Genetic screens for key transcriptional regulators of antiviral T cell immunity

描述(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，利用高通量T细胞对多种病毒感染的小鼠反应的遗传筛选。T细胞分化为各种效应细胞的能力和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组成部分。转录因子是这些分化过程的中心调节因子。识别调节不同途径的CD4和CD8T细胞分化的关键转录因子(TF)对于理解这些细胞的生物学意义重大。然而，非常清楚的是，TF并不是孤立起作用的，许多TF可能是T细胞分化途径的重要诱导者或抑制者。研究转铁蛋白网络生物学的最大挑战是，在受控条件下一次操作超过1个因子的实验操作通常并不可行，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，而对TF网络的大多数分析都是相关的或计算的。产生双基因和三基因敲除小鼠是非常耗时的。此外，令人信服的FANTOM研究强调了适度改变TF表达对于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，它继续偏见我们对淋巴细胞分化和功能的看法。由于概念、时间和金钱的限制，目前的实验方法不足以显著提高我们对CD4和CD8T细胞分化和功能以及总体淋巴细胞生物学的理解。因此，我们解决这一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，我们可以使用一种基于shRNAmir载体的新方法，同时调节和测试100个基因在体内抗病毒T细胞反应中的作用。我们已经建立了这个系统，现在能够在体内对原始的CD4或CDS T细胞进行遗传筛选，探索分化和功能。这三个项目大力追求对抗病毒CD4和CD8 T细胞的理解，主题联系在一起：什么转录因子调节这些细胞，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?