GA Treatment strategies for high-grade primary brain tumors such as glioblastoma multiforme (GBM) have failed to significantly and consistently extend survival. Recently, several studies have suggested that chemotherapy-induced tumor stress may increase tumor vulnerability to the immune reaponse. We have previously shown that expanded/activated gd T cells from healthy donors recognize NKG2D ligands expressed on malignant glioma and are cytotoxic to glioma cell lines, primary GBM explants, and

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... man glioma intracranial cell line xenografts placed in immunodeficient mice. In this report, we show standard therapies for GBM based on temozolomide (TMZ) increase the expression of stress-associated NKG2D ligands on TMZ-resistant glioma cells, potentially rendering them vulnerable to attack by gd T cells. As TMZ is also highly toxic to gd T cells, we genetically modified gd T cells in culture using a lentiviral vector encoding P140KMGMT, resulting in resistance to TMZ at concentrations up to 400mM. Genetic modification of gd T cells did not alter their phenotype or their ability to kill targets, as both non-modified and gene-modified gd T cells were highly cytotoxic to U87 cells and TMZ-resistant clones of SNB-19 (SNB-19 TMZ-R ) and U373 (U373 TMZ-R ) glioma cell lines in the presence of up to 400 mM TMZ. Importantly, gene modified gd T cells showed greater cytotoxicity to both U373 TMZ-R and SNB-19 TMZ-R cultures in the presence of TMZ, suggesting that TMZ exposed more targets to gd T cell lysis. These findings demonstrate that TMZ resistant gd T cells can be generated without impairing cellular functions, and the genetically modified cells efficiently kill TMZ-resistant human glioma cell lines in the presence of high concentrations of TMZ. These results provide a mechanistic basis for combining and timing chemotherapy and drug resistant cellular immunotherapy to treat GBM.