Background
The significant sequelae associated with life-long immunosuppression limits the clinical applicability of Vascularized Composite Allotransplantation (VCA). The establishment of tolerance protocols would render VCA a routine procedure. We here investigated a novel tolerance protocol using co-stimulation blockade combined with donor bone marrow infusion.
Methods
Fully MHC- and gender mismatched MGH miniature swine underwent heterotopic hind-limb transplantation. Recipient animals received a short course of tacrolimus monotherapy, +/− donor BM infusion (60x107cells/kg), and CTLA4Ig (abatacept). Short course tacrolimus only and untreated animals served as controls. Chimerism was assessed by SRY-gene PCR analysis. Alloreactivity against donor antigens was assessed in vitro using CFSE-based Mixed Lymphocyte Reaction (CFSE-MLR) assays. Robust immune tolerance in vivo was assessed by secondary skin grafting. Tolerance maintenance upon allograft removal was tested in vitro by CFSE-MLR. Donor-specific antibodies (DSA) production was assessed using flow cytometry.
Results
The abatacept-based immunomodulatory protocol with augmented donor BM infusion resulted in indefinite graft survival (>150 days, n = 4) whereas control and tacrolimus only groups rejected allografts at days 7+/−1 (n=3) and 31+/−1 (n=3) respectively. While only transient peripheral chimerism was observed, stable micro-chimerism in various graft and recipient tissues was found. An increase in intragraft Tregs was seen in tolerant animals as compared to controls. Tolerant animals displayed unresponsiveness to donor but not to third party allogeneic controls. Donor-matched secondary skin grafts were accepted whereas third-party grafts were acutely rejected indicating donor-specific tolerance. No chronic rejection or DSA were seen in tolerant recipients.
Conclusions
Combined costimulation blockade and donor BM cell infusion induced immune tolerance, allowing for immunosuppression-free long-term graft survival in a translational fully MHC-mismatched hind limb transplant model.