Single-molecule and super resolution investigations into the onco-immunogenic CCR5 receptor

Chemokine receptors play a key role in the inflammatory response, facilitating immune cell migration and recruitment via G-protein-mediated signalling triggered by chemokine recognition. Despite the immunogenic role that the CC chemokine receptor CCR5 plays in signal transduction, CCR5 has been implicated in the pathogenesis of a range of diseases,
notably acting as a key facilitator of the HIV-1 lifecycle. In addition, the CCR5-CCL5 chemotactic axis has been implicated in the formation of pro-tumorigenic microenvironments. Therapeutic targeting of CCR5 has seen the development of successful HIV-1 therapeutics considered for repurpose in cancer treatment. However, the advent of resistant HIV-1 strains necessitates our continued understanding of CCR5 behaviour and its interaction with binding partners. With several conformational pools being reported and little known of individual receptor characteristics, phenotypic investigations would benefit from advanced biophysical techniques capable of probing the receptor on a super-resolved and single-molecule level.

Structured illumination microscopy was used to visualise the super-resolved spatial distribution of CCR5 in adherent cells, revealing non-randomly distributed CCR5-enriched puncta across the membrane. Investigations employing a novel cell-line and imaging
technique, tailored to the single-molecule investigation of adherent cells, revealed the number of constituent receptors within individual puncta, indicating the presence of dimeric CCR5 sub-units.

Extension of these techniques to the study of CCL5, alongside the HIV-1 therapeutic Maraviroc and alternative prospective therapeutics, allowed the visualisation of CCL5 cell
association and ligand-induced effects on CCR5 presentation. Thereby affording insights into CCL5-induced internalisation and the interference of CCR5-targetting therapeutics on this process. These investigations provided evidence suggesting the CCL5-induced
reorganisation of CCR5 assemblies coinciding with internalisation and the non-interference of Maraviroc on redistribution despite its demonstrable antagonistic effect. Finally, this thesis presents findings from the attempted development of alternative CCR5-expressing cell lines tailored to assays of increased complexity and biological relevance.