Input encoding in T-cell receptor signaling

摘要 电池中的信号感测电路的性能通常与人工检测相当，甚至超过人工检测的水平 设备。了解这些电路如何在嘈杂的蜂窝环境中以最佳和稳健的方式运行将 提供对其基本设计原理的基本见解，并使我们能够重新调整这些电路的用途 用于细胞工程。介导T细胞检测抗原的信号通路尤其引人注目。 传感能力-它可以选择性地对抗原配体的几个拷贝做出反应，同时保留 在五个数量级范围内区分配基水平的能力。T细胞受体信号通路 能达到如此显著的选择性、灵敏度和动态范围，在其操作上尚不为人所知。至 为了解决这个问题，我们开发了一种新型的多路径荧光报告系统，该系统能够 第一次，同时实时跟踪下游的三个主要信号通路的活动 单细胞水平上的T细胞受体。在这里，我们将这位多途径的记者与量化直播相结合- 细胞成像、数学建模和微扰分析，以阐明T- 细胞配体感知，并确定它们在T细胞功能障碍中是如何被破坏的。首先，我们将(I)执行一项 单个通路和T细胞受体的输入/输出反应的系统、定量表征 订婚。这一特征将在单细胞级别执行，使用高的组合 吞吐量实时成像和计算图像分析。接下来，我们将(Ii)阐明监管反馈 这些响应背后的循环。为此，我们将对候选反馈进行数学建模 架构，然后进行迭代实验测试。最后，我们将(Iii)确定这些输入/输出是如何 使用活细胞成像技术与小鼠联合使用，状态对T细胞功能障碍感到不安 模特们。这些研究将对T细胞的抗原感知机制产生基本的见解， 产生了工程T细胞治疗癌症和其他疾病的指导原则。更广泛地说，这项工作 还将阐明哺乳动物信号电路体系结构和设计的基本原理，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.