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The intracellular pathway for the presentation of vitamin B–related antigens by the antigen-presenting molecule MR1

Nature Immunology volume 17, pages 531–537 (2016)Cite this article

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Abstract

The antigen-presenting molecule MR1 presents vitamin B–related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability.

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Figure 1: MR1 is recruited to the cell surface from a presynthesized intracellular pool.
Figure 2: Presentation of ligands produced by intracellular bacteria.
Figure 3: MR1 is stored as an immature protein in the ER.
Figure 4: Ligands promote MR1 folding and ER release.
Figure 5: Endocytosis and recycling of surface MR1.

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Acknowledgements

We thank T. Hansen (University of Washington) and W.J. Yankelevich (US Food and Drug Administration) for the gift of the 8F2.F9 hybridoma; L. Knodler (Washington State University College) for mCherry reagents; C. Dumont (University of Melbourne) for assistance with the internalization and recycling assays; H. Reid (Monash University) for assistance in cloning the MR1 K43R mutant; S. Londrigan (University of Melbourne) for assistance with the bronchial epithelial cells; and the Biological Optical Microscopy Platform (University of Melbourne) for microscopy expertise. S.B.G.E. is supported by an Early Career Research award from The University of Melbourne. This research was supported by the Australian National Health and Medical Research Council (NHMRC) (D.P.F., R.A.S., J.M., J.R. and J.A.V.) and the Australian Research Council (D.P.F. and J.R.).

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Author notes

  1. James McCluskey, Jamie Rossjohn and Jose A Villadangos: These authors jointly directed this work.

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Victoria, Australia

    Hamish E G McWilliam, Sidonia B G Eckle, Zhenjun Chen, Richard A Strugnell, James McCluskey & Jose A Villadangos

  2. Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    Hamish E G McWilliam, Alex Theodossis, Jacinta M Wubben & Jamie Rossjohn

  3. Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia

    Hamish E G McWilliam, Jacinta M Wubben & Jamie Rossjohn

  4. Australian Research Council (ARC) Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia

    Hamish E G McWilliam, Jacinta M Wubben & Jamie Rossjohn

  5. Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia

    Ligong Liu & David P Fairlie

  6. ARC Centre of Excellence in Advanced Molecular Imaging, The University of Queensland, Brisbane, Queensland, Australia

    Ligong Liu & David P Fairlie

  7. Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia

    Justine D Mintern & Jose A Villadangos

  8. Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK

    Jamie Rossjohn

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Contributions

H.E.G.M. undertook experiments, analyzed data and provided intellectual input. S.B.G.E., A.T., L.L., Z.C., J.M.W., D.P.F., R.A.S. and J.D.M. performed experiments, provided key reagents, analyzed data and/or provided intellectual input. J.M., J.R. and J.A.V. led the investigation and devised the project. H.E.G.M., J.R. and J.A.V. wrote the manuscript.

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Correspondence to James McCluskey, Jamie Rossjohn or Jose A Villadangos.

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Integrated supplementary information

Supplementary Figure 1 Production of the MAIT TCR tetramer

SDS-PAGE of E. coli-expressed TCR α- and β-chains of the MAIT TCR (upper panel). Non-reducing peak fraction lane 1, all fractions reducing lanes 3-8, including as a control 2 μg of reduced previously-generated irrelevant TCR (lane 2). SDS-PAGE of biotinylated and multimerised MAIT TCR (middle panel). Streptavidin alone (lane 1), TCR alone (lane 2), biotinylated TCR (lane 3) and multimerised TCR (lane 4). MonoQ anion exchange chromatography was used to purify biotinylated TCR (lower panel).

Supplementary Figure 2 MR1 and MHC class I surface expression kinetics in C1R.MR1 cells

MR1 cell surface expression (a) in C1R.MR1 cells over time measured with mAb 26.5 by flow cytometry, with MHC class I expression for comparison (b), and represented as geometric mean fluorescence intensity (gMFI) +/- standard error of the mean (s.e.m.). Cells were incubated with no ligand (upper graph) or with 10 μM Ac-6-FP (middle) or 5-OP-RU (lower), in the presence of brefeldin A, cycloheximide or without inhibitors.

Supplementary Figure 3 MR1-GFP remains immature until ligands promote maturation, similar to wild-type MR1

Analysis of Endo H-treated (+) or untreated (–) MR1-GFP precipitated from C1R cells transduced with MR1-GFP and cultured with or without 100 μM Ac-6-FP or 5-OP-RU for 6 hours. MR1-GFP was immunoprecipitated using anti-GFP agarose beads (GFP-Trap, Chromotek), and immunoblotted for GFP or β2m.

Supplementary Figure 4 MR1 trafficking schematic

In the steady state, MR1 predominantly resides as an immature protein in the endoplasmic reticulum (ER) in an unfolded conformation (1). Metabolite ligands derived from the extracellular medium or intracellular bacteria enter the ER via an unknown mechanism and load onto immature MR1 (2), where the charged lys43 (+) is neutralized and conformational folding occurs, including strong β2m association. Traffic through the Golgi apparatus results in further glycosylation (3). Mature MR1 remains at the cell surface for several hours (4) and is internalized to the endosomal compartment (5), with a low level of recycling back to the surface. MR1 may be degraded from the endosomes or directly from the ER (6).

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McWilliam, H., Eckle, S., Theodossis, A. et al. The intracellular pathway for the presentation of vitamin B–related antigens by the antigen-presenting molecule MR1. Nat Immunol 17, 531–537 (2016). https://doi.org/10.1038/ni.3416

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