Astrocyte cultures (control for toxicity). Targets were labeled with the membrane

Astrocyte cultures (control for toxicity). Targets were labeled with the membrane dye PKH26 (Sigma; St. Louis, MO). Expanded/ activated cd T cells were then added to the tubes at ratios of 0:1 (Background), 5:1, 10:1, 20:1 and 40:1 effectors/GBM targets, incubated for four hours at 37uC and 5 CO2, 1326631 washed once and resuspended in 1 ml HBSS. ToPro Title Loaded From File Iodide solution (20 ml) (Molecular Probes; Eugene, OR) was added prior to acquisition on the flow cytometer. Cytotoxicity was calculated as: (Toprolodide+PKH26+ events/total PKH26+ events) 6100. Single tubes were acquired for each experiment and duplicate experiments were performed as quality control.Genetic engineering of cd T cells does not alter their response to Zoledronic acid/IL-2 expansion or cytotoxic functionWe then tested whether genetic modification with the MGMT vector had an effect on the the proliferative or cytotoxic function of cd T cells (TMZ-transduced/resistant T cells – cdTMZ-R) in response to the Zoledronic acid and IL-2 expansion protocol. Two representative experiments using expanded/activated cd T cells from separate donors are shown. When comparing geneticallymodified cd T cells to unmodified cells we found no difference in the proliferative response, as all populations routinely yielded an expansion of cd T cells comprising 65 – 90 of the total lymphocyte population (Figure 4a and 4b). The cytotoxicity 1379592 of unmodified to modified cd T cells to the U87 glioma cell line was nearly equivalent at all E:T ratios (Figure 4c and 4d), verifying that cdTMZ-R genetically-modified T cell function is equivalent to that of unmodified cd T cells. For three separate donors, the expanded cells comprised approximately 2.0?2.06108 cells with transduced cell number yields generally less than unmodified cells due to cell loss during lentivirus transduction (Table 1). However, a 50 ml blood draw routinely yielded 26108 transduced cd T cells, which is sufficient for a therapeutic intracranial cell dose.Statistical analysisDescriptive statistics were used to characterize mean, standard deviation, and standard error of populations. For comparison of antigen expression, median fluorescence intensity (MFI) was obtained from single parameter histograms. Comparisons between groups was accomplished by single parameter t-test for differences between means and the Wilcoxon Signed-Rank test for differences in medians. A result was considered significant at a p value of 0.05.Results NKG2D Ligands are transiently upregulated on TMZresistant U87MG glioma cells after exposure to TMZIn this experiment, we sought to determine if TMZ exposure stresses a Title Loaded From File TMZ-resistant U87 culture that had not previously been exposed to TMZ, NKG2D ligand expression was examined at incremental time intervals following TMZ exposure. ChemotherDrug Resistant cd T Cell ImmunotherapyFigure 1. Transitory increase in stress-associated antigens on TMZ-resistant cell line U87 after exposure to TMZ. U87 cells were cultured to confluence and incubated in media containing 100 mM TMZ. Stress antigens were assessed at the time intervals noted on the x axis by flow cytometry and the increase in median fluorescence intensity over isotype control were calculated. Data are shown as percentage increase over unmanipulated U87 cells. SD of 3 experiments are shown. doi:10.1371/journal.pone.0051805.gGene-modified cd T cells function in the presence of TMZCytolytic function of MGMT-modified cd T cells was evaluated against TMZ-resistant clones of SN.Astrocyte cultures (control for toxicity). Targets were labeled with the membrane dye PKH26 (Sigma; St. Louis, MO). Expanded/ activated cd T cells were then added to the tubes at ratios of 0:1 (Background), 5:1, 10:1, 20:1 and 40:1 effectors/GBM targets, incubated for four hours at 37uC and 5 CO2, 1326631 washed once and resuspended in 1 ml HBSS. ToPro Iodide solution (20 ml) (Molecular Probes; Eugene, OR) was added prior to acquisition on the flow cytometer. Cytotoxicity was calculated as: (Toprolodide+PKH26+ events/total PKH26+ events) 6100. Single tubes were acquired for each experiment and duplicate experiments were performed as quality control.Genetic engineering of cd T cells does not alter their response to Zoledronic acid/IL-2 expansion or cytotoxic functionWe then tested whether genetic modification with the MGMT vector had an effect on the the proliferative or cytotoxic function of cd T cells (TMZ-transduced/resistant T cells – cdTMZ-R) in response to the Zoledronic acid and IL-2 expansion protocol. Two representative experiments using expanded/activated cd T cells from separate donors are shown. When comparing geneticallymodified cd T cells to unmodified cells we found no difference in the proliferative response, as all populations routinely yielded an expansion of cd T cells comprising 65 – 90 of the total lymphocyte population (Figure 4a and 4b). The cytotoxicity 1379592 of unmodified to modified cd T cells to the U87 glioma cell line was nearly equivalent at all E:T ratios (Figure 4c and 4d), verifying that cdTMZ-R genetically-modified T cell function is equivalent to that of unmodified cd T cells. For three separate donors, the expanded cells comprised approximately 2.0?2.06108 cells with transduced cell number yields generally less than unmodified cells due to cell loss during lentivirus transduction (Table 1). However, a 50 ml blood draw routinely yielded 26108 transduced cd T cells, which is sufficient for a therapeutic intracranial cell dose.Statistical analysisDescriptive statistics were used to characterize mean, standard deviation, and standard error of populations. For comparison of antigen expression, median fluorescence intensity (MFI) was obtained from single parameter histograms. Comparisons between groups was accomplished by single parameter t-test for differences between means and the Wilcoxon Signed-Rank test for differences in medians. A result was considered significant at a p value of 0.05.Results NKG2D Ligands are transiently upregulated on TMZresistant U87MG glioma cells after exposure to TMZIn this experiment, we sought to determine if TMZ exposure stresses a TMZ-resistant U87 culture that had not previously been exposed to TMZ, NKG2D ligand expression was examined at incremental time intervals following TMZ exposure. ChemotherDrug Resistant cd T Cell ImmunotherapyFigure 1. Transitory increase in stress-associated antigens on TMZ-resistant cell line U87 after exposure to TMZ. U87 cells were cultured to confluence and incubated in media containing 100 mM TMZ. Stress antigens were assessed at the time intervals noted on the x axis by flow cytometry and the increase in median fluorescence intensity over isotype control were calculated. Data are shown as percentage increase over unmanipulated U87 cells. SD of 3 experiments are shown. doi:10.1371/journal.pone.0051805.gGene-modified cd T cells function in the presence of TMZCytolytic function of MGMT-modified cd T cells was evaluated against TMZ-resistant clones of SN.