Identification of human preformed antibody targets in GTKO pigs
Identification of human preformed antibody targets in GTKO pigs
复制标题
DOI：
10.1111/j.1399-3089.2012.00695.x
复制
发表时间：
2012-03-01
影响因子：
3.9
通讯作者：
Tector, A. Joseph
中科院分区：
文献类型：
--
作者：
Burlak, Christopher;Wang, Zheng-Yu;Tector, A. Joseph
背景：尽管出现了α -半乳糖转移酶敲除（GTKO）猪，但人类预先形成的抗体仍能识别猪异种移植物。本研究检测了人预成型IgG和IgM抗体对GTKO猪肝抗原的潜在反应性。方法：测定人血清IgG、IgM、抗α -半乳糖抗体、抗非α -半乳糖抗体的浓度及对国产和GTKO成纤维细胞和肝窦内皮细胞（LSEC）的细胞毒性。我们使用先进的蛋白质组学技术在人血清中检测到针对GTKO猪肝细胞和组织样本的预形成抗体。通过MALDI TOF TOF质谱法鉴定预形成抗体的靶点，并通过共聚焦显微镜、免疫印迹和免疫沉淀进行验证。结果：针对GTKO成纤维细胞的人血清IgG含量为2.06 μ g/ml， IgM含量为0.013 μ g/ml。人IgG和IgM以剂量依赖的方式与GTKO LSEC结合，并具有细胞毒性。检测到357个人IgG和233个人IgM识别的蛋白点。人类IgG和IgM共有的蛋白有219种。质谱法鉴定出许多免疫反应蛋白，其中19种是肝细胞的膜蛋白。本研究中最重要的是α -烯醇化酶、CFTR和E-cadherin，它们在GTKO猪组织中含量丰富，并在GTKO LSEC表面表达。人IgG捕获α -烯醇化酶，CFTR和e -钙粘蛋白通过免疫沉淀验证蛋白质组学鉴定。结论：GTKO猪的多种膜抗原可被人IgG或IgM直接识别。进一步研究这些抗原在抗体介导的异种移植排斥反应中的作用是必要的。
Background: Human preformed antibodies continue to recognize porcine xenografts, despite the advent of alpha-galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver.Methods: Human serum was analyzed for the concentration of IgG, IgM, anti-alpha gal antibody, anti-non-agal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation.Results: Human serum used in this study contained 2.06 lg/ml IgG and 0.013 mu g/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose-dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were alpha-enolase, CFTR, and E-cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured alpha-enolase, CFTR, and E-cadherin by immunoprecipitation validating the proteomic identification.Conclusion: These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody-mediated xenograft rejection are necessary.