Screening for genes involved in CD8⁺ T cell cytotoxicity and characterisation of a super-killer
type: CreativeWork
description: Cytotoxic T Lymphocytes (CTL) are cells of the adaptive immune system that are responsible for the elimination of virally infected, tumorigenic or damaged cells through mechanisms that induce target cell lysis. The aim of this project was to identify genes that can impact CTL lytic capacity, using an unbiased high throughput immunophenotyping screen of murine single gene deletion lines from the Wellcome trust Sanger Institute (WTSI) as part of the 3i consortium. I first optimised the cytotoxicity assay to be used for high-throughput screening, considering how to minimise labour and cost per sample, whilst achieving optimal reproducibility. I went on to successfully test 431 mutant mouse lines, of which 12 were identified as hits using local WT controls. These hits identified new pathways and organelles required for the modulation of T cell function and included 8 genes that, when knocked out, resulted in a decrease in lytic capacity of CTL. These included: Ctr9, a component of the paf complex implicated in transcriptional initiation, RNA processing and histone modification; D6wsu163e, an orphan gene; Dffb, involved in the apoptosis and survival caspase cascade; Grsf1, involved with mtRNA expression; Rbm33, an RNA binding protein; Trappc9 and Trappc10, members of the TRAPP complex implicated in ciliogenesis and Usp30, involved in mitophagy and pexophagy. A further 4 genes were identified as increasing the lytic capacity of CTL and included: Arpc1b, involved in actin filament branching; Bach2, a transcriptional regulator; Lrrc8d, a component of the volume-regulated anion channel and Oaf, an orphan gene. Bach2 deficient murine CTL exhibited a marked increase in lytic capacity and was chosen for further investigation. Bach2 codes for a transcriptional regulator and its genetic locus contains a region capable of binding super enhancers, exerting major influences on T cell mediated immune regulation. The enhanced cytotoxic capacity of Bach2 deficient CTL was found to be i…
abstract: Cytotoxic T Lymphocytes (CTL) are cells of the adaptive immune system that are responsible for the elimination of virally infected, tumorigenic or damaged cells through mechanisms that induce target cell lysis. The aim of this project was to identify genes that can impact CTL lytic capacity, using an unbiased high throughput immunophenotyping screen of murine single gene deletion lines from the Wellcome trust Sanger Institute (WTSI) as part of the 3i consortium. I first optimised the cytotoxicity assay to be used for high-throughput screening, considering how to minimise labour and cost per sample, whilst achieving optimal reproducibility. I went on to successfully test 431 mutant mouse lines, of which 12 were identified as hits using local WT controls. These hits identified new pathways and organelles required for the modulation of T cell function and included 8 genes that, when knocked out, resulted in a decrease in lytic capacity of CTL. These included: Ctr9, a component of the paf complex implicated in transcriptional initiation, RNA processing and histone modification; D6wsu163e, an orphan gene; Dffb, involved in the apoptosis and survival caspase cascade; Grsf1, involved with mtRNA expression; Rbm33, an RNA binding protein; Trappc9 and Trappc10, members of the TRAPP complex implicated in ciliogenesis and Usp30, involved in mitophagy and pexophagy. A further 4 genes were identified as increasing the lytic capacity of CTL and included: Arpc1b, involved in actin filament branching; Bach2, a transcriptional regulator; Lrrc8d, a component of the volume-regulated anion channel and Oaf, an orphan gene. Bach2 deficient murine CTL exhibited a marked increase in lytic capacity and was chosen for further investigation. Bach2 codes for a transcriptional regulator and its genetic locus contains a region capable of binding super enhancers, exerting major influences on T cell mediated immune regulation. The enhanced cytotoxic capacity of Bach2 deficient CTL was found to be i…
author: Barton, Philippa
language: eng
keywords: BACH2, High throughput screening assays, Super-killer, Cytotoxic T Lymphocytes
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/09702084-3654-4169-968e-64d3705d8c18/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2020-04-16
date modified: 2026-07-22T22:23:57Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.58506