Amino acid sensing and phenotypic adaptation in T cells

描述(由申请人提供)：尽管人们对基础代谢控制重新产生了兴趣，但在包括mTOR和AMPK途径在内的每个关键途径中，关于细胞和上下文特定的改变和适应方面的知识存在很大差距。在这项建议中，我们将研究一种新的途径，将氨基酸感觉与T细胞代谢的下游变化联系起来，这些变化对T细胞的表型和功能有深远的影响。必需氨基酸的可获得性已成为调节T细胞生长和最终T细胞分化状态的中心代谢机制。抗原提呈细胞(APC)通过表达可降解精氨酸和色氨酸等必需氨基酸的诱导性酶来调节T细胞对氨基酸的生物利用度，并被假设在免疫微环境中限制T细胞的活动和增殖。暴露在氨基酸缺乏的微环境中的T细胞的代谢后果，以及下游对T细胞分化的影响目前尚不清楚。我们发现精氨酸耗竭在T细胞代谢中起着中心作用，而T细胞代谢与Th2反应为主的体内疾病直接相关。为了剖析氨基酸消耗抗原提呈细胞(APC)和T细胞之间的相互作用，我们建立了一个系统，在这个系统中，APC、T细胞和培养条件可以通过影响关键代谢检查点的遗传学和药物来操纵。我们的方法使我们能够评估接受APC介导的精氨酸限制的原代T细胞的新陈代谢，并可普遍用于研究任何氨基酸对T细胞和APC的影响。我们的初步数据表明，T细胞对精氨酸的感知涉及一种意想不到的机制，该机制与mTOR信号平行，选择性地关闭胆固醇/脂肪酸的生物合成，但同时维持糖酵解。在这项建议中，我们将研究T细胞代谢的两个要素。在目标1中，我们将定义精氨酸饥饿激活的信号通路的层次结构，精氨酸饥饿导致糖酵解的维持，但胆固醇/脂肪酸生物合成的消融。通过比较精氨酸饥饿的T细胞和分裂的T细胞的全基因组表达谱，我们发现精氨酸饥饿响应的主要下游途径是胆固醇/脂肪酸生物合成的关闭，这是通过外源精氨酸替代完全可逆的。我们将使用遗传、生化和化学方法来探索mTOR和AMPK及其相关途径如何将有关精氨酸水平的信息转化为下游代谢决策点。在目标2中，我们将确定包括mTOR和AMPK在内的关键代谢检查点之间的相互作用，从而导致辅助T细胞表型可塑性。我们发现，精氨酸饥饿的T细胞改变了它们的命运，采用了Tregs和Th17T细胞的特征。我们将使用创新的遗传方法来追踪T细胞的遗传命运，重点是标记有Foxp3-GFP的Treg样细胞。我们将基于Foxp3的表达来分离精氨酸饥饿培养中不同的T细胞群体，并使用这些细胞来了解T细胞如何利用代谢检查点来决定其表型。 公共卫生相关性：过度的T细胞反应是许多自身免疫综合征和与免疫反应失调相关的疾病的标志和驱动因素。我们的研究重点是通过感知微环境中氨基酸的耗竭来控制T细胞的增殖和表型。我们发现，氨基酸感应导致代谢途径驱动的T细胞命运的变化。因此，我们的研究对理解和治疗自身免疫综合征具有广泛的意义。

DESCRIPTION (provided by applicant): Despite the resurgence in interest in basic metabolic control, a large gap in knowledge concerns cell and context specific alteration and adaptation in each key pathway including the mTOR and AMPK pathways. In this proposal we will investigate a new pathway that links amino acid sensing with downstream alterations in T cell metabolism that have profound effects on T cell phenotype and function. The availability of essential amino acids has emerged as a central metabolic mechanism that regulates both T cell growth and the eventual T cell differentiation state. Antigen-presenting cells (APCs) regulate amino acid bioavailability to T cells via expression of inducible enzymes that degrade essential amino acids such as arginine and tryptophan, and have been hypothesized to restrict T cell activity and proliferation in immune microenvironments. The metabolic consequence to the T cells exposed to an amino acid-depleted microenvironment, and the downstream effects on T cell differentiation are at present poorly understood. We have discovered that arginine depletion has a central role in T cell metabolism that is directly relevant to in vivo diseases dominated by Th2 responses. To dissect the interplay between amino acid-depleting antigen presenting cells (APCs) and T cells we built a system where the APC, T cell and culture conditions can be manipulated by genetics and drugs that affect key metabolic checkpoints. Our approach has allowed us to evaluate metabolism in primary T cells undergoing APC-mediated arginine restriction, and can be used generally to investigate the consequences to both the T cell and APC for any amino acid. Our preliminary data suggests that arginine sensing by T cells involves an unanticipated mechanism that runs parallel to mTOR signaling to selectively shut down cholesterol/fatty acid biosynthesis but simultaneously maintain glycolysis. In this proposal we will investigate two elements of T cell metabolism. In Aim 1 we will define the hierarchy of signaling pathways activated by arginine starvation that leads to maintenance of glycolysis but ablation of cholesterol/fatty acid biosynthesis. Using genome-wide expression profiling of arginine-starved T cells compared to their dividing counterparts, we have discovered that the major downstream pathway responsive to arginine starvation is a shutdown of cholesterol/fatty acid biosynthesis that is completely reversible by exogenous arginine replacement. We will use genetic, biochemical and chemical methods to explore how the mTOR and AMPK and related pathways convert information about arginine levels into downstream metabolic decision points. In Aim 2 we will determine the interplay between key metabolic checkpoints including mTOR and AMPK that leads to helper T cell phenotypic plasticity. We have discovered that arginine-starved T cells change their fate to adopt characteristics of both Tregs and Th17 T cells. We will use innovative genetic approaches to track the genetic fate of T cells focusing on Treg-like cells marked with Foxp3-gfp. We will separate distinct T cell populations within arginine starved cultures based on Foxp3 expression and use these cells to understand how T cells use metabolic check points to make decisions about their phenotype. PUBLIC HEALTH RELEVANCE: Excessive T cell responses are a hallmark and driver of numerous autoimmune syndromes and diseases associated with disregulated immune responses. Our study focuses of a pathway that controls T cell proliferation and phenotype via sensing of amino acid depletion in microenvironments. We have found that amino acid sensing leads to metabolic pathway-driven changes in T cell fate. As such, our studies have wide implications for understanding and treating autoimmune syndromes.