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Vascularized composite allotransplantation (VCA) represents the most advanced option in the reconstructive spectrum for complex tissue loss. Most immune investigations have focused on the skin, considered the most immunogenic component. However, VCA comprises multiple heterogeneous tissues, each with distinct immunological characteristics. This complexity necessitates compartment-specific immune characterization. We aimed to identify tissue-specific molecular signatures to better understand VCA rejection.

A porcine heterotopic hindlimb transplantation model was used as a clinically representative VCA model. Animals were followed until end-stage rejection. Skin, muscle, and cartilage samples were collected at endpoint and compared with healthy naïve tissues. Innate and adaptive immune responses were assessed using histology, RNA sequencing, immunofluorescence, and electron microscopy.

Rejection-related changes were identified in skin, muscle, and cartilage, the latter previously considered relatively immune-privileged. Rejected cartilage demonstrated increased expression of innate immunity (neutrophils, complement deposition, macrophages) and adaptive immune markers (MHC-II and CD3⁺ T cells). Across all rejecting tissues, genes and pathways associated with innate immunity, including pattern recognition receptors, damage-associated molecular patterns, and antigen processing and presentation, were upregulated compared with healthy controls. Rejection was characterized by activation of alloreactive T cells, together with pro-inflammatory cytokine signaling and antigen-presenting cell activation. Although inflammatory infiltration and tissue destruction were more pronounced in skin and muscle than in cartilage, discordance between skin and muscle findings suggests that skin assessment alone may underestimate deeper tissue rejection.

VCA rejection involves multiple tissue compartments and is characterized by shared innate and adaptive immune activation with tissue-specific differences in severity. Comprehensive, compartment-based immune profiling may improve rejection monitoring and support the development of more precise diagnostic and therapeutic strategies, ultimately enhancing the long-term success and broader application of VCA.