Induction of B cell tolerance against Gal-alpha(1-3)Gal Ag in Cyclophosphamide (CP)-induced tolerance

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We have previously reported a method of tolerance induction that comprises an i.v. injection of 1x10^8 allogeneic spleen cells (SC) followed, 2 days later, by an i.p. administration of 200mg/kg of CP. By using this method, we were able to induce a long-lasting skin graft (SG) tolerance in H-2 identical combinations. By using alpha-Gal knockout mice which are preimmunized with alpha-Gal Ag and have anti-alpha Gal nAb, we evaluated the effectiveness of this tolerance system to induce B cell tolerance against Gal-alpha(1-3)Gal. <Method> alpha-Gal knockout (alpha-Gal KO ; H-2^<b/d>) and AKR (H-2^k) mice were used as recipients and donors. Group 1 : untreated alpha Gal KO mice. Group 2: alpha-Gal KO mice were injected with 1x10^8 AKR SC on day -2. Group 3 : alpha-Gal KO mice were injected with 1x10^8 AKR SC on day -2 and CP on day 0. The level of anti-alpha-Gal IgM, IgG1, IgG2a, IgG2b, IgG3 was measured by FACS. The kinetics of alpha-Gal nAb-producing Bcells were evaluated with flow cytomet … More ry. AKR heart grafts (HG) were transplanted 2 weeks after the treatments. Histological examination was performed in the transplanted HG. <Result> The production of anti-alpha-Gal IgM and IgG2a was increased two weeks after injection of AKR SC alone. However, the production of anti-alpha-Gal Ab was completely inhibited after treatment with SC and CP during the observation. AKR heart grafts were rejected within 7 days after transplantation in untreated or AKR SC injected alpha-Gal KO mice. Histological analysis showed the hemorrhage within the cardiac muscle and thromboembolism in the coronary artery, i.e., the evidence of humoral rejection. In recipients treated with SC and CP, on the other hand, survival of AKR HG were significantly prolonged and 70% of them survived over 100 days. Histological analysis showed no evidence of humoral rejection. <Conclusion> Our studies confirmed the effectiveness of our cyclophosphamide-induced tolerance system in the induction of humoral tolerance in alpha Gal-knockout mice. Less