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Bone marrow transplantation (BMT) may induce tolerance across xenogeneic barriers. We have established a xenogeneic BMT model where hamster BM is transplanted into splenectomized LEW rat recipients resulting in high levels of engraftment. Unfortunately, graft vs. host disease (GVHD) with severe dermatitis developed in all rat recipients. We were successful in treating or preventing the dermatitis of this xenogeneic GVHD by the use of the T-cell suppressant tacrolimus. However, this compound did not prevent the development of a fatal liver injury in the rat recipients. This study was designed to elucidate the pathogenesis of this liver injury appearing in T-cell suppressed rat recipients of hamster BM. Splenectomized and irradiated (10 Gy) LEW rats received 300 x 106 unfractionated hamster BM cells. These BMT recipients were divided in 3 groups: Group I recipients (n = 8) did not receive further immunosuppression. Group II animals (n = 10) received tacrolimus 1 mg/kg/d for 7 d. Group III recipients (n = 6) were given the same daily dose of tacrolimus on a long-term basis. Chimerism was detected by flow cytometry. Cytotoxicity of recipient's sera against rat and hamster lymph node cells was measured by complement-dependent cytotoxicity (CDC) test. Immunofluorescence was used to detect hamster antirat antibodies on several recipient organs. In Group I, 2 out of 8 animals engrafted (25%) and survived for a median of 21 d showing the severe dermatitis characteristic of GVHD. In group II (n = 10), 9/10 rat recipients engrafted (90%) and survival was increased to a median of 53.7 days. However, these surviving recipients developed fatal GVHD not different from that observed in Group I recipients. All animals in Group III (n = 6) engrafted and did not show the characteristic dermatitis of GVHD. Their survival, however, was shortened to a median of 30.3 d by a severe liver injury. This injury was characterized by hepatocyte necrosis in zones 1 and 2 with polymorphonuclear (PMN) cell infiltration. Deposits of hamster immunoglobulins were present around the necrotic areas and in the portal veins. Moreover, antirat antibodies appeared in the circulation. These antibodies were sensitive to dithiothreitol (DTT) treatment indicating that they were of the IgM class. This study shows that xenogeneic GVHD may have a dual presentation in the hamster-to-rat model: a classical cellular GVHD not distinct to the allogeneic one and a humoral GVHD affecting solely the recipient liver. The degree of humoral injury is potentiated by T-cell suppression.

Antigen pretreatment through the oral or intraportal route has been reported to suppress antibody formation or delayed-type hypersensitivity (DTH) response to the same antigens. However, this effect on allografted organs has not been well studied. In this study we evaluated the efficacy for suppression of antibody formation and DTH response to the allogeneic antigens and tried to prolong liver allograft survival in rats. Male ACI (RT1a) rats were used as donors and male BUF (RT1b) rats as recipients. Intraportal or intravenous injection of spleen cells (SPCs) (5 x 10(7)) was performed through the mesenteric vein or the tail vein, respectively. The anti-ACI DTH response was tested by ear challenge of ACI SPCs. Cytotoxic antibody was assessed by complement-dependent cytotoxicity assay. ACI rat liver was transplanted orthotopically to BUF rat by the cuff technique 10 days after SPC injection. Cytotoxic antibody titer rose to X2(6) to X2(8) at 7 to 10 days after intravenous injection of SPCs. However, intraportal injection of the cells rarely caused a rise in antibody titer and even strongly suppressed subsequent antibody formation induced by intravenous injection when given 10 days before. The DTH response was also suppressed by intraportal injection of SPCs, with a mean value of 0.18 +/- 0.13 mm versus 0.67 +/- 0.19 mm for controls or 0.46 +/- 0.04 mm with intravenous injection. Liver-allografted rats died between 9 and 11 days, averaging 10.1 +/- 0.7 days in the control group. All seven transplants injected intraportally with donor SPCs survived more than 100 days, whereas six of eight rats injected intravenously with donor SPCs died of bleeding from the surface of the liver grafts within 12 hours after grafting, with signs similar to those of hyperacute rejection. Four of five rats injected intraportally with F344 (third-party) SPCs died of acute rejection in the same way the control rats died. The liver allograft-bearing rats had permanently accepted ACI skin grafts when tested 60 days after liver transplantation but rejected F344 skin grafts in the normal fashion. Intraportal injection of donor SPCs markedly suppressed the antibody formation, as well as the DTH response, and completely blocked liver allograft rejection. Moreover, the liver allograft-bearing rats proved to be fully tolerant of the donor antigen. This method might be a promising modality for inducing donor-specific tolerance in liver transplantation.

In this study we investigated the effect of splenectomy in combination with cyclosporine (CsA) on survival of heterotopic cardiac xenografts from hamster to rat. A 12-fold prolongation of mean cardiac xenograft survival, to 41 days, was accomplished with the combined therapy. In both untreated controls and CsA-treated recipients rejection occurred in 3 days. Splenectomy by itself prolonged xenograft function to 5 days. Evidence of humoral-mediated rejection in this cross-species combination was given for the extensive thrombosis and hemorrhage in the subepicardial area, the appearance of lymphocytotoxic titers just before graft function ceased, and the presence of IgM deposits in subepicardial vessels of the xenograft. CsA by itself could not modify this pattern of rejection. Splenectomy decreased antibody formation significantly and rejection became more cellular in nature. The regimen of splenectomy in association with CsA suppressed antibody titers and produced a CsA dose-dependent prolongation of xenograft survival. Thus, a complementary or synergistic effect is the result of the immunosuppressive regimen of splenectomy and CsA in hamster-to-rat cardiac xenografts. In this study the effect of splenectomy in controlling the humoral response in concordant xenografts and its role in future clinical xenografting is emphasized.

Wistar (RT1bv1) rats transplanted orthotopically with ACI (RT1a) livers survive indefinitely without any immunosuppression, while heterotopic heart grafts or skin grafts are rejected acutely in this combination. Levels of alkaline phosphatase after liver allografting remain significantly higher than those found in controls receiving syngeneic grafts. We studied changes in immune responsiveness in rats receiving liver grafts. Local graft-versus-host reactivity was present at all times assayed. Delayed type hypersensitivity reactions were already positive 2 weeks after liver transplantation and increased in strength. Liver graft-bearing rats were subsequently grafted with donor or third-party skin. Third-party skin grafts survived significantly longer on liver-grafted rats than on untreated controls when grafted within the first week after grafting. Donor-type skin grafts survived longer than controls when grafted within the first 4 weeks after liver grafting, although the skin grafts were eventually rejected. Donor-type skin grafted more than 8 weeks after liver grafting was rejected acutely. In an adoptive transfer assay, ACI hearts survived significantly longer in Wistar rats given serum from Wistar donors 2-4 weeks after ACI liver grafts than in untreated controls. On the other hand, spleen cells obtained at any period after liver grafting were not capable of prolonging cardiac allograft survival after transfer to syngeneic recipients. Thus cellular responses to ACI antigen are not changed during the life-span of liver-grafted animals. Evidence suggests that a serum "enhancing" factor protects the donor liver from rejection in the initial period after liver transplantation. The long-term acceptance of liver grafts is discussed.