Combined treatment with regulatory T cells and vascularized bone marrow transplantation creates mixed chimerism and induces donor-specific tolerance to vascularized composite allografts without cytoreductive conditioning

Background: Cotreatment with regulatory T cells (T_reg) and conventional allogeneic bone marrow transplantation (BMT) successfully induced durable chimerism and tolerance to nonvascularized skin allografts without cytoreductive conditioning in mice. We sought to determine whether T _reg treatment combined with vascularized BMT (VBMT) could create mixed chimerism and induce tolerance to vascularized composite allografts (VCAs) without cytoreductive conditioning in rats. Methods: Recipient Lewis rats treated (day 0) with or without naturally sorted T_reg (3 × 10⁶) from Lewis rat spleen and lymph nodes received costimulation blockade (anti-CD154 monoclonal antibody, days 0 and 1 and CTLA-4 immunoglobin, days 2, 4, and 6), rapamycin (days -1, 0, and 2), and concurrent transplantation of fully mismatched allogeneic donor VCAs (day 0) from the Brown Norway rat hindlimb containing VBMT. The mixed chimerism level was assessed monthly using flow cytometry. Survival of VCAs and occurrence of graft-versus-host disease were assessed clinically and histologically. Results: The combination of T _reg and VBMT treatment led to long-term multilineage hematopoietic mixed chimerism (12-18%) and long-term donor-specific tolerance to VCAs (89% acceptance rate). Neither stable mixed chimerism nor VCA acceptance was observed in recipients without T_reg treatment. Graft-versus-host disease did not occur in the VBMT recipients. Conclusions: Cotreatment with T_reg and VBMT created stable mixed chimerism and induced long-term donor-specific tolerance to VCAs without requiring cytoreductive conditioning. This noncytoreductive T_reg-VBMT protocol has potential for clinical application in VCAs.