Plastic Surgery Research Council
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PSRC 60th Annual Meeting
Program and Abstracts

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Post Transplantation Cyclophosphamide Promotes Robust Immune Tolerance after Reconstructive Transplantation
Georg J. Furtmüller, M.D., Madeline L. Fryer, B.A., Byoungchol Oh, D.V.M., Ph.D., Sudipto Ganguly, Ph.D., Jessica Izzi, D.V.M., Jeffrey Dodd-o, MD, PhD, Giorgio Raimondi, Ph.D., Damon S. Cooney, M.D., Ph.D., WP Andrew Lee, M.D., Leo Luznik, M.D., Gerald Brandacher, M.D..
Johns Hopkins University, Baltimore, MD, USA.

Background: Reconstructive transplantation has become a viable treatment option for devastating tissue defects due to trauma, surgical resection or birth defects. However, the life-long need of immunosuppressive medications and their associated toxicities remain a major obstacle that curtails the wider use of vascular composite allografts. Thus, the central challenge for reconstructive transplantation is to develop novel treatment concepts to minimize/avoid immunosuppression and hence extend the benefits of these life-enhancing procedures to a wider patient population in need. In this study we investigated a clinically relevant tolerance inducting strategy by using a peri-transplant induction regimen and post transplant cyclophosphamide to avoid the need for post-transplant immuosuppression.
Methods: Murine skin, solid organ (heart), and vascularized composite allotransplantation (VCA, orthotopic hind limb) were performed across a full MHC mismatch barrier. Recipient animals were treated with a non-myeloablative dose of total body irradiation and monoclonal antibody-based T cell depletion 24 hours prior to transplantation. Donor bone marrow (BM) and splenocytes were injected at the time of transplantation. Three days post transplantation cyclophosphamide was administered. Post operatively, peripheral blood multi-lineage chimerism as well as Vβ-T cell receptor staining was performed over the observation period of 150 days. Donor-specific unresponsiveness was tested in-vitro by mixed lymphocyte reaction assays and in-vivo by secondary skin and solid organ allotransplantation.
Results: In untreated control groups (n=5) animals rejected skin grafts, solid organ grafts and hind limb transplants acutely by POD 14 (± 1 day), POD 9 (± 2 days), and 8 (± 1 day), respectively. The induction regimen extended skin graft survival to 32 ± 8 days (n=5). Additional donor BM augmentation lead to allograft survival of 150 days in 4 of 5 recipients. However, indefinite graft survival of >150 days was observed in all animals receiving the induction regimen and a VCA. Mixed chimerism analysis showed engraftment of donor BM in groups receiving BM at the time of skin transplantation (6.8% ± 3.1%). In groups receiving a VCA alone or an allograft augmented with donor BM and splenocytes, donor chimerism was detected at 22.51% ± 5.96% and 30.17% ± 8.72%, respectively. Vβ-T cell receptor staining showed decreased expression of Vβ 5.1/5.1 and Vβ 8.1/8.2 in animals receiving the full induction regimen compared to controls indicating a central selection and depletion mechanism for tolerance. In addition, long-term survivors showed donor-specific T cell unresponsiveness in-vitro as assessed by mixed lymphocyte reactions while demonstrated proliferation against third-party stimulators. In-vivo, tolerant animals challenged with secondary skin transplants accepted donor matched Balb/c skin while third-party FVB/N skin was acutely rejected. Donor-matched solid organ transplants were accepted long term of up to date 35 days post transplantation.
Conclusion: The results of this study demonstrate that a peri-transplant conditioning regimen and post-transplant cyclophosphamide induce robust tolerance and immunosuppression-free long-term allograft survival in a stringent fully MHC mismatched murine model of skin, heart, and vascularized composite allotransplantation. Results further underline the crucial role of the vascularized bone component in VCA and donor bone marrow augmentation in solid organ and skin transplantation for long-term graft acceptance.


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