Unveiling genes involved in the interaction between CD8+ T cells and antigen presenting cells across cytokine environments

揭示参与细胞因子环境中 CD8 T 细胞和抗原呈递细胞之间相互作用的基因

• 批准号: 2272896
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2272896
• 关键词: Unveiling; genes; involved; interaction; between

PhD project strategic theme: Understanding the rules of lifeThe human immune system is divided into two arms: innate and adaptive immunity. The innate immune system provides immediate host defence, while the adaptive immune system mounts a specific response to individual pathogens and it is regulated by B and T cells. CD8+ T cells are MHC Class I-restricted T lymphocytes involved the killing of cancerous cells and intracellular pathogens. Dendritic cells, macrophages and B-cells are referred to as professional antigen-presenting cells (APCs), which activate antigen-specific T cells during immune responses. While much is known about how APCs interact with T cells, it is still unclear how these interactions differ according to the pool of cytokines available in the environment. This project aims to unveil new genes and biological pathways involved in the CD8+:APC interaction in different cytokine environment. We will use CRISPR technology in high-throughput genome-wide genetic screens of CD8+ cells to introduce genetic perturbations across the genome and investigate their phenotypic consequences. We will establish a series of genome-wide (GW)-CRISPR knockout screens in primary CD8+ to investigate their interaction with APC in the presence or absence of TNF-a, IL12 and IFN-y. These cells will be sorted according to proliferation and exhaustion (measured by lack of proliferation and PD1 expression) levels and gRNAs will be sequenced and analysed to identify gene hits that are enriched in the population of interest. These experiments will identify, in an unbiased way, which genes contribute to the T cell-APC interaction in distinct cytokine milieu.GW-CRISPR screens will be followed by prioritization of approximately 20 response genes with the strongest enrichment scores in each cell-antibody context. T cells will be isolated and their genome edited by CRISPR/Cas9 to knockout these response genes. Cells will be exposed to inflammatory mediators or inhibitors and their transcriptome surveyed using single cell RNA-sequencing to obtain a high dimensional phenotype comparing wild-type and CRISPR-edited cells in the different conditions. Functional experiments will be designed to validate the biological pathways identified in CRISPR-screens. Taken together, these data will further our understanding how different cytokines interfere with the crosstalk between CD8+ and APC and as consequence their effector capabilities.

博士项目战略主题：了解生命的规则人类免疫系统分为两个武器：先天免疫和适应性免疫。先天性免疫系统提供直接的宿主防御，而适应性免疫系统对单个病原体产生特异性应答，并且它由B和T细胞调节。CD 8 + T细胞是参与杀死癌细胞和细胞内病原体的MHC I类限制性T淋巴细胞。树突状细胞、巨噬细胞和B细胞被称为专职抗原呈递细胞（APC），其在免疫应答期间激活抗原特异性T细胞。虽然关于APC如何与T细胞相互作用已经知道很多，但仍然不清楚这些相互作用如何根据环境中可用的细胞因子库而有所不同。该项目旨在揭示在不同细胞因子环境中参与CD 8+：APC相互作用的新基因和生物学途径。我们将在CD 8+细胞的高通量全基因组遗传筛选中使用CRISPR技术，在整个基因组中引入遗传扰动，并研究其表型后果。我们将在原代CD 8+中建立一系列全基因组（GW）-CRISPR敲除筛选，以研究它们在存在或不存在TNF-α、IL 12和IFN-γ的情况下与APC的相互作用。根据增殖和耗竭（通过缺乏增殖和PD 1表达测量）水平对这些细胞进行分选，并对gRNA进行测序和分析，以鉴定在目标群体中富集的基因命中。这些实验将以无偏的方式鉴定哪些基因在不同的细胞因子环境中有助于T细胞-APC相互作用。GW-CRISPR筛选之后将优先考虑在每个细胞-抗体环境中具有最强富集分数的大约20个应答基因。T细胞将被分离，并通过CRISPR/Cas9编辑其基因组以敲除这些应答基因。将细胞暴露于炎症介质或抑制剂，并使用单细胞RNA测序调查其转录组，以获得在不同条件下比较野生型和CRISPR编辑的细胞的高维表型。功能实验将被设计用于验证CRISPR筛选中鉴定的生物学途径。总之，这些数据将进一步我们了解不同的细胞因子如何干扰CD 8+和APC之间的串扰，并因此其效应器的能力。