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The Role Of Donor Antigen Persistance In Maintaining Immune Tolerance To A Vascularized Composite Allograft.
Angelo A. Leto Barone, MD, Zuhaib Ibrahim, MD, Saami Khalifian, BS, Karim A. Sarhane, MD, Georg Furtmüller, MD, Madeline Fryer, -, Leah Goldberg, BS, Eric Wimmers, MD, Lehao Wu, MD, Mohammed Alrakan, MD, Jaimie Shores, MD, Steven C. Bonawitz, MD, Justin M. Sacks, MD, Chad R. Gordon, MD, Giorgio Raimondi, Ph.D., Damon S. Cooney, MD, Ph.D., WP Andrew Lee, MD, Gerald Brandacher, MD.
Johns Hopkins, Baltimore, MD, USA.
PURPOSE: We have recently shown that indefinite graft survival can be achieved in a fully MHC mismatched swine hind limb transplantation model using a combined donor bone marrow infusion and co-stimulatory blockade regimen. The role of antigen persistence in the maintenance of tolerance in this setting, however, is still unknown. In this study we thus aimed to provide in vitro evidence supporting the clinical observation of tolerance and investigate whether donor-derived antigen persistence is necessary to maintain tolerance to a VCA.
METHODS: Recipients were conditioned using co-stimulatory blockade (CTLA4-Ig), BM infusion (60x107cells/kg) and a 30-day course of tacrolimus. Alloreactivity against donor and third-party antigens was assessed in vitro using Carboxyfluorescein Succinimidyl Ester-based mixed lymphocyte reaction (CFSE-MLR) assays. Graftectomy was performed in a tolerant animal on POD193 and donor-specific cellular responsiveness was assessed weekly thereafter. Upon return of responsiveness by MLR, the recipient underwent autologous, donor-matched and third party allogeneic (Yorkshire) STSGs and grafts were monitored daily to assess rejection. Serum samples were obtained weekly from POD 0 to 4 weeks following STSGs and donor-matched antibody production was assessed by flow cytometry.
RESULTS: Clinical immunosuppression free long-term survival was observed in all recipients (n=3). CFSE-MLR showed responsiveness against donor-matched peripheral blood mononuclear cells (PBMCs) before transplantation but unresponsiveness to donor while the viable graft was maintained. Response to third party allogeneic controls was maintained, confirming the ability of the conditioned animals to elicit a cellular response. Donor antigen specific tolerance was further confirmed in vivo by acceptance of donor-matched split-thickness skin grafts (STSG) and immunological competence assessed by third party allogeneic (Yorkshire) skin graft rejection. However, donor-specific responsiveness returned 5 weeks post-graftectomy. Subsequent donor-matched skin grafts were rejected by POD15 and third party Yorkshire grafts were rejected on POD7. The autologous control graft was viable at all time points. Baseline antibody levels decreased following allograft transplantation and remained low until secondary skin grafts were introduced. Following STSG, anti-donor and anti-Yorkshire antibody levels increased by week 2 (IgM) and weeks 3-4 (IgG). Antibody production was observed only following graftectomy and STSGs.
CONCLUSION: Our current conditioning regimen can induce immune tolerance in a fully MHC mismatched hind limb large animal VCA model. Such robust tolerance appears to be dependent on continuous presence of donor antigens. Furthermore, this data suggests that the return of the cellular response can trigger antibody production when a strong antigenic stimulus is present.
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