MOESM4 of Bi- and tri-valent T cell engagers deplete tumour-associated macrophages in cancer patient samples

Additional file 4. CD206- and FRβ BiTE-induced T cell-mediated killing of MDMs is dependent on the perforin pathway. T cells were co-cultured with autologous CFSE-stained MDMs and treated (or not) with the indicated BiTEs for 96 h, at which point % Live cells were calculated with Celigo image cytometry. For inhibition of perforin, T cells were pre-treated for 2 h with Concanamycin A (100 ng/mL), or an equivalent concentration of vehicle control (DMSO), then washed prior to BiTE addition. Inhibitors of Fas/FasL (anti-Fas antibody, clone ZB4, 2 μg/mL) and TRAIL (TRAIL-R1-Fc, 1 μg/mL) were added at the point of BiTE treatment and not removed. Data show mean ± SD of biological triplicates. Statistical significance was assessed by two-way ANOVA followed by Bonferroni post-hoc analysis, with each treatment being compared to the relevant “Mock” condition (*, P