VCA survival (>100 days, p<0.01). Likewise, CTLA4Ig (500 μg, i.p., on days 0, 2 and 4) plus RPM also induced long-term orthotopic VCA survival (>100 days, p<0.01). The success of either protocol required the presence of a bone-associated, radiation-sensitive component since removal of the long-bone or pre-transplant donor irradiation (800 cGy), prevented long-term allograft survival. Efficacy also required a T or B cell component since allograft rejection occurred when using Rag1-/- donors; and long-term allograft acceptance could not be restored by infusion of donor bone marrow cells peripherally at the time of engraftment (p<0.05). Use of a CXCR4 inhibitor (AMD3100) to mobilize donor bone marrow cells pre-transplant abrogated the efficacy of either protocol (p<0.01). Analysis of donor bone marrow showed ~40% of CD4+ T cells were Foxp3+ Treg cells, constituting the largest population of Tregs within the immune system. Lastly, donor Foxp3+ T-regulatory (Treg) depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed the diphtheria toxin receptor, restored rejection with either protocol, whereas without Treg depletion long-term survival was associated with an active tri-lineage bone marrow. Discussion Long-term VCA survival is possible across a full MHC disparity using costimulation blockade-based approaches. Surprisingly, the efficacy of costimulation blockade in these models depends on the presence of a population of radiation-sensitive, CXCR4+ Foxp3+ Tregs resident within donor bone marrow cells. The mechanisms by which these cells promote VCA survival post-transplant, including migration of donor Tregs to recipient lymphoid tissues, and the interactions of recipient lymphoid cells with donor bone marrow cells resident within long-bones, are under investigation. Department of Defense Wyss Foundation.