Chuheng Gou, Hong Zhang, Rui Ding, Quancheng Wang, Li Zhang, Xin Hong, Liqiang Zhao, Kefeng Dou, Xuan Zhang
{"title":"Human CD64 Alleviates Antibody-Mediated Rejection in Xenotransplantation.","authors":"Chuheng Gou, Hong Zhang, Rui Ding, Quancheng Wang, Li Zhang, Xin Hong, Liqiang Zhao, Kefeng Dou, Xuan Zhang","doi":"10.1111/xen.70110","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Xenotransplantation, using gene-edited pigs, represents an important approach to overcoming human organ shortages. A major obstacle to xenotransplantation is antibody-mediated rejection (AMR), which leads to xenograft injury by activating complement and effector cells through the fragment crystallizable domain (Fc) of donor-specific antibodies (DSAs). Therefore, we designed a strategy to express the human high-affinity IgG receptor (hCD64) on porcine endothelial cells to competitively bind IgG and protect xenografts from AMR.</p><p><strong>Methods: </strong>The lentiviral transduction of hCD64 into porcine aortic endothelial cells (PAEC) from wild-type and GTKO pigs was validated using quantitative reverse transcription (qRT)-PCR, Western blot, and flow cytometry. The effects of hCD64 transduction and activation on cell physiology were assessed using RNA sequencing. The IgG Fc-binding capacity of hCD64 was validated using flow cytometry and ELISA. Finally, the protective effect of hCD64 against complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in PAECs and GTKO PAECs was confirmed through apoptosis assays.</p><p><strong>Results: </strong>hCD64 was stably expressed at both mRNA and protein levels in PAEC<sup>hCD64</sup> and PAEC<sup>GTKO/hCD64</sup>, with both exhibiting normal physiological functions. PAEC<sup>hCD64</sup> and PAEC<sup>GTKO/hCD64</sup> bound free human IgG in a concentration-dependent manner. In contrast, hCD64 did not bind to human IgM or pig and mouse IgG. In the CDC assay, the survival of PAEC<sup>hCD64</sup> was significantly higher than that of PAEC<sup>NC</sup> (67.89% vs. 46.03%); moreover, the survival in GTKO PAEC had the same trend (85.18% vs. 71.09%). Similar results were obtained in the assay of ADCC: the survival rates of PAEC<sup>hCD64</sup> and PAEC<sup>NC</sup> were 67.27% and 44.95%, respectively, and the survival of PAEC<sup>GTKO/hCD64</sup> and PAEC<sup>GTKO/NC</sup> were 80.73% and 66.62%, respectively. Pre-saturation with high doses of human-derived mAbs did not abrogate the protective function of hCD64.</p><p><strong>Conclusion: </strong>hCD64 expression may partially protect xenografts from AMR through its ability to bind competitively with DSAs IgG Fc.</p>","PeriodicalId":23866,"journal":{"name":"Xenotransplantation","volume":"33 1","pages":"e70110"},"PeriodicalIF":3.8000,"publicationDate":"2026-01-01","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.70110","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: Xenotransplantation, using gene-edited pigs, represents an important approach to overcoming human organ shortages. A major obstacle to xenotransplantation is antibody-mediated rejection (AMR), which leads to xenograft injury by activating complement and effector cells through the fragment crystallizable domain (Fc) of donor-specific antibodies (DSAs). Therefore, we designed a strategy to express the human high-affinity IgG receptor (hCD64) on porcine endothelial cells to competitively bind IgG and protect xenografts from AMR.
Methods: The lentiviral transduction of hCD64 into porcine aortic endothelial cells (PAEC) from wild-type and GTKO pigs was validated using quantitative reverse transcription (qRT)-PCR, Western blot, and flow cytometry. The effects of hCD64 transduction and activation on cell physiology were assessed using RNA sequencing. The IgG Fc-binding capacity of hCD64 was validated using flow cytometry and ELISA. Finally, the protective effect of hCD64 against complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) in PAECs and GTKO PAECs was confirmed through apoptosis assays.
Results: hCD64 was stably expressed at both mRNA and protein levels in PAEChCD64 and PAECGTKO/hCD64, with both exhibiting normal physiological functions. PAEChCD64 and PAECGTKO/hCD64 bound free human IgG in a concentration-dependent manner. In contrast, hCD64 did not bind to human IgM or pig and mouse IgG. In the CDC assay, the survival of PAEChCD64 was significantly higher than that of PAECNC (67.89% vs. 46.03%); moreover, the survival in GTKO PAEC had the same trend (85.18% vs. 71.09%). Similar results were obtained in the assay of ADCC: the survival rates of PAEChCD64 and PAECNC were 67.27% and 44.95%, respectively, and the survival of PAECGTKO/hCD64 and PAECGTKO/NC were 80.73% and 66.62%, respectively. Pre-saturation with high doses of human-derived mAbs did not abrogate the protective function of hCD64.
Conclusion: hCD64 expression may partially protect xenografts from AMR through its ability to bind competitively with DSAs IgG Fc.
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.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
