Background. The antigenicity of skin is a major obstacle to expanding human composite tissue transplantation. For example, multiple rejection episodes of the skin have been noted in clinical hand transplant patients. We have previously demonstrated tolerance to vascularized musculoskeletal allografts in major histocompatibility complex (MHC)-matched miniature swine treated with 12 days of cyclosporine. This regimen did not reproducibly lead to tolerance to subsequent frozen donor skin grafts. However, such skin grafts did not have a primary vascular supply. The aim of this study was to determine if tolerance to limb allografts with a vascularized skin component could be achieved with MHC matching and a 12-day course of immunosuppression. Methods. Hind limb grafts harvested with a 100 cm² cutaneous paddle were transplanted heterotopically into six MHC-matched, minor antigen-mismatched miniature swine. All animals received a 12-day course of cyclosporine. One control animal was not immunosuppressed. Grafts were evaluated with biweekly biopsies and tissue viability determined by histologic analysis. To test for sensitization, frozen donor skin grafts were applied to all animals that survived to postoperative day 100. Results. All treated animals (n=6) were tolerant to their musculoskeletal allografts at the time of necropsy (>100 days) regardless of the status of the epidermis. One animal demonstrated tolerance to the skin for more than 180 days. The other five animals demonstrated prolonged survival of the epidermal portion of the graft. The control animal rejected the graft epidermis at 10 days postoperatively. Frozen donor skin grafts demonstrated accelerated rejection (<10 days) in three of the animals and led to simultaneous rejection of both the epidermis of the allograft and the skin graft in the long-term tolerant animal. The rejection of the skin grafts did not break tolerance to the musculoskeletal portion in any of the animals. Conclusions. All animals exhibited indefinite survival of the musculoskeletal portion of their allografts but only prolonged survival of the epidermis. The loss of the graft skin appears to be the result of an isolated immune reaction to the skin, and, in particular, the epidermis. This observation is further substantiated by the accelerated rejection of secondarily placed frozen donor skin grafts.

Transplant vasculopathy has not been systematically investigated in composite tissue allotransplantation (CTA). The impact of multiple acute rejections (ARs) on long-term graft outcomes in reconstructive transplantation remains unknown. This study in a rat hind-limb allotransplantation model systematically analyzes vasculopathy and tissue-specific pathological changes secondary to multiple AR episodes. LEW rats were transplanted with BN rat hind limbs and treated as follows: Group 1 (Iso): isografts. Group 2 (CsA): Cyclosporine (CsA) qd; Group 3 (mult AR): CsA and dexamethasone only when AR was observed. No AR was observed in Groups 1 and 2. Multiple AR were observed in Group 3, and each episode was completely reversed (clinically) with pulsed CsA + dexamethasone treatment. Group 3 animals demonstrated significant vascular lesions along with skin and muscle atrophy, upregulation of profibrotic gene expression and fibrosis when compared to Groups 1 and 2. In addition, allograft bone was sclerotic, weak and prone to malunion and nonunion. Interestingly, vasculopathy was a late finding, whereas muscle atrophy with macrophage infiltration was seen early, after only a few AR episodes. Taken together, multiple AR episodes lead to vasculopathy and tissue-specific pathology in CTA. This is the first evidence of ‘composite tissue vasculopathy and degeneration (CTVD)’ in CTA.

Strategies aiming at minimization or elimination of systemic immunosuppression are key immediate goals for clinical expansion of vascularized composite allotransplantation (VCA). We compared the in vitro and in vivo immunomodulatory efficacy of adipose-derived mesenchymal stem cells (AD-MSCs) and bone marrow (BM)-derived MSCs in a rat VCA model. Both cell types were tested in vitro for suppressor function using mixed lymphocyte reactivity assays. AD-MSCs or BM-MSCs were administered intravenously (1 × 10 or 5 × 10 cells/animal) to Lewis rat recipients of mismatched Brown Norway hindlimb transplants. Short course tacrolimus (FK-506) monotherapy was withdrawn at postoperative day 21. In vivo regulatory T-cell induction, peripheral blood chimerism, and microchimerism in lymphatic organs were analyzed. AD-MSCs and BM-MSCs exhibited strong dose-dependent suppressor function in vitro, which was significantly more pronounced for AD cells. In vivo, all animals revealed peripheral multi-lineage chimerism at four weeks (P < 0.01) independent of cell type and dosage. Regulatory T-cell levels were increased with both cell types, the most in AD-MSC groups. These immunomodulatory effects were only transient. MSC treatment resulted in long-term (>120 day) allograft survival in 47% of the animals, which correlated with durable microchimerism in BM and spleen. AD-MSCs and BM-MSCs exert immunomodulatory effects that prolong survival of immunogenic skin-bearing VCA grafts with short course (21 day) tacrolimus induction therapy. The in vivo findings in terms of allograft survival did not reflect superior immunomodulatory characteristics of AD-MSCs found in vitro.

Vascularized composite allotransplantation (VCA) encompasses face and limb transplantation, but as with organ transplantation, it requires lifelong regimens of immunosuppressive drugs to prevent rejection. To achieve donor-specific immune tolerance and reduce the need for systemic immunosuppression, we developed a synthetic drug delivery system that mimics a strategy our bodies naturally use to recruit regulatory T cells (T_reg) to suppress inflammation. Specifically, a microparticle-based system engineered to release the T_reg-recruiting chemokine CCL22 was used in a rodent hindlimb VCA model. These “Recruitment-MP” prolonged hindlimb allograft survival indefinitely (>200 days) and promoted donor-specific tolerance. Recruitment-MP treatment enriched T_reg populations in allograft skin and draining lymph nodes and enhanced T_reg function without affecting the proliferative capacity of conventional T cells. With implications for clinical translation, synthetic human CCL22 induced preferential migration of human T_reg in vitro. Collectively, these results suggest that Recruitment-MP promote donor-specific immune tolerance via local enrichment of suppressive T_reg.