# Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

## TL;DR

This study shows that mutations in MR1 affect how it presents different types of antigens to MAIT cells, with exogenous antigens being more sensitive to these changes.

## Contribution

The study reveals that MR1 mutations differentially impact exogenous and intracellular antigen presentation through distinct molecular mechanisms.

## Key findings

- MR1 mutations at amino acids 9-16 impair exogenous antigen presentation but preserve endosomal antigen presentation.
- MR1's interaction with B2M is critical for exogenous antigen presentation but not for intracellular antigens.
- Endosomal antigen presentation is less sensitive to B2M deficiency and may depend on MR1 availability.

## Abstract

The antigen presenting molecule major histocompatibility complex class I–related protein 1 (MR1) binds small molecule metabolites derived from the microbial riboflavin biosynthetic pathway and presents them at the cell surface for surveillance by MR1-restricted mucosal-associated invariant T cells (MAIT cells). MR1 ligands can originate in the extracellular space or in endosomal compartments that contain microbial pathogens. Distinct, complementary antigen processing and presentation pathways enable MR1 to survey diverse intracellular locations and present both exogenous and intracellular antigens. Here, we generated a panel of BEAS-2B MR1 KO cells reconstituted with MR1 proteins mutated at amino acids 9 to 16. The overexpressed mutated MR1 molecules differentially translocated to the cell surface in response to 6-formylpterin and differed in their ability to present mycobacterial antigens to MAIT cell clones. While they barely presented Mycobacterium smegmatis supernatant and other exogenous MAIT cell antigens, their ability to present antigens derived from mycobacterial infection and a 5-amino-6-D-ribitylaminouracil prodrug that requires endosomal processing remained largely intact. Protein coimmunoprecipitation and mass spectrometry-based proteomic analysis showed that mutated MR1 differentially associated with calnexin and β2-microglobulin (B2M). Knock down of B2M in cells overexpressing wildtype MR1 phenocopied the loss of exogenous antigen presentation but did not impact presentation of intracellular antigens. Thus, the MR1-mediated presentation of exogenous antigen appears to be limited by binding to B2M whereas a lower sensitivity to B2M deficiency suggests that MAIT cell activation via the endosomal antigen presentation pathway may be limited by the availability of MR1 itself.