Background – Colorectal cancer (CRC) is the second most common cause of cancer death. CD4+ T cells play an important role in anti-tumour immunity by promoting immune processes that can mediate tumour inhibition. CD4+ immunity to tumours, however, is not able to prevent tumour outgrowth. It is hypothesised that tumour outgrowth can occur due to weak recognition of tumour-derived epitopes by tumour-reactive T cell receptors (TCRs) and due to negative reg-ulation by inhibitory T cell molecules. In this study, CD4+ T cell responses to the oncofoetal antigen 5T4 are studied at the molecular level. The function of the co-inhibitory molecule LAG-3 is described biophysically and monoclonal antibodies which recognise LAG-3 were devel-oped. Results – Three 5T4-reactive CD4+ T cell clones were shown to recognise 5T4-derived pep-tides restricted to HLA-DR1. Each clone was sensitive to antigen and produced TH1 cytokines despite exhibiting weak recognition of cognate antigen. Subsequently, the structural character-istics of a 5T4-derived peptide epitope was described through x-ray crystallography which revealed insights into MHC-II presentation of peptides. Cell expressed LAG-3 was shown to interact with MHC-II at the cell surface and was characterised at the protein level using surface plasmon resonance (SPR) where LAG-3 bound MHC-II via an intermediate affinity interaction. Thirdly, through the immunisation of mice, anti-LAG-3 antibodies were cloned and character-ised in terms of their specificity and function. Conclusions – These studies demonstrate how tumour-specific CD4+ T cells can produce immune-stimulatory molecules in vitro yet exhibit weak engagement of cognate antigen. It is shown that peptide flanking residues of HLA-DR1 presented epitopes can contribute to peptide anchoring as well as form structural features that may influence TCR binding. It is shown that LAG-3 binds MHC-II at higher affinity than CD4 with implications in its inhibitory function. Finally, specific antibodies that bind LAG-3 have been characterised with potential for thera-peutic development.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:704925 |
| Date | January 2016 |
| Creators | MacLachlan, Bruce |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/98044/ |
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