Quantitative Modeling of Lymphocyte Signaling Pathways

为了进一步了解T细胞对抗原刺激的反应，我们试图建立与T细胞受体(TCR)激活相关的主要信号通路的激活的定量模型和模拟。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使用的方案。我们通过在细菌和/或哺乳动物细胞中以标记融合的形式表达小鼠的cDNA，建立了用于定量Raf1、MEK1+2、ERK1+2和KSR的蛋白质标准。在每种情况下都确定了最佳的表达和纯化方法，以获得可接受的纯蛋白质数量，用于定量Western blotting上的参考样本。已经对这些靶标的多种商业抗体来源进行了评估，以确定适合于免疫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)技术对蛋白质浓度和磷酸化的定量评估的适用性。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对刺激反应的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Western blotting获得的在5C.C7 CD4+T细胞中表达的MEK和ERK蛋白的数据都显示出很好的一致性。我们通过这些方法对蛋白质含量的估计将得到LSB细胞网络蛋白质组学部门进行的定量蛋白质组学分析的补充。这个项目的另一个关键要求是获得T细胞的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。这是通过用选择性染料对5C.C7 CD4+T细胞进行染色，并表达一组定位于细胞特定区域的亚细胞标记来实现的。在与NIAID RTB成像核心实验室的合作下，我们使用共聚焦显微镜和3D细胞重建软件来生成关于细胞形状特征和体积的可重复数据。我们已经确定了合适的抗血清来检测ERK级联蛋白ERK2、MEK2、RAF1、Grb2和磷酸化ERK2。这一点很重要，因为它提供了单细胞数据，并指示了蛋白质浓度和磷酸化反应的总体分布。ERK2代表从5C.C7小鼠分离的CD4+T细胞中表达的主要ERK亚型，我们正在使用该蛋白的磷酸化状态作为TCR激活模型的关键读数。我们已经用流式细胞术测定了ERK2在APC激活后的磷酸化动力学，并用Ekar报告器的FRET测量补充了这一数据，以提供ERK2磷酸化的空间数据。 今年，我们将这些研究扩展到杂交瘤细胞系(2B4)，该杂交瘤细胞株表达与从5C.C7转基因小鼠分离的原始细胞相同的TCR。从单个细胞中重新克隆2B4细胞系，以确定其中一个为CD4+的组分，当与相同的APC细胞系P13.9激活时，该细胞系具有一致的ERK2磷酸化和钙离子反应。这提供了一致的细胞供应，这些细胞表现出与原电池类似的响应特性。这也为信号报告的稳定表达提供了一个更好的模型系统，相应地，我们获得了定位于细胞质或细胞核的表达Ekar Erk报告基因的2B4系。这有可能为TCR用强肽和弱肽激动剂攻击后ERK活性提供重要的空间数据。 我们还发现2B4杂交瘤是一个有用的模型，用于分析单细胞钙释放对不同强度的TCR激活的反应。我们目前正在分析这些数据，以确定它可能如何为现有的TCR配体歧视模型提供信息。

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.