EGFR-negative cells were sensitized to bystander killing by T cell cytokines.

<p>(A) EGFR BiTE^®, T cells and NUGC4 cells (E:T ratio 10:1) were incubated in 96-well plates for 48 hours; supernatants containing T cells were either transferred directly (medium + cells) or clarified by centrifugation prior to transfer (medium only) to 96-well plates containing SW620 cells, or SW620 cells were directly treated with T cells and EGFR BiTE^® (no transfer control); N = 3, mean +/- sd (B) T cells + EGFR BiTE^® + NUGC4 cells were added to the top chamber of Transwell^® assays with 1μm and 5μm membranes; SW620 (or NUGC4 as control) cells were added to the bottom chambers. Percent cytotoxicity in the bottom chambers was determined with CellTiter-Glo^®. (C) SW620 cells were pre-treated for 24 hours +/- cytokines (10ng/ml IFNγ + 5ng/ml TNFα), then incubated for 24 hours with either resting T cells or BiTE^®-activated T cells. Cells were enumerated by nuclear count with cellular imaging; N = 4, mean +/- sd. Significance values: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.