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Irradiation and T Cell Depletion Augment Bone Marrow-derived Chimerism at the Cost of Morbidity and Mortality in a Non-human Primate Model of Vascularized Composite Allotransplantation
Philip S. Brazio, MD, Jinny S. Ha, MD, Raghava Munivenkatappa, MD, Raja Mohan, MD, Emile N. Brown, MD, Eduardo D. Rodriguez, MD, DDS, Stephen T. Bartlett, MD, Rolf N. Barth, MD.
University of Maryland, Baltimore, MD, USA.
The high immunogenicity of facial vascularized composite allografts (VCA) requires dependence on conventional immunosuppressive medications, incurring significant morbidity. Tolerance strategies using total body irradiation and infused bone marrow in human kidney recipients have allowed stable mixed chimerism and cessation of immunosuppression, while our group has demonstrated that the inclusion of vascularized bone marrow (VBM) prolongs survival of transplanted facial segments in nonhuman primates (NHP). To explore the potential for induction of chimerism and tolerance, we combined recipient preconditioning and VBM in a NHP model of face transplantation.
Five cynomolgus macaques underwent preconditioning with equine anti-human thymocyte immunoglobulin (Atgam), 7 Gy thymic irradiation, and 1.5 Gy total body irradiation (TBI). The two first animals received an additional 1.5 Gy TBI, a dose adopted from successful NHP kidney tolerance protocols. A heterotopic facial segment including skin, muscle, and hemimandible (bone marrow cell content ~50x106/kg animal weight) was then transplanted from an MHC class I mismatched donor. Immunosuppression was maintained with tacrolimus, with mycophenolate mofetil added after the start of immune reconstitution. Rejection episodes were not treated. Peripheral blood chimerism and T regulatory cells (Tregs) were measured using flow cytometry.
Specific depletion of lymphocytes was immediate and profound, with absolute CD3 and CD4 count nadirs of 27.5±42.9 and 25.3±40.7 by post-operative day (POD) 0-7 after single dose TBI (Figure 1). Preconditioning also caused gradual pancytopenia, with hematocrit, platelet, and total white blood cell count decreases of 48±19%, 79±17%, and 92±6% by POD 14-21. Cell counts began to recover to normal levels by POD 20-28. In contrast, in the double dose TBI subgroup cell counts fell more sharply and did not recover. Significant morbidity was observed in all, and three of the five animals reached endpoints due to weight loss or infection.
Although grafts in preconditioned recipients displayed no Banff grade II rejection, all had early endpoints due to technical failure or death (Figure 3). Preconditioning did augment chimerism vs. VBM alone, with transient macrochimerism in one animal and sustained chimerism up to 22.6% in another (Figure 4a). Tregs were highest after double dose TBI (Figure 4b).
Recipient pretreatment with irradiation and Atgam caused significant morbidity and mortality due to immune depletion and pancytopenia, even with reduced TBI dose. Despite benefits of increased chimerism and potentially tolerance, depletional conditioning requires further dose adjustment before it proves useable in clinical protocols.
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