Background: Whether mixed chimeras induced by nonmyeloablative conditioning are tolerant to challenge with donor allogeneic islet grafts is unknown. Here we investigate whether our nonmyeloablative, costimulation blockade-free and sirolimus (SRL)-based protocol could facilitate mixed chimerism via bone marrow transplantation (BMT) and induce islet allograft tolerance.
Methods: After low dose (1-3 Gy) total body irradiation (TBI, day -1), with or without prior lymphocyte depletion, C57BL/6 mice were transfused with 40 x 10(6) BALB/c bone marrow cells (day 0) and received SRL (3 mg/kg/day) for 4 weeks. Chimerism was monitored by flow cytometry and the recipients were rendered diabetic chemically and challenged with donor islets.
Results: Mixed chimerism was achieved in mice treated with TBI 3 Gy/SRL but it declined over time in 60% (9/15) of them. Long-term stable chimerism was established in 100% of recipients over 50 weeks with either antilymphocyte serum (ALS, 9/9), anti-CD4 (4/4), or anti-CD4 plus anti-CD8 (5/5) prior to BMT. TBI conditioning could be reduced to 1 Gy, with 90% (9/10) maintaining chimerism in the long-term. When TBI was substituted with cyclophosphamide (CTX) or busulfan (BUS), all mice remained chimeric in the long-term. The chimeras showed no proliferative response to donor antigen and accepted both first and second donor-specific islet grafts indefinitely while rejecting third-party grafts.
Conclusions: This data provides the first evidence that stable fully allogeneic chimeras induced with BMT after nonmyeloablative conditioning with SRL and lymphocyte-depleting antibodies exhibit robust donor-specific tolerance to islet grafts.