Metabolic barriers to T cell activation in clear cell renal cell carcinoma

摘要 利用免疫来消除癌细胞提供了巨大的新的治疗机会，包括 广泛使用的免疫检查点阻断(ICB)试剂。然而，这些方法受到了挑战，因为 肿瘤抑制抗肿瘤免疫并使其有效的多种机制 只有一小部分患者。我们已经证明，效应性T细胞需要高葡萄糖摄取率才能合成代谢 新陈代谢，现在很明显，癌细胞和肿瘤微环境(TME)破坏了抗肿瘤 免疫部分是通过代谢免疫抑制来实现的。为了解决免疫疗法的这一障碍，我们 从手术切除的人肾透明细胞标本检测肿瘤浸润性淋巴细胞(TIL) 癌(CcRCC)，一种ICB反应率中等的癌症，其特征是血管丢失 希佩尔-林道(VHL)肿瘤抑制药。这些肿瘤使我们能够证明葡萄糖和谷氨酰胺都是 当葡萄糖代谢促进效应T细胞时，谷氨酰胺的代谢受到抑制 T细胞效应器功能。目前尚不清楚哪些谷氨酰胺依赖的酶或代谢物会抑制T细胞，但 我们发现，谷氨酰胺酶缺乏改变了组蛋白甲基化，并减少了PI3K-Pik3ip3的表达。 抑制蛋白，抑制PI3K/mTORC1信号转导和炎性效应细胞因子的产生。 为了进一步探索谷氨酰胺抑制T细胞的机制，我们进行了体内CRISPR筛选 原发TIL和发现谷氨酰胺合成酶在所有谷氨酰胺代谢酶中损失最多 有效增加TIL积累量。我们还直接定义了特定细胞类型的葡萄糖和谷氨酰胺的使用 在TME中使用放射性标记正电子发射断层扫描示踪剂。与经典的华宝形成鲜明对比 代谢、肿瘤相关巨噬细胞()是葡萄糖的主要消耗细胞，其次是TIL， 和癌细胞，而癌细胞优先消耗谷氨酰胺。有趣的是，VHL的损失并没有增加 TIL和体内的葡萄糖摄取增加，而TIL和TIL的葡萄糖摄取增加。为了探索这些 在接受ICB治疗的患者中，我们接下来进行了高维CyTOF分析 从纵向队列患者开始治疗前和治疗3周后采集外周血。这种方法 特异性识别罕见但高度增殖的ICB反应性CD8和CD4T细胞 线粒体潜力。基于这些发现，我们假设肾细胞癌基因驱动新陈代谢 富含谷氨酰胺的免疫抑制TME抑制PI3K信号损害T细胞 效应器的分化和功能。我们将研究原代人肾细胞癌和小鼠肾细胞癌模型，以： (1)测试ccRCC TME中的营养物质和肿瘤遗传学如何影响TIL的功能和代谢；以及(2) 确定TME中的葡萄糖和谷氨酰胺代谢如何促进或抑制抗肿瘤免疫。 总之，这些研究将建立谷氨酰胺损害T细胞分化的机制，并测试新的 潜在的靶点是克服TME中的代谢免疫抑制，以提高抗肿瘤免疫。

SUMMARY Exploiting immunity to eliminate cancer cells offers tremendous new therapeutic opportunities, including the widely employed immune checkpoint blockade (ICB) agents. These approaches are challenged, however, by the multitude of mechanisms through which tumors can suppress anti-tumor immunity and render them effective in only a portion of patients. We have shown that effector T cells require high rates of glucose uptake for anabolic metabolism, and it is now apparent that cancer cells and the tumor microenvironment (TME) disrupt anti-tumor immunity in part through metabolic immune suppression. To address this barrier to immunotherapy, we examined tumor infiltrating lymphocytes (TIL) from surgically excised samples of human clear cell Renal Cell Carcinoma (ccRCC), a cancer with moderate rates of ICB response that is characterized by loss of the Von Hippel-Lindau (VHL) tumor suppressor. These tumors allowed us to show that both glucose and glutamine are available in the TME and while glucose metabolism promotes effector T cells, metabolism of glutamine restrains T cell effector function. It is unclear which glutamine-dependent enzymes or metabolites suppress T cells, but we found that Glutaminase-deficiency altered histone methylation and reduced expression of Pik3ip3, a PI3K- inhibitory protein that suppresses PI3K/mTORC1 signaling and production of inflammatory effector cytokines. To further explore mechanisms of T cell suppression by glutamine, we performed an in vivo CRISPR screen in primary TIL and found loss of Glutamine Synthetase among all glutamine-metabolizing enzymes to most effectively increase TIL accumulation. We also directly defined cell type-specific glucose and glutamine usage in the TME using radiolabeled Positron Emission Tomography tracers. In contrast to classic Warburg metabolism, tumor associated macrophages (TAM) were the dominant consumers of glucose, followed by TIL, and cancer cells, which instead preferentially consumed glutamine. Interestingly, loss of Vhl did not increase glucose uptake of RCC cells in vivo but instead increased glucose uptake in TIL and TAM. To explore these pathways in patients undergoing ICB therapy, we next performed high dimensional CyTOF analyses of peripheral blood from a longitudinal cohort of patients before and 3 weeks after start of therapy. This approach specifically identified rare but highly proliferative ICB-responsive CD8 and CD4 T cells with elevated mitochondrial potential. Based on these findings, we hypothesize that RCC genetics drive a metabolically immunosuppressive TME with abundant glutamine that suppresses PI3K signaling to impair T cell effector differentiation and function. We will study primary human ccRCC tumors and mouse RCC models to: (1) Test how nutrients in the ccRCC TME and tumor genetics influence TIL function and metabolism; and (2) Determine how glucose and glutamine metabolism in the TME promote or suppress anti-tumor immunity. Together, these studies will establish mechanisms by which glutamine impairs T cell differentiation and test new potential targets to overcome metabolic immune suppression in the TME to improve anti-tumor immunity.