Figure 8.

Disruption of SIRPα–CD47 interaction by hSIRPα-Fc enhances phagocytosis of AML but not normal hematopoietic targets. (a) SIRPα expression on human macrophages, untreated or stimulated with IFN-γ for 24 h and LPS for 1 h before flow cytometric analysis using anti–human SIRPα Ab. (b) Analysis of binding of NOR macrophages infected with lentiviral vectors expressing human SIRPα or NOD mouse SIRPα or empty vector (CEP), as indicated, to human CD47-Fc. (c) Flow cytometric analysis of CD47 expression on CD34⁺CD38⁻ cells from normal human adult BM, CB, and primary AML patient samples. The number of samples analyzed is indicated. CD47 mean fluorescence intensity (MFI) for each sample was normalized for cell size and CD47 expression level on lymphocytes from the same sample. Error bars indicate SE. *, P < 0.05 (ANOVA). (d) Summary of phagocytosis of human AML cells (AML9, AML19, AML22, AML23, and AML26) or normal mononuclear cells from human CB (n = 2) or BM (n = 4) by LPS/IFN-γ–stimulated human CB-derived macrophages with addition of IgG₄-Fc or hSIRPα-Fc as indicated. ***, P < 0.001 (ANOVA). (e) Phagocytosis of human AML (AML29) and CB mononuclear cells coincubated with LPS/IFN-γ–stimulated human CB-derived macrophages with addition of hSIRPα-Fc. *, P < 0.05 (Student’s t test). (d and e) The phagocytic index was determined by spinning disk confocal microscopy as the number of engulfed cells per 100 macrophages, with two to five replicates/sample. Bars indicate mean ± SD. (f) Peripheral blood hemoglobin concentration and neutrophil and platelet counts of NOD mice treated with NOD SIRPα-Fc (n = 4) or IgG₄-Fc control (n = 3) for 4 wk. (g) Percentage of Sca1⁺cKit⁺ cells in the Lin⁻ population (LSK) and CD150⁺CD48⁻ cells in the LSK fraction in the BM of NOD mice after 4 wk of treatment with NOD SIRPα-Fc or IgG₄-Fc control (n = 3 mice/group). (f and g) Bars indicate mean ± SD. No statistically significant differences were observed (Student’s t test).