Temozolomide is widely used as the first-line chemotherapeutic agent for malignant glioma. However, it can prolong the median survival and progression-free survival in glioblastoma patients by only a few months. Recently, it has been demonstrated that multiple human tumors express B7-H1 and B7-H4, immune inhibitory transmembrane glycoproteins, including glioblastoma. It has been suggested that tumor cells utilize these proteins as a potential mechanism of immunoressistance. Therefore, we sought to determine if glioma cells up-regulate B7-H1 and B7-H4 and promotes immunoressistance in glioma cells. We show by fluorescent activated cells sorting that T98G, human glioblastoma cell line, GBM157, GBM177, primary human glioblastoma cells, and GL26, murine glioma cell line, express both B7-H1 and B7-H4. We also demonstrate that both B7-H1 and B7-H4 were significantly up-regulated in surviving T98G, GBM157, and GL26 cells after 3-day temozolomide exposure. The standardized fluorescence index of B7-H1 protein after temozolomide (1000 mM) exposure significantly increased from 1.54 to 3.07 (p =0.0021) and that of B7-H4 significantly increased from 1.99 to 3.17 (p =0.0043) in surviving T98G cells. Real-time quantitative PCR also showed significant up-regulation in B7-H1 gene expression by 4.6 times (p < 0.0001) and in B7-H4 gene expression by 2.0 times (p = 0.0398) after 3-day temozolomide (1000mM) exposure in surviving T98G cells. In order to determine if up-regulation of B7-H1 promoted immunoressistance we co-culture surviving temozolamide exposed glioma cells with either CD4+ or CD8+ T-cells and determined secreted levels of IL-10 by enzyme-linked immunofluorescence assay and T-cell apoptosis, respectively. We show that CD4+ T-cells significantly up-regulate IL-10 secretion after co-culture with surviving temozolamide exposed glioma cells and that CD8+ T-cells apoptosis is also significantly increased as compared to control GL26 glioma cells. Taken together, these results suggest that up-regulation of B7-H1 and B7-H4 in glioma cells promotes glioma immunoressistance through the up-regulation of CD4+ T-cell IL-10 secretion and CD8+ T-cell apoptosis.
Citation Information: Clin Cancer Res 2010;16(7 Suppl):A22