System Dynamics of PD-1 Signaling in T Cells

T 细胞中 PD-1 信号传导的系统动力学

• 批准号: 10211871
• 负责人: William S Hlavacek
• 金额: $ 78.46万
• 依托单位: TRIAD NATIONAL SECURITY, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-03 至 2027-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211871
• 关键词: Accounting; Affinity; Antibodies; Antigen Presentation; Antigen-Presenting Cells; Antigens; Autoimmune Diseases; Autoimmunity; B7-DC antigen; Bioinformatics; Biological Assay; CD28 gene; CD8-Positive T-Lymphocytes; CD80 gene; CD86 gene; Cell Line; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Data; Disease; Engineering; Evaluation; Family; Fluorescence Microscopy; Formulation; Human; ITAM; Immune; Immune Targeting; Immune response; Immune system; Immunologic Surveillance; Immunotherapy; Individual; Innate Immune Response; Intervention; Investigation; Knowledge; Label; Ligands; Machine Learning; Mass Spectrum Analysis; Measurement; Measures; Mediating; Membrane; Methods; Modeling; Molecular; Monitor; NR0B2 gene; Oncology; PTPN11 gene; PTPN6 gene; Pattern; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Phosphotyrosine; Play; Population; Protein Dephosphorylation; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Protocols documentation; Reagent; Receptor Signaling; Role; SYK gene; Sampling; Signal Transduction; Signaling Protein; Site; Structural Models; System; Systems Biology; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Techniques; Testing; Therapeutic antibodies; Time; Toll-like receptors; Tyrosine Phosphorylation; Validation; Western Blotting; ZAP-70 Gene; adaptive immune response; arm; base; cancer cell; chronic infection; dectin 1; exhaustion; experimental study; immune activation; immune checkpoint; improved; interest; mathematical model; neoplastic cell; novel therapeutic intervention; particle; pathogen; pathogenic fungus; phosphoproteomics; predictive modeling; prevent; programmed cell death ligand 1; programmed cell death protein 1; receptor; recruit; response; single molecule

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.

项目摘要/摘要 自适应免疫反应受T细胞受体（TCR）信号的控制，这决定了命运和 T细胞的活性（助手，效应器等）。 TCR及其信号合作伙伴整合了抗原识别 信号和第二个信号，其中包含有关抗原表现的上下文的信息 发生。第二个信号可以是刺激性的或抑制性的：刺激信号对于T细胞至关重要 激活，而抑制信号（也称为检查点）负责T细胞耗尽和 抗原耐受性。当免疫系统的先天臂生成刺激性第二信号时 例如，通过Toll样受体的信号传导诱导B7-家庭配体B7-1（CD80）和B7-2的表达 （CD86）在抗原呈递细胞上。 B7-1/B7-2的表达表明抗原表现正在发生 在持续的先天免疫反应的背景下。 B7-1和B7-2被CD28（TCR）识别 有力增强TCR生成的T细胞激活信号的colecector。抑制性第二信号出现 在慢性刺激TCR信号传导过程中。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性引起的，但它们也可能是有害的。为了 例如，肿瘤细胞通常表达B7家庭配体B7-H1（PD-L1/CD274）和B7-DC（PD- L2/CD273），由PD-1识别，PD-1是抑制TCR生成的T细胞激活的TCR colecector 信号。 B7-H1/B7-DC表达传达了免疫特权对肿瘤细胞。由于这些和其他原因，这是 必须提高对检查点信号的基本理解。在这里，我们建议描述 PD-1调节的酪氨酸磷酸化的动力学在Jurkat E6-1，HUT 78和高104个细胞中的动力学， CRISPR工程细胞衍生自这些亲本细胞系和原代人CD8+细胞。我们将申请 定量质谱法（MS）获得无偏见的磷酸酪氨酸的图像 （PTYR）随着时间的推移，T细胞种群中有和没有PD-1/CD28共感染者信号传导的位置丰度随时间 以及跨条件。同时使用荧光显微镜和工程的SH2结构域亲和力 试剂，我们将表征用于TCR，CD28和PD-1的多站点磷酸化的单分子模式。 我们还将测量这些单个胞质信号伴侣的膜摄取寿命 受体。结果数据将用于驱动详细的公式和参数化 TCR信号传导的机械模型，占CD28和PD-1共激活的影响。虽然PD-1 被视为募集磷酸酶来抵消激活信号的磷酸酶的平台，我们将 评估有关PD-1如何产生阳性信号以进行T细胞激活的特定假设。 这些假设是由于PD-1的最佳特征信号伴侣是蛋白质的动机 酪氨酸磷酸酶SHP1和SHP2，已知可以在其他情况下促进细胞激活。 例如，介导抑制性PTYR位点的去磷酸化。模型预测将进行测试。