Project 2 - Chang

摘要--项目2--常： 在对微生物感染的免疫反应中，幼稚的CD8+T淋巴细胞可以产生终末效应器 提供急性宿主防御的细胞和在功能上不同的存储单元子集，从而提供耐用 豁免权。现在认识到，一个这样的子集，组织驻留存储单元(Trm)的产生 对粘膜和身体表面的保护必不可少。精疲力竭，免疫细胞未能达到或 维持最佳功能，是发生在慢性感染和癌症背景下的另一种结果。 先前的研究已经加深了我们对CD8+T细胞如何分化为Trm或耗尽细胞的理解， 但该领域面临着该项目试图克服的几个重要挑战。我们建议采取一项 不偏不倚的、表型标记不可知的方法，利用 创新的排序方法，可实现 同一单个细胞中蛋白质和转录组的同时测量 要克服这个问题 先前的研究在群体水平上进行转录图谱分析的固有局限性。我们 将利用体内基于shRNA的方法，使我们能够反复评估50个基因的功能 同时，从而克服了传统基因敲除导致的发现速度缓慢的问题 一次评估一个基因的模型。具体地说，我们将使用这些方法来实现以下目标 目的：(1)研究Trm细胞的分子异质性和功能重要性。 (2)通过重建幼稚细胞的转录路线图来阐明trm细胞的个体发育。 通过组织内和跨组织部位的渐进性分化状态；(3)在功能上及早识别 循环和驻留在组织中的CD8+T细胞的重要分子决定因素。一大强项 我们建议的是综合和协同设计，利用单个项目的优势和 核心主要调查员，生成为多个项目提供信息的数据集。我们预见到这种洞察力 我们的研究结果可能对疫苗的合理设计和 扭转疲惫状态的治疗性干预措施。

SUMMARY-PROJECT 2-CHANG: During an immune response to microbial infection, a naive CD8+ T lymphocyte can give rise to terminal effector cells that provide acute host defense and functionally distinct subsets of memory cells that provide durable immunity. Generation of one such subset, tissue-resident memory cells (Trm), is now recognized to be essential for protection at mucosal and body surfaces. Exhaustion, the failure of immune cells to achieve or maintain optimal function, is an alternative outcome that occurs in the setting of chronic infections and cancer. Prior studies have advanced our understanding of how CD8+ T cells differentiate into Trm or exhausted cells, but the field faces several important challenges that this Project seeks to overcome. We propose to take an unbiased, `phenotypic marker-agnostic' approach exploiting an innovative sequencing approach that enables simultaneous measurement of proteins and the transcriptome in the same single cells to overcome the inherent limitations of prior studies that have performed transcriptional profiling at the bulk population level. We will utilize an in vivo shRNA-based approach enabling us to reiteratively evaluate the function of 50 genes simultaneously, thereby overcoming the slow pace of discovery resulting from traditional genetic knockout models that evaluate one gene at a time. Specifically, we will use these approaches to achieve the following goals: (1) Investigate the molecular heterogeneity and functional importance of Trm cells within and among tissues; (2) Elucidate the ontogeny of Trm cells by reconstructing the transcriptomic roadmap from the naive state through progressive states of differentiation, within and across tissue sites; (3) Identify early, functionally important molecular determinants of exhausted circulating and tissue-resident CD8+ T cells. A major strength of our proposal is the integrated and synergistic design that exploits the strength of the individual Project and Core Principal Investigators, generating datasets that inform multiple Projects. We envision that insights resulting from our studies may have important implications for the rational design of vaccines and for therapeutic interventions that reverse the exhausted state.