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Title: Identifying regulators of cytotoxic T cell function through molecular and genetic screening
Author: Strege, Katharina
ISNI:       0000 0004 7968 407X
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Cytotoxic T lymphocytes (CTL) are crucial components of the adaptive immune system that kill infected and tumourigenic cells. CTL killing requires focused secretion of cytotoxic compounds from lytic granules. This process is known as degranulation. In this study, I aimed to establish the CRISPR-Cas9 gene editing technology in primary T cells and to optimise screening approaches to identify regulators of CTL killing. The first half of the thesis focuses on primary mouse CTL. The CRISPR technology was successfully optimised in CTL using Cas9-ribonucleoprotein complexes resulting in efficient CRISPR-mediated loss of target proteins. Genes encoding known mediators of CTL cytotoxicity, Rab27a Munc13-4 and Prf1, were targeted using CRISPR. The resulting samples were used to establish a flow cytometry-based assay that simultaneously measures CTL degranulation and target cell death. This assay enabled me to screen for mediators of CTL killing, while providing mechanistic insight by detecting degranulation. The screen was informed by a transcriptomic study that compared naive and effector CD8 T cells. 1803 significantly upregulated differentially expressed genes [log2(fold change) > 2] were identified. Functional annotation analysis and literature research were used to select genes for the targeted CRISPR screen, which highlighted the importance of HIF-1α and NFIL3 in CTL killing. The second half of the thesis focuses on primary human CTL. The combined degranulation and killing assay was further validated using patient-derived CTL, indicating its potential as a diagnostic test. I showed that the assay is suitable for mid-sized screens using a library of 64 compounds targeting the NF-κB signalling pathway. Further opportunities for increasing the scale of this screening technique are discussed. Finally, I successfully tested CRISPR using Cas9-ribonucleoprotein complexes in the human system. Additionally, stable Cas9 expression through lentiviral transduction was explored in primary CTL and related cell lines. This has the potential to allow selection of cells expressing the CRISPR machinery, providing a cleaner experimental system and the possibility of large-scale screening approaches. In summary, the techniques established in this thesis will be valuable for studying the genetics underlying CTL killing and the combined degranulation and killing assay furthermore shows great potential for diagnostic purposes.
Supervisor: Adams, David ; Griffiths, Gillian Sponsor: Medical Research Council ; Wellcome Sanger Institute
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Cytotoxic T cells ; CRISPR ; screening