The xenoantibody response and immunoglobulin gene expression profile of cynomolgus monkeys transplanted with hDAF-transgenic porcine hearts.
The xenoantibody response and immunoglobulin gene expression profile of cynomolgus monkeys transplanted with hDAF-transgenic porcine hearts.
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移植 hDAF 转基因猪心脏的食蟹猴的异种抗体反应和免疫球蛋白基因表达谱。
DOI：
10.1111/j.1399-3089.2007.00381.x
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发表时间：
2007
影响因子：
3.9
通讯作者：
Cramer,DonaldV
中科院分区：
文献类型：
--
作者：
Zahorsky-Reeves,JoanneL;Kearns-Jonker,MaryK;Lam,TuanT;Jackson,JeremyR;Morris,RandallE;Starnes,VaughnA;Cramer,DonaldV
翻译后摘要：背景：最近的工作表明，抗Galα1 - 3Gal（Gal）和抗非Gal异种抗体在灵长类动物体液排斥反应对人类衰变加速因子（hepatitis）转基因猪器官的作用。我们的实验室已经证明，人类和非人灵长类动物中的抗猪异种移植抗体由少量生殖系IgVH祖细胞编码。在这项研究中，我们扩展了我们的分析，以确定编码异种抗体的IgVH基因在免疫抑制的食蟹猴（Macaca fascicularis）移植hxB-转基因猪organs.Methods：三个免疫抑制的猴子进行异位心脏移植hxB猪心异种移植。三只动物中的两只给予GAS 914，这是一种聚赖氨酸衍生物，显示可与抗Gal异种抗体结合并中和它们。1只动物在术后第39天（POD）发生心脏排斥;另1只动物在POD 78发生移植心脏排斥。第三只猴在POD 36处以安乐死，但心脏未被排斥。在移植前和移植后的多个时间点从每只动物获得外周血白细胞（PBL）和血清。我们通过酶联免疫吸附试验（ELISA）分析了免疫应答，以确认移植物放置后是否诱导了抗Gal或抗非Gal异种抗体。然后产生并筛选免疫球蛋白重链基因（VH）cDNA文库。我们生成可溶性单链抗体（scFv），以建立克隆免疫球蛋白基因的结合特异性。结果：尽管使用了免疫抑制剂（包括使用聚合物GAS 914），但两只排斥心脏的动物显示细胞毒性抗猪红细胞（RBC）抗体和抗猪主动脉内皮细胞（PAEC）抗体水平升高。与移植前水平相比，未发生移植物排斥的猴的血清抗RBC、抗PAEC和抗Gal异种抗体下降。与VH 3 -11的等位基因具有高水平序列相似性的VH 3家族基因，命名为VH 3 - 11 cyno，在未给予GAS 914的猴中以升高的水平表达，并且其移植物直到POD 78才被排斥。还在该动物中诱导了针对N-乙酰乳糖胺（Gal表位的前体）的IgM而非IgG异种抗体。我们从这个新基因产生可溶性scFv以确定该抗体是否可以结合Gal碳水化合物，并且证明该蛋白能够阻断人血清异种抗体与Gal寡糖的结合，如先前用人VH 3 -11 scFv所示。结论：移植到食蟹猴中的转基因器官诱导由IgM和IgG异种抗体介导的抗Gal和抗非Gal异种抗体应答。在用GAS 914处理的动物中诱导高水平的抗非Gal异种抗体。在不存在GAS 914处理的情况下，诱导与Gal碳水化合物和N-乙酰乳糖胺结合的抗体。心脏保持跳动78天的动物证明了由结构上相关但不同于IGHV 311的种系祖细胞编码的抗体的使用增加。 该抗体与Gal碳水化合物结合，但在移植后第8天以高水平表达时不会诱导异种移植物的快速排斥反应。
Abstract:Background:Recent work has indicated a role for anti‐Galα1‐3Gal (Gal) and anti‐non‐Gal xenoantibodies in the primate humoral rejection response against human‐decay accelerating factor (hDAF) transgenic pig organs. Our laboratory has shown that anti‐porcine xenograft antibodies in humans and non‐human primates are encoded by a small number of germline IgVHprogenitors. In this study, we extended our analysis to identify the IgVHgenes encoding xenoantibodies in immunosuppressed cynomolgus monkeys (Macaca fascicularis) transplanted with hDAF‐transgenic pig organs.Methods:Three immunosuppressed monkeys underwent heterotopic heart transplantation with hDAF porcine heart xenografts. Two of three animals were given GAS914, a poly‐l‐lysine derivative shown to bind to anti‐Gal xenoantibodies and neutralize them. One animal rejected its heart at post‐operative day (POD) 39; a second animal rejected the transplanted heart at POD 78. The third monkey was euthanized on POD 36 but the heart was not rejected. Peripheral blood leukocytes (PBL) and serum were obtained from each animal before and at multiple time points after transplantation. We analyzed the immune response by enzyme‐linked immunosorbent assay (ELISA) to confirm whether anti‐Gal or anti‐non‐Gal xenoantibodies were induced after graft placement. Immunoglobulin heavy‐chain gene (VH) cDNA libraries were then produced and screened. We generated soluble single‐chain antibodies (scFv) to establish the binding specificity of the cloned immunoglobulin genes.Results:Despite immunosuppression, which included the use of the polymer GAS914, the two animals that rejected their hearts showed elevated levels of cytotoxic anti‐pig red blood cell (RBC) antibodies and anti‐pig aortic endothelial cell (PAEC) antibodies. The monkey that did not reject its graft showed a decline in serum anti‐RBC, anti‐PAEC, and anti‐Gal xenoantibodies when compared with pre‐transplant levels. A VH3 family gene with a high level of sequence similarity to an allele of VH3–11, designated VH3–11cyno, was expressed at elevated levels in the monkey that was not given GAS914 and whose graft was not rejected until POD 78. IgM but not IgG xenoantibodies directed atN‐acetyl lactosamine (a precursor of the Gal epitope) were also induced in this animal. We produced soluble scFv from this new gene to determine whether this antibody could bind to the Gal carbohydrate, and demonstrated that this protein was capable of blocking the binding of human serum xenoantibody to Gal oligosaccharide, as had previously been shown with human VH3–11 scFv.Conclusions:DAF‐transgenic organs transplanted into cynomolgus monkeys induce anti‐Gal and anti‐non‐Gal xenoantibody responses mediated by both IgM and IgG xenoantibodies. Anti‐non‐Gal xenoantibodies are induced at high levels in animals treated with GAS914. Antibodies that bind to the Gal carbohydrate and toN‐acetyl lactosamine are induced in the absence of GAS914 treatment. The animal whose heart remained beating for 78 days demonstrated increased usage of an antibody encoded by a germline progenitor that is structurally related, but distinct from IGHV311. This antibody binds to the Gal carbohydrate but does not induce the rapid rejection of the xenograft when expressed at high levels as early as day 8 post‐transplantation.