Genetic screens for key transcriptional regulators of antiviral T cell immunity

描述（由申请人提供）：我们的项目提出了一种高度集成的方法来揭示Tfh CD4 T细胞、Th1 CD4 T细胞、CTL CD8 T细胞和记忆CD8 T细胞的调节和分化的生物学，利用小鼠对多种病毒感染的T细胞应答的高通量遗传筛选。T细胞分化为各种效应细胞，以及分化为记忆细胞的能力，是对病原体和癌症的适应性免疫的重要部分。转录因子是这些分化过程的中心调节因子。鉴定调节CD4和CD8 T细胞分化的不同途径的关键转录因子（TF）对于理解这些细胞的生物学至关重要。然而，非常清楚的是，TF不单独起作用，许多TF可能是T细胞分化途径的重要诱导剂或阻遏物。研究TF网络生物学的最大挑战是在受控条件下一次操作超过1个因子的实验通常不可行，特别是在体内的原代细胞中。 因此，几十年来，一次关注一个基因一直是实验的必要性，并且TF网络的大部分分析都是相关的或计算的。双和三敲除小鼠的产生非常耗时。此外，引人注目的FANTOM研究强调了TF表达的适度变化对大多数细胞分化过程的重要性，而不是完全的开关。这一直是基因敲除小鼠的一个明显局限性，它继续使我们对淋巴细胞分化和功能的看法产生偏见。由于概念、时间和资金的限制，目前的实验方法不足以大大提高我们对CD4和CD8 T细胞分化和功能以及淋巴细胞生物学的理解。因此，我们解决这个严重问题的方法一直专注于产生一种实验方法，通过这种方法，我们可以使用一种新的基于shRNAmir载体的方法，平行调节和测试100个基因在体内抗病毒T细胞应答中的作用。我们已经建立了这个系统，现在能够在体内进行遗传筛选，在原代CD4或CDS T细胞中，探测分化和功能。这三个项目积极追求对抗病毒CD4和CD8 T细胞的理解，主题是：什么转录因子调节这些细胞以及它们是如何做到这一点的？

DESCRIPTION (provided by applicant): Our projects propose a highly integrated approach to revealing the biology of regulation and differentiation of Tfh CD4 T cells, Th1 CD4 T cells, CTL CD8 T cells, and memory CD8 T cells, making use of high throughput genetic screens of T cell response in mice to multiple viral infections. T cell differentiation into various effector cells,and the capacity to differentiate into memory cells, are important parts of adaptive immunity to pathogens and cancers. Transcription factors are central regulators of these differentiation processes. The identification of key transcription factors (TFs) regulating different pathways of CD4 and CD8 T cell differentiation have been central to understanding the biology of these cells. However, it is abundantly clear that TFs do not act in isolation and many TFs may be important inducers or repressors of a T cell differentiation pathway. The biggest challenge to studying TF network biology is that experimental manipulation of more than 1 factor at a time under controlled conditions has not been generally feasible, particularly in primary cells in vivo. Therefore, the focus on 1 gene at a time has been an experimental necessity for decades, and the large majority of analyses of TF networks have been correlative or computational. The generation of double and triple knockout mice is excessively time consuming. Furthermore, the compelling FANTOM study highlights the importance of moderate changes in TF expression for most cellular differentiation processes, not complete on-off switches. That has always been a clear limitation of knockout mice, and it continues to bias our perception of lymphocyte differentiation and function. Current experimental approaches are insufficient to dramatically advance our understanding of CD4 and CD8 T cell differentiation and function, and lymphocyte biology in general, due to conceptual, time, and monetary limitations. Therefore, our approach to this serious problem has been focused on generating an experimental approach whereby we can modulate and test 100 genes in parallel for their roles in antiviral T cell responses in vivo, using a new shRNAmir vector based approach. We have established this system, and are now able to perform genetic screens, in vivo, in primary CD4 or CDS T cells, probing differentiation and function. The three Projects vigorously pursue an understanding of antiviral CD4 and CD8 T cells, linked by the theme: what transcription factors regulate these cells and how do they do so?