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Engineered Drug Resistant γδ T Cells Kill Glioblastoma Cell Lines during a Chemotherapy Challenge: A Strategy for Combining Chemo- and Immunotherapy

Figure 5

TMZ-resistant clones of the GBM cell line (a) U373TMZ-R and (b) SNB-19TMZ-R were selected by incubation in increasing concentrations of TMZ over 60 days.

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 (γδTMZ-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-19TMZ-R showing less cell loss than U373TMZ-R. Addition of γδTMZ-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 γδTMZ-R is significantly less when assayed against SNB-19TMZ-R (c) with no TMZ in the media vs. γδTMZ-R against SNB-19TMZ-R in the presence of TMZ (p = 0.0085). Cytotoxicity was also trended greater against TMZ-resistant U373 with γδTMZ-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.

Figure 5