Tolerance to musculoskeletal allografts with transient lymphocyte chimerism in miniature swine.

BACKGROUND: Although transplantation of musculoskeletal allografts in humans is technically feasible, the adverse effects of long-term immunosuppression subject the patient to high risks for correcting a non-life-threatening condition. Achieving immunologic tolerance to musculoskeletal allografts, without the need for chronic immunosuppression, could expand the clinical application of limb tissue allografting. Tolerance to musculoskeletal allografts has been accomplished previously in miniature swine in our laboratory. Although stable, mixed chimerism has been suggested as the mechanism underlying long-term tolerance in a rat limb model, the mechanism of this tolerance induction has not been established. This report explores the possible relationship between hematopoietic chimerism and tolerance to musculoskeletal allografts in swine. METHODS: Twelve miniature swine underwent vascularized musculoskeletal allograft transplantation from histocompatibility complex (MHC) matched, minor antigen-mismatched donors. Eight animals received a 12-day coprse of cyclosporine, one of which was excluded due to subtherapeutic levels. Four recipients were not immunosuppressed. Serial biopsies to assess graft viability and flow cytometry to assess chimerism were performed. Donor and third-party skin grafts were placed on recipients with surviving allografts greater than 100 days to validate tolerance. RESULTS: Both groups developed early peripheral chimerism, but this chimerism became undetectable by postoperative day 19 in the cyclosporine group and by day 13 in the control group. Animals receiving cyclosporine developed permanent tolerance to their allografts, whereas those not receiving cyclosporine rejected their allografts in 6-9 weeks. Animals demonstrating tolerance to their bone allografts also demonstrated prolonged donor skin graft survival. CONCLUSIONS: Induction of tolerance to musculoskeletal allografts can be achieved in the MHC matched swine. Although hematopoietic chimerism is present in the immediate postoperative period, persistent, long-term chimerism does not seem to be necessary for maintenance of such tolerance.

Prolonged survival of musculoskeletal xenografts with combined cyclosporine and 15-deoxyspergualin.

This study was undertaken to evaluate the feasibility of performing vascularized musculoskeletal xenografts between mice and rats using immunosuppression. Vascularized musculoskeletal grafts were harvested from the hind limb of C57BL/6J (B6) mice, transplanted heterotopically into Lewis rats, and revascularized by microanastomoses of the graft artery and the recipient femoral artery and the graft vein to the recipient femoral vein. Recipient rats were divided into four groups. Group 1 received no immunosuppression (n = 10), group 2 was treated with cyclosporine (10 mg/kg/day; n = 10), group 3 was treated with 15-deoxyspergualin (5 mg/kg/day; n = 10), and group 4 received both cyclosporine and 15-deoxyspergualin (n = 10). Graft survival was directly examined on postoperative days 4, 7, and 14. In vitro assays were performed using mixed lymphocyte reactions and anti-donor cytotoxic antibody assays to assess the recipient's immune response. Grafts were examined by histology and immunohistochemistry. All grafts in group 1 were rejected by day 4. In groups 2 and 3, all grafts were rejected by day 7. In group 4, however, 8 of 10 recipients had viable grafts on day 14. Data from mixed lymphocyte reactions showed that cell-mediated immune responses were uniformly suppressed in groups 2, 3, and 4 compared with group 1. However, anti-donor antibody production was only partly suppressed in groups 2 and 3, suggesting that graft rejection was primarily caused by circulating cytotoxic anti-donor antibodies in groups 1, 2, and 3. Histologic observations in groups 1, 2, and 3 confirmed the important role of the humoral mechanism in xenograft rejection. Furthermore, immunohistochemical results demonstrated that the small vessels in the rejected grafts showed anti-rat immunoglobulin and complement depositions. Only a combination therapy of cyclosporine and 15-deoxyspergualin attenuated the rejection of xenografts.

Xenotransplantation model for vascularized musculoskeletal tissues in rodents.

The purpose of this study was to establish a model and to define the mechanism of rejection for the transplantation of vascularized musculoskeletal xenografts between C57BL/6j (B6) mice and Lewis rats. This was accomplished by using conventional skin xenografts to determine immunologic baseline data between these species and by performing musculoskeletal grafts from the B6 mice transplanted into Lewis rats. After the transplant, the xenografts were examined histologically and the recipients were assessed for immune reaction using in vitro assays to measure both cell-mediated and humoral responses. The results obtained from the skin xenografts showed activation of both cellular and humoral immunologic responses. All musculoskeletal xenografts were rejected between 3 and 4 postoperative days. Histologically, the grafts showed extensive vascular injury manifested by thrombosis and hemorrhage, suggesting an early humoral response. Anti-donor antibody production was detected in the recipient's sera soon after rejection of the xenogeneic tissue. The cell-mediated immune response, although detectable by the in vitro assays, was less pronounced than the humoral response and corroborated the histologic findings of mild lymphocyte infiltration in the rejected tissue. These results demonstrate that humoral rejection plays a predominant role in the rejection of vascularized musculoskeletal xenotransplants between concordant species. This mouse-to-rat vascularized xenograft model will be utilized for further studies on inducing tolerance to vascularized musculoskeletal xenografts.