Donor-specific tolerance induction in composite tissue allografts

Abstract

BACKGROUND: Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting would be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a strongly antigenic MHC mismatch without the need for long-term immunosuppression. METHODS: Chimeras were prepared using rat strains with strong MHC incompatibility [WF (RT1A u) + ACI (RT1A a) → WF, n = 13]. Syngeneic (WF) and allogeneic (ACI) bone marrow (BM) was harvested and T-cell depleted. Following confirmation of T-cell depletion by flow cytometry, a mixture of T-cell depleted syngeneic and allogeneic BM was injected into the recipient animals (all recipients pretreated with low-dose irradiation, 500 to 700 cGy). In addition, the recipient animals received a single dose of ALS (10 mg) 5 days prior to bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day prior to BMT to 10 days postoperatively. Rat chimeras were characterized by flow cytometry at 3 and 12 months after BM reconstitution and following hindlimb transplantation. RESULTS: Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 10 of 13 animals. Multilineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60%, there was no sign of limb rejection for the duration of the study. All animals with chimerism <20% developed moderate signs of rejection clinically and histologically. Gross motor and sensory reinnervation (weight bearing, toe spread) occurred at >60 days in 6 of 9 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n = 7). CONCLUSIONS: Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. This represents the first reliable model demonstrating rejection-free CTA survival in an adult animal without the long-term use of immunosuppressive agents across a strongly antigenic MHC mismatch.