Christopher Mallard

Vascularized Composite Allograft Tolerance Across MHC Barriers in a Large Animal Model

Vascularized composite allograft (VCA) transplantation can restore form and function following severe craniofacial injuries, extremity amputations or massive tissue loss. The induction of transplant tolerance would eliminate the need for long‐term immunosuppression, realigning the risk–benefit ratio for these life‐enhancing procedures. Skin, a critical component of VCA, has consistently presented the most stringent challenge to transplant tolerance. Here, we demonstrate, in a clinically relevant miniature swine model, induction of immunologic tolerance of VCAs across MHC barriers by induction of stable hematopoietic mixed chimerism. Recipient conditioning consisted of T cell depletion with CD3‐immunotoxin, and 100 cGy total body irradiation prior to hematopoietic cell transplantation (HCT) and a 45‐day course of cyclosporine A. VCA transplantation was performed either simultaneously to induction of mixed chimerism or into established mixed chimeras 85–150 days later. Following withdrawal of immunosuppression both VCAs transplanted into stable chimeras (n = 4), and those transplanted at the time of HCT (n = 2) accepted all components, including skin, without evidence of rejection to the experimental end point 115–504 days posttransplant. These data demonstrate that tolerance across MHC mismatches can be induced in a clinically relevant VCA model, providing proof of concept for long‐term immunosuppression‐free survival.

Transgenic Expression of Human CD47 Markedly Increases Engraftment in a Murine Model of Pig-to-Human Hematopoietic Cell Transplantation

Mixed chimerism approaches for induction of tolerance of solid organ transplants have been applied successfully in animal models and in the clinic. However, in xenogeneic models (pig‐to‐primate), host macrophages participate in the rapid clearance of porcine hematopoietic progenitor cells, hindering the ability to achieve mixed chimerism. CD47 is a cell‐surface molecule that interacts in a species‐specific manner with SIRPα receptors on macrophages to inhibit phagocytosis and expression of human CD47 (hCD47) on porcine cells has been shown to inhibit phagocytosis by primate macrophages. We report here the generation of hCD47 transgenic GalT‐KO miniature swine that express hCD47 in all blood cell lineages. The effect of hCD47 expression on xenogeneic hematopoietic engraftment was tested in an in vivo mouse model of human hematopoietic cell engraftment. High‐level porcine chimerism was observed in the bone marrow of hCD47 progenitor cell recipients and smaller but readily measurable chimerism levels were observed in the peripheral blood of these recipients. In contrast, transplantation of WT progenitor cells resulted in little or no bone marrow engraftment and no detectable peripheral chimerism. These results demonstrate a substantial protective effect of hCD47 expression on engraftment and persistence of porcine cells in this model, presumably by modulation of macrophage phagocytosis.

Prolonged Survival of Pig Skin on Baboons After Administration of Pig Cells Expressing Human CD47.

BackgroundSuccessful xenotransplantation will likely depend, in part, on the induction of immunological tolerance, because the high levels of immunosuppression otherwise required would likely have unacceptable side effects. Rapid clearance of administered porcine hematopoietic stem cells by primate macrophages has hampered previous attempts to induce tolerance through mixed hematopoietic chimerism across a pig-to-primate barrier. Phagocytosis is normally inhibited by binding of cell surface protein CD47 to macrophage signal regulatory protein &agr; receptors. However, pig CD47 has previously been shown to be ineffective in transducing signals through primate signal regulatory protein &agr;. MethodsMobilized peripheral blood hematopoietic cells from transgenic swine expressing high or low levels of human CD47 were infused into conditioned baboons at 3 time points over a 9-week period. Xenogeneic peripheral blood chimerism was assessed after each infusion. Split thickness skin grafts from the hematopoietic cell donor swine were placed on recipients 5 weeks after the last cell infusion and 7 weeks after the discontinuation of all immunosuppression to test immune response. ResultsThe level and duration of transient chimerism were substantially greater in baboons receiving hematopoietic cells from a pig expressing high levels of human CD47. Skin graft survival on high CD47 recipients was prolonged as well, in 1 case showing no signs of rejection at least 53 days after placement. ConclusionsProlongation of transient porcine chimerism via transgenic expression of human CD47 in a primate model is associated with an immune modulating effect, leading to markedly prolonged survival of donor swine skin xenografts that may be applicable to clinical solid organ xenotransplantation.

