Co-Stimulatory Blockade and Donor Bone Marrow Cell Infusion for Immunomodulation and Tolerance Induction in Vascularized Composite Allografts - A Large Animal Translational Trial

Abstract

Introduction: Particular Vascular Composite Allografts (VCA), such as hand or arm transplants, contain vascularized long bones that instantly and continuously produce bone marrow (BM) cells after engraftment and thus can be considered to be both a VCA and vascularized BM graft. The creation of hematopoietic chimerism through BM transplantation remains the most reliable method for inducing transplantation tolerance. However, the contribution of a vascularized bone graft in VCA towards such effects is largely unknown. Therefore, this study investigates the long-term effects of vascularized BM within VCA under costimulation blockade-based immunosuppression. Materials and methods: Complete MHC-mismatched miniature swine (n=12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30 or 60 × 106 cells/kg of whole, unmodified BM. The optimal dose of BM cell infusion was then applied to subsequent experiments evaluating the addition of CTLA4Ig. MGH miniature swine (n=24) underwent heterotopic hind limb transplantation and received a short course of 30 days of Tacrolimus monotherapy. Group 1 (control) received no treatment. Group 2 received FK506 only; Group 3 received irradiation and FK506; Group 4 received irradiation, BM infusion and FK506; Group 5 received FK506 and CTLA4-lg; Group 6 received irradiation, BM infusion, FK506 and CTLA4Ig; and Group 7 received BM infusion, FK 506 and CTLA4Ig fusion protein. Results: Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 ×106 cells/kg (P = .0001). Transplanted animals in Group 1 rejected the allograft 5 to 8 days post-transplant. Group 2 rejected the allograft (skin and muscle) 30-32 days post- transplant. Group 3 rejected the allograft (skin and muscle) 35-37 days post-transplant. Group 4 rejected the skin portion of the allograft at 50-53 days post-transplant. The remaining allograft components (muscle, bone, nerve, and vessel) survived indefinitely. Group 5 animals demonstrated significantly prolonged muscle survival beyond 150 days post-transplant; the skin component survived past 150 days in 3 out of 5 animals. Skin and muscle histology in all long-term surviving animals was rejection-free. Two animals in Group 5 developed parasitic diarrhea and were euthanized at post-operative day 45. Both skin and muscle components were rejection free at the time of euthanasia. All animals in Group 6 had unexpected weight loss leading to sudden death (n=1) or euthanasia (n=2). Group 7 is currently in progress with rejection-free survival beyond 52 days post-transplant. Conclusion: High-dose BM cell infusion results in stable levels of micro-chimerism. The addition of co-stimulatory blockade (CTLA4Ig) enabled us to optimize induction therapy, further reduce maintenance immunosuppression, and indefinitely prolong graft survival. Such targeted immunomodulatory protocols that combine BM cell-based strategies and biologics might facilitate immune tolerance and eliminate the need for multi-drug immunosuppression to maintain graft survival after vascularized composite allotransplants.