Anti-CD47 antibody increases phagocytosis through FcγR-independent and FcγR-dependent mechanisms and is not Mac-1 dependent. (A) CFSE⁺ TCL cells were incubated in vitro with B6H12 and F(ab′)2 fragment of B6H12 or isotype control in the presence of mBMDMs and hMDMs. (B) Same as panel A, using full-length and F(ab′)2 fragments of 2D3 and MIAP410. (C) CFSE⁺ TCL cells were incubated with WT or Fcer1g^−/− (KO) mBMDMs in the presence of the indicated antibodies. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (D-E) CFSE⁺ TCL cells were incubated with WT or Mac1^−/− (KO) mBMDMs in the presence of the indicated antibodies or F(ab′)2 fragment of B6H12. A representative experiment performed in triplicate with error bars representing SEM is shown. (F) On the left, phagocytosis of CFSE-labeled CTCL cells, Myla and HH by hMDMs in the presence of various antibodies including Moga at concentrations of 5 µg/mL is plotted. On the right, percent PI positive of the CFSE-labeled Myla and HH cells is plotted after culture with human NK cells in the presence of various antibodies at concentrations of 10 µg/mL. Shown are mean values of technical triplicates with error bars representing SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001 by 2-sided Welch t test. (G) Phagocytosis of Myla, HuT-78, MAC2A, and K-299 cells mediated by full-length B6H12-mIgG1κ, anti-HLA A,B,C mAb, W6/32 and their F(ab′)2 portions alone and in combination relative to isotype controls at 10 µg/mL (mIgG1κ for B6H12-mIgG1κ and mIgG2aκ for W6/32). Percentage of CFSE⁺ macrophages (of CD14⁺ hMDMs) is plotted. Shown are mean values of technical triplicates ± SEM. *P < .05; **P < .01; ***P < .001, ****P < .0001 by 2-sided Welch t test. KO, knockout; ns, not significant.