normoxic and hypoxic conditions, as well as in neurosphere medium as controls. Induction of pSTAT3 was determined by flow cytometry after overnight incubation. Expression of the cell surface markers MHC I, MHC II, CD80, CD86, CD163, and CD204 was determined by flow cytometry after 48 h of incubation. After 72 h of incubation, monocytes were harvested for phagocytosis assays, and supernatants were collected for cytokine ELISA. Flow cytometry FITC-conjugated anti-CD4 (RPA-T4) and APC-conjugated anti-CD8 (RPA-T8) antibodies were used for cell surface staining. Sub-analysis of the T cell populations was based on the gated surface expression of CD4 and CD8. To detect FoxP3 protein expression, the surface stained cells were further subjected to intracellular staining with PE-conjugated monoclonal antibodies to human FoxP3 (clone PCH101, eBiosciences) using staining buffers and conditions specified by the manufacturer. For intracellular cytokine staining, cells were stimulated for 6 h in the presence of 50 ng/ml phorbol myristate acetate (PMA), 500 ng/ml ionomycin (Sigma-Aldrich), and 2 mM monensin (GolgiStop, BD Sciences). Then the cells were incubated with FITC-conjugated anti-CD4 and APC-conjugated anti-CD8 (RPA-T8) antibodies for surface staining followed by intracellular staining using PE-conjugated anti-mouse IFN-c (4S.B3) antibody and FIX/PERM buffers (BD Pharmingen) according to the manufacturer’s instructions. For HIF1a intracellular staining, gCSCs were first fixed with cold 4% paraformaldehyde at room temperature for 10 minutes; following fixation, the cells were washed and permeabilized by 1% saponin on ice for 30 minutes, and then stained by APC-conjugated anti- human HIF-1a antibody (R&D Systems) for 45 minutes at room temperature in the dark. Intracellular p-STAT3 intracellular staining was performed as previously described [43]. Flow cytometry acquisition was done with a FACSCaliber (Becton Dickinson, San Diego, CA), and data analysis was with FlowJo software (TreeStar, Ashland, OR). Cell proliferation assay and regulatory T cell induction assay The conditioned medium from the gCSCs was added to 2 mM CFSE-labeled 36105 PBMCs/ml in the presence of 1 mg/ml pre- bound anti-CD3/anti-CD28 antibodies. After 72 h, T cell proliferation was measured by division of CFSE-labeled cells as recorded with flow cytometry. The inhibition of T cell proliferation was represented by the percentage decrease of CSFE+ divided T cells compared to neurosphere medium control. To detect FoxP3+ regulatory T cells, CD4 surface staining and then FoxP3 intracellular staining were performed on immune cells cultured for 96 h. Phagocytosis assay CD14+ monocytes were incubated with either normoxic or hypoxic gCSC-conditioned medium for 72 h. Positive control monocytes were incubated with neurosphere medium for 72 h. Cell viability was assessed by Trypan Blue exclusion, and live monocytes were transferred to a 96-well plate at a concentration of 1.06105 cells/well and incubated at 37uC in a humidified atmosphere containing 95% air/5% CO2 for 1 h to allow the cells to adhere. The cells were then incubated with pHrodo E. coli BioParticles (Invitrogen) suspended in PBS for 2 h at 37uC at atmospheric CO2 levels. Cells were examined using fluorescence microscopy, and phagocytosis was quantified by measuring the mean fluorescence intensity per cell and corrected for background Hypoxia Potentiates Glioma Immunosuppression using Adobe Photoshop CS software. Percent phagocytosis levels relative to the control were calculated for each experimental condition. Western Blot After exposure to normoxia and hypoxic conditions, gCSCs were harvested, pelleted by centrifugation, and rinsed with ice-cold PBS at 1,500-rpm for 5 min. The cells were lysed for 30 min in ice-cold lysis buffer (50-mM Tris–HCl [pH 8.0], 150-mM NaCl, 1-mM EDTA) containing 1% Triton-X-100 and phosphatase and protease inhibitors (Sigma-Aldrich). The lysates were centrifuged at 14,000- rpm for 10 min at 4uC. The supernatants were collected and quantified for protein content. Equal amounts of proteins (40-mg) were electrophoretically fractionated in 8% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis with specific antibodies against pSTAT3 (Tyr705), total STAT3 (both are from Cell Signaling Technology, Inc., Danvers, MA), HIF-1a (Novus Biologicals, Littleton, CO) and b-actin (Sigma-Aldrich). Autoradi- ography of the membranes was performed using Amersham ECL Western-blotting detection reagents (Amersham Biosciences). Statistical analysis All values were calculated as means and 95% confidence intervals (CIs) from at least three independent experiments. Student’s t test was used to test for differences in the means between two groups. P values of less than 0.05 were considered to be statistically significant. All statistical analyses were performed using the Statistical Package for the Social Sciences v.12.0.0 (SPSS, Chicago, IL). Error bars represent the s.d. Results Hypoxia enhances the production of immunosuppressive cytokines by the gCSCs To determine if hypoxia further potentiated gCSC elaborated immunosuppressive cytokines, the gCSCs (n = 4) were assayed for immunosuppressive cytokines by ELISA. Hypoxia enhanced immunosuppressive VEGF secretion in 3 out of 4 gCSCs (750– 1160 pg/106 cells/24 hours under normoxic conditions versus 1290–3300 pg/106 cells/24 hours under hypoxic conditions, an increase of 1.1–4.4X) and the monocyte chemokine attractant sCSF-1 in 2 out of 4 gCSCs (4.2–100 pg/106 cells/24 hours under normoxic conditions versus 5.6–114 pg/106 cells/24 hours, an increase of 1.1–1.7X). Trends were also observed showing hypoxia increasing the T cell apoptosis inducing cytokine galectin-3 secretion in 3 out of 4 gCSCs (4.2–1810 pg/106 cells/24 hours under normoxic conditions versus 88.9–8110 pg/106 cells/ 24 hours under hypoxic conditions, an increase of 4.5-101X), and the regulatory T cell chemokine attractant CCL-2 in 3 out of 4 gCSCs (5.6–761 pg/106 cells/24 hours under normoxic condi- tions versus 56.1–2465 761 pg/106 cells/24 hours under hypoxic conditions, an increase of 1.5–10.1X) (Fig. 1). Hypoxia induced variable responses in TGF-b1 production and failed to enhance production of MIC-1 (data not shown). Hypoxia augments the ability of gCSCs to inhibit T cell adaptive immunity To determine if hypoxia potentiates the immunosuppressive effects of gCSCs on T cell activation and proliferation, the gCSCs (n = 4) were placed under both normoxic and hypoxic conditions. All supernatants from gCSCs cultured under normoxic conditions inhibited T cell proliferation by approximately 43% (range: 29– PLoS ONE | www.plosone.org 3 January 2011 | Volume 6 | Issue 1 | e16195