Dissection of HLA-agnostic antibacterial T-cell responses

Thomas, Hannah Lian 2024. Dissection of HLA-agnostic antibacterial T-cell responses. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Tuberculosis, caused by Mycobacterium Tuberculosis is one of the biggest threats to humanity in the 21st century, with the rise of antibiotic-resistance causing great concern for the future. I aimed to investigate unconventional antibacterial T-cell responses towards multiple species of bacteria in the hope of finding novel T-cell subsets that do not rely on conventional mechanisms of antigen presentation. I generated B2M and BTN3A knockouts for the A549 and THP-1 antigen presenting cell (APC) lines and optimised methods for loading them with M. smegmatis, S.typhimurium and S. aureus. These allowed investigation into anti-bacterial T-cell subsets that were not restricted by the known MHC class I, MR1, CD1a-d and MHC class I-like molecules. PBMC from three healthy donors were primed with these bacteria-loaded APC and responsive T-cells were sorted and their T-cell receptors (TCRs) were sequenced. Clonotyping of these TCRs showed expected enrichment of Mucosal associated invariant T (MAIT) and Vγ9Vδ2 T-cells with no obvious novel subsets of pan-donor or pan-bacteria antibacterial T-cell responses. Based on this, in collaboration with the University of Oxford, we used a ligand discovery pipeline to search for bacteria-derived de novo MR1 ligands. This revealed two candidate ligands, one of which stimulated the A-F7 MAIT TCR and the MR1-restricted cancer reactive MC.7.G5 TCR. In the hope to find more TCRs reactive towards cancer and this candidate ligand I searched for more MC.7.G5-like TCRs. I found several T-cell lines across multiple donors that exhibited a bias towards the TRAJ42 gene usage that generated a conserved CDR3α motif CAXYGGSQGNLIF (X = 3-5 amino acids). I also found that the MAIT A-F7 TCR shows anti-cancer reactivity. My findings suggest several exciting avenues to confirm the de novo MR1-ligands and utilise our LC-MS ligand discovery pipeline for further bacterial and/or cancer ligand discovery.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Medicine
Date of First Compliant Deposit:	1 April 2025
Last Modified:	01 Apr 2025 15:29
URI:	https://orca.cardiff.ac.uk/id/eprint/177322

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