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Prolonged Vascularized Composite Allograft Survival and Multi-lineage Mixed Chimerism across a Full Major Histocompatibility Barrier in Swine
David A. Leonard, MBChB., Radbeh Torabi, MD., Christopher Mallard, BS, Alex Albritton, BS, Mark Randolph, MAS, Joseph Kurtz, PhD., David H. Sachs, MD., Curtis L. Cetrulo, Jr., MD..
Massachusetts General Hospital, Boston, MA, USA.
Vascularized composite allotransplantation offers restoration of complex anatomical and functional units, such as the face or hands, following devastating injury. Induction of transplant tolerance would significantly improve the risk-benefit ratio of these procedures. Skin presents a particularly stringent test of tolerance; previous preclinical studies have demonstrated epidermal rejection between days 35 and 50. Proof-of-principle for skin tolerance has been demonstrated by delayed transplantation into previously established mixed chimeras. The present study aimed to develop a clinically applicable day 0 protocol for induction of tolerance of VCA, including skin, in a preclinical large animal model.
Mismatched animals (SLAcc -> SLAaa) were selected from the MGH herd of MHC defined miniature swine. Recipient conditioning consisted of T-cell depleting CD3-immunotoxin (day -4 to -1), 200 cGy total body irradiation (two fractions, day -3 and -2), and 800 cGy thymic irradiation (day -2). Intravenous Cyclosporine A (CyA) was commenced on day -1, (target trough level of 400-800ng/ml) and tapered slowly from day 30 to 60. Gracilis myocutaneous VCA and bone marrow transplantation were performed simultaneously on day 0. VCA viability was monitored by clinical assessment, and regular biopsy. Chimerism was monitored by flow-cytometry. Immunological responses were assessed by measurement of anti-donor antibody in serum, antibody mediated cytotoxicity, mixed lymphocyte reaction (MLR) and cell mediated lympholysis (CML) assays. A control animal underwent VCA alone with neither conditioning nor bone marrow.
A total of 6x1010 bone marrow cells (3.4x109 cells/kg recipient weight) were transplanted. VCA warm ischemia time was 90 minutes. Day 6 biopsy revealed minimal perivascular cellular infiltrate with no evidence of rejection. VCA survival is currently 91 days, with no clinical evidence of rejection. A rejection episode commencing on day 46 was successfully treated with 5 days therapeutic dose CyA, after which taper was re-commenced. A second rejection episode commencing on day 62 resolved spontaneously by day 76 (figure 1). Control animals fully rejected their VCA by day 6.
Multi-lineage chimerism was achieved with myeloid chimerism of 80-90%, and CD3+ lymphocyte chimerism 2-4% (figure 2A).
Donor-specific unresponsiveness was demonstrated by MLR and CML assays (figure 2B, C). There was no evidence of anti-donor antibody production, nor antibody mediated cytotoxicity.
Two self-limiting episodes of cutaneous graft versus host disease (GvHD) were diagnosed, commencing on day 25 and day 56 respectively. There was no evidence of hepatic, gastrointestinal or bone marrow involvement, and resolution occured without treatment.
This protocol achieved mixed chimerism across a full MHC mismatch and significantly prolonged VCA survival, with in-vitro evidence of donor specific unresponsiveness. While continued follow-up is required to determine if tolerance has been achieved, this protocol represents a significant step toward development of a clinically applicable approach to tolerance induction for hand and face transplantation.
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