Quantitative Modeling of Lymphocyte Signaling Pathways

为了进一步了解T细胞对抗原刺激的反应，我们试图建立定量模型和模拟参与T细胞受体（TCR）激活的主要信号通路的激活。我们目前的实验模型是从5C中分离的CD4+ T细胞。我们已经建立了使用APC细胞系P13.9激活这些细胞的可重复的方案。我们通过在细菌和/或哺乳动物细胞中表达小鼠cdna作为标记融合物，生成了用于定量Raf1、Mek1+2、Erk1+2和Ksr的蛋白标准。在每种情况下确定了最佳表达和纯化方法，以提供可接受的纯蛋白量，用于定量western blots上加载参考样品。已经评估了这些靶点的多种商业抗体来源，以确定适合western和流式细胞术测量的抗血清。我们还评估了Luminex开发的xMAP（多分析物分析）技术用于定量评估蛋白质浓度和磷酸化的适用性。到目前为止，这已经为级联的几个组成部分提供了高质量的数据。我们打算使用上述所有方法来增加我们对刺激反应中蛋白质数量和蛋白质磷酸化程度的估计的信心。到目前为止，我们已经获得了5C中表达的Mek和Erk蛋白的数据。C7 CD4+ T细胞xMAP和定量western blotting结果一致。我们通过这些方法对蛋白质含量的估计将由LSB细胞网络蛋白质组学单位进行的定量蛋白质组学分析进行补充。该项目的另一个关键要求是获得对T细胞形态和几何特征的估计，以便准确计算细胞中信号蛋白的浓度。这是通过染色5C实现的。C7 CD4+ T细胞具有选择性染料和一组亚细胞标记的表达，这些亚细胞标记定位于细胞的特定区域。在与NIAID RTB成像核心实验室的合作中，我们使用共聚焦显微镜和3D细胞重建软件来生成细胞形状特征和体积的可重复数据。我们已经确定了适用于流式细胞术检测Erk级联蛋白ERK2、MEK2、Raf1、Grb2和phospho-ERK2的抗血清。这很重要，因为它提供了单细胞数据和蛋白质浓度和磷酸化反应的群体分布的指示。Erk2代表从5C分离的CD4+ T细胞中表达的主要Erk亚型。C7小鼠，我们使用该蛋白的磷酸化状态作为TCR激活模型的关键读数。我们通过流式细胞术确定了Erk2在APC激活时的磷酸化动力学，并使用EKAR报告器通过FRET测量来补充这些数据，以提供Erk2磷酸化的空间数据。

In order to further understanding of T cell responses to antigen stimulation, we seek to generate quantitative models and simulations of the activation of the major signaling pathways engaged upon T cell receptor (TCR) activation. Our current experimental model is CD4+ T cells isolated from 5C.C7 mice, and we have established reproducible protocols for activating these cells using the APC cell line P13.9. We have generated protein standards for quantification of Raf1, Mek1+2, Erk1+2 and Ksr by expressing the murine cDNAs as tagged fusions in bacterial and/or mammalian cells. The optimal expression and purification method has been identified in each case to give acceptable quantities of pure protein for loading reference samples on quantitative western blots. Multiple commercial antibody sources for these targets have been assessed to identify suitable antisera for both western and flow cytometry measurements. We have also assessed the suitability of the xMAP (multi-analyte profiling) technology developed by Luminex for quantitative assessment of protein concentration and phosphorylation. So far, this has provided high quality data for several components of the cascade. We intend to use all of the above approaches to increase confidence in our estimations of both protein number and degree of protein phosphorylation in response to stimulus. Thus far, the data we have obtained for the Mek and Erk proteins expressed in 5C.C7 CD4+ T cells by both xMAP and quantitative western blotting have shown good agreement. Our estimations of protein content by these methods will be complemented by quantitative proteomic analyses carried out by the LSB Cellular Networks Proteomics unit. Another key requirement in this project is to acquire an estimation of the morphology and geometric characteristics of the T cell to allow accurate calculations of the concentrations of the signaling proteins in the cell. This has been achieved through staining the 5C.C7 CD4+ T cells with selective dyes and expression of a panel of subcellular markers that localize to specific regions of the cell. In collaboration with the NIAID RTB imaging core laboratory, we have used confocal microscopy and 3D cell reconstruction software to generate reproducible data on cell shape characteristics and volume. We have identified suitable antisera for flow cytometry detection of the Erk cascade proteins ERK2, MEK2, Raf1, Grb2 and phospho-ERK2. This is important as it provides single cell data and an indication of the population distribution of protein concentrations and phosphorylation responses. Erk2 represents the major Erk isoform expressed in CD4+ T cells isolated from 5C.C7 mice, and we are using the phosphorylation status of this protein as a key readout in our modeling of TCR activation. We have determined the phosphorylation dynamics of Erk2 in response to APC activation by flow cytometry, and have complemented this data with FRET measurements using the EKAR reporter to provide spatial data on Erk2 phosphorylation. This year we have extended these studies to a hybridoma cell line (2B4) that expresses the same TCR as the primary cells isolated from 5C.C7 transgenic mice. The 2B4 cell line was re-cloned from single cells to identify a fraction that was CD4+ and gave consistent Erk2 phosphorylation and Ca2+ responses when activated with the same APC cell line P13.9. This provides a consistent supply of cells that show comparable response characteristics to the primary cells. It also provides a better model system for stable expression of signaling reporters, and accordingly, we have generated 2B4 lines that express the EKAR Erk reporter localized to either the cytoplasm or nucleus. This has the potential to provide important spatial data on Erk activity after TCR challenge with strong and weak peptide agonists. We have also found the 2B4 hybridoma to be a useful model for analysis of single cell Ca2+ release in response to varying strengths of TCR activation. We are currently analyzing this data to determine how it might inform existing models of TCR ligand discrimination.