Input encoding in T-cell receptor signaling

摘要 细胞中的信号感应电路的表现水平往往可以与人工检测相媲美，甚至超过 装置.了解这些电路如何在嘈杂的细胞环境中实现最佳和稳健的性能， 对它们的基本设计原理产生基本的见解，并将使我们能够重新使用这些电路 用于细胞工程。通过T细胞介导抗原检测的信号传导回路表现出特别显著的 传感能力-它可以选择性地响应甚至几个拷贝的抗原配体，同时保留 在五个数量级范围内区分配体水平的能力。T细胞受体信号电路 能达到如此卓越的选择性，灵敏度和动态范围，在他们的操作是不了解。到 为了解决这个问题，我们开发了一种新的多途径荧光报告系统， 第一次，同时实时跟踪三个主要信号通路下游的活动， 在单细胞水平上的T细胞受体。在这里，我们将联合收割机这一多途径报告与定量活 细胞成像，数学建模和扰动分析，以阐明T- 细胞配体传感，并确定它们如何在T细胞功能障碍中被破坏。首先，我们将（I）执行一项 单个通路和T细胞受体的输入/输出响应的系统定量表征 订婚这种表征将在单细胞水平上进行，使用高浓度的 吞吐量实时成像和计算图像分析。接下来，我们将（二）阐明监管反馈 这些反应背后的循环。为此，我们将对候选人反馈进行数学建模 架构，然后是迭代实验测试。最后，我们将（III）确定这些输入/输出 使用活细胞成像技术结合小鼠， 模型这些研究将产生对T细胞抗原感应机制的基本见解， 从而为T细胞工程治疗癌症和其他疾病提供指导原则。更广泛地说，这项工作 还将阐明哺乳动物信号电路的基本结构和设计原则，影响 跨不同领域的系统和信号生物学研究。

ABSTRACT Signal sensing circuits in cells often perform at a level that rivals or even exceeds that of man-made detection devices. Understanding how these circuits perform optimally and robustly in a noisy cellular environment will yield fundamental insights into their underlying design principles, and will enable us to re-purpose these circuits for cell engineering. The signaling circuit that mediates antigen detection by T-cells exhibits particularly striking sensing capabilities – it can selectively respond to even a few copies of antigenic ligand, while retaining an ability to distinguish ligand levels over a five order of magnitude range. How T-cell receptor signaling circuits can achieve such remarkable selectivity, sensitivity and dynamic range in their operation is not understood. To address this question, we have developed a novel multi-pathway fluorescent reporter system that enables, for the first time, simultaneous live tracking of the activity of the three primary signaling pathways downstream of the T-cell receptor at the single-cell level. Here, we combine this multi-pathway reporter with quantitative live- cell imaging, mathematical modeling and perturbation analysis to elucidate the control strategies underlying T- cell ligand sensing, and determine how they are disrupted in T-cell dysfunction. Firstly, we will (I) perform a systematic, quantitative characterization of input/output responses of individual pathways and T-cell receptor engagement. This characterization will be performed at the single-cell level, using a combination of high throughput live imaging and computational image analysis. Next, we will (II) elucidate regulatory feedback loops underlying these responses. To do so, we will perform mathematical modeling of candidate feedback architectures, followed by iterative experimental testing. Finally, we will (III) determine how these input/output states are perturbed upon T-cell dysfunction, using live-cell imaging techniques in conjunction with mouse models. These studies will generate fundamental insights into antigen sensing mechanisms by T-cells, yielding guiding principles for engineering T-cells to treat cancer and other diseases. More broadly, this work will also elucidate principles underlying architecture and design of mammalian signaling circuits, impacting systems and signaling biology studies across diverse fields.