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

项目摘要 产生抗体，改进它们的质量，创造持久的体液记忆是适应性免疫的关键部分。 免疫力疫苗抵御微生物的能力利用了这些过程的各个方面，但即使 对于一些批准的和有用的疫苗，需要更好的效力。因此，破译密钥至关重要 影响抗体（Ab）质量的细胞和分子过程。主要的努力是针对 确定细胞内传感器、中间代谢介质和代谢物本身 改变免疫细胞分化或功能。人们早就知道营养不良会破坏免疫系统， 抗感染的防御，以及大量的工作表明，蛋白质缺乏可能会降低有效的抗体， 应答营养供应与细胞内传感器（如mTORC 1）和编程有内在联系。 免疫细胞的细胞代谢。例如，孤立性蛋白质缺乏的实验模型 记录的氨基酸（a.a.）和较低的mTORC 1活性， 从营养不良的啮齿动物身上分离出的器官我们对B细胞中mTORC 1的研究发现， 被Raptor的B细胞限制性单倍体不足改变，部分降低的活性与 在蛋白质缺乏的情况下报告。此外，我们--以及其他类似的人--发现了 免疫后白色髓和淋巴滤泡中的杂色缺氧和生发中心的形成。 初步的体外和体内实验提供了证据，表明（a）生理浓度的谷氨酰胺 对于完全有效地转换为IgG 1和浆细胞分化是限制性的，和（B） 谷氨酰胺分解（谷氨酰胺转化为谷氨酸，然后转化为α-酮戊二酸）可能限制这些 流程.这些发现是这个应用程序的总体模型的前提：营养素可能是 存在于卵泡中，其浓度增加或进一步降低会改变抗体的性质 反应，因为它利用淋巴细胞增殖和功能。因此，在目标1中，我们将测试对Ab的影响 降低a.a.的反应提供给成熟B细胞或被成熟B细胞利用。目标2将确定以下方面的后果 其中谷氨酰胺供应或谷氨酰胺分解受到限制的B细胞的代谢和表观遗传编程， 单独或与降低的葡萄糖氧化能力一起使用。该模型的一个含义是，增加循环a.a. - 或 即使只是谷氨酰胺-也能提高免疫效果。在目标3中，我们将使用新发现的内分泌 一种检测高氨基酸血症是否会增加间质谷氨酰胺并产生更大Ab应答的方法，或 体液记忆作为实验的一个新的方面，我们将利用最先进的成像技术 质谱法（IMS）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，我们会（i）提供 国际监测系统应用于生物问题的突破性技术进展，（二）阐明一个长期存在的问题 在营养和免疫力的关系，和（iii）确定一种新的方法，提高抗体反应。

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.