Objective: Liver xenotransplantation using genetically-engineered pigs offers a potential solution to the global organ shortage. However, non-human primate (NHP) recipients exhibit limited survival, necessitating an in-depth investigation of underlying mechanisms.
Methods: Nine genetically-engineered pig-to-NHP orthotopic liver xenotransplantations were performed. Recipients' clinical and immunological parameters were monitored longitudinally. Graft tissues were analyzed via immunohistochemistry (IHC) at various time points. Single-cell RNA sequencing characterized immune cell subsets and activation in peripheral blood mononuclear cells (PBMCs) and graft tissues from three recipients with extended survival.
Results: Recipients of Triple-knockout (GTKO/CMAHKO/β4GalN2KO/hCD55/hTBM) livers survived 5, 5, and 3 days, respectively, compared to ≤2 days for GTKO-only (GTKO or GTKO/hCD55/hCD59/hTBM) livers. In longer-surviving recipients, coagulation parameters and platelet counts remained stable for the first three days but declined thereafter. Shorter-surviving recipients exhibited severe coagulopathy and thrombocytopenia immediately post-transplant. Grafts from shorter-surviving recipients exhibited extensive necrosis and red blood cell destruction, whereas those from longer-surviving recipients showed focal necrosis, minimal T cell infiltration, and limited antibody and complement deposition. However, grafts and PBMCs from longer-surviving recipients showed increased macrophages (CD163+, CD5L+) and NK cells (CCL5+, GNLY+), and activated dendritic cells (PTPRC+, SKAP1+), accompanied by a significant rise in IL-6 levels in recipient peripheral blood. The IL-6 surge coincided with cardiac and renal damage, ultimately leading to recipient death.
Conclusions: Triple-knockout pig donors could mitigate early coagulopathy and thrombocytopenia in NHP recipients, extending survival. However, innate immune cell activation and the associated IL-6-mediated pro-inflammatory response contribute to recipient mortality. Targeting these pathways may improve outcomes in liver orthotopic xenotransplantation.