Amino acid sensing and phenotypic adaptation in T cells

描述（由申请人提供）：尽管对基础代谢控制的兴趣重新抬头，但在每个关键途径（包括mTOR和AMPK途径）中的细胞和环境特异性改变和适应方面存在很大的知识缺口。在这项提案中，我们将研究一种新的途径，将氨基酸传感与T细胞代谢的下游改变联系起来，对T细胞的表型和功能产生深远的影响。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，并且已经被假设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微环境的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。我们已经发现精氨酸消耗在T细胞代谢中具有中心作用，其与Th 2应答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在原代T细胞进行APC介导的精氨酸限制的代谢，并可以通常用于调查的后果，T细胞和APC的任何氨基酸。我们的初步数据表明，T细胞的精氨酸传感涉及一种与mTOR信号传导平行的意料之外的机制，以选择性地关闭胆固醇/脂肪酸生物合成，但同时维持糖酵解。在这个提议中，我们将研究T细胞代谢的两个要素。在目标1中，我们将定义由精氨酸饥饿激活的信号通路的层次结构，该信号通路导致糖酵解的维持但胆固醇/脂肪酸生物合成的消融。使用精氨酸饥饿的T细胞的全基因组表达谱与它们的分裂对应物相比，我们发现响应于精氨酸饥饿的主要下游途径是胆固醇/脂肪酸生物合成的关闭，其通过外源性精氨酸替代是完全可逆的。我们将使用遗传学，生物化学和化学方法来探索mTOR和AMPK以及相关途径如何将精氨酸水平的信息转化为下游代谢决策点。在目标2中，我们将确定关键代谢检查点之间的相互作用，包括mTOR和AMPK，导致辅助T细胞表型可塑性。我们已经发现，甘氨酸饥饿的T细胞改变它们的命运，以采用T细胞和Th 17 T细胞的特征。我们将使用创新的遗传方法来跟踪T细胞的遗传命运，重点是标记有Foxp 3-gfp的Treg样细胞。我们将根据Foxp 3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。 公共卫生相关性：过度的T细胞应答是与失调的免疫应答相关的许多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检测微环境中的氨基酸消耗来控制T细胞增殖和表型的途径。我们已经发现，氨基酸传感导致T细胞命运的代谢途径驱动的变化。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.