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: 8336249
• 负责人: Rajat Varma
• 金额: $ 118.88万
• 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/8336249
• 关键词: Affect; Affinity; Agonist; Amino Acid Sequence; Antigen-Presenting Cells; Antigens; Binding; Bioinformatics; Biological; Biophysics; CD28 gene; CD80 gene; CREB1 gene; Calcium; Calcium Signaling; Cell Nucleus; Cell membrane; Cell surface; Cells; Chimeric Proteins; Chinese Hamster Ovary Cell; Collaborations; Complex; Computer Simulation; Computer software; Cytokine Receptor Binding; Cytokine Receptors; Cytoplasmic Tail; Diffuse; Diffusion; Dose; Event; Family; Fluorescence Microscopy; Future; Genetic Transcription; Glass; Goals; Guanosine Triphosphate Phosphohydrolases; Hormone Receptor; Hour; ITAM; In Vitro; Indium; Individual; Infection; Influenza; Intercellular adhesion molecule 1; Interleukin 2 Receptor Gamma; Interleukin-15; Interleukin-2; Interleukin-4; Interleukin-7; Interleukin-9; Joints; Kinetics; Lead; Length; Ligand Binding; Ligands; Link; Lipid Bilayers; Lipids; MAP Kinase Gene; MAPK14 gene; Measurement; Membrane; Methods; Mitogen-Activated Protein Kinase Inhibitor; Modeling; Mus; NF-kappa B; Nature; Pathway interactions; Pattern; Peptide Sequence Determination; Peptide/MHC Complex; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Postdoctoral Fellow; Proteins; Reaction; Receptor Signaling; Recruitment Activity; Relative (related person); Role; Signal Pathway; Signal Transduction; System; T cell differentiation; T-Cell Receptor; T-Lymphocyte; Thick; Time; Transcription Factor AP-1; Transgenic Mice; Transgenic Organisms; Transmembrane Domain; Work; base; biological systems; cell type; cellular imaging; cytokine; inhibitor/antagonist; interest; mathematical model; member; promoter; receptor; research study; response; rho; trafficking; transcription factor

In 2011, we made progress in the following projects: TCR signaling in response to partial agonists, Fos induction in T cells, cross talk among common gamma chain family of cytokine receptors in T cells and developing a method of tuning diffusion coefficients of proteins in glass supported lipid bilayers. Additionally we initiated a new project on studying the role of RhoH in TCR triggering. 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. We have now further biochemically determined that these low potency ligands lead to the phosphorylation of Erk albeit at low levels. 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 phosphorylation 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 peptide-MHC complexes, ICAM-1 and CD80. We find that the low potency ligands do not recruit LAT to the TCR microclusters and only poorly recruit Zap-70. We are currently refining our results to understand the nature of recruitment of Grb2 and working towards expressing Sos in T cells which has been a significant challenge. 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 Ras dependent MAPK and Calcium dependent CamK-Creb pathway 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 by binding SRE element in the Fos promoter. On the other hand active CREB binds to the CRE element in the fos promoter. Calcium and MAPK signaling occurs downstream of TCR, however, how strength of TCR signaling affects the efficacy of these signaling pathway as well as their relative activity is not known. We find that Fos induction occurs with distinct kinetics depending on the strength of TCR stimulation. The kinetics of Fos induction is more rapid in response to ligands that cause calcium signaling. MAPK inhibitors have a differential effect on Fos induction depending on the strength of TCR stimulation. Weaker affinity ligands are more affected by p38 inhibitors than the higher affinity ligands. We are currently exploring the contribution of the CREB pathway in Fos induction. The common gamma chain family of cytokine receptors (IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21) shares the gamma chain for signaling. Since many receptors are expressed on T cells at the same time, it is not clear how these receptors share the gamma chain and if it ever is limiting for signaling. Binding curves that reflect receptor occupancy and dose response curve representing functional response are rarely aligned in biological systems. For example, in the hormone receptor system, the EC-50 of biological response occurs at receptor occupancy less than 50%, a phenomenon known as the Strickland effect. We have explored the relationship between receptor occupancy and STAT phosphorylation for the common-gamma-chain family of cytokine receptors in nave mouse T cells. We find that EC50 for STAT phosphorylation occurs at receptor occupancy of less than 3%. To explain this, we invoke a serial triggering mechanism, where in a few cytokine bound receptors cause phosphorylation of multiple STAT molecules by sequentially engaging many gamma chains. Consistent with this proposal we find that the kinetics of STAT phosphorylation are slow, requiring at least 10 minutes to saturate. We further postulate that STAT phosphorylation reaches saturation at low receptor occupancy at least due to two mechanisms; one, the creation of transiently inactive SOCS-1 bound gamma chain molecules which limit the availability of gamma chains harboring Jak3 molecules that can be activated and two, the activation of phosphatases that negatively regulate signaling via Jak kinases. A consequence of this postulate is the observation of cross-inhibition among IL-4, IL-7 and IL-21 when cells are stimulated sequentially with these sets of cytokines. We are collaborating with the lab of Dr. Martin Meier-Schellersheim who is using detailed computer simulations of the signaling cascades downstream of the cytokine receptors to explore the plausibility of this model and analyze the contributions of membrane-bound and cytoplasmic reactions to the overall response kinetics. 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 of differing length and expressed them in CHO cells. These molecules were not GPI anchored and trafficked to the cell surface. We could purify them and incorporate them in glass supported lipid bilayers and observed that they were mobile. We are now characterizing their diffusion coefficient. RhoH is a member of the Rho family of GTPases, but is unusual in that it does not have a GTPase activity, but instead has an ITAM like motif which binds Zap70. We have initiated a project to understand what role it plays in TCR triggering. Does it help bring Zap70 to the membrane as is currently thought? RhoH is also implicated in negatively regulating Rac activity by competing for phosphoinositide lipids. How do these two functions of RhoH play out in TCR triggering? This project is in collaboration with the lab of Dr. Martin Meier-Schellersheim who will incorporate these observations in the context of a mathematical model. We have hired a joint postdoc for this project.

