Abstract: Combined Co-Stimulatory Blockade and Donor Bone Marrow Cells Induce Robust Immune Tolerance in a Fully SLA-Mismatched Swine Hind Limb Transplant Model (Plastic Surgery 2013)

Monday, October 14, 2013: 10:45 AM

Zuhaib Ibrahim, MD , Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD

Angelo Alberto Leto barone, MD , Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Karim A Sarhane, MD, MSc , Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD

Joani Christensen, MD , Plastic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Georg Furtmuller, MD , Plastic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Mohammed Alrakan, MD , Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD

Saami Khalifian, BS , Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Eric Wimmers, MD , Plastic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Stefan Schneeberger, MD , Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Steven C. Bonawitz, MD , Plastic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Jaimie T Shores, MD , Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Damon S Cooney, MD, PhD , Plastic and Reconstructive Surgery, Johns Hopkins Medical Institutions, Baltimore, MD

Kakali Sarkar, PhD , Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD

Justin M. Sacks, MD , Plastic Surgery, The Johns Hopkins Hospital, Baltimore, MD

W. P. Andrew Lee, MD , Plastic Surgery, Johns Hopkins University School of Medicine, Baltimore, MD

Gerald Brandacher, MD , Plastic and Reconstructive Surgery, Johns Hopkins University, Baltimore, MD

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PURPOSE: Vascularized Composite Allografts (VCA), such as hand and upper extremity transplants, contain vascularized bone marrow (BM) and a BM niche representing a constant source of donor-derived stem cells and hence can favor chimerism and tolerance induction (1). This study investigates the immunological effects of vascularized BM within VCA under co-stimulation blockade-based regimen and its impact on allograft survival and tolerance induction.

METHODS AND MATERIALS: Fully MHC- and gender mismatched MGH miniature swine (n=20) underwent heterotopic hind-limb transplantation containing intact vascularized BM component (Figure 1). Recipient animals received a short course (30 days) of tacrolimus monotherapy with or without donor BM infusion (60x10⁶cells/kg), and CTLA4Ig. Short course tacrolimus only and untreated animals served as controls. Chimerism was assessed by SRY-1 qRT-PCR analysis. Sequential skin and muscle biopsies were performed for histology. Alloreactivity against donor antigens was assessed in vitro using CFSE-based mixed lymphocyte reaction assays. Challenge with secondary skin grafts was utilized to demonstrate robust immune tolerance in vivo.

RESULTS: The co-stimulation blockade based immunomodulatory protocol resulted in indefinite graft survival (>150 days) in 3 out of 5 animals whereas control and tacrolimus only groups rejected allografts at days 7+/-1 and 29+/-2 respectively (Figure 2). Combined costimulation blockade with augmented donor BM infusion resulted in indefinite graft survival in 2 out of 3 animals (>150 days). Long-term survivors demonstrated only transient peripheral but stable micro-chimerism in various graft and recipient tissues including skin, lymph node, bone marrow, and spleen. CFSE-MLR data showed unresponsiveness to donor but not to third party allogeneic controls. Secondary skin grafting demonstrated advanced rejection of third party grafts on day 7 while donor-matched grafts were accepted indicating donor-specific immune tolerance. There was no evidence of donor specific antibody formation in long-term survivors. Donor unresponsiveness in MLR was lost 5 weeks post-graftectomy, which demonstrated that persistent antigenic stimulation was required for operational tolerance.

CONCLUSION: Combined costimulation blockade and donor BM cell infusion can induce robust immune tolerance in a fully MHC mismatched hind limb transplant model. Such targeted immunomodulatory protocols might eliminate the need for long-term multi-drug immunosuppression after reconstructive transplantation.

Figure 1: Schematic diagram: Osteomyocutaneous flap is transplanted to a subcutaneous pocket with externalized skin component.

Figure 2: Kaplan-Meier survival curve demonstrating rejection free survival of skin component of VCA

23043 Combined Co-Stimulatory Blockade and Donor Bone Marrow Cells Induce Robust Immune Tolerance in a Fully SLA-Mismatched Swine Hind Limb Transplant Model