Exploiting metabolic vulnerabilities of CD4 T cell subsets to control inflammatory disease

总结 炎症性疾病通常由效应CD 4 T细胞（Teff）的不适当反应驱动。IL 17、IFNγ或 双重产生的多功能效应Th 1、Th 17或Th17.1 T细胞可以变得与抑制性T细胞失衡， Treg CD 4 T细胞在各种疾病环境中的作用，包括炎症性肠病（IBD）。一种关键的治疗方法 因此，努力将免疫平衡向耐受性转变的目的是选择性抑制Teff， 促进Treg。我们已经表明，Teff和Treg亚群利用不同的代谢程序， T细胞生物学的基本特征。在这里，我们探讨一碳（1C）代谢和相关的 微环境因素调节炎症性疾病中的CD 4 T细胞。1C代谢整合了多种 为蛋氨酸和嘌呤的从头合成提供中间体， 服用抗叶酸药物用1C代谢酶聚焦的IBD中的原代T细胞的体内CRISPR筛选 gRNA文库将线粒体酶亚甲基-四氢叶酸脱氢酶2（MTHFD 2）鉴定为 对于效应T细胞增殖和炎症是条件性必需的。MTHFD 2在T细胞中上调， 各种炎症条件，而MTHFD 2缺陷损害Teff，MTHFD缺陷Treg有 增加小鼠和人T细胞中FoxP 3的表达。与代谢检查点的作用一致 在炎症方面，MTHFD 2缺乏可预防IBD和其他炎性疾病。机械地说， MTHFD 2抑制抑制mTORC 1活性，可能通过减少蛋氨酸和/或中断嘌呤 合成.重要的是，局部营养素在1C代谢和T细胞命运中发挥关键作用。为了量化T细胞进入 针对体内营养物质，我们建立了基于正电子发射断层扫描（PET）示踪剂的方法， 并测量体内的营养吸收。这些研究表明，在T细胞葡萄糖摄取急剧增加， 炎症此外，IBD通常与叶酸缺乏有关。膳食叶酸对T细胞的影响 然而，1C代谢、mTORC 1信号传导和命运尚不清楚。因为炎症与 发烧和酶是温度依赖性的，我们还测试了发烧条件对T细胞代谢的影响。我们发现 发热导致Teff和线粒体活性氧（ROS）产生的细胞因子增加 特别是在Th 1细胞中，令人惊讶的是，这导致了Tp 53依赖性细胞凋亡。这些发现支持 假设1C代谢是限制性的，并作为代谢检查点，以整合局部营养素， 通过MTHFD 2和mTORC 1信号传导提供新的免疫代谢靶点， 调节效应和调节性T细胞。为了验证这一点，我们将：（1）测试MTHFD 2作为一种 mTORC 1信号传导和效应T细胞所必需的甲硫氨酸和核苷酸合成中的限制酶;和 (2)确定营养和微环境因素如叶酸和发烧如何影响效应子和 调节性T细胞代谢和炎症。这些研究将测试MTHFD 2并确定新的 本发明涉及有助于或可用于治疗炎性疾病的免疫代谢机制和靶点。

SUMMARY Inflammatory diseases are often driven by inappropriate responses of effector CD4 T cells (Teff). IL17, IFNγ, or dual-producing polyfunctional effector Th1, Th17, or Th17.1 T cells can become imbalanced with suppressive Treg CD4 T cells in a variety of disease settings, including inflammatory bowel diseases (IBD). A key therapeutic objective in efforts to shift the immunologic balance towards tolerance, therefore, is to selectively inhibit Teff and promote Treg. We have shown that Teff and Treg subsets utilize different metabolic programs that represent fundamental features of T cell biology. Here we explore one carbon (1C) metabolism and related microenvironmental factors to modulate CD4 T cells in inflammatory diseases. 1C metabolism integrates multiple nutrient inputs to provide intermediates for de novo methionine and purine synthesis and is commonly targeted with anti-folate drugs. An in vivo CRISPR screen of primary T cells in IBD with a 1C metabolism enzyme-focused gRNA library identified the mitochondrial enzyme Methylene-tetrahydrofolate Dehydrogenase 2 (MTHFD2) as conditionally essential for effector T cell proliferation and inflammation. MTHFD2 was upregulated in T cells a variety of inflammatory conditions and while MTHFD2-deficiency impaired Teff, MTHFD-deficient Treg had increased FoxP3 expression in both mouse and human T cells. Consistent with a role as a metabolic checkpoint on inflammation, MTHFD2-deficiency protected against IBD and other inflammatory diseases. Mechanistically, MTHFD2 inhibition suppressed mTORC1 activity, possibly through reduced methionine and/or interrupted purine synthesis. Importantly, local nutrients play key roles in 1C metabolism and T cell fate. To quantify T cell access to nutrients in vivo, we established Positron Emission Tomography (PET) tracer-based methods to directly image and measure nutrient uptake in vivo. These studies showed a sharp increase in T cell glucose uptake in inflammation. In addition, IBD is often associated with folate-deficiency. The effects of dietary folate on T cell 1C metabolism, mTORC1 signaling, and fate, however, are unclear. Because inflammation is associated with fevers and enzymes are temperature-dependent, we also tested fever conditions on T cell metabolism. We found fever led to increased cytokine production from Teff and mitochondrial Reactive Oxygen Species (ROS) specifically in Th1 cells that, surprisingly, led to Tp53-dependent apoptosis. These findings support the hypothesis that 1C metabolism is limiting and serves as a metabolic checkpoint to integrate local nutrients and physical conditions through MTHFD2 and mTORC1 signaling to provide new immunometabolic targets to modulate effector and regulatory T cells. To test this, we will: (1) Test the role and mechanism of MTHFD2 as a limiting enzyme in methionine and nucleotide synthesis essential for mTORC1 signaling and effector T cells; and (2) Determine how nutrient and microenvironmental factors such as folate and fever influence effector and regulatory T cell metabolism and inflammation. These studies will test MTHFD2 and identify new immunometabolic mechanisms and targets that contribute to or may be exploited to treat inflammatory disease.