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.

针对传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日益强大和系统的分析 在这种分化过程中发生的变化已经确定了可以发挥作用的大量基因和途径 调节 T 细胞分化以应对急性感染。现在的挑战是将功能归因于 每个基因已被确定为细胞状态广泛重编程的一部分，该重编程发生在 CD8+ 和 CD4+ T 细胞分化。该提案旨在利用 Cas9/CRISPR 介导的功能丧失 体内遗传筛选，系统地识别控制细胞分化的关键基因 CD8+ 和 CD4+ T 细胞响应急性感染。 我们开发了一个基因筛选平台，该平台使用汇集的体内 CRISPR 介导的缺失 功能遗传筛选，以识别正向或负向调节效应器和发育的基因 记忆群体。我们已经使用这个平台进行基于测序的混合文库 反卷积并行测试 CD8+ T 细胞中 100 多个基因响应急性感染的功能。 这种原理验证筛选成功地恢复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，我们现在建议扩展该系统以识别和验证 小鼠模型和人类细胞中 T 细胞对急性感染反应的调节因子，目的如下： 目标 1：从急性期 CD8+ 和 CD4+ T 细胞的 scRNA-seq 图谱中筛选候选调节因子 使用 scRNA-seq 配置文件进行感染。我们将使用大规模并行单细胞 RNA-seq 来深入分析 急性感染期间抗原特异性 CD8+ 和 CD4+ T 细胞，并使用计算模型（带有 Core B）来 确定候选监管机构。从这个丰富的数据集中，我们将识别出一组潜在的调节分子 将在《目标2》中上映。 目标 2：进行 CRISPR 汇总筛选，以确定 CD8+ 和 CD4+ 命运和功能的调节因子。我们 将使用汇集的 CRISPR/Cas9 介导的遗传筛选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，以揭示 启动并维持效应器和记忆分化的基因；指引记忆命运的承诺； 控制血统选择；或调节细胞因子分泌的特定模式的获得。 目标 3：验证在小鼠模型和人类 T 细胞中调节 CD8 和 CD4 分化的候选药物 细胞。为了验证这些候选调节剂，我们将使用一系列广泛的细胞免疫学测定 体内和体外使用基因组编辑或传统的基因敲除分别探测它们的功能 可用的。我们将通过 CRISPR/Cas9 介导的原代 CD8+ 和 来自健康人类受试者的 CD4+ T 细胞，以测试其调节作用在人类 T 细胞中是否保守。