Introduction IL-17 plays an important role in autoimmunity, promoting autoimmunity, inflammation and invasion in multiple sclerosis, rheumatoid arthritis and type I diabetes. The role of IL-17 in cancer is unclear, however, as there are few studies examining IL-17 protein expression in cancer. We therefore examined IL-17 protein expression in human breast cancer and modelled its potential biological significance in vitro.

Little is known of the regulation of interleukin-23 secretion in dendritic cells (DC) despite its importance for human Th17 responses. Here we show for first time that the Ataxia Telangiectasia Mutated (ATM) pathway, involved in DNA-damage-sensing, acts as an IL-23 repressor. Inhibition of ATM with the highly-selective antagonist, KU55933, markedly increased IL-23 secretion human monocyte-derived DC (moDC) and freshly isolated myeloid DC (myDC). In contrast, inhibiting the closely related mammalian target of rapamycin (mTOR) had no effect on IL-23. Priming naïve CD4+ T-cells with ATM-inhibited DC increased Th17 responses over and above those obtained with mature DC. Whilst ATM-blockade increased the abundance of p19, p35 and p40 mRNA, IL-12p70 secretion was unaffected. In order to further examine a role for ATM in IL-23 regulation we exposed DC to low doses of ionizing radiation. Exposure of DC to X-rays resulted in ATM phosphorylation and a corresponding depression of IL-23. Importantly, ATM-inhibition with KU55933 prevented radiation-induced ATM phosphorylation and abrogated the capacity of X-rays to suppress IL-23. To explore how ATM repressed IL-23 we examined a role for ER-stress responses by measuring generation of the spliced form of X-box protein-1 (XBP1s), a key ER-stress transcription factor. Inhibition of ATM increased the abundance of XBP1s mRNA and this was followed 3hr later by increased peak p19 transcription and IL-23 release. In summary, ATM-activation or inhibition respectively inhibited or augmented IL-23 release. This novel role of the ATM pathway represents a new therapeutic target in autoimmunity and vaccine development.

To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.