Lack of cross-sensitization between α-1,3-galactosyltransferase knockout porcine and allogeneic skin grafts permits serial grafting

Background The current standard of care for burns requiring operative treatment consists of early burn excision and autologous split-thickness skin grafting. However, in large burns, sufficient donor sites may not be available to achieve total coverage, necessitating temporary coverage with allogeneic human cadaver skin grafts or synthetic skin substitutes. A previous study from this laboratory demonstrated that skin grafts from alpha-1,3 galactosyltransferase knockout (GalT-KO) miniature swine enjoyed survival comparable to that of allogeneic skin grafts in baboons. Methods In the present study, we have evaluated the immune response against sequential GalT-KO and allogeneic skin grafts to determine whether such serial grafts could extend the period of temporary wound coverage before definitive grafting with autologous skin. Results We report that rejection of primary GalT-KO skin grafts led to an anti-xenogeneic humoral response with no evidence for sensitization to alloantigens nor acceleration of rejection of allogeneic skin grafts. Similarly, presensitization with allogeneic skin did not lead to accelerated rejection of xenogeneic skin. Conclusions These data suggest that GalT-KO skin grafts could provide an early first-line treatment in the management of severe burns that would not preclude subsequent use of allografts, and that serial grafting of GalT-KO skin and allogeneic skin could potentially be used to provide an extended period of temporary burn wound coverage.

The gracilis myocutaneous free flap in swine: An advantageous preclinical model for vascularized composite allograft transplantation research

Vascularized composite allotransplantation (VCA) has become a clinical reality, prompting research aimed at improving the risk‐benefit ratio of such transplants. Here, we report our experience with a gracilis myocutaneous free flap in Massachusetts General Hospital miniature swine as a preclinical VCA model. Fourteen animals underwent free transfer of a gracilis myocutaneous flap comprised of the gracilis muscle and overlying skin, each tissue supplied by independent branches of the femoral vessels. End‐to‐end anastomoses were performed to the common carotid artery and internal jugular vein, or to the femoral vessels of the recipients. Thirteen of fourteen flaps were successful. A single flap was lost due to compromise of venous outflow. This model allows transplantation of a substantial volume of skin, subcutaneous tissue, and muscle. The anatomy is reliable and easily identified and harvest incurs minimal donor morbidity. We find this gracilis myocutaneous flap an excellent pre‐clinical model for the study of vascularized composite allotransplantation.

Genetically modified porcine split-thickness skin grafts as an alternative to allograft for provision of temporary wound coverage: preliminary characterization

Temporary coverage of severely burned patients with cadaver allograft skin represents an important component of burn care, but is limited by availability and cost. Porcine skin shares many physical properties with human skin, but is susceptible to hyperacute rejection due to preformed antibodies to α-1,3-galactose (Gal), a carbohydrate on all porcine cells. Our preliminary studies have suggested that skin grafts from α-1,3-galactosyltransferase knock out (GalT-KO) miniature swine might provide temporary wound coverage comparable to allografts, since GalT-KO swine lack this carbohydrate. To further evaluate this possibility, eight non-human primates received primary autologous, allogeneic, GalT-KO, and GalT+xenogeneic skin grafts. Additionally, secondary grafts were placed to assess whether sensitization would affect the rejection time course of identical-type grafts. We demonstrate that both GalT-KO xenografts and allografts provide temporary coverage of partial- and full-thickness wounds for up to 11 days. In contrast, GalT+xenografts displayed hyperacute rejection, with no signs of vascularization and rapid avulsion from wounds. Furthermore, secondary GalT-KO transplants failed to vascularize, demonstrating that primary graft rejection sensitizes the recipient. We conclude that GalT-KO xenografts may provide temporary coverage of wounds for a duration equivalent to allografts, and thus, could serve as a readily available alternative treatment of severe burns.

The Effect of MHC Antigen Matching Between Donors and Recipients on Skin Tolerance of Vascularized Composite Allografts

The emergence of skin‐containing vascularized composite allografts (VCAs) has provided impetus to understand factors affecting rejection and tolerance of skin. VCA tolerance can be established in miniature swine across haploidentical MHC barriers using mixed chimerism. Because the deceased donor pool for VCAs does not permit MHC antigen matching, clinical VCAs are transplanted across varying MHC disparities. We investigated whether sharing of MHC class I or II antigens between donors and recipients influences VCA skin tolerance. Miniature swine were conditioned nonmyeloablatively and received hematopoietic stem cell transplants and VCAs across MHC class I (n = 3) or class II (n = 3) barriers. In vitro immune responsiveness was assessed, and VCA skin‐resident leukocytes were characterized by flow cytometry. Stable mixed chimerism was established in all animals. MHC class II–mismatched chimeras were tolerant of VCAs. MHC class I–mismatched animals, however, rejected VCA skin, characterized by infiltration of recipient‐type CD8+ lymphocytes. Systemic donor‐specific nonresponsiveness was maintained, including after VCA rejection. This study shows that MHC antigen matching influences VCA skin rejection and suggests that local regulation of immune tolerance is critical in long‐term acceptance of all VCA components. These results help elucidate novel mechanisms underlying skin tolerance and identify clinically relevant VCA tolerance strategies.

