High Resolution Studies of Immune Cell Signaling

免疫细胞信号转导的高分辨率研究

• 批准号: 8745472
• 负责人: Rajat Varma
• 金额: $ 71.61万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 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/8745472
• 关键词: Address; Affect; Affinity; Agonist; Algorithms; Autoimmunity; Binding; Biological Assay; Calcium; Cell Line; Cell Surface Receptors; Cell surface; Cells; Collaborations; Complex; Computer Simulation; Cytokine Receptors; Cytokine Signaling; Data; Discrimination; Dose; Event; Exhibits; Family; Family member; Fluorescence Microscopy; Goals; Image; Image Analysis; Immediate-Early Genes; Immune; Immune response; Interleukin 2 Receptor Gamma; Interleukin-15; Interleukin-2; Interleukin-4; Interleukin-7; Interleukin-9; Kinetics; Laboratories; Lead; Life; Ligand Binding; Ligands; Major Histocompatibility Complex; Maps; Mediating; Mitogen-Activated Protein Kinases; Modeling; Molecular; Mus; Nature; PTPRC gene; Pathway interactions; Peptides; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Postdoctoral Fellow; Proteins; Receptor Activation; Receptor Signaling; Regulation; Resolution; Role; STAT protein; Scanning; Signal Pathway; Signal Transduction; Systems Biology; T-Cell Receptor; T-Lymphocyte; TLR2 gene; TLR4 gene; Testing; Time; Toll-like receptors; Translating; Work; base; computerized tools; cytokine; density; design; insight; macrophage; novel; receptor; receptor binding; receptor expression; release of sequestered calcium ion into cytoplasm; response

In 2013, we made progress in the following projects: Pre-existing nature of T Cell Receptor (TCR) microclusters, TCR signaling in response to low potency ligands, role of RhoH in TCR signaling 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 un-stimulated cells, indicating that the mechanisms leading to microcluster formation do not require ligand binding. These preexisting microclusters are stabilized by low-potency ligand engagement, including positively selecting ligands, and the density of stabilized TCR microclusters increases with ligand potency without a change in the number of TCRs in a microcluster. In characterizing their composition, we found that such microclusters exclude the phosphatase CD45, and contain the signaling adapters LAT and Grb2. Signaling through these microclusters in response to high concentrations of low-potency ligands results in low-grade signals. The utilization of preexisting microclusters by pMHC for TCR triggering is a novel mechanism that may explain the sensitivity and fidelity exhibited by the TCR. This work has been submitted and is under review. The current paradigm of TCR ligand discrimination is based on the kinetic proofreading model, which assumes a singular and sequential signaling cascade downstream of the TCR. To test this assumption, we identified doses of the agonist and a low potency TCR ligand that induced the same amount of the immediate early gene, Fos. Analysis of TCR proximal signals revealed that calcium fluxes in response to these doses of the agonist and low potency ligand were also identical; however, the high dose of the low potency ligand led to higher MAP kinase responses. ERK activation in response to the agonist ligand was calcium independent while it was calcium dependent in response to the low potency ligand. Total internal reflection fluorescence microscopy demonstrated lack of significant Zap70 kinase recruitment to TCR microclusters engaging low potency ligand, leading us to propose the lack of Zap70 involvement. Reducing Zap70 expression in T cells specifically affected calcium fluxes in response to the agonist. The existence of a Zap70 independent pathway downstream of TCR stimulation by low potency ligand suggests that ligand discrimination is accompanied by structural changes in the TCR complex that causes induction of distinct signaling pathways downstream of the two types of ligands. We continued our studies exploring the role of RhoH in TCR signaling. We find that RhoH localizes to TCR microclusters. RhoH is a protein that has been shown to interact with both Zap70 and the kinase Lck. The amount of RhoH present in TCR microclusters is correlated with the amount of Zap70 in TCR microclusters, allowing us to hypothesize that perhaps RhoH is stabilized in microclusters by Zap70 binding. Anke Knauf, a postdoctoral fellow working on this project developed a new algorithm to detect TCR microclusters, reducing the amount of time required to analyze the images. 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 signals. Using quantitative flow cytometric analysis, we quantified cell surface receptor expression, STAT phosphorylation dose-responses and kinetics, and crosstalk among cytokine-induced signals. We found that, even though gamma chain would be limiting if each private chain were pre-associated with it, signal-induced recruitment of gamma chain to ligated private chains would not lead to a shortage of gamma chain for physiological cytokine concentrations. We observed strikingly lower EC50 values for STAT phosphorylation than would be expected from the receptor binding curves. The kinetics of signaling through these receptors was relatively slow, taking several minutes to reach saturation. IL-7 pre-exposure led to cross inhibition of IL-4 and IL-21 responses at receptor occupancies where gamma chain would not be limiting. An assay was designed to determine the fraction of each STAT protein phosphorylated in response to IL-7, IL-4 and IL-21. This assay revealed that IL-7 exerted the maximum negative regulation on STAT phosphorylation that correlated with its ability to cause cross-inhibition. Additionally, over expressing the gamma chain relieved the IL-7 mediated cross-inhibition of IL-4 and IL-21 responses. 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. 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 cell surface distribution of TLR2 and TLR4 in mouse macrophage cell lines.

