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.

在2013年，我们在以下项目中取得了进展：T细胞受体（TCR）微量群体的预先存在性质，TCR信号对低效力配体的响应，RHOH在TCR信号传导中的作用以及Comma gamma细胞因子受体家族中的跨性别脉冲链。 TCR信号传导事件有助于自我和外国肽负载的主要组织相容性复合物（PMHC）发生在TCR微量群体中。我们发现，一些TCR微簇存在于未刺激的细胞中，表明导致微簇形成的机制不需要配体结合。这些先前存在的微簇通过低功能配体的互动稳定，包括积极选择配体，而稳定的TCR微量群体的密度随着配体效力而增加，而微量簇中的TCR数量也会改变。在表征它们的组成时，我们发现这种微量群体排除了磷酸酶CD45，并包含信号适配器LAT和GRB2。通过这些微簇的信号响应高浓度的低功率配体会导致低级信号。 PMHC通过PMHC对TCR触发的先前存在的微簇的利用是一种新型机制，可以解释TCR所表现出的灵敏度和忠诚度。这项工作已提交并正在审查中。 TCR配体歧视的当前范式基于动力学校对模型，该模型假设TCR下游的单数和顺序信号级联。为了测试这一假设，我们确定了激动剂的剂量和低效力TCR配体，这些剂量诱导了相同量的早期基因FOS。 TCR近端信号的分析表明，对这些激动剂和低效力配体剂量的钙通量也相同。但是，高剂量的低效力配体导致更高的MAP激酶反应。响应激动剂配体的ERK激活是独立的，而钙依赖于低效力配体的钙。总内反射荧光显微镜表明缺乏对TCR微量群体吸引低效力配体的显着ZAP70激酶募集，这使我们提出缺乏ZAP70参与。降低T细胞中ZAP70表达的特异性影响了钙通量对激动剂的响应。低效力配体的TCR刺激下游的ZAP70独立途径的存在表明配体歧视伴随着TCR复合物中的结构变化，从而导致两种配体的下游诱导不同的信号通路。 我们继续研究RHOH在TCR信号传导中的作用。我们发现RHOH本地化为TCR微簇。 RHOH是一种已显示与ZAP70和激酶LCK相互作用的蛋白质。 TCR微簇中存在的RHOH量与TCR微量群体中的ZAP70量相关，从而使我们可以假设RHOH可能通过ZAP70结合在微量群体中稳定下。该项目的博士后研究员Anke Knauf开发了一种新算法来检测TCR微量群体，减少了分析图像所需的时间。 使用细胞因子特异性α链结合使用Common Gamma链的细胞因子，通过受体复合物通过受体复合物通过受体复合物（IL-2，IL-4，IL-9，IL-15和IL-21）信号的伽马链家族的细胞因子（IL-2，IL-4，IL-9，IL-15和IL-21）的细胞因子。鉴于其共同的性质，我们想知道，当多个细胞因子信号同时需要时，伽马链是否可以限制。使用定量的流式细胞仪分析，我们量化了细胞表面受体表达，统计磷酸化剂量 - 子弹和动力学，以及细胞因子诱导的信号之间的串扰。我们发现，即使伽马链与它预先相关，伽马链也将限制，但信号诱导的伽马链募集到连接的私有链不会导致生理细胞因子浓度的伽马链短缺。我们观察到与受体结合曲线所期望的STAT磷酸化的EC50值明显低。通过这些受体信号传导的动力学相对较慢，需要几分钟才能达到饱和。 IL-7暴露前导致对伽马链不会受到限制的受体占用物的IL-4和IL-21反应的跨抑制作用。设计了一个测定方法来确定响应IL-7，IL-4和IL-21磷酸化的每个Stat蛋白的分数。该测定法表明，IL-7对统计磷酸化的最大负调节与其引起交叉抑制的能力相关。此外，超过表达伽马链的IL-7介导的IL-4和IL-21响应的交叉抑制作用。 我们与马丁·梅尔·塞勒斯海姆（Martin Meier-Schellersheim）的实验室合作有广泛的合作。他的小组正在通过普通的细胞因子受体家族开发信号传导的计算模型。他们开发了新的计算工具，使他们可以在模型中扫描多个参数并将其映射到动力学和剂量响应数据。这些模型为我们提供了宝贵的见解，即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。 作为系统生物学实验室中协作努力的一部分，以研究受体（TLR）信号传导，与Iain Fraser组合作，我们已经开始对小鼠巨噬细胞系中TLR2和TLR4的细胞表面分布进行想象。