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 获得性免疫反应受T细胞受体(TCR)信号支配，TCR信号决定命运和 T细胞的活动(辅助者、效应者等)。TCR及其信号伙伴整合了抗原识别 信号和第二信号，它们携带关于抗原呈递的上下文的信息 发生的。第二种信号既可以是刺激性的，也可以是抑制性的：刺激性信号对T细胞是必不可少的 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。刺激的第二信号是由免疫系统的先天手臂产生的，当 例如，通过Toll样受体的信号诱导B7家族配体B7-1(CD80)和B7-2的表达 (CD86)表达于抗原提呈细胞。B7-1/B7-2的表达表明正在发生抗原提呈 在持续的先天免疫反应的背景下。B7-1和B7-2被TCR CD28识别 能有效增强TCR产生的T细胞激活信号的辅助受体。抑制性第二信号出现 在慢性刺激TCR信号的过程中。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/CD274)和B7-DC(PD-DCs)。 L2/CD273)，可被PD-1识别，PD-1是一种TCR辅助受体，可抑制TCR产生的T细胞激活 信号。B7-H1/B7-DC的表达向肿瘤细胞传递免疫豁免。出于这些和其他原因，它是 当务之急是提高我们对检查站信号的基本理解。在这里，我们建议将 PD-1调节Jurkat E6-1、HUT 78和TALL-104细胞酪氨酸磷酸化的动力学 CRISPR工程细胞来源于这些亲本细胞系和原代人类CD8+细胞。我们会申请 定量质谱学(MS)以获得无偏的、几乎全面的磷酸酪氨酸图像 T细胞群体中有无PD-1/CD28共受体信号的(PTyr)位点丰度随时间的变化 并且跨越各种条件。同时，使用荧光显微镜和工程SH2结构域亲和 试剂，我们将表征TCR，CD28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胞质信号伙伴的膜募集寿命 感受器。所产生的数据将用于驱动详细的 TCR信号机制模型解释了CD28和PD-1共激活的影响。虽然PD-1 被视为一个招募磷酸酶的平台，它抵消了来自激酶的激活信号，我们将 评估关于PD-1如何潜在地产生T细胞激活的积极信号的具体假设。 这些假说的动机是这样一个事实，即PD-1最典型的信号伙伴是蛋白质 酪氨酸磷酸酶，SHP1和SHP2，已知在其他环境中通过促进细胞激活， 例如，介导抑制pTyr位点的去磷酸化。模型预测将得到检验。