Human CD64 Alleviates Antibody-Mediated Rejection in Xenotransplantation

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.

antibody‐dependent cell‐mediated cytotoxicity (ADCC); antibody‐mediated rejection (AMR); complement‐dependent cytotoxicity (CDC); human CD64 (hCD64); xenotransplantation.