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: 8555950
• 负责人: Rajat Varma
• 金额: $ 81.39万
• 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/8555950
• 关键词: Affect; Affinity; Agonist; Antigen-Presenting Cells; Antigens; Binding; Biochemical; Biological Models; Biophysics; Book Chapters; CD80 gene; CREB1 gene; Calcium; Calcium Signaling; Cell membrane; Cell surface; Cells; Collaborations; Complex; Computer Simulation; Cyclic AMP Response Element; Cytokine Receptors; Cytoplasmic Tail; Dependence; Diffusion; Dominant-Negative Mutation; Dose; Event; Family; Family member; Feedback; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Future; GTP Binding; Generations; Genes; Glass; Goals; Guanosine Triphosphate Phosphohydrolases; Histocompatibility Antigens Class I; Immediate-Early Genes; Immune; In Vitro; Individual; Infection; Influenza; Intercellular adhesion molecule 1; Interleukin 2 Receptor Gamma; Interleukin-15; Interleukin-2; Interleukin-4; Interleukin-7; Interleukin-9; Kinetics; Ligand Binding; Ligands; Lipid Bilayers; Lipids; MAPK14 gene; MAPK8 gene; MHC Class I Genes; Manuscripts; Maps; Measurement; Measures; Membrane; Methods; Microscope; Mitogen-Activated Protein Kinases; Modeling; Molecular Conformation; Monitor; Mus; NF-kappa B; Nature; Pathway interactions; Pattern; Peptide/MHC Complex; Peptides; Phosphorylation; Phosphotransferases; Process; Proteins; Publishing; Receptor Signaling; Recruitment Activity; Relative (related person); Resolution; Role; Serum Response Element; Signal Pathway; Signal Transduction; Signaling Molecule; Solutions; Sorting - Cell Movement; Source; Spectrum Analysis; Surface; Surface Antigens; Synapses; System; T cell differentiation; T-Cell Receptor; T-Lymphocyte; Techniques; Thymocyte Development; Time; Total Internal Reflection Fluorescent; Transcription Factor AP-1; Transgenic Mice; Transgenic Organisms; Tyrosine Phosphorylation; Vaccinia; Vaccinium; Virus; Work; Writing; base; cellular imaging; interest; member; mutant; promoter; receptor; research study; response; rho; transcription factor

In 2012, we made progress in the following projects: TCR signal transduction in response to partial agonists, Fos induction in T cells, feedback between calcium signals and Map kinase signals downstream of TCR stimulation and its influence on cFos induction, cross talk among common gamma chain family of cytokine receptors in T cells and role of RhoH in TCR signaling. Additionally in collaboration with the labs of Jon Yewdell and Jack Bennink we demonstrated peptide specific clustering of MHC class I molecules at the surface of virally infected antigen presenting cells. Due to the diverse nature of TCR and peptide loaded MHC complexes, TCRs from different cells bind their cognate peptide-MHC complexes (MHCp) with varying affinities. To understand signaling downstream of TCR in response to ligands of varying affinity we employ the model system consisting of T cells from a TCR transgenic mouse (AND) stimulated with altered peptide ligands. As the potency of peptides is reduced, the cells ability to cause calcium signaling is compromised while activation of MAP kinases is preserved. Upon binding peptide MHCp, TCRs undergo micron scale clustering in the plasma membrane called TCR microclusters. TCR microclusters act as a biochemical unit associated with proximal TCR signaling where various signaling molecules are recruited. 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 as expected that high potency ligands recruit Zap70, Lat and Grb2 to the microclusters. One of the medium potency ligands causes the phosphorylation and recruitment of Lat to the microclusters without the recruitment of Zap70. We are exploring the role of other kinases that maybe responsible for the phosphorylation of Lat. One of the ligands we have studied leads to the generation of Erk signals in the absence of calcium signals. These Erk signals are correlated with the specific recruitment of Grb2 to the TCR microclusters in the absence of Lat recruitment. Our results have demonstrated a qualitative difference in TCR signaling in response to MHCp of varying affinity. Immediate early genes such as cFos and Egr-1 are dependent on calcium and MAP kinase signaling via, CRE (cyclic AMP response element) and SRE (serum response element) in their promoters respectively. We have explored feedback between calcium and MAP kinase