Project 1: CRISPR screens to discover regulators of CD8 and CD4 cell fates and function

对感染病原体的保护性免疫需要CD8+和CD4+T细胞的协调分化。 对转录、表观遗传和代谢变化的日益强大和系统化的描述 在这种分化过程中发生的基因和途径已经确定了一个巨大的目录，这些基因和途径可能发挥作用 在调节T细胞分化以应对急性感染方面。现在的挑战是将函数归因于 已被确认为细胞状态广泛重新编程的一部分的每个基因 CD8+和CD4+T细胞分化。该提案寻求使用功能丧失的CAS9/CRISPR中介 在体内进行基因筛选，系统地识别控制细胞分化的关键基因 CD8+和CD4+T细胞对急性感染的反应。 我们已经开发了一个基因筛查平台，它使用汇集的体内CRISPR介导的缺失- 功能基因筛选以识别积极或消极调节效应器和 记忆群体。我们已经使用这个平台进行了基于测序的池化文库 去卷积，以平行测试CD8+T细胞中100多个基因对急性感染的反应。 该原理验证筛成功地回收了效应器的已知正负调节因子 差异化。在这些初步研究的基础上，我们现在建议扩展这一系统，以识别和验证 在小鼠模型和人类细胞中调节T细胞对急性感染的反应的目的如下： 目的1：从急性加重期CD8+和CD4+T细胞的scRNA-seq谱中筛选候选调节剂 使用scRNA-seq图谱的感染。我们将使用大规模并行单细胞rna-seq来深入剖析 在急性感染期间抗原特异性CD8+和CD4+T细胞，并使用计算模型(具有核心B)来 确定候选监管机构。从这个丰富的数据集中，我们将识别一组潜在的调节分子 将在《目标2》中上映。 目的2：进行联合CRISPR筛查，以确定CD8+和CD4+的调节因子的命运和功能。我们 将使用集合CRISPR/Cas9介导的遗传筛查来调查数千个基因的功能 调节CD8+和CD4+T细胞的分化。我们将使用定义的表型进行筛选，这将揭示 启动和维持效应器和记忆分化的基因；记忆命运承诺的指南； 管理血统选择；或调节特定细胞因子分泌模式的获得。 目的3：在小鼠模型和人类T细胞中验证调节CD8和CD4分化的候选基因 细胞。为了验证这些候选调节剂，我们将使用一套广泛的细胞免疫分析在 在体内和体外使用基因组编辑或传统的基因敲除来单独探测它们的功能 可用。我们将用CRISPR/Cas9介导的初级CD8+和CD8+基因编辑来补充这些研究 来自健康受试者的CD4+T细胞，以测试它们的调节作用是否在人类T细胞中保守。

Protective immunity to infectious pathogens requires the coordinated differentiation of CD8+ and CD4+ T cells. Increasingly powerful and systematic profiling of the transcriptional, epigenetic, and metabolic changes that occur during this differentiation has identified an immense catalog of genes and pathways that could play a role in regulating T cell differentiation in response to acute infection. The challenge is now to ascribe functions to each of the genes that have been identified as part of the broad reprogramming of cell state that occurs during CD8+ and CD4+ T cell differentiation. This proposal seeks to use loss-of-function Cas9/CRISPR-mediated genetic screens in vivo to systematically identify the critical genes that govern the differentiation of CD8+ and CD4+ T cells in response to acute infection. We have developed a genetic screening platform that uses pooled, in vivo CRISPR-mediated loss-of- function genetic screens to identify genes that positively or negatively regulate the development of effector and memory populations. We have already used this platform to conduct pooled libraries with sequencing-based deconvolution to test in parallel the function of over 100 genes in CD8+ T cells responding to acute infection. This proof-of-principle screen successfully recovered known positive and negative regulators of effector differentiation. Building on these initial studies, we now propose to extend this system to identify and validate regulators of T cell response to acute infection in mouse models and human cells in the following aims: Aim 1: Curate candidate regulators from scRNA-seq profiles of CD8+ and CD4+ T cells during acute infection using scRNA-seq profiles. We will use massively parallel single-cell RNA-seq to deeply profile antigen-specific CD8+ and CD4+ T cells during acute infection and use computation modeling (with Core B) to identify candidate regulators. From this rich dataset we will identify a set of potential regulatory molecules that will be screened in Aim 2. Aim 2: Perform pooled CRISPR screens to identify regulators of CD8+ and CD4+ fate and function. We will use pooled CRISPR/Cas9 mediated genetic screens to survey the function of thousands of genes in regulating differentiation of CD8+ and CD4+ T cells. We will screen using defined phenotypes that will uncover genes that: initiate and sustain effector and memory differentiation; guide to a memory fate commitment; govern lineage choice; or regulate the acquisition of specific patterns of cytokine secretion. Aim 3: Validate candidates that regulate CD8 and CD4 differentiation in mouse models and human T cells. To validate these candidate regulators we will use an extensive set of cellular immunological assays in vivo and in vitro to probe their function individually using genome editing or conventional knockouts where available. We will complement these studies with CRISPR/Cas9 mediated gene editing in primary CD8+ and CD4+ T cells from healthy human subjects to test whether their regulatory role is conserved in human T cells.