Induction of permanent mixed chimerism and skin allograft tolerance acrossfully MHC-mismatched barriers by the additional myelosuppressive treatments in mice primed with allogeneic spleen cells followed by cyclophosphamide

A pure method of drug (cyclophosphamide plus busulfan)-induced skin allogra ft tolerance in mice that can regularly overcome fully H-2-mismatched barri ers in mice has been established. The components of the method are i.v. adm inistration of 1 x 10(8) allogeneic spleen cells on day 0, i.p. injection o f 200 mg/kg CP and 25 mg/kg busulfan on day 2, and i.v. injection of T cell -depleted 1 x 10(7) bone marrow cells from the same donor on day 3, Recipie nt B10 (H-2(b); IE-) mice prepared with this conditioning developed donor-s pecific tolerance, and long-lasting survival of skin allografts was shown i n almost of the recipient mice. In the tolerant B10 mice prepared with new conditioning, stable multilineage mixed chimerism was observed permanently, and IE-reactive V beta 11(+) T cells were reduced in periphery as seen in untreated B10.D2 (H-2(d); IE+) mice. The specific tolerant state was confir med by the specific abrogation against donor Ag in the assays of CTL activi ty and MLR and donor-specific acceptance in the second skin grafting. These results demonstrated that the limitation of standard protocol of cyclophos phamide-induced tolerance, which have been reported by us since 1984, can b e overcome by the additional treatments with the myelosuppressive drug busu lfan, followed by 1 x 10(7) T cell-depleted bone marrow cells. To our knowl edge, this is the first report to induce allograft tolerance with a short c ourse of the Ag plus immunosuppressive drug treatment without any kind of m Abs (pure drug-induced tolerance).