signaling and found that upon inhibiting calcium signals in T cells, Erk responses are increased and JNK and p38 responses are diminished. On the other hand, inhibiting Erk caused decreased calcium signals while, inhibiting p38 caused increased calcium signals. We have further studied the influence of calcium and MAP kinase signaling in the induction of immediate early genes cFos and Egr-1. We find that both these genes are absolutely dependent on calcium signaling for their induction and are only partially dependent on MAP kinases. Their calcium dependence is exerted via CREB phosphorylation which is calcium dependent. Additionally, dominant negative CREB inhibits cFos 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 is ever limiting for signaling. We have quantified the numbers of each of the members of this family on the surface of nave polyclonal T cells and find that a simple model where each of these receptors are paired with the gamma chain is not possible as the gamma chain is not abundant enough. Intriguingly, dose response curves show that the STAT phosphorylation is saturated at receptor occupancies of less than 1 per cent, suggesting that only a small fraction of receptors need to be pre-associated with the gamma chain for full signaling. We further found that IL-7 is able to inhibit signaling in response to IL-4 and IL-21 when cells are sequentially stimulated with IL-7 and IL-4 or IL-21. We are exploring the biochemical mechanisms behind this competition as it is not a straightforward competition for gamma chains. RhoH is a member of the Rho family of GTPases, but is unusual in that it does not have GTPase activity, and is constitutively GTP bound. RhoH is indispensable for TCR signaling as RhoH deficient mice have defective thymocyte development. RhoH is regulated by tyrosine phosphorylation and is thought to regulate the membrane localization of Lck and Zap70. We have found that RhoH localizes to TCR microclusters in a signal strength dependent manner. We have found that mutants of RhoH that cannot be phosphorylated have altered amounts in the cSMAC of immune synapses. These experiments will allow us to understand how RhoH regulates the concentration of Zap70 and Lck in TCR microclusters. We have an extensive on going collaboration with the lab of Martin Meier-Schellersheim, wherein we are building computational models of signaling via the common gamma chain family of cytokine receptors and the role of RhoH in TCR signaling. This collaboration will help us refine our experiments and the experimental results will in turn refine the computational models. In collaboration with the labs of Jon Yewdell and Jack Bennink we have studied the clustering of MHC class I proteins on the surface of antigen presenting cells using total internal reflection fluorescence microscopy. The clustering of MHC class I molecules has been known for a while. We, however, found that clusters of MHC class I were peptide specific when source antigens were presented by vaccinia or vesicular stomatis virus. The intracellular distribution of these peptide loaded MHC molecules were also distinct, suggesting that the cells had a mechanism of sorting MHC molecules based on the peptide presented and this sorting dependent on the cytoplasmic domain of MHC class I molecules. This work is now in press. In collaboration with the lab of Stephen Lockett at NCI, we have set up a new microscope to perform Fluorescence Correlation Spectroscopy. This technique will help us monitor molecular interactions in the plasma membrane and determine the diffusion coefficient of proteins in solution and in the plasma membrane. We can also monitor the fluctuations in FRET signals at time resolution of a few microseconds on this microscope. This will allow us to measure calcium transients in cells as well as measure conformation changes in proteins. We published a video article describing a method of transfecting T cells and imaging them using TIRF microscopy. The article has a detailed description of setting up a two channel simultaneous acquisition TIRF microscope and has been helpful for a lab at MIT where they are in the process of replicating the system. We also wrote a book chapter describing different fluorescence techniques to study events in the plasma membrane. In the near future we shall be submitting three manuscripts covering the three projects described.

