Hypoxia Potentiates Glioma Immunosuppression Figure 1. Hypoxia increased immunosuppressive cytokine production in gCSCs. Under hypoxic conditions, there was an increase in production of: VEGF in 3 of 4 gCSCs, galectin-3 in 3 out of 4 gCSCs, sCSF-1 in 3 of 4 gCSCs, and CCL-2 in 3 of 4 gCSCs, as assessed by ELISA. Error bars are derived from three separate experiments representing the deviation of the associated means between experiments throughout the manuscript. * P,0.05. doi:10.1371/journal.pone.0016195.g001 65%). Supernatants from gCSCs cultured under conditions of hypoxia further inhibited T cell proliferation represented by division of CFSE labeled T cells by approximately 64% (range: 52–78%, P = 0.04 comparing hypoxia to normoxia, Fig. 2A). Because hypoxia is likely to enhance the induction of regulatory T cells, we tested supernatants from gCSCs grown under normoxic conditions and found that they caused expansion of the number of CD4+FoxP3+ regulatory T cells in healthy donor PBMCs by 236% (range: 34%–400%). However, when cultured under hypoxic conditions, the gCSCs further increased the induction of regulatory T cells by 342% (range: 55%–512%, P = 0.05 comparing hypoxia to normoxia, Fig. 2B). To assess for functional activity of T cell suppression, IFNc production in the CD3/CD28 stimulated T cells were measured after culture with supernatants from gCSCs exposed to normoxic and hypoxic conditions. We found that hypoxic gCSCs caused a significant decrease of the IFNc+ T cells compared to normoxia (51–74% vs 24–53%, P = 0.02, Fig. 2C). Hypoxia enhances the ability of gCSCs to inhibit phagocytosis in monocytes To determine whether hypoxia-stressed gCSCs would alter the expression of MHC and co-stimulatory molecules on monocytes, which give rise to macrophages, we compared monocytes that were exposed to gCSC-conditioned medium under hypoxia and normoxic conditions and then analyzed surface markers by flow cytometry. Hypoxia-stressed gCSCs did not alter the expression of CD80, CD86, MHC I, MHC II, cytotoxic T-lymphocyte antigen (CTLA)-4, or B7-H1 on monocytes in comparison with those cultured in normoxic conditions (data not shown), indicating that hypoxia did not alter the gCSC-mediated expression of inflam- matory markers on the monocytes. To determine whether the ability of gCSCs to inhibit phagocytosis in monocytes was increased under hypoxic condi- tions, phagocytosis assays were performed on monocytes exposed to medium conditioned by gCSCs under normoxic and hypoxic conditions. Under normoxic conditions, the gCSC supernatants (n = 4) inhibited phagocytosis in monocytes (Fig. 3A) (to 47%–59% of the level seen in positive control monocytes exposed to neurosphere medium alone; P = 0.0005). The inhibition of phagocytosis was further enhanced to a statistically significant degree under hypoxic conditions (to 26%–39% of positive control) compared with supernatants from gCSC cultured under normoxic conditions (P = 0.005, paired t-test) in all four gCSCs tested (Fig. 3B). PLoS ONE | www.plosone.org 4 January 2011 | Volume 6 | Issue 1 | e16195