Human CD64 Alleviates Antibody‐Mediated Rejection in Xenotransplantation

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 PAEC hCD64 and PAEC GTKO/hCD64 , with both exhibiting normal physiological functions. PAEC hCD64 and PAEC GTKO/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 PAEC hCD64 was significantly higher than that of PAEC NC (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 hCD64 and PAEC NC were 67.27% and 44.95%, respectively, and the survival of PAEC GTKO/hCD64 and PAEC GTKO/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.