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: 9523362
• 负责人: Arlene H. Sharpe
• 金额: $ 50.66万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9523362
• 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 Simulation; 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; Immunosuppressive Agents; In Vitro; Individual; Knock-out; Libraries; Lymphocytic choriomeningitis virus; Mediating; Memory; 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; metabolic profile; 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CD 8+和CD 4 + 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一系列研究已经确定了一个巨大的基因和途径目录， 在调节T细胞分化对急性感染的反应。现在的挑战是将职能分配给 每一个基因都被确定为细胞状态广泛重编程的一部分， CD 8+和CD 4 + T细胞分化。该提案寻求使用功能丧失的Cas9/CRISPR介导的 在体内进行遗传筛选，以系统地鉴定控制细胞分化的关键基因， CD 8+和CD 4 + T细胞对急性感染的反应。 我们已经开发了一种遗传筛选平台，该平台使用合并的体内CRISPR介导的缺失- 进行遗传筛选以鉴定正或负调节效应子发育的基因， 记忆人口我们已经使用这个平台进行了基于测序的池库， 通过解卷积来并行测试响应急性感染的CD 8 + T细胞中超过100个基因的功能。 该原理验证筛选成功地回收了已知的效应子的正和负调节子。 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 在小鼠模型和人细胞中对急性感染的T细胞应答的调节剂，目的如下： 目的1：从急性期CD 8+和CD 4 + T细胞的scRNA-seq谱中筛选候选调节因子。 使用scRNA-seq图谱进行感染。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CD 8+和CD 4 + T细胞，并使用计算建模（使用Core 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控分子， 将在目标2中进行筛选。 目的2：进行合并的CRISPR筛选以鉴定CD 8+和CD 4+命运和功能的调节剂。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的功能， 调节CD 8+和CD 4 + T细胞的分化。我们将使用定义的表型进行筛选， 启动和维持效应子和记忆分化的基因;指导记忆命运的承诺; 支配谱系选择;或调节细胞因子分泌的特定模式的获得。 目的3：在小鼠模型和人T细胞中寻找调节CD 8和CD 4分化的候选物 细胞为了验证这些候选调节剂，我们将使用一组广泛的细胞免疫学测定， 使用基因组编辑或常规敲除分别在体内和体外探测它们的功能， available.我们将通过CRISPR/Cas9介导的基因编辑来补充这些研究， 来自健康人类受试者的CD 4 + T细胞，以测试它们的调节作用在人类T细胞中是否是保守的。