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

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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.