Hypoxia Potentiates Glioma Immunosuppression Figure 2. Hypoxia enhances the gCSC-mediated immunosuppression on human T cells. A. When healthy donor peripheral blood mononuclear cells (PBMCs) were cultured in the presence of supernatant medium from cultures of each of the gCSCs, T cell proliferation was inhibited, as demonstrated by fluorescence-activated cell sorting (FACS) analysis of CD3+ T cell carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling that measures T cell proliferation. For each of the matched gCSCs under hypoxic conditions, T cell proliferation was further inhibited compared to normoxia. Representative FACS histograms or dot plots are shown on the left, and the summary bar graphs of percentage changes are shown on the right. T cells cultured with neurosphere cell medium served as a control. B. The supernatants from the gCSCs induced an increase in the number of FoxP3+ regulatory T cells in the gated CD4+ T cells, which is further enhanced under conditions of hypoxia. C. The inhibition of IFNc production in CD3+ T cells was enhanced under hypoxic condition compared to normoxia. * P,0.05. doi:10.1371/journal.pone.0016195.g002 Hypoxia enhances pSTAT3 and HIF-1a activity To ascertain if hypoxia increases gCSC expression of pSTAT3 and HIF-1a, the gCSCs were exposed to hypoxia and normoxic conditions and the expression levels of pSTAT3 and HIF-1a were determined by flow cytometry analysis. Hypoxia augmented the expression of both pSTAT3 (Fig. 4A and B) and HIF-1a (Fig. 4C and 4D) by 19,87% (average 43%) and 28,86% (average 62%), respectively. Western blot analysis showed a similar increase of pSTAT3 and HIF-1a expression, and no change for total STAT3 (Fig. 4E), indicating the increase of pSTAT3 were not due to general STAT3 changes. WP744 and WP1066 affect pSTAT3 and HIF-1a activity To determine if we could reverse the hypoxia-induced immunosuppression, we first assessed the ability of WP744 and WP1066 to reverse hypoxia-induced expression of HIF-1a. WP744 was a more potent inhibitor of HIF-1a and reduced expression by 53% under conditions of hypoxia, compared to 25% HIF-1a reduction by WP1066 (Figure 5A and 5B), thereby essentially abolishing the hypoxia-induced amounts of HIF-1a A similar but less degree inhibition of HIF-1a was observed under conditions of normoxia, confirming that these agents possessed HIF-1a inhibitory properties. However, we found WP744 enhanced pSTAT3 expression, but WP1066 inhibited its expres- sion under both conditions of normoxia and hypoxia (Figure 5C and 5D). There were up-regulation of pSTAT3 expression by 25%–184% (P = 0.02, paired t-test) in all 4 gCSCs under hypoxia (Fig. 5D), in contrast to the inhibitory effect of WP1066 (Fig. 5D) (decrease 24%–68% under hypoxia alone, P = 0.0005, paired t- test), indicating that WP1066 possess inhibitory activity of STAT3 and HIF-1a, whereas WP744 only inhibits HIF-1a. Inhibition of pSTAT3 and HIF-1a reverses hypoxia induced immunosuppression To assess the impact of WP1066 and WP744 on cytokine secretion in hypoxia-exposed gCSC, the gCSCs were treated with each inhibitor under conditions of hypoxia. WP1066 reduced galectin-3 (43%–79%, P = 0.02) and sCSF-1 (0%–24%, P = 0.05) production in all four hypoxia-exposed gCSCs, VEGF production in 3/4 gCSCs (23%–67%, P = 0.08), and CCL-2 production in 2/ 4 gCSCs (16%–46%, P = 0.04). The gCSCs in which WP1066 did not affect CCL-2 production (gCSC2 and gCSC4) produced only very low levels of CCL-2 under either normoxic or hypoxic conditions. WP744 reduced galectin-3 (79%–98%, P = 0.09) in all four gCSCs, and VEGF in 3/4 gCSCs (23%–41%, P = 0.04), but increased sCSF-1 production (105%–159%, P = 0.05), and had no Figure 3. The ability of gCSCs to inhibit phagocytosis in monocytes is enhanced by hypoxic conditions. A. Supernatants from gCSCs cultured in normoxic and hypoxic conditions inhibited phagocytosis of fluorescent microbeads (red) in monocytes exposed to the supernatants for 72 h, with hypoxic supernatants inhibiting phagocytosis to a greater extent than normoxic supernatants for all 4 gCSCs tested. Representative high- power fluorescent microscope images (40X) of monocytes exposed to (i) neurosphere medium alone as a positive control, (ii) normoxic gCSC supernatant, and (iii) hypoxic gCSC supernatants. Cell nuclei were stained with DAPI (blue). B. *P,0.05 indicates a significant difference between phagocytosis inhibition by hypoxic versus normoxic gCSC supernatants. doi:10.1371/journal.pone.0016195.g003 PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16195