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

我们之前报道过一种耐受诱导方法，包括静脉注射1x10^8个异体脾细胞（SC）， 2天后，通过腹腔注射200mg/kg CP。通过这种方法，我们能够在H-2相同的组合中诱导持久的皮肤移植（SG）耐受。采用α -Gal Ag预免疫和α -Gal nAb抗免疫的α -Gal敲除小鼠，我们评估了该耐受系统诱导B细胞对α - α (1-3)Gal耐受的有效性。方法以> α - gal敲除（α - gal KO; H-2^<b/d>）和AKR （H-2^k）小鼠为受体和供体。第一组：未经治疗的alpha Gal KO小鼠。第2组：α - gal - KO小鼠于第2天注射1 × 10^8 AKR SC。第3组：α - gal - KO小鼠第2天注射1 × 10^8 AKR SC，第0天注射CP。采用流式细胞仪检测抗α - gal IgM、IgG1、IgG2a、IgG2b、IgG3的水平。用流式细胞仪观察α - gal nab生成b细胞的动力学。治疗2周后移植更多的AKR心脏移植物（HG）。<结果>单独注射AKR SC两周后，抗α - gal IgM和IgG2a的产生增加。然而，在观察期间，SC和CP处理后，抗α - gal Ab的产生完全被抑制。AKR心脏移植物在未治疗或注射AKR SC的α - gal KO小鼠移植后7天内发生排斥反应。组织学分析显示心肌出血和冠状动脉血栓栓塞，即体液排斥反应的证据。另一方面，在接受SC和CP治疗的受体中，AKR HG的生存期明显延长，70%的患者存活超过100天。组织学分析未发现体液性排斥反应。我们的研究证实了我们的环磷酰胺诱导耐受系统在诱导α - gal敲除小鼠体液耐受方面的有效性。少

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