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High Resolution Studies of Immune Cell Signaling

免疫细胞信号转导的高分辨率研究

基本信息

批准号：
8946426
负责人：
Rajat Varma
金额：
$ 52.29万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8946426
关键词：
1-Phosphatidylinositol 3-Kinase Address Affinity Agonist Autoimmunity CD28 gene CD80 gene Cell Line Cells Characteristics Chemotaxis Collaborations Complex Computer Simulation Cytokine Receptor Binding Cytokine Receptors Cytokine Signaling Data Dictyostelium Discrimination Dose Endocytosis Event Exclusion Family Family member Feedback Fluorescence Microscopy France Goals Image Immune Immune response Immunology Incubated Interleukin 2 Receptor Gamma Interleukin 7 Receptor Interleukin-15 Interleukin-2 Interleukin-4 Interleukin-7 Interleukin-9 Ions Journals Kinetics Knock-in Mouse Laboratories Life Ligand Binding Ligands Major Histocompatibility Complex Manuscripts Maps Mediating Metals Microscopy Modeling Molecular Mus Mutation Nature Outcome PTPRC gene Peptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Process Publishing Reading Receptor Activation Receptor Signaling Resolution Role STAT5A gene Scanning Signal Pathway Signal Transduction Speed Surface Synapses System Systems Biology T Cell Receptor Signaling Pathway T-Cell Receptor T-Lymphocyte TLR2 gene TLR4 gene Time Toll-like receptors Transgenic Mice Translating Universities Work cellular imaging computerized tools cytokine density insight interleukin-21 receptor molecular imaging prevent receptor release of sequestered calcium ion into cytoplasm response

项目摘要

In 2014, we continued to make progress in the following projects: Pre-existing nature of T Cell Receptor (TCR) microclusters, TCR signaling in response to low potency ligands and cross talk among common gamma chain family of cytokine receptors. TCR signaling events that contribute to discrimination between self and foreign peptide-loaded Major Histocompatibility Complexes (pMHC) occur in TCR microclusters. We found that some TCR microclusters are present in unstimulated murine T cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These preexisting microclusters increase in absolute number following engagement by low-potency ligands. This increase is accompanied by an increase in cell spreading, with the result that the density of TCR microclusters on the surface of the T cell is not a strong function of ligand potency. In characterizing their composition, we observed a constant number of TCRs in a microcluster, constitutive exclusion of the phosphatase CD45, and preassociation with the signaling adapters LAT and Grb2. The existence of TCR microclusters prior to ligand binding in a state that is conducive for the initiation of downstream signaling could in part explain the rapid kinetics with which TCR signal transduction occurs. This work has been published in the Journal of Immunology. We asked the question whether TCR signaling in response to low-potency ligands is qualitatively different from agonist stimulation. To address this question we used a Fos-GFP transgenic mouse as a read out of TCR signaling and identified doses of agonist and low potency ligands that gave rise to similar amount of Fos induction and analyzed TCR proximal signaling events, such as calcium fluxes, Map kinase activation, PLCg1 phosphorylation, LAT phosphorylation, Zap70 phosphorylation and CD3zeta phosphorylation. We did not find evidence for a qualitatively different TCR signaling pathway in response low potency ligand stimulation. These studies led to the observation that there exists a metal ion dependent negative feedback acting on TCR proximal signaling events that is specific to CD28 engagement by CD80. We are following up on these observations and a manuscript describing these results will soon be submitted. Cytokines of the gamma chain family (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) signal through receptor complexes using cytokine-specific alpha chains in combination with the common gamma chain. Given its shared nature, we wondered whether the gamma chain could be limiting when required simultaneously by multiple cytokine bound receptors. Using quantitative flow cytometric analysis, we found that the gamma chain is outnumbered by cytokine specific private chains on the surface of nave T cells by 5:1. We further found that signaling via common gamma chain cytokines is very sensitive requiring between 1 and 10 private chains occupied, suggesting that not many gamma chains are required for signal transduction. The limiting nature of the gamma chain was revealed when it was found that pre-incubating cells with IL-7 prevents efficient signaling via IL-4 and IL-21 receptors. The extent of IL-7 mediated cross-inhibition of IL-4 and IL-21 responses correlated with the ratio of IL-7R to the gamm chain. We ruled out specific endocytosis of gamma chain and induction of STAT specific phosphatases as mechanisms responsible for cross-inhibition. T cells derived from mice carrying a knock-in mutation in IL7-receptor (Y449F) do not cause STAT5 phosphorylation or PI3-kinase activation in response to IL-7, yet, in these T cells, IL-7 pre-incubation inhibited IL-4 responses. These results suggest that IL-7 induced redistribution of gamma chains limits access to it by other gamma chain cytokine receptors. We are in the process of submitting this manuscript. We have an extensive on going collaboration with the lab of Martin Meier-Schellersheim. His group is developing computational models of signaling via the common gamma chain family of cytokine receptors. They have developed new computational tools that allow them to scan multiple parameters in the model and map them on to kinetics and dose response data. These models are providing us with valuable insights in to the mechanism by which IL-7 mediates its cross-inhibitory effect on other gamma chain family members. In another on going collaboration, we are investigating the mechanisms of chemotaxis in dictyostelium. For this purpose, we have developed a high-speed confocal imaging system. We are using this to map receptor distribution and signaling outcome in response to chemo-attractant in real time. We have an on going collaboration with the labs of Drs. Laurent Limozin and Kheya Sengupta at the Mediterranean University at Marseille, France, to study the topography of the immune synapse using reflection interference contrast microscopy. We have studied the effect of ligand mobility on the spreading characteristics of T cells. A manuscript describing these results is under review at the Biophysical journal. As part of a collaborative effort within the Laboratory of Systems Biology to study Toll Like Receptor (TLR) signaling, in collaboration with Iain Fraser's group we have begun to image the cellular response to single and multiple TLR stimulations in multiple cell lines. Imaging the molecular players downstream of TLR2 and TLR4 stimulation is giving valuable insights into the intracellular signaling events.

