Amino acid sensing and phenotypic adaptation in T cells

描述（由申请人提供）：尽管人们对基本代谢控制的兴趣重新兴起，但在包括 mTOR 和 AMPK 途径在内的每个关键途径中的细胞和环境特异性改变和适应方面，知识存在很大差距。在本提案中，我们将研究一种将氨基酸传感与 T 细胞代谢下游变化联系起来的新途径，这些变化对 T 细胞表型和功能具有深远的影响。必需氨基酸的可用性已成为调节 T 细胞生长和最终 T 细胞分化状态的中心代谢机制。抗原呈递细胞 (APC) 通过表达可降解必需氨基酸（如精氨酸和色氨酸）的诱导酶来调节 T 细胞的氨基酸生物利用度，并被假设可限制免疫微环境中的 T 细胞活性和增殖。目前，对暴露于氨基酸耗尽的微环境中的 T 细胞的代谢后果以及对 T 细胞分化的下游影响知之甚少。我们发现精氨酸耗竭在 T 细胞代谢中起着核心作用，与 Th2 反应主导的体内疾病直接相关。为了剖析氨基酸消耗抗原呈递细胞 (APC) 和 T 细胞之间的相互作用，我们建立了一个系统，其中 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经历 APC 介导的精氨酸限制的原代 T 细胞的代谢，并且通常可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 信号传导平行，选择性地关闭胆固醇/脂肪酸生物合成，但同时维持糖酵解。在本提案中，我们将研究 T 细胞代谢的两个要素。在目标 1 中，我们将定义由精氨酸饥饿激活的信号通路的层次结构，该通路导致糖酵解的维持，但胆固醇/脂肪酸生物合成的消除。通过对精氨酸饥饿 T 细胞与分裂细胞的全基因组表达谱进行比较，我们发现，对精氨酸饥饿做出反应的主要下游途径是胆固醇/脂肪酸生物合成的关闭，而这种关闭可以通过外源精氨酸替代来完全逆转。我们将利用遗传、生化和化学方法来探索mTOR和AMPK及相关通路如何将精氨酸水平信息转化为下游代谢决策点。在目标 2 中，我们将确定导致辅助 T 细胞表型可塑性的关键代谢检查点（包括 mTOR 和 AMPK）之间的相互作用。我们发现精氨酸饥饿的 T 细胞会改变其命运，采用 Tregs 和 Th17 T 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，重点关注标记有 Foxp3-gfp 的 Treg 样细胞。我们将根据 Foxp3 表达在精氨酸饥饿培养物中分离不同的 T 细胞群，并使用这些细胞来了解 T 细胞如何使用代谢检查点来决定其表型。 公共健康相关性：过度的 T 细胞反应是许多与免疫反应失调相关的自身免疫综合征和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消耗来控制 T 细胞增殖和表型的途径。我们发现氨基酸传感会导致代谢途径驱动的 T 细胞命运变化。因此，我们的研究对于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.