Deciphering the metabolic signature of CD4+ T helper cells that express dual master transcription factors.

重要性：免疫疗法近年来在癌症和自身免疫领域都取得了成功。特别是，使用T细胞的临床试验已经彻底改变了肿瘤（Th1细胞）和自身免疫性青少年糖尿病（Treg细胞）的治疗。这些试验的主要缺陷仍然是体内T细胞的持久性。最近的报道将缺乏持久性归因于这些试验中使用的T细胞的生物能量学。因此，迫切需要了解炎症与调节性T细胞的生物能量学特征，以改善临床护理。背景：CD4+ T细胞是适应性免疫的重要介质，根据其转录因子谱可分为不同的亚型。表达Tbet转录因子的CD4+ T辅助细胞称为Th1细胞、Th2 （GATA3+）、Th17 （RORgt+）或Tregs （FoxP3+）细胞。每个亚群都有独特的功能，由各自的转录因子决定，Tbet驱动IFNg细胞因子的产生，从而促进炎症反应，FoxP3在调节细胞中灌输抑制功能。T细胞受体（TCR）的激活以及共刺激信号对于辅助性T细胞分化也是至关重要的，这导致代谢重塑和获得有氧糖酵解。具有不同效应功能的CD4+ T辅助亚群的分化伴随着代谢的额外变化，以满足其生物能量需求。因此，与倾向于脂质氧化的treg细胞相比，Th1细胞具有独特的代谢特征、线粒体能量学和糖酵解谱（强糖酵解）。我们和其他人已经证明Tbet+Th1细胞也可以表达FoxP3并获得调节性表型，但保持Tbet的表达。在Th1细胞中通过PD-1/PDL-1（程序性死亡1通路）提供共抑制信号，稳定FoxP3表达和Tbet表达。这些表达双重对立转录因子的细胞的代谢特征是未知的。在本提案中，我们旨在阐明Tbet+FoxP3+细胞的代谢特征，并确定代谢如何在小鼠炎症性疾病模型中与其功能相关。方法：一种独特的资源可供我们使用，它是一种命运定位报告鼠菌株，它驱动Tbet和FoxP3，因此可以根据转录因子表达分离Tbet+FoxP3+ T细胞。我们的目标是从WT小鼠中分离出这些细胞，使用Seahorse XF Analyzer来分析它们的代谢谱，以评估细胞的实时细胞呼吸和对代谢调节剂的反应。该分析同时重复测量了分别代表线粒体氧化磷酸化（OXPHOS）和糖酵解的耗氧率（OCR）和细胞外酸化率（ECAR）（分别通过氧和质子（pH）浓度的变化来测量）。此外，Tbet+的CD4+ T辅助细胞在体外PDL-1存在或不存在的情况下都会扩增为Tbet+FoxP3+细胞。如前所述，我们将比较这些扩增细胞与第0天WT Tbet+FoxP3+分选细胞的代谢谱和体外效应表型。将测试Tbet+FoxP3+细胞的体内功能，以确定功能是否与该亚群中的特定代谢特征相关。培训机会：参与本项目的学生将接受生物化学和免疫学跨学科领域的培训，包括小鼠疾病模型和命运映射报告，体外细胞免疫和生化分析以及多参数流式细胞术的使用。这个博士学位的跨学科方面将大大提高学生未来的职业前景。除了参加每周的专业课题研究会议、期刊俱乐部和研究所研究项目外，学生还将参与研究生院的技能发展项目。

Importance:Immunotherapy has shown success in recent years in the field of both cancer and autoimmunity. In particular, clinical trials using T cells have revolutionised treatment of tumour (Th1 cells) and autoimmune juvenile diabetes (Treg cells). The major pitfall of these trials remains in the area of in-vivo T cell persistence. Recent reports have attributed the lack of persistence to the bioenergetics of T cells utilised in these trials. Hence there is an immediate need for understanding the bioenergetics profile of inflammatory versus regulatory T cells for improving clinical care.Background: CD4+ T cells are important mediators of adaptive immunity and are classified into various subtypes based on their transcription factor profile. CD4+ T helper cells that express Tbet transcription factor are called Th1 cells, Th2 (GATA3+), Th17 (RORgt+) or Tregs (FoxP3+) cells. Each subset has a unique function as determined by the respective transcription factor with Tbet driving the production of IFNg cytokine and hence promoting inflammatory responses and FoxP3 instills suppressive function in Tregulatory cells. Activation of the T cell receptor (TCR) along with co-stimulatory signalling is also critical for T helper cell differentiation, which results in metabolic remodelling and acquiring aerobic glycolysis. Differentiation of CD4+ T helper subsets with diverse effector functions is accompanied by additional changes in metabolism to meet their bioenergetic demands. Hence, Th1 cells have a unique metabolic signature, mitochondrial energetics and glycolytic profile (strongly glycolytic) as compared to Tregs that prefer oxidation of lipids. We and others have shown that Tbet+Th1 cells can also express FoxP3 and acquire a regulatory phenotype but maintain Tbet expression. Providing co-inhibitory signals through PD-1/PDL-1 (programmed death 1pathway) in Th1 cells stabilizes FoxP3 expression and Tbet expression. The metabolic signature of these cells that express dual opposing transcription factors is unknown.In this proposal we aim to elucidate the metabolic signature of Tbet+FoxP3+ cells and identify how metabolism co-relates to their function in mouse models of inflammatory diseases.Methodology: A unique resource that is available to us is a fate mapping reporter murine strain that drives both Tbet and FoxP3 and hence allow isolating Tbet+FoxP3+ T cells based on their transcription factor expression. We aim to isolate these cells from WT mice to analyze their metabolic profile by using the Seahorse XF Analyzer to evaluate real time cellular respiration of the cells and response to metabolic modulators. This analysis takes simultaneous repeated measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) that are representative of mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis respectively (measured by changes in oxygen and proton (pH) concentration respectively). In addition, CD4+ T helper cells that are Tbet+, will be expanded in vitro in the presence/absence of PDL-1 to become Tbet+FoxP3+ cells. As before we will compare the metabolic profile and in vitro effector phenotype of these expanded cells to day 0 WT Tbet+FoxP3+ sorted cells. The in-vivo function of Tbet+FoxP3+ cells will be tested to determine if function co-relates to specific metabolic signature in this subset.Training opportunities: The students involved in this project will be trained in cross-disciplinary fields of biochemistry and immunology which include murine models of disease and fate mapping reporters, in vitro cellular immunological and biochemical assays and use of multi-parameter flow cytometry. The strong inter-disciplinary aspect of this PhD will significantly enhance the student's future career prospects. As well as taking part in the weekly specialist subject research meetings, journal clubs and institute research programs, the student will engage with the Graduate School's Skills Dev Prog.