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

人体免疫系统分为两部分：先天免疫和适应性免疫。先天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反应，并由B细胞和T细胞调节。CD8+ T细胞是MHC i类限制性T淋巴细胞，参与杀死癌细胞和细胞内病原体。树突状细胞、巨噬细胞和b细胞被称为专业抗原呈递细胞（apc），它们在免疫反应中激活抗原特异性T细胞。虽然人们对apc如何与T细胞相互作用了解甚多，但仍不清楚这些相互作用如何根据环境中可用的细胞因子池而有所不同。本项目旨在揭示不同细胞因子环境下参与CD8+:APC相互作用的新基因和生物学途径。我们将在CD8+细胞的高通量全基因组遗传筛选中使用CRISPR技术，以在基因组中引入遗传扰动并研究其表型后果。我们将在原发CD8+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-a、il - 12和IFN-y时与APC的相互作用。这些细胞将根据增殖和衰竭（通过缺乏增殖和PD1表达来测量）水平进行分类，并对grna进行测序和分析，以确定在感兴趣的群体中富集的基因命中。这些实验将以公正的方式确定哪些基因在不同的细胞因子环境中促进T细胞- apc相互作用。GW-CRISPR筛选之后，将对每种细胞抗体环境中富集分数最高的大约20个应答基因进行优先排序。将分离T细胞，并通过CRISPR/Cas9编辑其基因组以敲除这些应答基因。细胞将暴露于炎症介质或抑制剂中，并使用单细胞rna测序对其转录组进行调查，以获得在不同条件下比较野生型和crispr编辑细胞的高维表型。将设计功能性实验来验证crispr筛选中确定的生物学途径。综上所述，这些数据将进一步加深我们对不同细胞因子如何干扰CD8+和APC之间的串扰以及它们的效应能力的理解。

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.