Genome wide association studies have identified GPR65 risk loci with many immune-mediated diseases, including ankylosing spondylitis; however, its pathogenic role remains unknown. The inflamed synovial joint has a low pH signature, therefore understanding the role of this lymphocyte-expressed, proton-sensing GPCR in the context of joint inflammation is important.
With no commercial reagents available, I set out to generate monoclonal antibodies specific to human GPR65, with the aim to use them as tools in further studies. Phage display technology paired with novel antigen design, and multiple immunisation strategies proved challenging and largely unsuccessful in my hands. Despite terminal sera titres binding GPR65-expressing HEK cells, antigen-specificity was lost upon screening against primary cells.
In parallel, I have explored receptor expression on PBMC subsets and interrogated the biology of GPR65. Upon stimulation, cellular activation and pro-inflammatory cytokine release was suppressed from cells that express the highest levels GPR65 at pH6.5, compared to those at neutral pH. Due to their receptor expression profile, response to extracellular pH changes and involvement in disease, I focused on mucosal-associated invariant T (MAIT) cells. Significant partial recovery of pH-dependent IFNγ inhibition was observed, at transcriptional and protein levels, when silencing GPR65 in this cell subset; whilst at neutral pH, a pro-inflammatory role for GPR65 activity was proposed, with an increase of IL-17A production from TCR-stimulated MAIT cells detected
