Quantitative Modeling of Lymphocyte Signaling Pathways

为了进一步了解T细胞对抗原刺激的反应，我们试图生成参与T细胞受体（TCR）活化的主要信号通路的活化的定量模型和模拟。 我们目前的实验模型是从5C.C7小鼠分离的CD 4 + T细胞，并且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方案。我们通过在细菌和/或哺乳动物细胞中表达作为标记融合物的鼠cDNA，产生了用于定量Raf 1、Mek 1 +2、Erk 1 +2和Ksr的蛋白标准品。 已在每种情况下确定了最佳表达和纯化方法，以获得可接受量的纯蛋白质，用于在定量蛋白质印迹上加载参比样品。 已经评估了这些靶标的多种商业抗体来源，以鉴定用于Western和流式细胞术测量的合适抗血清。 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。 我们打算使用所有上述方法来增加我们对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，我们通过xMAP和定量蛋白质印迹法获得的5C.C7 CD 4 + T细胞中表达的Mek和Erk蛋白的数据显示出良好的一致性。我们通过这些方法对蛋白质含量的估计将得到PSIIM蛋白质组学小组进行的定量蛋白质组学分析的补充。该项目的另一个关键要求是获得T细胞形态和几何特征的估计，以准确计算细胞中信号蛋白的浓度。 这是通过用选择性染料染色5C.C7 CD 4 + T细胞并表达定位于细胞特定区域的一组亚细胞标志物来实现的。 与NIAID RTB成像核心实验室合作，我们使用共聚焦显微镜和3D细胞重建软件来生成有关细胞形状特征和体积的可重复数据。我们已经确定了用于流式细胞术检测Erk级联蛋白ERK 2、MEK 2、Raf 1、Grb 2和磷酸化ERK 2的合适抗血清。 这是重要的，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。 Erk 2代表了从5C.C7小鼠分离的CD 4 + T细胞中表达的主要Erk同种型，我们使用该蛋白的磷酸化状态作为TCR活化建模的关键读数。 我们已经确定了Erk 2的磷酸化动力学响应APC激活的流式细胞术，我们目前正在补充这个数据与FRET测量使用EKAR报告提供空间数据Erk 2磷酸化。

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 PSIIM Proteomics group. 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 we are currently complementing this data with FRET measurements using the EKAR reporter to provide spatial data on Erk2 phosphorylation.