Introduction: The application of Vascularized Composite Allograft (VCA) transplantation is limited by the need for immunosuppression. Tolerance induction protocols using stem cell transplantation can eliminate this requirement but the engraftment of these cells can lead to Graft-Versus-Host-Disease (GVHD). In this current study, we have modified our protocol to allow for transient donor engraftment after VCA transplantation and with selective rejection of donor stem cells. Methods: 5 Haploidentical canine recipients received a non-myeloablative conditioning regimen of 350 cGy TBI, mobilized donor stem cells (PBMC) and VCA transplantation followed by a limited course of immunosuppression (MMF for 56 days and Cyclosporine for 70 days). Peripheral blood chimerism was evaluated by PCR techniques weekly. Peripheral blood cytokine expression was evaluated by flow cytometry. VCA rejection was followed clinically and confirmed histologically after routine biopsies. Results: All 5 animals tolerated the conditioning regimen. One rejected the PBMC at 4 weeks post transplantation and went on to reject the VCA transplant following the cessation of immunosuppression. One dog fully engrafted and remains tolerant to the VCA allograft (POD 168). 3 dogs demonstrated initial engraftment of the PBMC (6 – 14 weeks) rejected the donor cells after cessation of immunosuppression without acute rejection of their donor VCAs. One of these dogs was euthanized for persistent fevers at pod 147 with no sign of rejection. No dog developed GVHD. Early cytokine analysis in tolerant versus non-tolerant animals reveals a significantly elevated IL-4 and IL-10 compared to baseline, suggesting a Th2 profile during induction of tolerance. Conclusions: In this study we demonstrate that our non-myeloablative protocol allows for selective rejection of donor stem cells and elimination of GVHD risks without acute rejection of the VCA transplant. Transient donor cell chimerism or engraftment is required to induce tolerance, but after induction, maintenance of tolerance may not be dependent on chimerism. Early after conditioning the establishment of aTh2 profile appears to contributing to the anti-inflammatory environment.

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