Murine CD47 expressed on the surface of human hepatocytes protects from murine macrophage action in vitro and in vivo

Macrophages play an important role in the rejection of transplanted xenogeneic cells. Recently, is has been shown that species-specific interaction between CD47 on target cells and SIRPα on macrophages inactivates macrophages and reduces phagocytosis. In this study we use lentiviral gene transfer to express murine CD47 in human cell lines to test whether they become less susceptible for phagocytosis by murine macrophages in vitro. In order to show the anti-phagocytotic effect of murine CD47 in vitro, we introduced murine CD47 into HepG2 cells by lentiviral gene delivery and performed phagocytotic assays with mouse RAW264.7 macrophage cell line. We were able to demonstrate that RAW-macrophages engulfed HepG2 negative controls efficiently while only a small fraction of macrophages acted on HepG2 cells expressing murine CD47. Live imaging experiments showed that phagocytotic events of control HepG2 cells were more than doubled in comparison to those of HepG2 cells expressing murine CD47. Edu-based proliferation assays were performed to rule out the possibility that murine CD47 expressing cells actually may have a proliferative advantage.

We also transduced primary human hepatocytes with murine CD47-expressing lentiviral vectors and transplanted them into Rac/γc(-/-) – uPAtg (+/+) mice. To determine engraftment success during the lifetime of transplanted mice, we monitored human albumin serum levels in transplanted mice. Overall engraftment success of primary human hepatocytes expressing murine CD47 increased significantly in comparison to control primary human hepatocytes.

Summing up, we were able to show that human cells expressing murine CD47 are less likely to be engulfed and phagocytosed by mouse macrophages in vitro. In vivo we show considerable improvement of engraftment of primary human hepatocytes expressing murine CD47 transplanted intrasplenically into Rac/γc(-/-) uPAtg (+/+) mice in comparison to control hepatocytes.

CD47 - gene therapy - phagocytosis - xenotransplantation