Donor-specific immunological tolerance using high doses of donor bone marrow cells (BMC) has been demonstrated in mixed chimerism-based tolerance induction protocols; however, the development of graft versus host disease (GVHD) remains a risk.Full-thickness BALB/c skin allografts were transplanted onto C57BL/6 mice. Mice were treated with anti-CD4 and anti-CD8 mAbs on day 0, + 2, + 5, + 7 and + 14 along with low dose busulfan (5 mg/kg, i.p.) on day +5. Human amnion-derived multipotent progenitor cells (AMPCs) and unfractionated donor bone marrow cells (5 x 10⁵) were co-infused intravenously on day +7. Chimerism and clonal cell deletion were evaluated using flow cytometry. Donor-specific tolerance was tested by donor and third-party skin grafting and mixed leukocyte reaction (MLR). AMPC + BMC co-infusion with minimal immunosuppression led to stable, mixed, multilineage macrochimerism and long-term allograft survival (> 300 days). Mixed lymphoid and myeloid donor-recipient macrochimerism (35-70%) was observed without GvHD. Donor-reactive T cells were clonally deleted and a 2.7-3.2-fold increase in CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) was observed in the spleen. Tolerant mice subsequently accepted second donor, but not third-party (C3H/HEJ), skin grafts and recipient splenocytes failed to react with allogeneic donor cells indicating donor-specific immunological tolerance was achieved. Conditioned mice treated identically with AMPCs alone or BMC alone lost their skin grafts 25-40 days post grafting. We conclude that the co-infusion of low dose donor BMC + AMPCs without prolong cytoreductive recipient conditioning can induce indefinite survival of skin allografts via mechanisms involving the establishment of a multilineage macrochimeric state principally through clonal deletion of alloreactive T cells and peripherally induced CD4⁺Foxp3⁺ Tregs. We have demonstrated that the co-infusion of low numbers of donor unfractionated bone marrow cells with human amnion-derived progenitor cells 7 days post allograft transplantation facilitates high level macrochimerism induction and allograft tolerance.

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