Introduction

MR1 is a unique non-classical class I molecule in that 1) it is monomorphic, 2) very little MR1 is on the cell surface and 3) it presents small molecule, non-peptidic antigens, some of which are derived from the riboflavin biosynthesis pathway. In airway epithelial cells (BEAS-2Bs) expressing MR1GFP, MR1 resides in the endoplasmic reticulum (ER) and in discrete intracellular vesicles. Currently, little is known about the precise trafficking of native MR1 or about MR1 recycling.

Methods

We used BEAS-2Bs and A549 MR1 knockout cells as our antigen presenting cells. Our exogenous antigen was filtered Mycobacterium smegmatis (Msmeg) supernatant. Antigen presentation was measured via IFN-γ release by MR1-restricted T cells (MR1Ts). For experiments utilizing MR1GFP, we used the plasmid TET-MR1GFP, which has a doxycycline inducible promoter to control MR1GFP synthesis.

Results

BEAS-2Bs treated overnight with the MR1 antagonist 6-formylpterin (6-FP) demonstrated an augmented response to Msmeg supernatant compared to controls. To understand the mechanism of this “boosting” effect, we transfected A549 MR1 knockout cells with TET-MR1GFP to see if new protein synthesis accounted for the increased response. We found that 6-FP pretreatment, even in the absence of new protein synthesis, resulted in more IFN-γ release by MR1Ts in response to Msmeg supernatant. Using flow cytometry we found that 6-FP treated cells have more MR1, indicating that the possible mechanism of 6-FP “boosting” is that MR1 is rescued from degradation and is then recycled for re-use.

Conclusions

Our data suggest that 6-FP rescues MR1 from degradation and that 6-FP loaded MR1 can be reloaded with exogenous mycobacterial antigen. This indicates the existence of a recycling pathway for MR1 and shows that MR1 can be loaded with antigen even when MR1 is outside of the ER.