在2011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细胞中常见的γ链中的脉冲链链中的细胞因子受体家族在T细胞中的传播以及开发一种调整玻璃支持脂质双层蛋白质蛋白质系数的方法。此外，我们启动了一个新项目，以研究RHOH在TCR触发中的作用。 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。现在，我们已经进一步确定这些低效力配体导致ERK的磷酸化，尽管其水平低。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，而无需募集或磷酸化。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。我们发现，低效力配体不会招募LAT到TCR微量群体，而仅招募ZAP-70。我们目前正在完善我们的结果，以了解GRB2募集的性质，并致力于在T细胞中表达SOS，这是一个重大挑战。 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的RAS依赖性MAPK和依赖性CAMK-CREB途径的控制。已知活性ERK是通过磷酸化的三元复合物因子ELK-1，SAP-1和NET的磷酸化成员，这些因子ELK-1，SAP-1和NET通过结合FOS启动子中的SRE元件来引起FOS的转录。另一方面，活性CREB与FOS启动子中的CRE元件结合。钙和MAPK信号传导发生在TCR的下游，但是，TCR信号的强度如何影响这些信号传导途径的功效以及它们的相对活性尚不清楚。我们发现，根据TCR刺激的强度，FOS诱导具有独特的动力学。 FOS诱导的动力学对导致钙信号传导的配体的响应更快。 MAPK抑制剂对FOS诱导具有差异影响，具体取决于TCR刺激的强度。与较高的亲和力配体相比，亲和力弱的配体受P38抑制剂的影响更大。我们目前正在探索CREB途径在FOS诱导中的贡献。 细胞因子受体（IL-2，IL-4，IL-7，IL-9，IL-15和IL-21）的常见伽马链家族共享用于信号传导的伽马链。由于许多受体同时在T细胞上表达，因此尚不清楚这些受体如何共享伽马链以及它是否限制了信号传导。反映了代表功能反应的受体占用率和剂量反应曲线的结合曲线在生物系统中很少排列。例如，在激素受体系统中，生物学反应的EC-50发生在受体占用率小于50％的情况下，这种现象称为Strickland效应。我们已经探索了中神小鼠T细胞中的普通γ链细胞因子受体家族的受体占用和统计磷酸化之间的关系。我们发现，用于统计磷酸化的EC50在受体占用率小于3％时发生。为了解释这一点，我们调用了一种串行触发机制，其中在几个细胞因子结合的受体中，通过依次与许多伽马链接合来引起多个Stat分子的磷酸化。与该提案一致，我们发现统计磷酸化的动力学很慢，需要至少10分钟才能饱和。我们进一步指出，在低受体占用率下，统计磷酸化至少由于两个机制而达到饱和。第一，创建瞬时无效的SOCS-1结合伽马链分子，限制了具有可以激活的JAK3分子的伽马链的可用性，而两个分子的磷酸酶激活了通过JAK激酶对信号进行负调节信号传导的激活。这一假设的结果是，当用这些细胞因子依次刺激细胞时，IL-4，IL-7和IL-21之间的交叉抑制作用。我们正在与Martin Meier-Schellersheim博士的实验室合作，他正在使用细胞因子受体下游的信号级联反应的详细计算机模拟来探索该模型的合理性，并分析膜结合和细胞质反应对整体反应反应动力学的贡献。 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，我们发现在CHO细胞中表达时，它们最终成为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，其中包含不同长度的CD28的TM结构域，并在CHO细胞中表达它们。这些分子未锚定并运输到细胞表面。我们可以将它们净化并将其纳入玻璃支撑的脂质双层，并观察到它们是流动的。我们现在正在表征它们的扩散系数。 RhoH是Rho GTPases家族的成员，但不寻常的是，它没有GTPase活性，而是具有绑定ZAP70的ITAM类似的ITAM。我们已经启动了一个项目，以了解其在TCR触发中的作用。它是否有助于将ZAP70带到膜上？ RHOH还涉及通过竞争磷酸肌醇脂质来负面调节RAC活性。 RHOH的这两个功能如何在TCR触发中发挥作用？该项目与Martin Meier-Schellersheim博士的实验室合作，该实验室将在数学模型的背景下纳入这些观察结果。我们已为该项目雇用了联合博士后。