Effects of Transient Donor Chimerism on Rejection of MHC-Mismatched Vascularized Composite Allografts in Swine

Background: Despite encouraging outcomes in vascularized composite allograft (VCA) transplantation, the risks of chronic immunosuppression limit widespread applicability. It has been suggested that infusion of donor bone marrow along with the VCA may reduce the level of immunosuppression required to prevent clinical VCA rejection. However, no clear evidence has yet been presented to confirm the role of donor bone marrow in the prevention of rejection. In this study we investigated the immunologic effects of concurrent bone marrow transplantation in a large animal VCA model. Methods: MGH miniature swine (n=4) received a non-myeloablative conditioning regimen consisting of low-dose total body irradiation, T-cell depletion, a short course of Cyclosporine A, with or without varying doses of donor bone marrow cells in combination with a complete MHC-mismatched VCA. Animals were monitored daily for signs of rejection or graft versus host disease. Chimerism levels were assessed using flow cytometry and in vitro assays were performed to assess for donor-specific responses. Results: Transient chimerism was prolonged with increased bone marrow cell doses and total body irradiation. While animals that received BMC infusions did not have significantly prolonged VCA acceptance following cessation of immunosuppression compared to animals that received conditioning without BMCs, they demonstrated better early clinical outcomes and demonstrated donor-specific unresponsiveness during the presence of detectable chimerism. Conclusions: Detectable mixed chimerism following bone marrow transplantation and VCA mitigates donor-specific responses and acute rejection episodes, but does not appear to be sufficient for tolerance induction.

Skin grafts from genetically modified α-1,3-galactosyltransferase knockout miniature swine: A functional equivalent to allografts

Burn is associated with a considerable burden of morbidity worldwide. Early excision of burned tissue and skin grafting of the resultant wound has been established as a mainstay of modern burn therapy. However, in large burns, donor sites for autologous skin may be limited. Numerous alternatives, from cadaver skin to synthetic substitutes have been described, each with varying benefits and limitations. We previously proposed the use of genetically modified (alpha-1,3-galactosyl transferase knockout, GalT-KO) porcine skin as a viable skin alternative. In contrast to wild type porcine skin, which has been used as a biologic dressing following glutaraldehyde fixation, GalT-KO porcine skin is a viable graft, which is not susceptible to loss by hyperacute rejection, and undergoes graft take and healing, prior to eventual rejection, comparable to cadaver allogeneic skin. In the current study we aimed to perform a detailed functional analysis of GalT-KO skin grafts in comparison to allogeneic grafts for temporary closure of full thickness wounds using our baboon dorsum wound model. Grafts were assessed by measurement of fluid loss, wound infection rate, and take, and healed appearance, of secondary autologous grafts following xenograft rejection. Comparison was also made between fresh and cryopreserved grafts. No statistically significant difference was identified between GalT-KO and allogeneic skin grafts in any of the assessed parameters, and graft take and function was not adversely effected by the freeze-thaw process. These data demonstrate that GalT-KO porcine grafts are functionally comparable to allogeneic skin grafts for temporary closure of full thickness wounds, and support their consideration as an alternative to cadaver allogeneic skin in the emergency management of large burns.

Topical Delivery of Immunosuppression to Prolong Xenogeneic and Allogeneic Split-Thickness Skin Graft Survival

Abstract Cadaveric skin allograft is the current standard of treatment for temporary coverage of large burn wounds. Porcine xenografts are viable alternatives but undergo α-1,3-galactose (Gal)–mediated hyperacute rejection and are lost by post-operative day (POD) 3 because of naturally occurring antibodies to Gal in primate recipients. Using baboons, we previously demonstrated that xenografts from GalT-KO swine (lacking Gal) provided wound coverage comparable with allografts with systemic immunosuppression. In this study, we investigate topical immunosuppression as an alternative to prolong xenograft survival. Full-thickness wounds in baboons were created and covered with xenogeneic and allogeneic split-thickness skin grafts (STSGs). Animals were treated with slow-release (TyroSphere-encapsulated) topical formulations (cyclosporine-A [CSA] or Tacrolimus) applied 1) directly to the STSGs only, or 2) additionally to the wound bed before STSG and 1). Topical CSA did not improve either xenograft or allograft survival (median: treated grafts = 12.5 days, control = 14 days; P = 0.27) with similar results when topical Tacrolimus was used. Pretreatment of wound beds resulted in a significant reduction of xenograft survival compared with controls (10 vs 14 days; P = 0.0002), with comparable results observed in allografts. This observation was associated with marked reduction of inflammation on histology with Tacrolimus and not CSA. Prolongation of allograft and xenograft survival after application to full-thickness wound beds was not achieved with the current formulation of topical immunosuppressants. Modulation of inflammation within the wound bed was effective with Tacrolimus pretreatment before STSG application and may serve as a treatment strategy in related fields.