Major histocompatibility complex class I-related protein 1 molecule (MR1) presents vitamin B metabolites that are synthesised by wide range of microbes. Presentation of MR1-metabolite complexes by cells which encounter bacteria activate the highly abundant innate-like T cells, mucosal associated invariant T (MAIT) cells which then secrete inflammatory cytokines and perform cytotoxic activity to clear the infection. When the infection is resolved and the metabolite antigen is no longer present, cell-surface MR1 is internalised and mostly degraded. This is hypothesised to be a crucial step in terminating the MAIT cell response. However, the cellular machinery that controls the internalisation of MR1 is not known.
To discover this machinery, we employed a genome-wide CRISPR-Cas9 library screen. We identified adaptor protein complex 2 (AP2) as a potential regulator of this process. To verify the significance of AP2 in MR1 internalisation, we deleted the AP2A1 protein in C1R cells, using CRISPR-Cas9 gene editing. These cells showed significantly reduced MR1 internalisation rate, resulting in a higher surface expression level. Impaired MR1 internalisation in AP2A1-deleted cells leads to the accumulation of MR1-ligand complex on the cell surface, in turn causing prolonged activation of MAIT cells even 24 hours after metabolite antigen exposure. Interestingly, the MR1 cytoplasmic tail does not contain the canonical sorting motif common to other AP2 substrates. In conclusion, MR1 internalisation is regulated by AP2 using a novel recognition motif. These results open the possibility of manipulating the internalisation of MR1 without affecting the trafficking properties of most AP2-regulated membrane proteins.