Long-term Tolerance Toward Haploidentical Vascularized Composite Allograft Transplantation in a Canine Model Using Bone Marrow or Mobilized Stem Cells.

BACKGROUND: The development of safe and reliable protocols for the transplantation of the face and hands may be accomplished with animal modeling of transplantation of vascularized composite allografts (VCA). Previously, we demonstrated that tolerance to a VCA could be achieved after canine recipients were simultaneously given marrow from a dog leukocyte antigen-identical donor. In the present study, we extend those findings across a dog leukocyte antigen mismatched barrier. METHODS: Eight recipient dogs received total body irradiation (4.5 cGy), hematopoietic cell transplantation (HCT), either marrow (n = 4) or granulocyte-colony stimulating factor mobilized peripheral blood stem cells (n = 4), and a VCA transplant from the HCT donor. Post grafting immunosuppression consisted of mycophenolate mofetil (28 days) and cyclosporine (35 days). RESULTS: In 4 dogs receiving bone marrow, 1 accepted both its marrow transplant and demonstrated long-term tolerance to the donor VCA (>52 weeks). Three dogs rejected both their marrow transplants and VCA at 5 to 7 weeks posttransplant. Dogs receiving mobilized stem cells all accepted their stem cell transplant and became tolerant to the VCA. However, 3 dogs developed graft-versus-host disease, whereas 1 dog rejected its stem cell graft by week 15 but exhibited long-term tolerance toward its VCA (>90 weeks). CONCLUSIONS: The data suggest that simultaneous transplantation of mobilized stem cells and a VCA is feasible and leads to tolerance toward the VCA in a haploidentical setting. However, there is a higher rate of donor stem cell engraftment compared with marrow HCT and an increase in the incidence of graft-versus-host disease.

Simultaneous transplantation of hematopoietic stem cells and a vascularized composite allograft leads to tolerance.

BACKGROUND: We have previously demonstrated that tolerance to a vascularized composite allograft (VCA) can be achieved after the establishment of mixed chimerism. We test the hypothesis that tolerance to a VCA in our dog leukocyte antigen-matched canine model is not dependent on the previous establishment of mixed chimerism and can be induced coincident with hematopoietic cell transplantation (HCT). METHODS: Eight dog leukocyte antigen-matched, minor antigen mismatched dogs received 200 cGy of radiation and a VCA transplant. Four dogs received donor bone marrow at the time of VCA transplantation (group 1), whereas a second group of four dogs did not (group 2). All recipients received a limited course of postgrafting immunosuppression. All dogs that received HCT and VCA were given donor, third-party, and autologous skin grafts. RESULTS: All group 1 recipients were tolerant to their VCA (>62 weeks). Three of the four dogs in group 2 rejected their VCA transplants after the cessation of immunosuppression. Biopsies obtained from the muscle and skin of VCA from group 1 showed few infiltrating cells compared with extensive infiltrates in biopsies of VCA from group 2. Compared with autologous skin and muscle, elevated levels of CD3+ FoxP3+ T-regulatory cells were found in the skin and muscle obtained from the VCA of HCT recipients. All group 1 animals were tolerant to their donor skin graft and promptly rejected the third-party skin grafts. CONCLUSION: These data demonstrated that donor-specific tolerance to all components of the VCA can be established through simultaneous nonmyeloablative allogeneic HCT and VCA transplantation protocol.

Tolerance to vascularized composite allografts in canine mixed hematopoietic chimeras.

BACKGROUND: Mixed donor-host chimerism, established through hematopoietic cell transplantation (HCT), is a reproducible strategy for the induction of tolerance toward solid organs. Here, we ask whether a nonmyeloablative conditioning regimen establishing mixed donor-host chimerism leads to tolerance of antigenic vascularized composite allografts. METHODS: Stable mixed chimerism was established in dogs given a sublethal dose (1-2 Gy) total body irradiation before and a short course of immunosuppression after dog leukocyte antigen-identical marrow transplantation. Vascularized composite allografts from marrow donors were performed after a median of 36 months (range, 4-54 months) after HCT. RESULTS: All marrow recipients maintained mixed donor-host hematopoietic chimerism and accepted vascularized composite allografts for periods ranging between 52 and 90 weeks; in turn, marrow donors rejected vascularized composite allografts from their respective marrow recipients within 18 to 29 days. Biopsies of muscle and skin of vascularized composite allografts from mixed chimeras showed few infiltrating cells compared with extensive infiltrates in biopsies of vascularized composite allografts from marrow donors. Elevated levels of CD3+ FoxP3+ T-regulatory cells were found in skin and muscle of vascularized composite allografts of mixed chimeras compared with normal tissues. In mixed chimeras, increased numbers of T-regulatory cells were found in draining compared with nondraining lymph nodes of vascularized composite allografts. CONCLUSIONS: These data suggest that nonmyeloablative HCT may form the basis for future clinical applications of solid organ transplantation and that T-regulatory cells may function toward maintenance of the vascularized composite allograft.

A preclinical canine model for composite tissue transplantation.

The feasibility of composite tissue allografts (CTAs) has been demonstrated by the successful transplantation of the hand, abdomen, and face. However, the survival of these transplants depends on immunosuppression. Our laboratory is interested in achieving tolerance to decrease the risks associated with the use of chronic immunosuppression. The purpose of this experiment was to develop a large-animal model for CTA. Four canine flaps were autotransplanted to examine the use of a myocutaneous rectus flap based on the deep inferior epigastric vessels. Five CTA transplants were performed between dog leukocyte antigen (DLA)-identical littermates without posttransplant immunosuppression. The allografts were followed clinically and underwent routine biopsies. The anatomic dissections and autotransplants were all successful and revealed that the flap could be divided into two separate components. Skin was perfused by the superficial epigastric artery. Rectus muscle was perfused by the deep inferior epigastric system. This allowed the allografts to be transplanted as muscle or skin or with both components based on the external iliac artery and veins. The DLA-identical littermates rejected the allografts in 15 to 30 days. This study demonstrated the versatility of the myocutaneous rectus flap for use in canines as CTA models.