Introduction: Organ transplantation is currently the best therapy for patients with end-stage organ failure, and genetically-modified pigs are a promising solution to cover the huge world-wide demand for organs. To understand the immune rejection mechanisms in the xenograft, ex vivo organ perfusion systems and single cell RNA sequencing technologies (scRNAseq) can provide detailed insights into humoral and cellular immune responses.
Methods: Kidneys of genetically-modified 4x knockout (4xKO) pigs, carrying inactivations of GGTA1, CMAH, B4GALNT2 and SLAI, and wildtype pigs were perfused for 6 hours with human blood. Renal injury, complement deposition, coagulation, immune cell infiltration and single cell analysis of endothelial cells from 4xKO and wildtype control pigs were assessed by immunohistochemistry, coagulation assays and scRNAseq data analysis.
Results: Histological analysis and scRNAseq revealed overall strongly reduced immune reactions in the 4xKO kidneys compared to the wildtype controls. A compartment-specific endothelial activation could be shown with cortical and endothelial cells being more affected. Differential gene expression analysis revealed a downregulation of inflammatory-, apoptotic- and innate signaling pathways.
Conclusion: Inactivations of xenoantigens has become a valuable approach to reduce rejection mechanisms. ScRNAseq data will be very useful to identify previously unknown antigens. The 4xKO genotype already revealed strongly reduced endothelial activation and immune responses that will be decisive to enable clinical xenotransplantation and long-term graft survival.