2013年，我们在以下几个项目上取得了进展：T细胞受体(TCR)微簇的预先存在性质、低效配体对TCR信号的响应、RhoH在TCR信号中的作用以及常见的细胞因子受体伽马链家族之间的串扰。 TCR微簇中的TCR信号事件有助于区分自体和外源多肽负载的主要组织相容性复合体(PMHC)。我们发现在未受刺激的细胞中存在一些TCR微簇，这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通过低效配体结合(包括正向选择配体)来稳定，稳定的TCR微簇的密度随着配体效力的增加而增加，而微簇中TCR的数量不变。在鉴定它们的组成时，我们发现这些微簇不包括磷酸酶CD45，而包含信号适配器LAT和Grb2。通过这些微簇对高浓度的低效配体做出反应的信号会导致低级别的信号。PMHC利用预先存在的微团簇来触发TCR是一种新的机制，可以解释TCR表现出的敏感性和保真度。这项工作已经提交，正在审查中。 目前的TCR配基识别范式是基于动力学校对模型，该模型假设TCR下游有一个单一的、顺序的信号级联。为了验证这一假设，我们确定了剂量的激动剂和低效力的TCR配体，它们诱导了等量的即刻早期基因Fos。对TCR近端信号的分析表明，这些剂量的激动剂和低效配体对钙离子的反应也是相同的；然而，高剂量的低效配体导致更高的MAP激酶反应。ERK对激动剂配体的激活不依赖于钙离子，而对低效价配体则依赖于钙离子。全内反射荧光显微镜显示，与低效配体结合的TCR微簇缺乏显著的ZAP70激酶募集，因此我们提出了ZAP70缺乏参与的观点。减少T细胞中ZAP70的表达会影响激动剂对钙离子的反应。低效配体刺激TCR下游存在一条ZAP70独立通路，提示配体识别伴随着TCR复合体的结构变化，从而导致两种配体下游不同的信号通路诱导。 我们继续我们的研究，探索RhoH在TCR信号中的作用。我们发现RhoH定位于TCR微团簇。RhoH是一种已被证明与ZAP70和激酶Lck相互作用的蛋白质。TCR微簇中RhoH的含量与TCR微簇中ZAP70的数量相关，这使得我们能够假设RhoH可能通过ZAP70结合稳定在微簇中。参与该项目的博士后Anke Knauf开发了一种新的算法来检测TCR微星团，减少了分析图像所需的时间。 伽马链家族的细胞因子(IL-2、IL-4、IL-7、IL-9、IL-15和IL-21)通过使用细胞因子特异性的阿尔法链与共同的伽马链结合的受体复合体来传递信号。考虑到伽马链的共同性质，我们想知道当多种细胞因子信号同时需要时，伽马链是否会受到限制。使用定量的流式细胞术分析，我们量化了细胞表面受体的表达，STAT磷酸化的剂量反应和动力学，以及细胞因子诱导的信号之间的串扰。我们发现，尽管如果每个专用链都预先与伽马链相关联，那么伽马链将是有限的，但通过信号诱导将伽马链招募到连接的专用链上并不会导致生理细胞因子浓度的伽马链短缺。我们观察到STAT磷酸化的EC50值明显低于受体结合曲线的预期。通过这些受体传递信号的动力学相对较慢，需要几分钟才能达到饱和。IL-7预先暴露导致在受体占位处对IL-4和IL-21反应的交叉抑制，在该受体中，伽马链不受限制。设计了一种分析方法，以确定每个STAT蛋白对IL-7、IL-4和IL-21的响应而被磷酸化的比例。本实验表明，IL-7对STAT磷酸化的负性调节作用最大，这与其引起交叉抑制的能力有关。此外，过表达的伽马链缓解了IL-7对IL-4和IL-21反应的交叉抑制。 我们正在与Martin Meier-Schellersheim的实验室进行广泛的合作。他的团队正在开发通过常见的细胞因子受体伽马链家族传递信号的计算模型。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，并将它们映射到动力学和剂量响应数据上。这些模型为我们提供了对IL-7介导其对其他伽马链家族成员的交叉抑制作用的机制的有价值的见解。 作为系统生物学实验室内研究Toll样受体(TLR)信号的合作努力的一部分，我们与Iain Fraser的团队合作，已经开始成像TLR2和TLR4在小鼠巨噬细胞系中的细胞表面分布。