Necessity for Genetic Induction of Recipient CD47 to Prevent Macrophage-Mediated Xenograft Rejection through CD47-SIRPα Inhibitory Signaling

Abstract

Background: Signal regulatory protein (SIRP)α is a critical immunoinhibitory receptor expressed on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. We have previously proven that the interspecies incompatibility of CD47 causes in vitro phagocytosis of porcine xenogeneic cells by human macrophages. In this study, we used an in vivo mouse model to investigate whether genetic manipulation of rat insulinoma cells for mouse CD47 expression could inhibit macrophage-mediated xenograft rejection. Methods: To determine whether rat CD47 can interact with mouse SIRPa, we compared SIRPa tyrosine phosphorylation in mouse macrophages after interaction of the macrophages with mouse or rat red blood cells (RBCs). To determine whether expression of mouse CD47 on rat insulinoma (INS-1E) cells could confer protection against phagocytosis by mouse macrophages, we generated mouse CD47-expressing INS-1E (mCD47-INS-1E) cells. The phagocytotic activity of mouse macrophages towards INS-1E cells was evaluated by in vivo assays. Carboxyfluorescein succinimidyl ester (CFSE)-labeled mCD47-INS-1E cells and control vector-transfected INS-1E (cont-INS-1 E) cells were injected into the peritoneal cavity of Rag2-/-gc-/- mice that had streptozotocin-induced diabetes and lacked T, B, and natural killer (NK) cells. The blood glucose levels were intermittently monitored using a blood glucose test meter. To further investigate whether inhibition of CD47-SIRPα signaling would abrogate the effect of genetic manipulation of CD47 expression, we injected anti-mouse SIRPa monoclonal antibody (mAb) into the peritoneal cavity of Rag2-/-gc-/- mice that had streptozotocin-induced diabetes. Results: Western blotting showed that incubation of mouse macrophages with mouse RBCs and mCD47-INS-1E cells caused significant SIRPa tyrosine phosphorylation. However, SIRPa tyrosine phosphorylation was not induced in mouse macrophages incubated with rat RBCs and cont-INS-1E cells, similar to that observed in the control macrophages incubated with medium alone. Mouse CD47 expression on INS-1E cells radically reduced the susceptibility of these cells to phagocytosis by mouse macrophages. Diabetic Rag2-/-gc-/- mice became normoglycemic immediately after receiving mCD47-INS-1E cells. In contrast, the mice that received cont-INS-1E cells failed to achieve normoglycemia. However, mice that had received mCD47-INS-1E cells and were injected with anti-mouse SIRPa mAb failed to achieve normoglycemia, similar to that observed in mice that had received cont-CD47-INS-1E cells. Conclusions: These results show that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of recipient CD47 on donor cells for generating SIPRα-inhibitory signals on recipient macrophages is necessary to prevent macrophage-mediated xenograft rejection.