Background: VCA is a viable treatment option for devastating tissue defects. However, the life-long need for immunosuppression curtails the wider use of VCA. Unique VCA-features such as an intragraft vascularized bone marrow compartment may favor tolerance induction. Methods: Skin, heart, and orthotopic hind limb were performed across a full MHC mismatch barrier in wild type and thymextomized animals. Recipient animals were treated with a non-myeloablative dose of TBI and a T-cell depleting antibody 24 hours prior to transplantation. In selected groups, donor BM and splenocytes (DBM) were injected at the time of transplantation. CyP was administered on POD 3 (PTCy). Chimerism, Vβ analysis, MLR's and 2[deg] transplants were performed. Donor- and host-derived Treg depletion pre and post transplantation was performed. Results: Controls (n=5) rejected skin, SOT and VCAs acutely with a mean survival of 14 ± 1, 9 ± 2, and 8 ± 1 days, respectively. The combination of PTCy and DBM prolonged VCA and non-VCA survival indefinitely in 7/8 skin, 7/8 heart, however, in VCA DBM was not required to achieve 100% long term (150 days) allograft survival N=(20/20). PTCy-treated thymectomized recipients (N=6, >110 days) did not reject the VCA, compared to untreated controls (N=3, MST 8d). Mixed chimerism was shown in skin/heart transplantation plus DBM (6.8% ± 3.1%), in VCA recipients ± DBM at 22.51% ± 5.96% and 30.17% ± 8.72%, and at 24.96 ± 4.12 in thymectomized recipients. Vβ-TCR staining showed decreased expression of donor-specific TCRs in wild type animals and MLRs showed donor-specific unresponsiveness with robust 3^rd party reactivity in long-term survivors. In-vivo, tolerant animals challenged with 2[deg] skin transplants accepted donor matched Balb/c skin (250 days) while 3^rd party FVB/N skin was acutely rejected (15 ± 2 days). In VCA, donor/recipient-derived Treg depletion did not abrogate graft tolerance. Chimerism and graft loss ensued after recipient Treg depletion on POD-2, not POD30&60 in skin + DBM recipients (N=4, MST 23d). Conclusion: Tolerance in this VCA model as achieved by post-transplant cyclophosphamide-based treatment is supported by the intragraft vascularized donor bone marrow component and recipient T regulatory cells.

CITATION INFORMATION: Furtmüller G, Oh B, Fryer M, Ganguly S, Malek V, Zhou X, Dodd-o J, Raimondi G, Cooney D, Brayton C, Lee W, Luznik L, Brandacher G. The Intragraft Vascularized Bone Marrow Component and Recipient T Regulatory Cells Facilitate Tolerance Induction After Post Transplant High Dose Cyclophosphamide Treatment in VCA. Am J Transplant. 2017;17 (suppl 3).

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