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Background. Recently, we have described a drug (cyclophosphamide [CP] plus busulfan [BU])-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers. Using this method, we have investigated whether or not this regimen can prolong the survival of heart allograf

We elucidated the possible role of chimerism in skin and heart allograft tolerance using cyclophosphamide (CP)-induced tolerance. When C3H (H-2 k ; Thyl.2, Mls-1 b ) mice were i.v. primed with 1 × 10 8 spleen cells (SC) from H-2 matched AKR (H-2 k ; Thy1.1, Mls-1 a ) mice and then treated i.p. with 200 mg/kg of CP, the survivals of both AKR skin grafts and heart grafts (HG) were permanently prolonged in a tolerogen-specific fashion. After this treatment, a minimal degree of mixed chimerism, the clonal destruction of Mls-1 a -reactive CD4 + Vβ6 + T cells in the periphery, and the clonal deletion of Vβ6 + thymocytes were all observed. When AKR SC and 100 mg/kg CP were used for conditioning, the AKR HG were permanently accepted, but the survival of the AKR skin grafts was only mildly prolonged. The clonal destruction of CD4 + Vβ6 + T cells in the periphery and the intrathymic clonal deletion of Vβ6 + thymocytes were induced in both the SC and the 100 mg/kg CP-treated C3H mice. A minimal degree of mixed chimerism was detectable at 4 and 12 weeks after AKR SC and 100 mg/kg CP treatment, and still did not disappear at 40 weeks. The degree of mixed chimerism induced with SC and 100 mg/kg CP was significantly lower than that with SC and 200 mg/kg CP during the observation. No posttransplant cardiac allograft vasculopathy (CAV) was observed to develop, while both the Th1 type (interferon-y) and Th2 type (interleukin-4 and -10) cytokine expressions decreased in the AKR HG of the tolerant C3H mice treated with both AKR SC plus 200 mg/kg CP, and AKR SC plus 100 mg/kg CP. A second set of skin grafts from donor AKR mice survived for more than 100 days in a tolerogen-specific fashion in all C3H mice treated with AKR SC and 200 mg/kg CP and also accepted the AKR HG for over 200 days, while 80% of the C3H mice treated with AKR SC and 100 mg/kg CP and accepted the AKR HG for more than 200 days. These results strongly suggested the following conclusions: 1) the degree of chimerism can strongly influence the induction of skin and heart allograft tolerance, 2) posttransplant CAV does not develop in the donor HG maintained by chimerism-based CP-induced tolerance, 3) the mRNA expression of both Th1 and Th2 type cytokine decreased in the donor HG maintained by chimerism-based CP-induced tolerance, and 4) the induction of skin allograft tolerance is more difficult than the prevention of posttransplant CAV.

An essential role of murine CD4 + T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4 + and CD8 + T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2 b ) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3±(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2 k ) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4 + T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8 + T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8 + T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8 + T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4 + T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8 + T cells also are capable of rejecting discordant human skin xenografts.

Background. The method for mouse vascularized heart transplantation have been described using suture and cuff techniques. Technical problems have limited its widespread use. Here, we describe our method of modified cervical heterotopic transplantation with the cuff technique. Methods. By using a smaller Teflon cuff (external diameter 0.6 mm, internal diameter 0.4 mm) and superfine-tip forceps, it became possible to directly pull the edge of the carotid artery and evert the proximal end of the artery over the cuff. Similarly, the external jugular vein could be easily everted over a 22-gauge cuff with this direct pulling method. Results. By these modifications, the operation time was reduced. It usually takes 20 min for the donor harvest, 15 min for preparation of the cervical vessels, and 15 min for anastomosis. All procedures from the donor harvest through skin closure of the recipient mice can be completed within 1 hr, and ischemic time is within 25-40 min. Conclusions. This method can be used to investigate cyclophosphamide-induced tolerance and mechanisms of reperfusion injury.

Background.Injection of allo-spleen cells (SC) followed by a single dose of cyclophosphamide (CP) can induce tolerance of tumor and/or skin allografts in mice. To minimize the damage caused by CP, fractionation of CP that can establish long-lasting skin graft survival, stable mixed chimerism, and in

Recently, we have described a drug (cyclophosphamide [CP] plus busulfan [BU])-induced skin allograft tolerance in mice that can regularly overcome fully H-2-mismatched barriers. Using this method, we have investigated whether or not this regimen can prolong the survival of heart allografts and inhibit the development of posttransplant cardiac allograft vasculopathy (CAV).The components of the method are intravenous administration of 1 x 108 allogeneic spleen cells on day 0, intraperitoneal injection of 200 mg/kg of CP and 30 mg/kg of BU on day 2, and intravenous injection of T cell-depleted 1 x 107 allogeneic bone marrow cells from the same strain of mice on day 3. Heart grafting was performed on day 28. Chimerism in peripheral blood was followed by flow cytometric analysis, and histological analysis was performed at various times after grafting.In a fully major histocompatability complex (MHC)-mismatched combination of B10.D2 (H-2d, IE+)--B10 (H-2b, IE-), stable, multilineage-mixed chimerism was observed permanently. B10.D2 heart grafts were accepted permanently in a donor-specific manner, and posttransplant CAV did not develop.These results demonstrated that the drug-induced tolerance recently established by us can regularly induce a long-lasting heart allograft tolerance without development of CAV.