Induction Of Delayed Immune Tolerance After Reconstructive Transplantation By Combining Donor Bone Marrow Transplantation And High-dose Cyclophosphamide Treatment
YINAN GUO1, Franka Messner, M.D.1, Byoung Chol Oh, D.V.M. ; Ph.D1, Georg J. Furtmüller, M.D.1, W.P. Andrew Lee, M.D.1, Damon S. Cooney, M.D. ; Ph.D1, Leo Luznik, M.D.2, Gerald Brandacher, M.D.1.
1Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2Division of Hematologic Malignancies, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA.
PURPOSE:Developing novel treatment concepts to minimize/avoid immunosuppression by induction of immune tolerance represents a primary goal in the field of transplantation. Immunosuppression-free allograft survival has been achieved in several animal models as well as in humans in living-related combined kidney and donor bone marrow transplantation by inducing mixed hematopoietic chimerism. However, success of this concept relies on extensive pre-transplant recipient preconditioning which is not feasible in VCA. Many VCAs though inherently contain vascularized donor bone marrow and thus a vital bone marrow niche home to donor-derived hematopoietic progenitor cells facilitating chimerism induction. In this study we therefore explored a novel approach to induce delayed immune tolerance subsequent to conventional immunosuppressive treatment combining high-dose cyclophosphamide treatment and donor bone marrow transplantation.
ETHODS:Orthotopic hind limb transplantation from Balb/c to C57BL6 mice is performed across a full MHC mismatch barrier. Recipient animals are assigned to a course of long-term treatment with conventional mTOR inhibitor-based immunosuppression. Induction treatment comprises non-myeloablative total body irradiation (TBI) and T-cell depletion and a single dose of cyclophosphamide (Cy) on POD 30 combined with donor bone marrow transplantation (dBMT) in selected groups. Animal survival, donor bone marrow engraftment, and frequency of memory T cells are assessed via flow cytometry on a weekly basis prior and after the application of the delayed tolerance regimen.
RESULTS: Untreated animals rejected their grafts acutely within 8±1 days. In treated animals, allograft survival was maintained over 30 days with conventional immunosuppression (Rapamycin) followed by Cy +/- dBMT which prolonged graft survival to 76.5d (±25.89d) without dBMT and 83.6d (±15.89d) with dBMT. Mixed chimerism levels in animals without dBMT were 7.17% (±4.22%) on POD7 and 2.15% (±1.09%) POD30. Comparatively, chimerism levels were at 1.70% (±1.08%) and 3.77% (±2.17%) on POD 7 and 30, respectively, for animals with dBMT on POD 30. Multi-lineage mixed chimerism persisted after PT/Cy with declining levels at the time of allograft rejection. CD8+ effector memory T cell (Tmem) frequency after transplantation in animals with and without dBMT on POD 7 was (dBMT-: 4.65% (±8.38%); dBMT+: 1.7% (±0.19%)) and POD 30 (dBMT-: 7.22% (±2.65%); dBMT+: 2.31% (±0.94%)). Of note, after induction treatment, the proportion of CD8+Tmem cells in groups with or without dBMT increased to 28.08%±20.90% and 41.08%±21.85%, respectively, representing a trend which negatively correlated with graft survival.
Delayed application of combined high dose cyclophosphamide and donor bone marrow transplantation following a long-term course of conventional immunosuppression leads to extended yet not indefinite allograft survival despite the presence of multi-lineage mixed chimerism. Further studies will focus on studying memory T cell barriers to achieve delayed immune tolerance in this murine model of VCA.
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