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.
CONCLUSION: 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.