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.x
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发表时间：
2004-03-01
影响因子：
3.9
通讯作者：
McKenzie, IFC
中科院分区：
文献类型：
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作者：
Loveland, BE;Milland, J;McKenzie, IFC
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，可保护异种移植物免受补体介导的排斥反应，如本文使用非免疫抑制狒狒和异位CD46转基因猪肾异种移植物所示。本报告是一个精心设计的转基因，使高水平的CD46表达。通过转染和生产转基因猪系来验证新的CD46小基因。检测猪淋巴细胞对抗体和补体介导的裂解的抗性，表征转基因组织的CD46表达，并将肾脏移植到狒狒体内而不进行免疫抑制。测量抗Galalpha（1，3）Gal表位（抗GAL）血清抗体的吸收。转基因猪在所有组织中表达高水平的CD46，特别是血管内皮，通过三代稳定表达，易于通过转基因外周血单核细胞（PBMC）的流式细胞术监测。用抗体预致敏的转基因PBMC对人补体介导的裂解具有高度抵抗性，而人补体介导的裂解容易裂解正常猪PBMC。正常的猪肾移植到非免疫抑制的成年狒狒体内，没有冷缺血，平均存活时间为3.5小时（n = 7），而转基因移植物（n = 9），以类似于24小时的时间间隔收获，要么肉眼正常（在29，48和68小时），要么表现出有限的肉眼损伤（中位数> 50小时）。移植的转基因肾的显微镜评估显示仅局部肾小管梗死，其他地方有存活的肾组织，没有内皮肿胀或多晶型粘附和淋巴细胞浸润，从3天开始。凝血病不是一个功能的组织学在四个肾脏没有拒绝，并在移植后48小时或更晚进行评估。狒狒抗GAL血清抗体滴度在移植前很高，在一个广泛分析的受体中，在5.5小时内降低了8倍。数据表明，尽管存在抗体和补体沉积，但单个CD46转基因可控制未治疗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具有高效功能，控制经典和旁路途径补体激活，这表明它可能是保护异种移植物的首选补体调节剂。
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-Galalpha(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 similar to24-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 similar to8-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.