Patterning of Transcription Factor Activity in T cells During Influenza Infection

流感感染期间 T 细胞转录因子活性的模式

• 批准号: 8157026
• 负责人: Rajat Varma
• 金额: $ 99.52万
• 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/8157026
• 关键词: Address; Affect; Affinity; Biochemical; Bioinformatics; CD28 gene; CD80 gene; Chimeric Proteins; Cytokine Receptors; Cytoplasmic Tail; Diffuse; Diffusion; Dose; Energy Transfer; Family; Fluorescence; Future; Gene Expression; Glass; Goals; Hand; Image; Immune system; In Vitro; Infection; Influenza; Interleukin 2 Receptor Gamma; Interleukin 4 Receptor; Ligand Binding; Link; MAP Kinase Gene; MAPK14 gene; Molecular; Pattern; Peptide/MHC Complex; Physiological; Receptor Signaling; Resolution; Sum; Surface; T cell differentiation; T-Lymphocyte; Thick; Transmembrane Domain; ZAP-70 Gene; base; cellular imaging; ras Guanine Nucleotide Exchange Factors; transcription factor

In 2010, we made progress in the following projects: TCR signaling in response to partial agonists, Fos induction in T cells, quantitative analysis of cytokine receptor expression levels on T cells and developing tools to study gene expression and tune diffusion coefficients of proteins in glass supported lipid bilayers. We have an interest in understanding the signaling pathways activated downstream of TCR in response to low potency MHC-peptide complexes. Based on our previous work we have observed that there are a class of TCR ligands that generate Ras signals in the absence of calcium signals. Our hypothesis to explain this observation is that these Ras signals are generated by recruitment of Grb2-Sos module to the TCR complex in microclusters without recruitment or phophorylation of LAT. We have now made several observations to support our hypothesis. We have visualized the recruitment of GFP tagged LAT, Zap70 and Grb2 transfected in in-vitro activated AND TCR transgenic T cells using Total Internal Reflection Fluorescence microscopy (TIRFM) of cells interacting with glass supported lipid bilayers containing lipid anchored peptid-MHC complexes, ICAM-1 and CD80. We setup a two channel simultaneous imaging system for these experiments so that there is no delay between the TCR and GFP images. We find that the low potency ligands do not recruit LAT to the TCR microclusters and only poorly recruit Zap-70. On the other hand we see a specific recruitment of Grb2 to TCR microclusters generated in response to the low potency ligands over background. We have written a software program that in an automated way extracts quantitative information about the no. of microclusters and the relative amounts of fluorescence associated with it. Since Grb2 can recruit Sos which then acts as a Guanine nucleotide exchange factor for Ras, this explains the generation of Ras signals in the absence of Calcium signals in this setting. We want to understand the relationship between TCR engagement events at the cell surface and activation of transcription factors in the nucleus. For these experiments we have obtained transgenic mice that express a fusion protein between the transcription factor Fos and GFP under the control of the Fos promoter. Fos is not expressed in T cells in the basal state and is induced upon signaling. We have crossed these mice to AND TCR transgenic mice and have begun to study the induction of Fos using single cell imaging in response to ligands of varying strengths. We find that Fos is induced within 20 minutes of interaction with antigen and continues to accumulate for three hours after which the expression plateaus presumably due to degradation of Fos. Given that it