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

项目 1：通过 CRISPR 筛选发现 CD8 和 CD4 细胞命运和功能的调节因子

• 批准号: 10207349
• 负责人: Arlene H. Sharpe
• 金额: $ 86.54万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-05 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10207349
• 关键词: Adaptive Immune System; Animals; Antigens; Autoimmunity; Biological Assay; Bone Marrow; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; CRISPR screen; CRISPR/Cas technology; Catalogs; Cell Compartmentation; Cells; Chimera organism; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Computer Models; Data Set; Development; Foundations; Gene Deletion; Gene Expression Profiling; Gene Library; Genes; Genetic Screening; Genomics; Guide RNA; Hematopoietic stem cells; Human; Immune; Immune system; Immunity; Immunology procedure; In Vitro; Individual; Knock-out; Libraries; Lymphocytic choriomeningitis virus; Mediating; Memory; Metabolic; Pathway interactions; Pattern; Phenotype; Play; Population; Regimen; Reporter; Role; Surveys; System; T cell differentiation; T cell response; T-Lymphocyte; Testing; Transgenic Animals; Transgenic Organisms; acute infection; base; cytokine; epigenetic profiling; experimental study; genome editing; human subject; in vivo; innovation; loss of function; mouse model; novel; pathogen; promoter; reconstitution; response; single-cell RNA sequencing; vaccination strategy; vector

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.

对感染性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越来越强大且系统的分析 在这种差异期间发生的情况已经确定了可能发挥作用的基因和途径的巨大目录 在调节急性感染的T细胞分化方面。挑战现在是将功能归功于 每个被识别为在细胞状态的广泛重编程的一部分的基因 CD8+和CD4+ T细胞分化。该建议旨在使用功能丧失CAS9/CRISPR介导的 在体内遗传筛选，以系统地识别控制的关键基因 CD8+和CD4+ T细胞响应急性感染。 我们已经开发了一个基因筛查平台，该平台使用汇总的体内CRISPR介导的损失 功能遗传筛选以识别积极或负调节效应子发展的基因和 记忆群体。我们已经使用了这个平台来进行基于测序的汇总库 反卷积在与急性感染反应的CD8+ T细胞中100多个基因的功能。 此原则屏幕成功恢复了已知的效应阳性和负调节剂 分化。在这些初步研究的基础上，我们现在建议扩展该系统以识别和验证 在以下目的中，小鼠模型和人类细胞中T细胞对急性感染的反应调节剂： AIM 1：急性期间CD8+和CD4+ T细胞的SCRNA-SEQ轮廓的策展候选调节剂 使用SCRNA-SEQ剖面感染。我们将使用大量平行的单细胞RNA-Seq到深度剖面 急性感染期间抗原特异性CD8+和CD4+ T细胞，并使用计算建模（与核心B） 确定候选监管机构。从这个丰富的数据集中，我们将确定一组潜在的调节分子 将在AIM 2中进行筛选。 AIM 2：执行合并的CRISPR屏幕，以识别CD8+和CD4+命运和功能的调节器。我们 将使用合并的CRISPR/CAS9介导的遗传筛选来调查数千个基因在 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 基因：启动和维持效应子和记忆分化；记忆命运承诺的指南； 控制血统的选择；或调节细胞因子分泌特定模式的获取。 AIM 3：验证在小鼠模型和人t中调节CD8和CD4分化的候选者 细胞。为了验证这些候选调节剂，我们将在 体内和体外，使用基因组编辑或常规敲除分别探测其功能，其中 可用的。我们将使用CRISPR/CAS9介导的基因编辑在原发性CD8+和 来自健康人受试者的CD4+ T细胞测试其调节作用是否在人类T细胞中保守。