Manipulating & imaging nutrient micro-milieux as B cells effect humoral immunity

项目摘要 产生抗体，提炼抗体的性质，创造持久的体液记忆是适应性的关键部分 豁免权。疫苗抵御微生物的能力利用了这些过程的每一个方面，但即使是 对于一些批准的和有用的疫苗来说，效果需要更好。因此，解密密钥是至关重要的 影响抗体(Ab)质量的细胞和分子过程。主要努力针对的是 鉴定细胞内感受器、中间代谢介体和代谢物的适当方式 改变免疫细胞的分化或功能。人们很早就知道营养不良会破坏免疫力 对感染的防御，一系列研究表明，蛋白质缺乏可能会降低有效抗体 回应。营养物质的供应本质上与细胞内的传感器有关，如mTORC1和编程 免疫细胞中的细胞代谢。例如，分离蛋白缺乏的实验模型有 记录的静脉氨基酸浓度下降(A.A.)并降低新鲜血液中mTORC1的活性 从营养不良的啮齿动物身上分离出器官。我们对B细胞中mTORC1的研究发现抗体反应是 被B细胞限制性的Raptor单倍体功能不全改变，部分活动减少的程度与 据报道，在蛋白质剥夺的背景下。此外，我们和其他人同时发现了 免疫后白髓和淋巴滤泡呈斑点状缺氧，生发中心形成。 初步的体外和体内实验证明：(A)谷氨酰胺在生理浓度下 非炎性间质，限制了完全有效地转换为IgG1和浆细胞分化，以及(B) 谷氨酰胺分解(谷氨酰胺转化为谷氨酸，然后是α-酮戊二酸)对这些可能是有限的 流程。这些发现是这一应用的总体模型的前提：营养物质可能是 在卵泡中存在的浓度增加或进一步减少会改变抗体的性质 当它利用淋巴细胞的增殖和功能时，它会产生反应。因此，在目标1中，我们将测试对Ab的影响 减少AA的反应。供应成熟的B细胞或由其利用。目标2将确定以下方面的后果 限制谷氨酰胺供应或谷氨酰胺分解的B细胞的代谢和表观遗传编程， 单独或葡萄糖氧化能力降低。该模型的一个含义是增加了循环AA。-或 即使只是谷氨酰胺-也可以提高免疫效果。在目标3中，我们将使用一种新发现的内分泌 测试高氨基酸血症是否会增加间质谷氨酰胺并产生更强的抗体反应或 体液记忆。作为实验的一个新方面，我们将利用成像技术的最新发展 质谱仪(IMS)用于评估选定淋巴微环境中的谷氨酰胺和选定代谢物 实验动物。拟议研究的预期结果和影响是：(I)我们将提供 IMS应用于生物学问题的突破性技术进展，(Ii)阐明一个长期存在的问题 在营养和免疫的关系方面，以及(3)确定一种增强抗体反应的新方法。

Project Summary Generating antibodies, refining their qualities, and creating durable humoral memory are crucial parts of adaptive immunity. The capacity of vaccines to protect against microbes draws on each facet of these processes, but even for some approved and useful vaccines the efficacy is needs to be better. Accordingly, it is vital to decipher key cellular and molecular processes that affect antibody (Ab) qualities. Major efforts are directed toward the identification of ways in which intracellular sensors, mediators of intermediary metabolism, and metabolites proper alter immune cell differentiation or function. It has long been known that malnutrition undermines immune defenses against infection, and a body of work has suggested that protein deficiency may decrease effective Ab responses. Nutrient supply is intrinsically linked to intracellular sensors such as mTORC1 and programming of cellular metabolism in immune cells. For instance, experimental models of isolated protein deficiency have documented decreases in venous concentrations of amino acids (a.a.) and lower mTORC1 activity in freshly isolated organs from the malnourished rodents. Our work on mTORC1 in B cells found antibody responses to be altered by B cell-restricted haplo-insufficiency of Raptor, with partially reduced activity similar in magnitude to that reported in the setting of protein deprivation. Moreover, we – and others in parallel – uncovered evidence of variegated hypoxia in the white pulp and lymphoid follicles after immunization and formation of germinal centers. Preliminary in vitro and in vivo experiments provide evidence that (a) glutamine, at the physiological concentration of non-inflamed interstitia, is limiting for fully efficient switching to IgG1 and for plasma cell differentiation, and (b) glutaminolysis (the conversion of glutamine to glutamate, and then α-ketoglutarate) can be limiting for these processes. These findings are the premise for the overarching model of this application: that nutrients may be present in follicles at concentrations where either increases or further decreases alter the nature of the antibody response as it draws on lymphocyte proliferation and function. Accordingly, in Aim 1 we will test the impact on Ab responses of reducing a.a. supply to or utilization by mature B cells. Aim 2 will identify consequences for metabolic and epigenetic programming of the B cells in which glutamine supply or glutaminolysis are restricted, alone or with reduced glucose oxidation capacity. An implication of the model is that increased circulating a.a. – or even just glutamine – could enhance outcomes of immunization. In Aim 3, we will use a newly identified endocrine approach to test if hyperaminoacidemia increases interstitial glutamine and yields greater Ab responses or humoral memory. As a novel facet of the experiments, we will leverage a state-of the-art development in imaging mass spectrometry (IMS) to assess glutamine and selected metabolites in selected lymphoid micro-environments of experimental animals. The expected outcome & impact of the proposed studies are that we will (i) provide a ground-breaking technical advance in application of IMS to biological problems, (ii) elucidate a long-standing issue at the nexus of nutrition and immunity, and (iii) identify a novel means of boosting antibody responses.