takes about 6-7 minutes for GFP to mature, it is likely that the kinetics of Fos induction are even faster. Fos expression is controlled by MAPK activity in various cell types. Active Erk is known to phosphorylate members of the ternary complex factors, Elk-1, Sap-1 and Net which cause the transcription of Fos. MAPK signaling occurs downstream of TCR, however, how strength of TCR signaling affects the efficacy of this signaling pathway is not known. We find that Fos induction follows distinct kinetics depending on whether the TCR signals cause calcium signaling or not. The kinetics of Fos induction is more rapid in response to ligands that cause calcium signaling. We dont think that this has anything to do with calcium signaling but more to do with how Ras is activated in the two scenarios. Expectedly we find that Fos induction can be blocked by Erk and p38 inhibitors. These experiments will help us understand the hierarchy of MAPK signaling in T cells. We have also generated constructs of NFAT and p65 fused to tag-RFP (a red fluorescent protein) which we are simultaneously expressing in these cells so that we can follow the dynamics of two transcription factors at the same time. We are using endogenous promoters to express these transcription factors in T cells so that they are expressed at physiological levels and we are learning a lot regarding the requirements of expression of p65 in T cells. These reagents will allow us to address multiple questions concerning the single cell dynamics of transcription factors which cannot be addressed using biochemical analysis. How multiple subunit containing receptors such as antigen receptors whose ligand binding subunit is different from the signal transducing subunit, communicate information from ligand binding to signal transduction is not known. Fluorescence Resonance Energy Transfer (FRET) is a fluorescence phenomenon in which fluorophores transfer energy among themselves only when they are in molecular proximity of each other. Hence if FRET measurements are done at high time resolution, it offers the possibility of studying the dynamics of individual subunits within a receptor complex. The T cell receptor is very complex as it contains multiple subunits, and hence to establish a proof of principle approach we wanted to study a more simplified system which is equally relevant to the immune system. So we decided to investigate cytokine receptors belonging to the common gamma chain family. Before setting out to do the experiment it was important first to determine the physiological amounts of subunit of this family (IL2Ralpha, IL2Rbeta, IL15Ralpha, IL7Ralpha, IL4Ralpha, IL21Ralpha and the gamma chain) on the surface of naive and activated T cells. Unexpectedly we find that the levels of gamma chain are limiting when compared to the sum of all the other chains that it can pair up with. We are intrigued by this result and are exploring the significance of this in terms of signaling via these receptors. For example this may mean that under competing levels of certain cytokines one could potentially observe competition. We indeed find that when we have saturating amounts of IL-7 the cells are unable to signal via the IL-4 receptor. When we combine these experiments with FRET measurements we will be able to answer several questions: Is association between subunits of cytokine receptor governed by the cytokine driven affinity conversion model? What are the mechanisms of triggering of cytokine receptors and what is the speed with which they can be triggered? Diffusion of MHC molecules in the plasma membrane of