Dissecting the pathways controlling tunable responses to TCR signaling

解剖控制对TCR信号的可调反应的通路 摘要 刺激T细胞受体(TCR)导致激活，这一过程包括T细胞的变化 代谢、存活、增殖、细胞因子反应、迁移行为和效应器 功能。T细胞的生死以及谱系决定在很大程度上取决于 TCR信号的强度。我们之前的研究已经证明，转录因子IRF4 在CD8T细胞中通过TCR刺激而上调，并且达到的最大水平是 依赖于TCR通过Tec激酶ITK的信号强度。反过来，IRF4促进T细胞 以剂量依赖的方式分化为大量增殖的抗病毒效应细胞。然而， 对TCR刺激如何产生动态范围的反应的机械论洞察 目前缺乏不同的基因表达模式。我们的初步研究表明，技术 激酶ITK是TCR信号的可调节成分的焦点。此前的研究表明， 并不是所有的TCR信令都需要ITK；相反，在没有ITK的情况下，TCR信令非常重要 减少了。从这些研究中，很难看出ITK的明确功能，因为T的某些方面 在没有ITK的情况下，细胞激活看起来是正常的，而其他T细胞功能则显著 受伤了。我们当前的数据，使用IRF4上调作为TCR调优的示例，提供了一个 理解这些明显差异的框架。这些研究表明，变异 在抗原密度和TCR亲和力方面可以在激活后不久调节基因表达模式 幼稚的CD8 T细胞。解剖这些通路表明，两个第二信使产生了 通过激活ITK的主要底物磷脂酶C-γ，协同调节全有或全无 (数字)与分级(模拟)对TCR信号强度变化的响应。相对平衡 这些不同的输入决定了哪些响应表现出对TCR的最大范围的可调谐 发信号。根据这些数据，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 调节转录因子激活的钙反应。此外，我们建议ITK- TCR信号强度的依赖调节控制已建立的基因表达程序 在TCR刺激后的数小时内，并因此影响激活细胞的分化途径。 为了检验这些假设，我们建议确定TCR信号强度的变化如何导致 整体和单细胞基因表达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通过T之后的第一波转录激活来建立的 细胞刺激。我们还将评估ITAM磷酸化的幅度是否调节 ITK依赖的TCR信号的动态范围。最后，我们将检查是否完全动态 需要一系列依赖ITK的TCR信号才能产生广泛的病原体- 对主要感染病原体的变种提供增强反应的特定T细胞。 这些实验将对改变的下游后果提供重要的见解 在TCR信号强度上，允许更知情的方法来操纵T细胞激活途径 在疫苗和免疫治疗领域的应用。

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.