TMZ-resistant clones of the GBM cell line (<i>a</i>) U373^TMZ-R and (<i>b</i>) SNB-19^TMZ-R were selected by incubation in increasing concentrations of TMZ over 60 days.

<p>The cell lines were labeled with PKH-26 and incubated for 4 hours in the presence of 100 µM TMZ alone (upper panel) and with P140KMGMT transduced γδ T cells (γδ^TMZ-R) at a 10∶1 effector:target ratio. The culture was then labeled with ToPro Iodide and acquired for flow cytometric phenotyping. A minimum of 5000 PKH26+ events was acquired to insure statistical validity of the data. All plots gated on PKH-26+ target cells. Note that the cloned cell lines are resistant to killing in media supplemented with TMZ with SNB-19^TMZ-R showing less cell loss than U373^TMZ-R. Addition of γδ^TMZ-R results in much greater incorporation of ToPro Iodide after 4 h incubation suggesting that the increased cytotoxicity is overwhelmingly due to genetically modified γδ T cells. Dose-dependent cytotoxicity of γδ^TMZ-R is significantly less when assayed against SNB-19^TMZ-R (c) with no TMZ in the media vs. γδ^TMZ-R against SNB-19^TMZ-R in the presence of TMZ (p = 0.0085). Cytotoxicity was also trended greater against TMZ-resistant U373 with γδ^TMZ-R as effectors as well when the assay was conducted in the presence of TMZ (p = .0875). These assays were conducted as separate experiments from different donors.