Objective: Clinical islet transplantation involves infusion of pancreatic islets into hepatic portal vein and is associated with instant blood mediated inflammatory reaction (IBMIR). IBMIR characterized by non-specific inflammatory reaction and is a major culprit for significant loss of islet mass. Myeloid cells play central role in IBMIR. CD47/SIRPα pathway serves as an important innate immune checkpoint with demonstrated function in negatively regulating various innate immune cells, including neutrophils, platelets, macrophages, involved in IBMIR. We hypothesized that the presence of CD47 protein on islet surface will improve their engraftment in the liver by mitigating IBMIR.
Methods: A synthetic gene containing the extracellular domain of mouse CD47 C-terminus fused to the core of streptavidin (SA)-CD47 was expressed in insect cells. The chimeric SA-CD47 protein was purified and characterized structurally and functionally. The SA-CD47 protein was transiently displayed on the surface of biotinylated cells and islets by taking the advantage of high affinity interaction between biotin and streptavidin. In vitro loop assay and marginal mass islet transplant settings were used to assess the role of CD47 in mitigating IBMIR and islet engraftment. Immunohistochemistry, quantitative RT-PCR, and flow cytometry were used for immune profiling.
Results: Rat splenocytes engineered with SA-CD47 protein evaded phagocytosis by mouse macrophages, providing direct evidence for the efficacy of SA-CD47 transiently displayed on the cell surface in modulating innate immunity. SA-CD47-engineered islets showed morphological, functional, and metabolic competence at the same levels as naïve unmanipulated islets. Islets engineered with SA-CD47 protein showed intact morphology with significantly less CD11b+ neutrophil/ granulocytes infiltration in an in vitro loop assay, simulating IBMIR in vivo, as compared with control islets. Most importantly, SA-CD47-enginered islets exhibited enhanced engraftment and function as compared with streptavidin-engineered control islets recipients in a syngeneic marginal mass intraportal islet transplantation model. Enhanced engraftment was associated with intragraft downregulation of cellular and molecular culprits of IBMIR as assed by flow cytometry, and qRT-PCR.
Conclusion:  CD47 serves as an important target to mitigate IBMIR as an effective means of overcoming peri-transplant loss of islet grafts with significant clinical implications.