Immunobiology and Physiology of Liver xenograft in the Decedent - Abstract Recent advances in genetic engineering have enabled significant modifications in porcine organs, such as the removal of glycan sequences linked to hyperacute rejection, inactivation of porcine endogenous retroviruses, and expression of human proteins to mitigate inflammation and immune responses. Transplantation of genetically modified pig kidneys and hearts into non-human primates (NHPs) and limited human studies have demonstrated physiological function with tailored immunosuppression. However, liver xenotransplantation in NHPs faces physiological and immunological challenges, including severe thrombocytopenia, metabolic incompatibilities, and the need for a deeper understanding of the immune response to liver xenografts. We propose using human brain-dead donors (decedents) to investigate liver xenograft physiology and early immune responses. Our decedent model employs an extracorporeal liver cross-circulation system, where the liver xenograft is connected to the decedent via venous bypass lines, allowing systemic circulation and observation of interactions between the liver xenograft and human physiology. This model facilitates monitoring xenograft function and collecting specimens for studying immune responses, hematological dysregulations, and metabolic functions. We hypothesize that decedent studies can accurately replicate the early post-transplant experience in humans, thereby identifying uniquely human immune, metabolic, and pharmacological responses. Aim 1 focuses on defining early innate and adaptive immune responses to genetically modified liver xenografts. Advanced technologies, including spatial transcriptomics and single-nucleus RNA sequencing, will generate comprehensive datasets to identify biomarkers of xenoimmune responses. Cell type specific cell-free DNA studies will enhance the monitoring of immune cell activation and liver xenograft injury, providing qualitative and quantitative insights into adaptive xenoimmune responses. Aim 2 is designed to elucidate physiological compatibilities in liver xenotransplantation and evaluate therapeutic interventions. First, we will assess liver xenografts' metabolic and detoxification functions using non- radioactive tracers and metabolomic assays to profile phenotypes that may be associated with xenograft dysfunction, impact decedent metabolic derangements, and affect xenoimmune response. Second, we will rigorously test our hypotheses that thrombocytopenia in xenotransplant recipients is driven by either antibody- mediated platelet phagocytosis, complement-mediated platelet lysis, platelet apoptosis, or porcine molecules inducing platelet agglutination and activation. These mechanisms for the thrombocytopenia will be analyzed ex vivo, while their reversibility will be tested in vivo using clinically approved therapeutics. The study outcomes in a human decedent model are expected to provide insights into early xenograft immune activation, peripheral biomarkers of xenoimmune response, metabolic compatibility, and potential pharmacological solutions for hematological dysregulations.
