Infused Bone Marrow Fails to Prolong Vascularized Composite Allograft Survival in Nonhuman Primates

Abstract

Introduction: Clinical protocols for hand and face vascularized composite allografts (VCA) have included varying amounts donor bone marrow cell (BMC) infusion after lymphocyte depletion as an immunomodulatory strategy. We previously demonstrated that vascularized bone marrow (VBM) protects facial VCA from rejection in unconditioned nonhuman primates. To investigate the potential immunologic benefit of infused BMC, we substituted infused BMC for VBM in the transplanted facial segment. Methods: Three cynomolgus macaques underwent heterotopic facial segment transplantation with mandibular segment removed, followed by central venous infusion of BMC from the same MHC-mismatched donor on postoperative day 1. Bone marrow cells were collected from the donor lumbar vertebrae following euthanasia. Cells counts and viability were confirmed using trypan blue and automated complete blood count. Donor BMC were infused at 200 million cells per kg of recipient weight, a standard for bone marrow transplantation. CD34+ hematopoeitic stem and progenitor cells were identified using a modified ISHAGE gating strategy. Immunosuppression was maintained with tacrolimus and mycophenolate mofetil. Rejection episodes were not treated. Chimerism was measured in blood samples using flow cytometry. Results: Mean cell content of the mandibular segments set aside before transplantation was 48.2 million cells/kg of donor weight. Hematopoeitic stem and progenitor cells comprised 5.63% of vertebral bone marrow BMC and 7.60% of mandibular BMC. Animals in the previous VCA+VBM treatment group had no graft loss while on immunosuppression. Additionally, graft survival was longer vs. VCA alone (205-430 vs. 9-42 days), as was rejection-free survival (44-358 vs. 7-15 days). In the VCA + BMC group all animals developed acute rejection (Banff grade I or higher) by day 14. One progressed to grade IV rejection and graft loss by day 28, while another resolved but progressed to chronic rejection by day 50 and endpoint at day 115 for post-transplant lymphoproliferative disease with chronic rejection. Only one animal receiving BMC demonstrated transient macrochimerism days 85-98. Chimerism was similarly observed transiently in only one animal receiving VCA without either VBM or BMC. In contrast, 3 of 4 animals with VBM demonstrated macrochimerism.Figure: [Graft Survival]Conclusion: Despite larger cell counts, BMC infusion failed to prolong facial VCA survival compared to VBM. These data support that VBM is a more effective immunomodulatory strategy than infusion of donor BMC without depletional pre-conditioning. The addition of irradiation and T cell depletion therapies to VBM may further enhance the amount and durability of chimerism, and potentially allow for the development tolerance.