2014年，我们继续在以下项目中取得进展：T细胞受体（TCR）微簇的预先存在性质，TCR对低效力配体的反应信号传导以及细胞因子受体的常见γ链家族之间的串扰。有助于区分自身和外源肽负载的主要组织相容性复合物（pMHC）的TCR信号传导事件发生在TCR微簇中。我们发现，一些TCR微簇存在于未受刺激的小鼠T细胞中，表明导致微簇形成的机制不需要配体结合。这些预先存在的微簇在被低效力配体接合后绝对数量增加。这种增加伴随着细胞扩散的增加，结果是T细胞表面上TCR微簇的密度不是配体效力的强函数。在表征其组成时，我们观察到恒定数量的TCR在微簇中，组成性排除磷酸酶CD 45，并与信号适配器LAT和Grb 2预关联。TCR微簇在配体结合之前以有利于下游信号传导起始的状态存在可以部分解释TCR信号转导发生的快速动力学。这项工作发表在Journal of Immunology上。我们提出了这样一个问题，即对低效配体的TCR信号传导是否与激动剂刺激有质的不同。为了解决这个问题，我们使用Fos-GFP转基因小鼠作为TCR信号传导的读出，并鉴定引起相似量的Fos诱导的激动剂和低效配体的剂量，并分析TCR近端信号传导事件，例如钙通量、Map激酶活化、PLCg 1磷酸化、LAT磷酸化、Zap 70磷酸化和CD 3 zeta磷酸化。我们没有发现在低效力配体刺激反应中存在定性不同的TCR信号传导途径的证据。这些研究导致观察到存在作用于TCR近端信号传导事件的金属离子依赖性负反馈，其特异于CD 80与CD 28的接合。我们正在对这些观察结果采取后续行动，不久将提交一份描述这些结果的手稿。 γ链家族的细胞因子（IL-2、IL-4、IL-7、IL-9、IL-15和IL-21）通过受体复合物使用精氨酸特异性α链与共同γ链的组合发出信号。鉴于其共有的性质，我们想知道当多种细胞因子结合受体同时需要时，γ链是否会受到限制。使用定量流式细胞术分析，我们发现，在幼稚T细胞的表面上，γ链的数量超过细胞因子特异性私有链的数量5：1。我们进一步发现，通过常见的γ链细胞因子的信号传导非常敏感，需要占据1至10条私有链，这表明信号转导不需要很多γ链。当发现用IL-7预孵育细胞阻止通过IL-4和IL-21受体的有效信号传导时，揭示了γ链的限制性性质。IL-7介导的IL-4和IL-21应答的交叉抑制程度与IL-7 R与γ链的比率相关。我们排除了γ链的特异性内吞作用和STAT特异性磷酸酶的诱导作为交叉抑制的机制。来源于携带IL-7-受体敲入突变（Y 449 F）的小鼠的T细胞不引起STAT 5磷酸化或PI 3-激酶活化以响应IL-7，然而，在这些T细胞中，IL-7预孵育抑制IL-4应答。这些结果表明，IL-7诱导的γ链重新分布限制了其他γ链细胞因子受体对其的接近。我们正在提交这份手稿。我们与Martin Meier-Schellersheim的实验室进行了广泛的合作。他的团队正在开发通过细胞因子受体的常见γ链家族进行信号传导的计算模型。他们开发了新的计算工具，使他们能够扫描模型中的多个参数，并将其映射到动力学和剂量反应数据上。这些模型为我们提供了有价值的见解，IL-7介导其对其他γ链家族成员的交叉抑制作用的机制。在另一个正在进行的合作中，我们正在研究在dictyosteopathy的趋化机制。为此，我们研制了一种高速共焦成像系统。我们正利用这一点来绘制受体分布和信号传导结果，以响应真实的时间的化学引诱剂。我们与法国马赛的地中海大学的Laurent Limozin和Kheya Sengupta博士的实验室正在进行合作，使用反射干涉对比显微镜研究免疫突触的拓扑结构。我们研究了配体迁移率对T细胞铺展特性的影响。一份描述这些结果的手稿正在《生物物理学》杂志上进行审查。作为系统生物学实验室研究Toll样受体（TLR）信号传导的合作努力的一部分，我们与Iain Fraser的小组合作，开始在多个细胞系中对单个和多个TLR刺激的细胞反应进行成像。对TLR 2和TLR 4刺激下游的分子参与者进行成像，为细胞内信号传导事件提供了有价值的见解。