antigen presenting cells affects how they trigger TCRs. To study this phenomenon using glass supported bilayers, we are trying to develop a system where we can tune the diffusion coefficient of molecules in the bilayer. If transmembrane anchored molecules are incorporated in bilayers, their cytoplasmic tail interacts with glass and gets stuck and hence they don't diffuse. We are exploring the possibility that if we incorporate a cytoplasmic tail deleted protein, then it would have reduced interaction with glass and hence may diffuse slower than a lipid anchored protein. We could then modulate the thickness of the bilayer using different lipids and thereby tune the diffusion coefficients of the incorporated proteins. We first made several truncated CD80 molecules, however, we found that they ended up becoming GPI anchored when expressed in CHO cells. Using bioinformatics software that would predict whether a protein sequence is likely to be GPI linked or not we found that the transmembrane domain of CD28 when truncated is not likely to be GPI-anchored. Using this we have generated several CD80 molecules containing the TM domain of CD28. We will first test if these are GPI anchored or not and then purify them and test their mobility in glass supported lipid bilayers.

在2010年，我们在以下项目中取得了进展：TCR信号传导响应部分激动剂，T细胞的FOS诱导，T细胞上细胞因子受体表达水平的定量分析以及开发研究基因表达的工具，研究基因表达和调整玻璃支持的脂质双层中蛋白质的扩散系数。 我们有兴趣理解对低效力MHC肽复合物响应TCR下游激活的信号传导途径。根据我们以前的工作，我们观察到，在没有钙信号的情况下，有一类TCR配体会产生RAS信号。我们解释这一观察结果的假设是，这些RAS信号是通过将GRB2-SOS模块募集到微量群体中的TCR复合物中产生的，而无需募集或phophophoration的LAT。现在，我们已经进行了几次观察以支持我们的假设。我们使用总内部反射荧光显微镜（TIRFM）与玻璃支撑的脂质双层相互作用的细胞中，在体外激活和TCR转基因T细胞中转染GFP标记的LAT，ZAP70和GRB2的募集。我们为这些实验设置了两个同时的成像系统，以使TCR和GFP图像之间没有延迟。我们发现，低效力配体不会招募LAT到TCR微量群体，而仅招募ZAP-70。另一方面，我们看到特定的GRB2募集到TCR微量群体中，响应于背景上的低效力配体而产生的。我们编写了一个软件程序，该程序以自动化的方式提取有关NO的定量信息。微簇及其相关的荧光相对量。由于GRB2可以募集SOS，然后作为RAS的鸟嘌呤核苷酸交换因子，因此在这种情况下没有钙信号的情况下解释了RAS信号的产生。 我们想了解细胞表面的TCR参与事件与细胞核中转录因子的激活之间的关系。对于这些实验，我们获得了在FOS启动子控制下转录因子FOS和GFP之间表达融合蛋白的转基因小鼠。 FOS在基底状态的T细胞中不表达，并在信号传导时诱导。我们已经越过这些小鼠和TCR转基因小鼠，并开始使用单细胞成像来研究FOS的诱导，以响应各种强度的配体。我们发现，在与抗原相互作用的20分钟内诱导了FOS，并继续积聚三个小时，然后表达高原可能是由于FOS的降解。鉴于GFP成熟大约需要6-7分钟，因此FOS诱导动力学的速度可能更快。 FOS表达受到各种细胞类型中的MAPK活性控制。已知活性ERK是导致FOS转录的三元复合因子ELK-1，SAP-1和NET的磷酸化成员。 MAPK信号传导发生在TCR的下游，但是，TCR信号的强度如何影响该信号通路的功效。我们发现，FOS诱导遵循不同的动力学，具体取决于TCR信号是否引起钙信号传导。 FOS诱导的动力学对导致钙信号传导的配体的响应更快。我们认为这与钙信号传导无关，而与在两种情况下激活RAS的方式有关。预计我们发现FOS诱导可以被ERK和P38抑制剂阻止。这些实验将有助于我们了解T细胞中MAPK信号的层次结构。 我们还生成了NFAT和p65的构建体与TAG-RFP（红色荧光蛋白）融合，我们在这些细胞中同时表达，因此我们可以同时遵循两个转录因子的动力学。我们正在使用内源性启动子在T细胞中表达这些转录因子，以便它们在生理水平上表达，并且我们在T细胞中p65表达的要求学习了​​很多。这些试剂将使我们能够解决有关转录因子的单细胞动力学的多个问题，这些问题无法使用生化分析来解决。 含有多个亚基含有配体结合亚基的抗原受体的受体与信号转导亚基的抗原受体如何，从配体结合到信号转导的信息传达信息。 荧光共振能量转移（FRET）是一种荧光现象，仅当它们彼此邻近时，荧光团之间只有它们之间的转移能量。因此，如果在较高时间分辨率时进行了FRET测量，它提供了研究受体复合物中各个亚基的动力学的可能性。 T细胞受体非常复杂，因为它包含多个亚基，因此要建立一种原理方法的证明，我们想研究一个与免疫系统同样相关的更简化的系统。因此，我们决定研究属于普通伽马链家族的细胞因子受体。在进行实验之前，重要的是首先要确定该家族的亚基的生理量（IL2RALPHA，IL2RBETA，IL15RALPHA，IL7RALPHA，IL7RALPHA，IL4RALLPHA，IL4RALPHA，IL21RALPHA和gamma链）在NAIVE和活跃T细胞的表面上。出乎意料的是，与它可以配对的所有其他链的总和相比，伽马链的水平是限制的。我们对这个结果很感兴趣，并正在通过这些受体进行信号传导的意义。例如，这可能意味着在某些细胞因子的竞争水平下，人们可能会观察到竞争。我们确实发现，当我们拥有饱和量的IL-7时，细胞无法通过IL-4受体发出信号。当我们将这些实验与FRET测量结果结合在一起时，我们将能够回答几个问题：由细胞因子驱动的亲和力转换模型支配的细胞因子受体的亚基之间的关联吗？触发细胞因子受体的机制是什么，可以触发它们的速度是多少？ MHC分子在抗原呈递细胞的质膜中的扩散会影响它们触发TCR的方式。为了使用玻璃支持的双层研究这种现象，我们正在尝试开发一个系统，在该系统中我们可以调整分子在双层中的扩散系数。如果将跨膜锚定的分子掺入双层中，则其细胞质尾巴与玻璃相互作用并卡住，因此它们不会扩散。我们正在探讨如果我们结合了细胞质尾巴删除的蛋白质，那么它将减少与玻璃的相互作用，因此比脂质锚定蛋白的相互作用可能会慢得多。然后，我们可以使用不同的脂质调节双层的厚度，从而调整掺入蛋白的扩散系数。我们首先制作了几个截短的CD80分子，但是，我们发现它们最终在CHO细胞中表达时锚定了GPI。使用生物信息学软件，可以预测蛋白质序列是否可能是链接的，我们发现当截短时CD28的跨膜结构域不太可能是GPI锚定的。使用它，我们生成了几个CD80分子，其中包含CD28的TM结构域。我们将首先测试这些是GPI是否锚定的，然后净化它们并在玻璃支撑的脂质双层中测试其迁移率。