The necessity of both allogeneic antigens and stem cells for cyclophosphamide-induced skin allograft tolerance in mice.

We have reported that in an H-2 identical murine combination of AKR/J (AKR, H-2k, Thy1.1) and C3H/HeJ (C3H, H-2k, Thy 1.2), specific tolerance to C3H skin in AKR mice is induced only when both intravenously (i.v.) 1 x 10(8) viable C3H spleen cells and, two days later, intraperitoneally (i.p.) 200 mg/kg cyclophosphamide (CP) have been given. To further examine this mechanism of tolerance, we used 2000R-irradiated C3H spleen cells as an antigen source and bone marrow cells depleted of Thy1.2+ cells and Ia+ cells as a stem cell source. When a mixture of 1 x 10(8) irradiated spleen cells and 3 x 10(7) bone marrow cells was used as tolerogen and 200 mg/kg CP was administered two days later, a profound and specific long-lasting tolerance was induced. This tolerant state, however, was less profound than that induced with spleen cells plus CP. When the number of irradiated spleen cells was fixed at 1 x 10(8), the tolerant state was dose-dependent on the quantity of bone marrow cells. On the other hand, when the number of bone marrow cells was fixed at 1 x 10(6), tolerance induction depended on the dosage of irradiated spleen cells. Tolerance induced with irradiated spleen cells plus bone marrow cells and CP was tolerogen specific. Tolerance was never induced when the bone marrow cells had been irradiated with 2000R prior to injection. Transfer experiments showed that the tolerant state, in its acute phase, appeared to be predominantly based on reduction of functionally reactive cells. The prolongation of skin allograft survival in tolerant mice could not be attributed directly to suppressor cells, nor was any evidence of suppressive factor induction observed. In the chronic phase, however, the importance of the suppressive mechanisms appeared to be relatively increased. EPICS analysis of the thymocytes using fluorescein-conjugated anti-Thy1.1 and anti-Thy1.2 antibodies showed that a minimal degree of mixed chimerism had been established in the tolerant mice. Moreover, both T cells and Ia+ cells had beneficial effects on the induction of tolerance. We conclude that in the tolerance induced by spleen cells plus CP, histocompatibility antigens expressed on the surface of the spleen cells were essential to the antigen-stimulated cell destruction mechanism. Stem cells contained in the spleen cells also appeared to be crucial for maintaining tolerance by establishing a minimal degree of mixed chimerism.