Phagocytotic activity of human reticulo-endothelial macrophages toward porcine xenogeneic cells is independent on α-gal epitopes but their cytotoxicity is partially dependent on α-gal epitopes

P1087 Aims: Although host macrophages are known to contribute at least in part to rapid clearance of transplanted xenogeneic cells, the mechanisms underlying macrophage-induced killing of xenogeneic cells has not been fully addressed. The aim of this study was to identify the precise role of human macrophages in the rejection of porcine xenogeneic cells. Methods: By high-gradient magnetic sorting, human peripheral macrophages (P-macrophages) were separated from volunteers’ peripheral blood and reticulo-endothelial macrophages (RE-macrophages) were separated from the perfusion effluents of liver allografts for clinical liver transplantation. Phagocytotic and cytolytic activities of those macrophages toward porcine cells were evaluated by in vitro assay that included neither antibodies nor complements. Carboxyfluorescein succinimidyl ester (CFSE)-labeled human syngeneic RBCs (Auto-RBCs), ABO-imcompatible RBCs (ABO-RBCs) and xenogeneic porcine RBCs (Xeno-RBCs) were incubated with the human P- and RE-macrophages in RPMI 1640 medium containing 15% controlled processed serum replacement for 4 hours. The macrophages that had phagocytosed the RBCs were identified as CFSE-labeled cells by FCM analysis. Cytolytic activity was quantified by calculating LDH levels in each culture supernatant. To assess the significance of α-galactosyl xenoantigens (α-Gal) in phagocytotic and cytolytic activity of human macrophages, α -Gal epitopes on the Xeno-RBCs were eliminated before the assays by using of endo-β -galactosidase C, which cleaves the Galβ 1-4GlcNAc linkage. Results: Phagocytotic and cytolytic activities of human P-macrophages toward Xeno-RBC were feeble. In contrast, human RE-macrophages spontaneously phagocytosed Xeno-RBCs with no stimulation, but not Auto-RBCs and ABO-RBCs. Elimination of α -Gal epitopes on the Xeno-RBCs did not prevent phagocytotic activity of RE-macrophages, suggesting that such phagocytosis was initiated by the recognition of xenoantigens other than α -Gal. Human RE-macrophages had marked cytolytic activity toward Xeno-RBCs but toward neither Auto-RBCs nor ABO-RBCs. Elimination of α -Gal epitopes on the Xeno-RBCs partially prevented cytolytic activity of RE-macrophages. Conclusions: Phagocytotic activity of RE-macrophages toward xenogeneic cells was independent on α -Gal epitopes but their cytotoxicity was partially dependent on α -Gal epitopes, suggesting that regulation of RE-macrophages in human recipients might be needed to achieve long-term acceptance of porcine xenografts even if α -Gal knockout pigs were used.