Rotation 1: CD8+ Tregs: the key to poor vaccine responses in the aged?

t细胞是一种高度迁移的细胞，在淋巴器官和血液之间以一种基本静止的状态不断循环。T细胞通过其T细胞受体（TCR）激活后，T细胞经历深刻的表型转换，进入高功能的激活状态。这些活化的T细胞的迁移能力进一步扩大，进入非淋巴组织的能力增强，包括内部（如肝脏）和屏障（如肠道）组织。在这些高度多样化的微环境中，T细胞需要在高能量状态下保持高效的功能。事实上，免疫激活是人体所能进行的最耗费能量的活动之一。已知T细胞的激活会改变它们的代谢特征和依赖性；代谢谱/依赖性的进一步变化很可能会发生在非淋巴组织中，然而由于在组织环境中研究T细胞的技术限制，这方面的研究很少。该项目汇集了组织免疫学（Liston实验室）和代谢学（Trefely实验室）的专业知识，研究组织T细胞的代谢需求。我们将使用Liston实验室最近开发的FlowCode系统，用于大规模并行流式细胞术的体内CrispR筛选。该系统允许在体内测试小的CrispR文库（几百个基因），使用流式细胞术表型分析，提供高度的灵敏度，独特的功能读出，并使工作能够在小的组织常驻人群中进行。以代谢为重点的CrispR文库将被克隆到FlowCode逆转录病毒文库中，并被转导到成熟的T细胞中。体内转移提供了一组蛋白质表位条形码T细胞，每个细胞都具有独特的代谢基因敲除。我将测试代谢缺陷对循环淋巴细胞室内不同T细胞亚型（CD4 Tconv、CD8、Treg和naïve/活化/记忆亚群）的相对影响，以确定细胞类型特异性需求。我还将使用基于组织的提取和条形码来确定哪些代谢基因改变了组织驻留，在不同的组织范围内。最后，我将用饮食（高脂肪饮食）和传染病（流感）挑战小鼠，以确定这些组织代谢环境的变化是否会改变代谢途径的分子依赖性。在细胞类型/组织类型/代谢背景矩阵中确定的遗传控制网络将在代谢物水平上使用基于先进的液相色谱-质谱（LC-MS）的代谢组学分析方法进行解剖。这些发现将揭示组织T细胞的基因代谢规则，并为代谢困难组织中有效的T细胞治疗的分子需求提供信息。

BBSRC strategic theme: Bioscience for an integrated understanding of healthT cells are highly migratory cells that continually circulate between lymphoid organs and the blood in a largely quiescent state. Upon activation of T cells through their T cell receptor (TCR), T cells undergo a profound phenotype switch, entering an activated state of high functionality. These activated T cells have a further expansion of their migratory capacity, with elevated ability to enter non-lymphoid tissues, including both internal (e.g. liver) and barrier (e.g. gut) tissues. Across these highly diverse microenvironments, T cells need to maintain highly efficient function, at a high energetic state. Indeed, immune activation is among the most energetically expensive activity the body is capable of. The activation of T cells is known to change their metabolic profiles and reliance; it is likely that further changes in metabolic profiles/reliance will occur in non-lymphoid tissues, however this is poorly studied due to the technical limitations of studying T cells within the tissue environment.This project brings together expertise in tissue immunology (Liston lab) and metabolism (Trefely lab) to study the metabolic requirements of tissue T cells. We will use the FlowCode system, recently developed by the Liston lab for massively parallel flow cytometry-based CrispR screens in vivo. This system allows testing of small CrispR libraries (several hundred genes) in vivo, using flow cytometry phenotypic assays, providing a high degree of sensitivity, unique functional read-outs, and enabling the work to be performed on the small tissue-resident population. A metabolic-focused CrispR library will be cloned into the FlowCode retrovirus library and transduced into mature T cells. Transfer in vivo provides for a protein epitope-barcoded set of T cells, each with a unique metabolic gene knockout. I will test the relative impact of the metabolic deficiencies on different T cell subtypes (CD4 Tconv, CD8, Treg; and naïve/activated/memory subsets) within the circulating lymphoid compartment, to identify cell type-specific requirements. I will also use tissue-based extraction and barcoding to determine which metabolic genes alter tissue residency, across a diverse range of tissues. Finally, I will challenge mice with dietary (high fat diet) and infectious (flu) challenges, to determine whether these changes to the metabolic context of the tissues alters the molecular reliance of metabolic pathways. Identified genetic control networks across the cell type / tissue type / metabolic context matrix will then be dissected at the metabolite level using cutting-edge liquid chromatography-mass spectrometry (LC-MS) based methods for metabolomic analysis.These findings will unravel the gene-metabolism rules of tissue T cells, and inform as to the molecular requirements of effective T cell-based therapeutics in metabolically-challenged tissues.