System Dynamics of PD-1 Signaling in T Cells

项目总结/摘要 适应性免疫应答由T细胞受体（TCR）信号传导控制，其决定免疫应答的命运和免疫应答。 T细胞的活性（辅助细胞、效应细胞等）。TCR及其信号传导伙伴整合了抗原识别 信号和第二信号，其携带关于抗原呈递被 正在发生。第二信号可以是刺激性的或抑制性的：刺激性信号对T细胞是必不可少的。 激活，而抑制性信号（也称为检查点）负责T细胞耗竭， 抗原耐受性刺激性第二信号由免疫系统的先天臂产生， 例如，Toll样受体的信号传导诱导B7家族配体B7-1（CD 80）和B7-2的表达 （CD 86）在抗原呈递细胞上。B7-1/B7-2的表达表明发生了抗原呈递 在先天免疫反应的背景下。B7-1和B7-2被CD 28识别，CD 28是TCR 在一些实施方案中，所述受体是有效增强TCR产生的T细胞活化信号的辅助受体。抑制性第二信号出现 在TCR信号传导的慢性刺激过程中。它们对于限制附带损害很重要 由免疫反应和避免自身免疫引起，但它们也可能是有害的。为 例如，肿瘤细胞通常表达B7家族配体B7-H1（PD-L1/CD 274）和B7-DC（PD-L1/CD 274）。 L2/CD 273），其被PD-1识别，PD-1是抑制TCR产生的T细胞活化的TCR辅助受体 信号. B7-H1/B7-DC表达表达对肿瘤细胞的免疫赦免。由于这些和其他原因， 我们必须提高对检查点信号的基本理解。在这里，我们建议描述 Jurkat E6-1、HuT 78和TALL-104细胞中PD-1调节的酪氨酸磷酸化的动力学， 源自这些亲本细胞系的CRISPR工程化细胞和原代人CD 8+细胞。我们将应用 定量质谱法（MS），以获得磷酸酪氨酸的无偏，几乎全面的图片 随时间推移，T细胞群体中存在和不存在PD-1/CD 28辅助受体信号传导的（pTyr）位点丰度 和各种条件。同时，使用荧光显微镜和工程化SH 2结构域亲和力， 试剂，我们将表征TCR，CD 28和PD-1的多位点磷酸化的单分子模式。 我们还将测量这些细胞的单个胞质信号伙伴的膜募集寿命。 受体。由此产生的数据将用于驱动详细的 TCR信号传导的机制模型解释了CD 28和PD-1共活化的作用。虽然PD-1 被视为一个平台，招聘的磷酸酶，抵消激活信号的激酶，我们将 评估关于PD-1如何可能产生T细胞活化的阳性信号的具体假设。 这些假设的动机是这样一个事实，即PD-1的最佳表征的信号伴侣是蛋白质 酪氨酸磷酸酶，SHP 1和SHP 2，已知它们在其它情况下通过促进细胞活化， 例如，介导抑制性pTyr位点的去磷酸化。模型预测将得到验证。

PROJECT SUMMARY/ABSTRACT Adaptive immune responses are governed by T cell receptor (TCR) signaling, which determines the fates and activities of T cells (helper, effector, etc.). The TCR and its signaling partners integrate antigen-recognition signals and second signals, which carry information about the context in which antigen presentation is occurring. Second signals can be either stimulatory or inhibitory: stimulatory signals are essential for T cell activation, whereas inhibitory signals (also called checkpoints) are responsible for T cell exhaustion and antigen tolerance. Stimulatory second signals are generated by the innate arm of the immune system when, for example, signaling by Toll-like receptors induces expression of the B7-family ligands B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells. Expression of B7-1/B7-2 indicates that antigen presentation is occurring within the context of an ongoing innate immune response. B7-1 and B7-2 are recognized by CD28, a TCR coreceptor that potently enhances TCR-generated T-cell activation signals. Inhibitory second signals arise during the course of chronic stimulation of TCR signaling. They are important for limiting the collateral damage caused by an immune response and avoidance of autoimmunity, but they can also be deleterious. For example, tumor cells commonly express the B7-family ligands B7-H1 (PD-L1/CD274) and B7-DC (PD- L2/CD273), which are recognized by PD-1, a TCR coreceptor that inhibits TCR-generated T-cell activation signals. B7-H1/B7-DC expression conveys immune privilege to tumor cells. For these and other reasons, it is imperative that we improve our basic understanding of checkpoint signaling. Here, we propose to characterize the dynamics of PD-1-regulated tyrosine phosphorylation in Jurkat E6-1, HuT 78, and TALL-104 cells, CRISPR-engineered cells derived from these parental cell lines, and primary human CD8+ cells. We will apply quantitative mass spectrometry (MS) to obtain an unbiased, nearly comprehensive picture of phosphotyrosine (pTyr) site abundances with and without PD-1/CD28 coreceptor signaling in populations of T cells over time and across conditions. Concurrently, using fluorescence microscopy and engineered SH2 domain affinity reagents, we will characterize single-molecule patterns of multisite phosphorylation for TCR, CD28, and PD-1. We will also measure membrane-recruitment lifetimes for individual cytosolic signaling partners of these receptors. The resulting data will be used to drive the formulation and parameterization of a detailed mechanistic model for TCR signaling accounting for the effects of CD28 and PD-1 coactivation. Although PD-1 is viewed as a platform for recruitment of phosphatases that counteract activation signals from kinases, we will evaluate specific hypotheses about how PD-1 could potentially generate positive signals for T-cell activation. These hypotheses are motivated by the fact that the best characterized signaling partners of PD-1 are protein tyrosine phosphatases, SHP1 and SHP2, which are known to promote cell activation in other contexts by, for example, mediating the dephosphorylation of inhibitory pTyr sites. Model predictions will be tested.