Reporter immunogenicity as a defined rejection antigen: a noninvasive platform for skin transplant immunobiology.

BACKGROUND: Preclinical transplantation models largely rely on macroscopic or terminal assessments of graft fate, limiting longitudinal evaluation of immune rejection and tolerance induction. Reporter proteins such as enhanced green fluorescent protein (eGFP) and firefly luciferase enable real-time, non-invasive imaging of transplanted tissues, but their utility in immunocompetent hosts is constrained by their immunogenicity as non-murine xenoantigens capable of eliciting robust adaptive immune responses. METHODS: Full-thickness skin grafts from CAG-eGFP-luciferase donor mice were transplanted onto genetically tolerant (NoGlow+) or non-tolerant (NoGlow-) littermate recipients. Graft viability was monitored longitudinally by fluorescence and bioluminescence imaging. The impact of pharmacologic immunosuppression on anti-reporter immune responses and graft signal retention was evaluated in wild-type recipients. Multiplex transplantation experiments, in which multiple grafts were placed on individual animals, were performed to enable direct comparison of biomaterial-based interventions. RESULTS: Reporter antigens served as defined, trackable targets of antigen-specific immune rejection in non-tolerant recipients, with progressive loss of graft signal correlating with rejection kinetics. Tolerant NoGlow+ recipients maintained stable reporter signal throughout the observation period. Pharmacologic immunosuppression mitigated anti-reporter immune responses and preserved bioluminescent signal in wild-type hosts. Multiplex transplantation within individual animals enabled side-by-side comparison of biomaterial therapies without invasive tissue sampling or early euthanasia. CONCLUSIONS: These findings establish a fluorescent and bioluminescent reporter-based skin graft platform that provides quantitative, non-invasive, longitudinal readouts of transplant viability and immune rejection. This system offers a flexible and tractable framework for studying transplant immunobiology and accelerating preclinical evaluation of immunomodulatory and biomaterial-based strategies aimed at improving graft outcomes.