Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide

Abstract

A new method of cyclophosphamide (CP)-induced skin allograft tolerance in mice that can regularly overcome fully allogeneic (major H-2 plus non-H-2) antigen barriers in mice has been established. The components of the method are intravenous or intraperitoneal administration of 50-100 μg of anti-Thy-1.2 mAb on day -1, intravenous injection of 90 x 106 allogeneic spleen cells mixed with 30 x 106 allogeneic bone marrow cells from the same donor on day 0, and intraperitoneal injection of 200 mg/kg CP on day 2. In each of four fully allogeneic donor→recipient combinations, including C3H/HeJ (C3H; H-2(k))→C57BL/6J(B6; H-2b), B6→C3H, BALB/cByJ (BALB; H-2(d))→B6, and BALB→C3H, long-lasting survival of skin allografts was induced in most of the recipient mice. The specific tolerant state induced was dependent on the doses of the antibody and bone marrow cells used. The optimal timing of CP treatment to induce tolerance was found to be 1-3 d after the stimulating cell injection. Treatment with the anti-Thy-1.2 antibody together with CP on day 2 after the cell injection on day 0 also induced profound tolerance. In the B6 mice made tolerant of C3H with antibody, C3H spleen cells plus C3H bone marrow cells, and then CP, a minimal degree of stable mixed chimerism was established and the antitolerogen (C3H) immune responses examined here, including delayed footpad reaction (DFR), CTL activity, and capacity for antibody production against donor-strain antigens were abrogated in a tolerogen-specific manner. From cell transfer experiments, the mechanism of tolerance could be largely attributed to reduction of effector T cells reactive against the tolerogen, and strong suppressive influences that might prolong skin allograft survival directly were not detected in the tolerant mice. Moreover, pretreatment with anti-Thy-1.2 antibody or anti-L3T4 (CD4) antibody was more effective than pretreatment with anti-Lyt-1 (CD5) antibody or anti-Lyt-2 (CD8) antibody as an initial step in tolerance induction. These results suggest that permanent tolerance to fully allogeneic skin grafts may be induced because antibody given before the stimulating cell injection reduces the number of reactive T cells in the recipient mice. This antibody treatment may facilitate an antigen-stimulated destruction of responding and thus proliferating cells with CP by preventing a possibly less proliferative, more rapid maturation of reactive T cells or by destroying residual effector T cells. The injection of bone marrow cells mixed with spleen cells appear to have facilitated maintenance of the tolerant state by establishing a state of stable mixed chimerism in the tolerant mice. This methodology, which induces within a very short interval a long-lasting tolerant state in immunologically mature mice, may possibly be adaptable to production of full immunological tolerance across major plus minor histocompatibility barriers in other mature mammals, e.g., lower primates and even humans. If this proves to be the case, it could greatly facilitate organ transplantation without the need to constantly depress immunologic reactivities, as is now needed in the practice of organ and tissue transplantation in humans.