P52: Glioma-derived soluble factors pose strong chemoattractants and partially change the cytotoxic activity of IFN-alpha-induced dendritic cells

Abstract

Dendritic cells (DCs) are the most potent antigen-presenting cells, which play crucial role in initiation and maintenance of immune response. However, DCs of high-grade glioma patients are characterized by some functional impairment including reduced TNF α -mediated antitumor cytotoxic activity and low migratory activity. Tumor-produced soluble molecules can profoundly affect the differentiation and maturation of DCs and influence the functional activity of DCs. The aim of the present study was to determine the effect of supernatants of primary glioma cell lines on the migration and the cytotoxic activity of healthy donor DCs. The study included 11 healthy donors and 7 patients with histologically verified glioma Grade II (2 patients) and glioma Grade IV (5 patients). DCs were generated from peripheral blood monocytes of healthy donors in the presence of GM-CSF and IFN-alpha (IFN-DCs) followed by the addition of lipopolysaccharide (LPS Escherichia coli). Primary glioma cell cultures from tissue samples of studied patients were obtained by mechanical and enzymatic (0.3% collagenase IA) disaggregation followed by culturing in DMEM/F12 medium containing 10% FCS. 7-day supernatants were collected upon reaching the cellular subconfluence. Migratory capacity of donor IFN-DCs in response to supernatants (v/v 25%) of primary glioma cell cultures was measured in trans-well culture chamber. DCs that migrated to the bottom chamber with medium alone as a control or with supernatants were counted by FACS analysis using BD calibration beads. Cytotoxic activity of donor IFN-DCs against tumor cell line HEp-2 was studied using MTT-assay for 24 h at a ratio of DC: HEp-2 1:1. IFN-DCs were preincubated for 2 h with supernatants of primary glioma cell cultures. Migration index of donor IFN-DCs to lymphoid chemokine CCL19 (100 ng/ml) was an average of 6.5 ± 2.1. Migration capacity of IFN-DCs towards concentration gradient of primary glioma culture supernatants was significantly higher. Supernatants of low-grade glioma cultures as well as of high-grade glioma cultures effectively induced chemotaxis of IFN-DCs. Migration index in response to concentration gradient of primary glioma line-derived soluble factors (6 cell lines) ranged from 32.6 ± 9 to 48.4 ± 15.5. To assess the effect of supernatants of primary lines on the cytotoxic activity of IFN-DCs four primary high-grade glioma lines were used. The cytotoxicity of intact LPS-stimulated donor IFN-DCs against HEp-2 cells was an average of 34.8 ± 7.4%. Supernatant pretreating of IFN-DCs reduced the cytotoxic activity of DCs in 8 out of 16 experiments (with an average of 43%). In other 8 cases, supernatants of high-grade glioma lines, in contrast, possessed a mild stimulating activity and enhanced the cytotoxicity of DCs on average about of 34%. Thus, tumor cells in glioma patients produce chemoattractants capable of providing migration of DCs to tumor. At the same time glioma-derived soluble factors change the cytotoxic activity both ways. One can suppose thereupon that impairment of DC cytotoxic activity in glioma patients occurs not only due to tumor cell impact but because of unknown mechanisms.