EGFR-negative bystander cells were lysed by BiTE^®-activated T cells when co-cultured with EGFR-positive cells.

<p>NUGC4 (EGFR-positive) and SW620 (EGFR-negative) cells were mixed in various ratios and incubated with T cells (E:T ratio 10:1) and EGFR BiTE^® in duplicate plates. Thousands of cells/well were analyzed, with good agreement between replicate plates. This result was reproducible (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183390#pone.0183390.s003" target="_blank">S2C Fig</a>). After 48 hours, cells were stained and analyzed as describe in Materials and Methods. Cytotoxicity of (A) EGFR-positive and (B) EGFR-negative cells was measured by nuclear count (N = 2, all data points shown). (C) IFNγ and (D) TNFα were measured using commercially available MSD assays (N = 3, mean +/- sd). (E) T cells from quadruplicate wells were combined and percent CD69+/CD25+ cells determined by flow cytometry.