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Tolerance Induction with Post Transplantation High-Dose Cyclophosphamide relies on Recipient-derived Immune Regulation and Donor Bone Marrow in a Murine Model of Vascularized Composite Allotransplantation
Georg J. Furtmüller, M.D., Ph.D., Byoungchol Oh, D.V.M., Ph.D., Madeline L. Fryer, B.A., M.S., Paul Akre, M.S., Sudipto Ganguly, Ph.D., Jeffrey Dodd-o, M.D., Ph.D., Veronika Malek, B.A., Giorgio Raimondi, Ph.D., W. P. Andrew Lee, M.D., Leo Luznik, M.D., Gerald Brandacher, M.D..
Johns Hopkins University, Baltimore, MD, USA.

PURPOSE: Developing novel treatment concepts to minimize/avoid immunosuppression by induction of immune tolerance represents the prime task in the field of transplantation. In the clinic to date, this was achieved in highly selected patients in clinical trials of kidney transplantation combined with concomitant stem cell or bone marrow transplantation. Vascularized composite allotransplantation (VCA) is evolving as viable treatment modality for patients suffering from functional and esthetic defects to craniofacial structures, extremities and urogenital tissues. In certain VCAs transplantation of vascularized bone marrow may be inherently part of the allograft designs (i.e. hands and faces) and thereby representing a unique opportunity in combining bone marrow and VCA.
METHODS: Murine skin, heart, and hind limb (VCA) transplants were performed across a full MHC mismatch barrier. Recipients treatment comprised non-myeloablative TBI and T-cell depletion and a single dose of post-transplant cyclophosphamide (PTCy). Donor BM and splenocytes (DBM) were injected at the time of transplantation. Post-transplant multi-lineage and, Foxp3 chimerism as well as Vβ-TCR staining was performed. Donor-specific unresponsiveness was tested by MLR and by 2° skin and solid organ transplantation (SOT). Mechanistic studies were undertaken using transgenic mice (DEREG and Foxp3-DTR) to investigate the role of regulatory T cells (Tregs). VCA was performed in animals thymectomized prior to receiving PTCy and the transplant to highlight the role of the thymus in induction and maintenance of tolerance in this murine model.
RESULTS: Untreated animals rejected skin grafts, SOT and VCA acutely within 14 ±1 days, 9 ±2 days, and 8±1 days, respectively. The treatment regimen extended skin and SOT graft survival (32 ± 8; 65±4, respectively). Additional DBM augmentation lead to allograft survival of >150 days in skin and SOT. However, indefinite graft survival of >150 days was observed in all animals receiving the induction regimen and a VCA ± DBM. In groups receiving a VCA ± DBM, donor chimerism was detected at 22.51% ± 5.96% and 30.17% ± 8.72%, respectively. Prior or post transplantation depletion of recipient-derived Tregs in VCA recipients did not lead to abrogation of tolerance. In recipients of skin graft + DBM, chimerism was not achieved after recipient-derived Treg depletion prior to transplantation (POD-2) and consequently the allografts were rejected (MST = 23.5 days, N=4). In-vitro, Vβ-T cell receptor staining indicates clonal deletion as an additional central tolerance mechanism. However, transplantation into a thymectomized host did not abrogate long-term allograft survival (N=6). All long-term survivors showed donor-specific T cell unresponsiveness in-vitro (MLR) while demonstrated proliferation against 3rd party stimulators. In-vivo, tolerant animals accepted donor-matched secondary skin, while 3rd party FVB/N skin was acutely rejected. Donor-matched hearts were accepted long-term.
CONCLUSION: Taken together, robust tolerance and immunosuppression-free long-term allograft survival can be achieved with PTCy in stringent fully MHC mismatched murine models of skin, heart, and vascularized composite allotransplantation. Stable multi lineage chimerism and recipient derived regulatory T cells play a critical role in maintaining tolerance in this model, in particular in the peritransplant time period.


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