Waters, Erin Andrea; (2023) The role of ligand and receptor dimerisation in control of CD28 and CTLA4 function. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The CD28-CTLA4 system is central to the regulation of T cell responses, with dysregulation associated with autoimmune disease and cancer progression. CD28 and CTLA4 are homodimeric T cell receptors providing co-stimulatory and inhibitory functions, respectively, through their shared ligands, CD80 and CD86. Whilst CD86 exists as a monomer, CD80 can form non-covalent dimers in the cell membrane and is therefore able to form higher-order lattice structures with CTLA4, providing an avidity advantage and a unique membrane organization compared to CD86. CD80 also interacts in cis with PDL1 at the CD80 dimer interface, although the function of this interaction is unknown. Functionally, CTLA4 removes CD80 and CD86 from opposing cells, targeting ligand for destruction via transendocytosis. However, why two ligands are required for this regulation, and how the properties of ligand:CTLA4 interactions contribute to this mechanism are poorly understood. We removed the avidity influence on receptor-ligand interactions by creating mutants unable to form functional dimers. We demonstrate that following transendocytosis, CD80 remains bound to CTLA4, resulting in ubiquitination and reduced CTLA4 availability. We show this is due to CD80:CTLA4 lattice formation, as dimer disruption by mutation or presence of PDL1 averts CTLA4 ubiquitination. We show loss of avid CD80:CTLA4 paradoxically enhances sensitivity to CTLA4 mediated transendocytosis, as the ability of CD80 to dissociate from CTLA4 following transendocytosis is augmented, permitting CTLA4 recycling and further transendocytosis - a feature normally seen with CD86. Finally, we detected signs of altered CD28 engagement resulting from mutant CD80 co-stimulation indicating monomeric ligands may have improved CD28 signalling. Our results provide further detail on CTLA4 transendocytosis, where dimerisation and avidity of ligand interactions, (including those regulated by PDL1), are significant in altering the fate of CTLA4, and influencing CD28 mediated co-stimulation.

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