Evaluation of Complement-Dependent Cytotoxicity Assays for Gene-Edited Pig-to-Human Xenotransplantation.

IF 3.3 4区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Xenotransplantation Pub Date : 2025-01-01 DOI:10.1111/xen.70012

Hao Feng, Man Zhang, Qiangbing Xia, Jiaxiang Du, Tao Li, Song Chen, Yi Wang, Dengke Pan, Lan Zhu, Gang Chen

{"title":"Evaluation of Complement-Dependent Cytotoxicity Assays for Gene-Edited Pig-to-Human Xenotransplantation.","authors":"Hao Feng, Man Zhang, Qiangbing Xia, Jiaxiang Du, Tao Li, Song Chen, Yi Wang, Dengke Pan, Lan Zhu, Gang Chen","doi":"10.1111/xen.70012","DOIUrl":null,"url":null,"abstract":"Background: Gene-edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene-edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement-dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s).Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat-inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one-step CDC method was established using pooled normal human sera (NHS) without the addition of exogenous complement.Results: There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement-mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement-mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one-step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b-9 deposition on pig PBMCs was positively correlated with the expression level of hCD55.Conclusion: The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene-edited pig-to-human xenotransplantation.","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"32 1","pages":"e70012"},"PeriodicalIF":3.3000,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Xenotransplantation","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1111/xen.70012","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"MEDICINE, RESEARCH & EXPERIMENTAL","Score":null,"Total":0}

引用次数: 0

Abstract

Background: Gene-edited pigs for xenotransplantation usually contain one or more transgenes encoding human complement regulatory proteins (CRPs). Because of species differences, human CRP(s) expressed in gene-edited pigs may have difficulty inhibiting the activation of exogenous rabbit complement added to a complement-dependent cytotoxicity (CDC) assay. The use of human complement instead of rabbit complement in CDC experiments may more accurately reflect the actual regulatory activity of human CRP(s).

Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from one GTKO pig and two GTKO/hCD55 pigs with a high or low level of hCD55 expression. After incubation of heat-inactivated normal human sera (HINHS) with porcine PBMCs, CDC levels were measured after the addition of commercial rabbit complement or human complement. In addition, a modified one-step CDC method was established using pooled normal human sera (NHS) without the addition of exogenous complement.

Results: There was no significant difference in the binding of IgM/IgG to PBMCs from the three pigs. Both rabbit and human complement mediated a significant cytotoxic effect on GTKO pig PBMCs (98.97% vs. 82.73%). Even the high expression of hCD55 only had a very limited inhibitory effect on rabbit complement-mediated cytotoxicity (81.70% vs. 98.97%). However, regardless of whether the expression level was high or low, hCD55 had a very remarkable inhibitory effect on human complement-mediated cytotoxicity (2.94% and 23.83% vs. 82.73%; p < 0.01). Similar results were obtained using the modified one-step CDC method. In addition, the inhibitory effect of hCD55 on C3c and C5b-9 deposition on pig PBMCs was positively correlated with the expression level of hCD55.

Conclusion: The use of human complement instead of rabbit complement in CDC assays can better reflect the actual cytotoxic effect of human xenoantibodies against pig PBMCs expressing human CRP(s), and thus may have potential application to gene-edited pig-to-human xenotransplantation.

查看原文

微信好友朋友圈 QQ好友复制链接

本刊更多论文

基因编辑猪到人异种移植中补体依赖性细胞毒性测定的评价。

背景：用于异种移植的基因编辑猪通常含有一个或多个编码人类补体调节蛋白（CRPs）的转基因。由于物种差异，在基因编辑的猪中表达的人CRP可能难以抑制添加到补体依赖性细胞毒性（CDC）试验中的外源性兔补体的激活。在CDC实验中使用人补体代替兔补体可以更准确地反映人CRP的实际调节活性。方法：分别取1只GTKO猪和2只hCD55高表达或低表达的GTKO/hCD55猪外周血单个核细胞（PBMCs）。将热灭活的正常人血清（HINHS）与猪PBMCs孵育后，在加入商业兔补体或人补体后测定CDC水平。此外，建立了一种改进的一步CDC方法，使用汇集的正常人血清（NHS），不添加外源性补体。结果：3只猪的IgM/IgG与外周血单核细胞的结合无显著差异。兔和人补体对GTKO猪PBMCs均有显著的细胞毒作用（分别为98.97%和82.73%）。即使高表达的hCD55对补体介导的兔细胞毒性的抑制作用也非常有限（81.70% vs. 98.97%）。然而，无论表达水平高低，hCD55对人补体介导的细胞毒性均有非常显著的抑制作用(2.94%和23.83% vs. 82.73%；p结论：在CDC检测中使用人补体代替兔补体能更好地反映人异种抗体对表达人CRP的猪PBMCs的实际细胞毒作用，因此可能在基因编辑的猪-人异种移植中具有潜在的应用价值。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文去求助

来源期刊

Xenotransplantation 医学-医学：研究与实验

CiteScore

6.80

自引率

15.40%

发文量

审稿时长

>12 weeks

期刊介绍： Xenotransplantation provides its readership with rapid communication of new findings in the field of organ and tissue transplantation across species barriers.The journal is not only of interest to those whose primary area is xenotransplantation, but also to veterinarians, microbiologists and geneticists. It also investigates and reports on the controversial theological, ethical, legal and psychological implications of xenotransplantation.