Screening for genes involved in CD8⁺ T cell cytotoxicity and characterisation of a super-killer
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 independent of changes to either signalling or degranulation but potentially the result of larger lytic granules containing elevated levels of lytic proteins. Whilst investigating Bach2 deficient splenocyte samples, I observed the presence of an increased proportion of central memory cells prior to stimulation, compared to WT samples. Cells stimulated from a central memory phenotype exhibited an increased lytic capacity when compared to cells stimulated from a naïve phenotype, which again appeared to be due to an increase in the size of lytic granules. This was interesting due to the fact that although memory cells are known to confer greater resistance to infection upon antigen re-encounter, the mechanisms responsible for this are largely unknown. It was concluded that the increased number of central memory cells in Bach2 deficient splenocyte samples contributed to but did not fully account for the increased cytotoxicity initially observed. I was also keen to expand and improve upon potential screening techniques. The incucyte Imaging platform was therefore employed as a means for increasing the speed and accuracy of high-throughput screening of mutant CTL. In order to optimise this screening method, I tested mice with mutations in genes relevant to NK cell development and function. The incucyte assay was able to provide a more accurate and efficient method for assessing CTL cytotoxicity and was used to assess a further 54 mutant lines from which 2 genes were identified as hits using local WT controls. These hits included Arpc1b, as identified in the previous screen, as well as Far1. Far1 is a fatty acid reductase that localises to peroxisomes and Far1 deficient CTL exhibited a decreased lytic capacity. These screens allowed for the identification of either defective or “super-killer” CTL and many of the hits identified were orphan genes, or genes with very little known about them, providing a rich resource for further investigation.
BACH2, High throughput screening assays, Super-killer, Cytotoxic T Lymphocytes
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