Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non‐immunosuppressed baboons
Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non‐immunosuppressed baboons
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DOI：
10.1046/j.1399-3089.2003.00103_11_2.x
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发表时间：
2004-03
影响因子：
3.9
通讯作者：
B. Loveland;J. Milland;P. Kyriakou;B. Thorley;D. Christiansen;M. Lantéri;Mark Regensburg;M. Duffield;A. J. French;L. Williams;L. Baker;M. Brandon;P. Xing;D. Kahn;I. Mckenzie
中科院分区：
文献类型：
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作者：
B. Loveland;J. Milland;P. Kyriakou;B. Thorley;D. Christiansen;M. Lantéri;Mark Regensburg;M. Duffield;A. J. French;L. Williams;L. Baker;M. Brandon;P. Xing;D. Kahn;I. Mckenzie
摘要：人膜辅因子蛋白(CD46)控制补体激活，当作为转基因充分表达时，可以保护异种移植物免受补体介导的排斥反应，如在非免疫抑制的狒狒和异位CD46转基因猪肾移植中所显示的那样。这份报告是一种精心设计的转基因，能够实现CD46的高水平表达。通过转基因猪品系的导入和生产，验证了一个新的CD46微型基因。猪的淋巴细胞对抗体和补体介导的裂解的抵抗力进行了测试，转基因组织的CD46表达进行了鉴定，并将肾脏移植到了没有免疫抑制的狒狒身上。测定抗半乳糖α(1，3)半乳糖表位(抗半乳糖)血清抗体的吸收。转基因猪在所有组织中均高水平表达CD46，尤其是血管内皮细胞，并在三代中稳定表达，这一点很容易通过转基因外周血单个核细胞(PBMC)的流式细胞术进行监测。用抗体预致敏的转基因PBMC对人补体介导的裂解高度抵抗，而人补体介导的裂解很容易裂解正常猪的PBMC。将无冷缺血的正常猪肾移植到非免疫抑制的成年狒狒体内，存活时间中位数为3.5h(n=7)，而在∼间隔24小时收获的转基因移植肾(n=9)，要么大体正常(29、48和68h)，要么表现出有限的大体损伤(中位数>；50h)。对移植的转基因肾脏的显微镜评估显示，从3天开始，只有局灶性肾小管梗死，其他地方有存活的肾组织，没有内皮肿胀或淋巴细胞的多形性黏附和渗透。在移植后48小时或以后评估的4个未被排斥的肾脏中，凝血障碍不是组织学特征。在移植前，∼抗体滴度很高，在一个广泛分析的受体中，CD46在5.5h内降低了8倍。这些数据表明，尽管存在抗体和补体沉积，但单一的CD46转基因控制了未经处理的狒狒的超急性肾移植排斥反应。表达水平、组织分布和体外功能测试表明，CD46具有高效的功能，控制着经典途径和替代途径的补体激活，这表明它可能是保护异种移植的补体调节因子。
Abstract: Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement‐mediated rejection, as shown here using non‐immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high‐level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement‐mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti‐Galα(1,3)Gal epitope (anti‐GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre‐sensitized with antibody were highly resistant to human complement‐mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non‐immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at ∼24‐h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median > 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti‐GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced ∼8‐fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.