在2012年，我们在以下项目中取得了进展：TCR信号转导对部分激动剂，T细胞的FOS诱导，钙信号和MAP激酶信号之间的反馈TCR刺激的下游及其对CFOS诱导的影响，T细胞中Cytokine受体在TCR ohOH中的Comma Chain thema comma链中的交叉言论以及TCR信号在TCR信号中的作用。此外，在与乔恩·尤德尔（Jon Yewdell）和杰克·本克（Jack Bennink）的实验室合作的情况下，我们证明了在病毒感染的抗原呈现细胞表面MHC I类分子的肽特异性聚类。 由于TCR和肽负载的MHC复合物的多样性，来自不同细胞的TCR结合其同源肽MHC复合物（MHCP）的亲密关系。 为了理解TCR下游的信号传导，响应于不同亲和力的配体，我们采用了由TCR转基因小鼠的T细胞组成的模型系统（并被改变的肽配体刺激）。随着肽的效力降低，导致钙信号传导的细胞能力被损害，同时保留了MAP激酶的激活。结合肽MHCP后，TCR在称为TCR微量群体的质膜中经历微米尺度聚类。 TCR微量群体充当与近端TCR信号传导相关的生化单位，其中募集了各种信号分子。 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，高效力配体招募了ZAP70，LAT和GRB2到微量群体。中等效力配体之一导致LAT磷酸化和募集到微量群体，而无需募集ZAP70。我们正在探索可能导致LAT磷酸化的其他激酶的作用。我们研究的配体之一导致在没有钙信号的情况下产生ERK信号。这些ERK信号与在没有LAT募集的情况下将GRB2的特定募集到TCR微量群体相关。我们的结果表明，响应于不同亲和力的MHCP，TCR信号传导存在质量差异。 立即的早期基因（例如CFOS和EGR-1）分别取决​​于钙和MAP激酶信号传导通过其启动子中的CRE，CRE（环状AMP响应元件）和SRE（血清响应元件）。我们已经探索了钙和MAP激酶信号传导之间的反馈，发现在抑制T细胞中的钙信号后，ERK反应会增加，JNK和P38响应减少。另一方面，抑制ERK导致钙信号降低，而抑制p38导致钙信号增加。我们进一步研究了钙和MAP激酶信号传导对诱导早期基因CFO和EGR-1的影响。我们发现，这两个基因绝对取决于钙信号的诱导，仅部分取决于MAP激酶。它们的钙依赖性是通过钙依赖性的CREB磷酸化施加的。另外，显性负CREB抑制CFO诱导。 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。我们已经在中殿多克隆T细胞表面量化了该家族的每个成员的数量，并发现一个简单的模型，其中每个受体与伽马链配对是不可能的，因为伽马链不够丰富。有趣的是，剂量反应曲线表明，在受体占用率不到1％的受体占用物上，统计磷酸化饱和，这表明只有一小部分受体才需要与伽马链预先相关以进行完全信号传导。我们进一步发现，当用IL-7和IL-7和IL-4或IL-21依次刺激细胞时，IL-7能够抑制对IL-4和IL-21的信号传导。我们正在探索这项竞争背后的生化机制，因为它不是伽马链的直接竞争。 RhoH是Rho GTPases家族的成员，但不寻常的是，它没有GTPase活性，并且是组成型GTP结合的。对于TCR信号，RHOH是必不可少的，因为RHOH缺乏小鼠的胸腺细胞发育不良。 RHOH受酪氨酸磷酸化调节，被认为调节LCK和ZAP70的膜定位。我们发现，RHOH以信号强度依赖性方式定位于TCR微量群体。我们发现，在免疫突触的CSMAC中，无法磷酸化的RHOH突变体改变了量。这些实验将使我们能够了解RHOH如何调节TCR微量群体中ZAP70和LCK的浓度。 我们在与Martin Meier-Schellersheim的实验室合作方面有广泛的合作，在其中，我们正在通过Comman Gamma链的细胞因子受体家族构建信号传导的计算模型，以及RHOH在TCR信号传导中的作用。这项合作将有助于我们完善实验，而实验结果反过来将完善计算模型。 与乔恩·尤德尔（Jon Yewdell）和杰克·本尼克（Jack Bennink）的实验室合作，我们使用总内反射荧光显微镜研究了MHC I类蛋白在抗原呈现细胞表面的聚类。 MHC I类分子的聚类已有一段时间了。但是，我们发现，当通过离子或囊泡气孔病毒表示源抗原时，MHC I类的簇是特异性的。这些肽负载的MHC分子的细胞内分布也很明显，这表明这些细胞具有基于介绍的肽对MHC分子进行分类的机制，并且该分类取决于MHC I类分子的细胞质结构域。这项工作现在正在印刷中。 与NCI的Stephen Lockett的实验室合作，我们建立了一个新的显微镜，以执行荧光相关光谱。该技术将有助于我们监测质膜中的分子相互作用，并确定蛋白在溶液和质膜中的扩散系数。我们还可以在此显微镜上的几微秒的时间分辨率下监测FRET信号中的波动。这将使我们能够测量细胞中的钙瞬变以及测量蛋白质的构象变化。 我们发表了一篇视频文章，描述了一种使用TIRF显微镜对T细胞转染和对其进行成像的方法。该文章有一个详细的描述，即同时设置两个通道TIRF显微镜，并对MIT的实验室很有帮助，它们正在复制系统。我们还撰写了一本书章节，描述了不同的荧光技术来研究质膜中的事件。在不久的将来，我们将提交三个涵盖所述三个项目的手稿。