ApyFigure 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 22948146 100 mM TMZ alone (upper panel) and with P140KMGMT transduced cd T cells (cdTMZ-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 cdTMZ-R results in much greater incorporation of ToPro Iodide after 4 h incubation suggesting that the increased cytotoxicity is overwhelmingly due to genetically modified cd T cells. Dose-dependent cytotoxicity of cdTMZ-R is significantly less when assayed against SNB-19TMZ-R (c) with no TMZ in the media vs. cdTMZ-R against SNB-19TMZ-R in the presence of TMZ (p = 0.0085). Cytotoxicity was also trended greater against TMZ-resistant U373 with cdTMZ-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 purchase BI 78D3 different donors. doi:10.1371/journal.pone.0051805.g19 and U373 cell lines constitutively express high levels of surface NKD2D ligands ULBP-2 and ULBP-3 (data not shown) as well as MIC-A for U373, suggesting that the additive effect of TMZ on cd T cell-based cytotoxicity may be partially mediated by nonpeptide ligands [51]. Besides inducing tumor associated stress molecules, chemotherapy can also augment immunotherapy in several ways, such as by enhancing the persistence of tumor reactive T lymphocytes and by increasing tumor trafficking of tumor responsive T cells, and by modulating immunosuppressive factors [52]. Thus administration of chemotherapy prior to cellular immunotherapy can modulate an immune environment that can be beneficial to the infused immune effector cells, such as cd T cells. It has been shown that chemotherapy treatments can facilitate the rapid infiltration of large numbers of cd T cells into tumors and prior to invasion of Tc1 cells [53]. Furthermore,temozolomide based chemotherapy has been shown to decrease the population of Fox-P3+ regulatory T cells, which provides an environment to further enhance the immune 15755315 response [54]. Therefore, rapidly emerging evidence supports the crucial contribution of the innate immune system to the anti-tumorigenicity of conventional chemotherapy-based cancer treatments [22,26,49]. In the context of GBM therapy, in order to access the chemotherapy derived window of opportunity of tumor vulnerability it may be beneficial to place a high concentration of cd T cells at the tumor site and to protect these effector cells, by gene transfer of MGMT, from the cytotoxic effects of TMZ chemotherapy, which would otherwise reduce or abrogate their function. In the present study, we successfully demonstrated two key aspects that are essential to the success of such a localized and a passive immunotherapy approach to target GBM: i) the order Anlotinib genetic engineer-Drug Resistant cd T Cell Immunotherapying of cd T cells and their expansion to concentrations sufficient for a therapeutic dose based on previous studies of cd T cell therapy of human xenografts in immunodeficient mice [35], and i.ApyFigure 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 22948146 100 mM TMZ alone (upper panel) and with P140KMGMT transduced cd T cells (cdTMZ-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 cdTMZ-R results in much greater incorporation of ToPro Iodide after 4 h incubation suggesting that the increased cytotoxicity is overwhelmingly due to genetically modified cd T cells. Dose-dependent cytotoxicity of cdTMZ-R is significantly less when assayed against SNB-19TMZ-R (c) with no TMZ in the media vs. cdTMZ-R against SNB-19TMZ-R in the presence of TMZ (p = 0.0085). Cytotoxicity was also trended greater against TMZ-resistant U373 with cdTMZ-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. doi:10.1371/journal.pone.0051805.g19 and U373 cell lines constitutively express high levels of surface NKD2D ligands ULBP-2 and ULBP-3 (data not shown) as well as MIC-A for U373, suggesting that the 
additive effect of TMZ on cd T cell-based cytotoxicity may be partially mediated by nonpeptide ligands [51]. Besides inducing tumor associated stress molecules, chemotherapy can also augment immunotherapy in several ways, such as by enhancing the persistence of tumor reactive T lymphocytes and by increasing tumor trafficking of tumor responsive T cells, and by modulating immunosuppressive factors [52]. Thus administration of chemotherapy prior to cellular immunotherapy can modulate an immune environment that can be beneficial to the infused immune effector cells, such as cd T cells. It has been shown that chemotherapy treatments can facilitate the rapid infiltration of large numbers of cd T cells into tumors and prior to invasion of Tc1 cells [53]. Furthermore,temozolomide based chemotherapy has been shown to decrease the population of Fox-P3+ regulatory T cells, which provides an environment to further enhance the immune 15755315 response [54]. Therefore, rapidly emerging evidence supports the crucial contribution of the innate immune system to the anti-tumorigenicity of conventional chemotherapy-based cancer treatments [22,26,49]. In the context of GBM therapy, in order to access the chemotherapy derived window of opportunity of tumor vulnerability it may be beneficial to place a high concentration of cd T cells at the tumor site and to protect these effector cells, by gene transfer of MGMT, from the cytotoxic effects of TMZ chemotherapy, which would otherwise reduce or abrogate their function. In the present study, we successfully demonstrated two key aspects that are essential to the success of such a localized and a passive immunotherapy approach to target GBM: i) the genetic engineer-Drug Resistant cd T Cell Immunotherapying of cd T cells and their expansion to concentrations sufficient for a therapeutic dose based on previous studies of cd T cell therapy of human xenografts in immunodeficient mice [35], and i.