Introduction: Porcine genome editing has revolutionized xenotransplantation by allowing the first pig-to-human heart transplantations. However, the mechanisms driving the immune response in heart xenografts transplanted to humans remain poorly understood.
Methods: We analyzed two heart xenografts transplanted from 10-gene-edited pigs into brain-dead human recipients. Biopsies at 66-hour post-reperfusion were studied using a multimodal phenotyping approach combining: morphological evaluation, immunophenotyping, ultrastructural assessment, automated quantification of multiplex immunofluorescence staining and gene expression profiling. Xenografts before implantation, wild-type pig hearts with and without ischemia injury as well as wild-type pig hearts with brain death and ischemia reperfusion were used as controls.
Results: Both xenografts showed evidence of endothelial activation and mild microvascular inflammation without capillary C4d deposition. Immune infiltrates were mainly composed of CD15+ and CD68+ innate immune cells (A). Ultrastructural assessment showed endothelial swelling with occasional intravascular leucocytes. Deep-learning based automated multiplex immunofluorescence analysis confirmed that microvascular inflammation was primarily associated with CD15+ and CD68+ innate immune cells (B). Finally, both xenografts showed increased expression of genes associated with monocyte/macrophage activation, neutrophil activation, interferon-gamma response, natural killer cell burden, endothelial activation, apoptosis and injury repair (C). This phenotype was absent in control pig hearts, in ischemia and brain death models.
Conclusion: Multimodal phenotyping of pig-to-human heart xenografts revealed a pattern of mild humoral xeno-immune response, characterized by mild innate microvascular inflammation, endothelial activation, and molecular signature of antibody-mediated rejection. Integration of this approach into future clinical trials could facilitate real-time graft monitoring, mitigate rejection risk, and enhance xenotransplant outcomes.