Characterizing the Immune Response in Pig-to-Human Heart Xenografts Using a Multimodal Diagnostic System

BACKGROUND: Porcine genome editing has revolutionized xenotransplantation, recently enabling the first pig-to-human heart xenotransplants. However, the xenoimmune response in heart xenografts remains largely unexplored. This study aimed to precisely characterize the xenoimmune response and injury in 2 heart xenografts transplanted from 10-gene-edited pigs into brain-dead human recipients. METHODS: We analyzed xenograft biopsy specimens 66 hours after reperfusion using a multimodal phenotyping approach combining morphological evaluation, immunophenotyping, ultrastructural assessment, automated quantification of multiplex immunofluorescence staining, and gene expression profiling. Xenografts before implantation and wild-type pig hearts with and without ischemia/reperfusion injury and brain death 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. 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. Both xenografts showed increased expression of genes and pathways associated with monocyte/macrophage activation, neutrophil activation, interferon-gamma response, natural killer cell burden, endothelial activation, apoptosis, and injury repair. This phenotype was absent in all control pig hearts independent of ischemia/reperfusion injury and brain death. CONCLUSIONS: Multimodal phenotyping of pig-to-human heart xenografts revealed early signs of xenoimmune response, characterized by mild innate microvascular inflammation, endothelial activation, and a molecular signature characteristic of antibody-mediated rejection. Developing such a precision diagnostic system could improve graft monitoring in future clinical settings.