Table_1_Hypoxia Inducible Factor 1α Inhibits the Expression of Immunosuppressive Tryptophan-2,3-Dioxygenase in Glioblastoma.docx

Abnormal circulation in solid tumors results in hypoxia, which modulates both tumor intrinsic malignant properties as well as anti-tumor immune responses. Given the importance of hypoxia in glioblastoma (GBM) biology and particularly in shaping anti-tumor immunity, we analyzed which immunomodulatory genes are differentially regulated in response to hypoxia in GBM cells. Gene expression analyses identified the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2) as the second most downregulated gene in GBM cells cultured under hypoxic conditions. TDO2 catalyses the oxidation of tryptophan to N-formyl kynurenine, which is the first and rate-limiting step of Trp degradation along the kynurenine pathway (KP). In multiple GBM cell lines hypoxia reduced TDO2 expression both at mRNA and protein levels. The downregulation of TDO2 through hypoxia was reversible as re-oxygenation rescued TDO2 expression. Computational modeling of tryptophan metabolism predicted reduced flux through the KP and lower intracellular concentrations of kynurenine and its downstream metabolite 3-hydroxyanthranilic acid under hypoxia. Metabolic measurements confirmed the predicted changes, thus demonstrating the ability of the mathematical model to infer intracellular tryptophan metabolite concentrations. Moreover, we identified hypoxia inducible factor 1α (HIF1α) to regulate TDO2 expression under hypoxic conditions, as the HIF1α-stabilizing agents dimethyloxalylglycine (DMOG) and cobalt chloride reduced TDO2 expression. Knockdown of HIF1α restored the expression of TDO2 upon cobalt chloride treatment, confirming that HIF1α controls TDO2 expression. To investigate the immunoregulatory effects of this novel mechanism of TDO2 regulation, we co-cultured isolated T cells with TDO2-expressing GBM cells under normoxic and hypoxic conditions. Under normoxia TDO2-expressing GBM cells suppressed T cell proliferation, while hypoxia restored the proliferation of the T cells, likely due to the reduction in kynurenine levels produced by the GBM cells. Taken together, our data suggest that the regulation of TDO2 expression by HIF1α may be involved in modulating anti-tumor immunity in GBM.实体瘤的异常循环会导致缺氧，缺氧可同时调控肿瘤固有恶性特性与抗肿瘤免疫应答。鉴于缺氧在胶质母细胞瘤（glioblastoma, GBM）生物学中的重要作用，尤其是其对抗肿瘤免疫的塑造作用，我们分析了胶质母细胞瘤细胞在缺氧刺激下发生差异表达的免疫调节基因。基因表达分析显示，免疫抑制酶色氨酸-2,3-双加氧酶（tryptophan-2,3-dioxygenase, TDO2）是缺氧培养的胶质母细胞瘤细胞中第二大下调基因。TDO2可催化色氨酸氧化为N-甲酰犬尿氨酸，这是色氨酸沿犬尿氨酸通路（kynurenine pathway, KP）降解的第一步，也是限速步骤。在多种胶质母细胞瘤细胞系中，缺氧均可在mRNA与蛋白水平下调TDO2的表达。缺氧介导的TDO2下调具有可逆性，复氧即可恢复TDO2的表达水平。色氨酸代谢的计算模型预测，缺氧条件下犬尿氨酸通路的代谢通量会降低，细胞内犬尿氨酸及其下游代谢物3-羟基邻氨基苯甲酸的浓度也会下降。代谢检测验证了上述预测结果，证实该数学模型可准确推断细胞内色氨酸代谢物的浓度。此外，我们发现缺氧诱导因子1α（hypoxia inducible factor 1α, HIF1α）可在缺氧条件下调控TDO2的表达：缺氧诱导因子稳定剂二甲基乙二酰基甘氨酸（dimethyloxalylglycine, DMOG）与氯化钴均可下调TDO2的表达；而敲低HIF1α则可在氯化钴处理后恢复TDO2的表达，证实HIF1α可调控TDO2的表达。为探究这一新型TDO2调控机制的免疫调节效应，我们将分离的T细胞与表达TDO2的胶质母细胞瘤细胞分别在常氧与缺氧条件下进行共培养。常氧条件下，表达TDO2的胶质母细胞瘤细胞可抑制T细胞增殖；而缺氧则可恢复T细胞的增殖能力，这可能是由于胶质母细胞瘤产生的犬尿氨酸水平降低所致。综上，我们的研究结果表明，HIF1α对TDO2表达的调控可能参与了胶质母细胞瘤的抗肿瘤免疫调节。