Dissecting the pathways controlling tunable responses to TCR signaling

剖析控制 TCR 信号传导可调反应的途径

• 批准号: 10074912
• 负责人: LESLIE JOAN BERG
• 金额: $ 2.99万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-01-24 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10074912
• 关键词: ATAC-seq; Affinity; Alleles; Antigens; Antiviral Agents; Behavior; Biochemical; Biological Assay; CD8-Positive T-Lymphocytes; Calcium; Categories; Cell physiology; Cells; Cellular Metabolic Process; Cessation of life; Chromatin; Chronic; Cytokine Receptors; Data; Dose; Effector Cell; Equilibrium; Exhibits; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genes; Genetic Transcription; Hour; IRF4 gene; ITAM; Immediate-Early Genes; Immune response; Immunity; Immunotherapy; Impairment; Infection; Infectious Agent; Knock-in; Lead; Life; Link; Listeria monocytogenes; Memory; Microbe; Mus; Nuclear; Outcome; Ovum; Pathogenicity; Pathway interactions; Pattern; Peptide/MHC Complex; Phosphorylation; Population; Process; Proliferating; Receptor Signaling; Second Messenger Systems; System; T cell differentiation; T cell response; T-Cell Activation; T-Cell Activation Pathway; T-Cell Development; T-Cell Receptor; T-Lymphocyte; T-cell receptor repertoire; TEC Protein Tyrosine Kinase; Testing; Transcriptional Activation; Transgenic Mice; Treatment Efficacy; Tyrosine; Up-Regulation; Variant; Virus Diseases; analog; autoreactive T cell; base; cancer therapy; cytokine; density; digital; experimental study; insight; migration; pathogen; phospholipase C gamma; programs; response; single-cell RNA sequencing; thymocyte; transcription factor; transcriptome sequencing; vaccinology

Dissecting the pathways controlling tunable responses to TCR signaling Summary Stimulation of the T-cell receptor (TCR) leads to activation, a process that includes changes in T cell metabolism, survival, proliferation, cytokine responsiveness, migration behavior, and effector functions. Life versus death, as well as lineage decisions of T cells are determined in large part by the strength of TCR signaling. Our previous studies have demonstrated that the transcription factor IRF4 is upregulated by TCR stimulation in CD8 T cells, and that the maximum level of IRF4 achieved is dependent on the strength of TCR signaling via the Tec kinase ITK. In turn, IRF4 promotes T cell differentiation into massively proliferating antiviral effector cells in a dose-dependent manner. Yet, mechanistic insight into how TCR stimulation produces a dynamic range of responses defined by distinct gene expression patterns is currently lacking. Our preliminary studies indicate that the Tec kinase ITK is a focal point for the tunable component of TCR signaling. Previous studies showed that ITK is not required for all TCR signaling; instead, in its absence, TCR signaling is significantly reduced. From these studies, the clear function of ITK was difficult to discern, as some aspects of T cell activation appeared normal in the absence of ITK, whereas other T cell functions were greatly impaired. Our current data, using IRF4 upregulation as an example of TCR tuning, provide a framework to understand these apparent discrepancies. These studies have revealed that variations in antigen density and in TCR affinity can modulate gene expression patterns shortly after activation of naive CD8 T cells. Dissecting these pathways shows that the two second messengers generated by activation of phospholipase C-γ, the major substrate of ITK, cooperate to regulate all-or-nothing (digital) versus graded (analog) responses to changes in TCR signal strength. The relative balance of these differing inputs determines which responses exhibit the broadest range of tunability to TCR signaling. Based on these data, we hypothesize that the magnitude of ITK activity is determined by the multiplicity of ITAM phosphorylation at the TCR, and that variations in ITK activity tune the calcium response that regulates transcription factor activation. Further, we propose that ITK- dependent tuning of TCR signal strength controls a program of gene expression that is established within hours after TCR stimulation, and thus impacts the differentiation pathways of activated cells. To test these hypotheses, we propose to determine how variations in TCR signal strength lead to graded versus digital expression of entire programs of gene expression at the global and single cell level, and how these patterns are established by the first wave of transcriptional activation following T cell stimulation. We will also assess whether the magnitude of ITAM phosphorylation regulates the dynamic range of ITK-dependent TCR signaling. Finally, we will examine whether the full dynamic range of ITK-dependent TCR signaling is required to generate a broad repertoire of pathogen- specific T cells that provide enhanced responses to variants of the primary infecting pathogen. These experiments will provide important insights into the downstream consequences of alterations in TCR signal strength, allowing a more informed approach to manipulating T cell activation pathways for applications in the fields of vaccinology and immunotherapy.

剖析控制TCR信号传导的可调反应的途径 总结 T细胞受体（TCR）的刺激导致活化，这一过程包括T细胞的变化， 代谢、存活、增殖、细胞因子反应性、迁移行为和效应物 功能协调发展的生与死，以及T细胞的谱系决定在很大程度上取决于T细胞的功能。 TCR信号强度。我们以前的研究表明，转录因子IRF 4 在CD 8 T细胞中通过TCR刺激上调，并且达到的IRF 4的最大水平是 这取决于经由Tec激酶ITK的TCR信号传导的强度。反过来，IRF 4促进T细胞 在一些实施方案中，本发明提供了一种以剂量依赖性方式诱导抗病毒效应细胞分化成大量增殖的抗病毒效应细胞的方法。然而， 对TCR刺激如何产生由以下定义的动态范围的响应的机械见解 目前缺乏独特的基因表达模式。我们的初步研究表明 激酶ITK是TCR信号传导的可调组分的焦点。先前的研究显示 ITK不是所有TCR信号传导所必需的;相反，在其不存在时，TCR信号传导显著降低。 降低从这些研究中，ITK的明确功能很难辨别，因为T 在缺乏ITK的情况下，细胞活化似乎正常，而其他T细胞功能则大大降低。 受损我们目前的数据，使用IRF 4上调作为TCR调节的一个例子，提供了一个新的方法。 来理解这些明显的差异。这些研究表明， 在抗原密度和TCR亲和力中，可以在活化后不久调节基因表达模式 幼稚的CD 8 T细胞。对这些通路的分析表明， 通过激活磷脂酶C-γ，ITK的主要底物，协同调节全或无 （数字）与分级（模拟）对TCR信号强度变化的响应。之间相对平衡 这些不同的输入决定了哪种响应对TCR表现出最宽的可调谐范围 发信号。基于这些数据，我们假设ITK活性的大小由以下因素决定： TCR处ITAM磷酸化的多样性，以及ITK活性的变化调节了 调节转录因子激活的钙反应。此外，我们建议ITK- TCR信号强度的依赖性调节控制基因表达程序， 在TCR刺激后的数小时内，细胞的增殖和分化，从而影响活化细胞的分化途径。 为了验证这些假设，我们建议确定TCR信号强度的变化如何导致 在整体和单个细胞中基因表达的整个程序的分级表达与数字表达 水平，以及这些模式是如何建立的第一波转录激活后T 细胞刺激我们还将评估ITAM磷酸化的程度是否调节了细胞的增殖。 ITK依赖性TCR信号传导的动态范围。最后，我们将研究是否完全动态 一系列ITK依赖性TCR信号传导是产生广泛的病原体库所必需的， 特异性T细胞，其对主要感染病原体的变体提供增强的应答。 这些实验将提供重要的见解下游的后果改变 在TCR信号强度，允许更明智的方法来操纵T细胞活化途径 用于疫苗学和免疫治疗领域。