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Fig. 2 | Journal for ImmunoTherapy of Cancer

Fig. 2

From: Bi- and tri-valent T cell engagers deplete tumour-associated macrophages in cancer patient samples

Fig. 2

CD206- and FRβ-targeting BiTEs activate primary human T cells to kill autologous M2-polarised macrophages. a Schematic representations of CD206- and FRβ-targeting BiTEs. b, Western blot analysis of supernatants from HEK293A cells 48 h after transfection with BiTE expression plasmids. Blots were probed with a mouse anti-His primary antibody, followed by an HRP-conjugated anti-mouse secondary antibody. c Human MDMs were polarised as indicated, stained with CFSE, and treated with T cells (10:1 E:T ratio) and increasing concentrations of BiTEs. Macrophage killing was assessed 96 h later by propidium iodide staining and Celigo image cytometry. d MDMs were stained with CFSE and treated with the indicated concentrations of BiTE in the presence or absence of T cells (10:1 E:T ratio). 96 h later, cytotoxicity was assessed by propidium iodide staining and analysis with a Celigo image cytometer. e T cell activation in the presence or absence of target cells was assessed by flow cytometric measurement of CD25 expression 96 h after BiTE addition. Data show mean ± SD of biological triplicates (c, d and e). Statistical analysis was performed by two-way ANOVA with Bonferroni post-hoc tests comparing with the relevant “Mock” condition (d and e) (*, P < 0.05; **, P < 0.01; ***, P < 0.001)

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