The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti-CD154 mAb, nondepleting anti-CD4 combined to either depleting or nondepleting anti-CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long-term allograft survival in both combinations. Nevertheless, robust donor-specific tolerance, defined by the acceptance of a fresh donortype skin allograft and simultaneous rejection of thirdparty grafts, required initial CD8 + T-cell depletion. Mixed donor-recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL-2, IFN-c and TNF-a in MLC with donor antigen while significant alloreactivity persisted against third-party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor-recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8 + T-cell depletion and costimulation/coreceptor blockade.

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