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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要适应性免疫反应由 T 细胞受体 (TCR) 信号传导控制，它决定了命运和 T 细胞的活动（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，它们携带有关抗原呈递的环境的信息发生。第二信号可以是刺激性的，也可以是抑制性的：刺激性信号对于 T 细胞至关重要激活，而抑制信号（也称为检查点）负责 T 细胞耗竭和抗原耐受性。当以下情况时，免疫系统的先天臂会产生第二刺激信号：例如，Toll 样受体的信号传导可诱导 B7 家族配体 B7-1 (CD80) 和 B7-2 的表达 (CD86) 作用于抗原呈递细胞。 B7-1/B7-2 的表达表明正在发生抗原呈递在持续的先天免疫反应的背景下。 B7-1和B7-2被TCR CD28识别有效增强 TCR 生成的 T 细胞激活信号的辅助受体。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。为了例如，肿瘤细胞通常表达 B7 家族配体 B7-H1 (PD-L1/CD274) 和 B7-DC (PD- L2/CD273)，可被 PD-1 识别，PD-1 是一种抑制 TCR 生成的 T 细胞激活的 TCR 辅助受体信号。 B7-H1/B7-DC 表达向肿瘤细胞传递免疫特权。由于这些和其他原因，我们迫切需要提高对检查点信号的基本理解。在这里，我们建议表征 Jurkat E6-1、HuT 78 和 TALL-104 细胞中 PD-1 调节的酪氨酸磷酸化动态，源自这些亲本细胞系和原代人类 CD8+ 细胞的 CRISPR 工程细胞。我们将申请定量质谱 (MS) 以获得磷酸酪氨酸的无偏差、近乎全面的图像随着时间的推移，T 细胞群中存在和不存在 PD-1/CD28 辅助受体信号传导的 (pTyr) 位点丰度以及跨条件。同时，使用荧光显微镜和工程化的 SH2 结构域亲和力试剂，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质信号伴侣的膜募集寿命。受体。所得数据将用于驱动详细的制定和参数化 TCR 信号传导的机制模型解释了 CD28 和 PD-1 共激活的影响。虽然PD-1 被视为招募抵消激酶激活信号的磷酸酶的平台，我们将评估关于 PD-1 如何可能产生 T 细胞激活的阳性信号的具体假设。这些假设的动机是 PD-1 的最佳特征信号传导伙伴是蛋白质酪氨酸磷酸酶、SHP1 和 SHP2，已知它们在其他情况下可通过以下方式促进细胞活化：例如，介导抑制性 pTyr 位点的去磷酸化。将测试模型预测。