T-cell activation by transitory neo-antigens derived from distinct microbial pathways

Subjects

Abstract

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids1,2,3,4. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells5,6, an abundant population of innate-like T cells in humans7,8,9. However, the genesis of these small organic molecules and their mode of presentation to MAIT cells by the major histocompatibility complex (MHC)-related protein MR1 (ref. 8) are not well understood. Here we show that MAIT-cell activation requires key genes encoding enzymes that form 5-amino-6-d-ribitylaminouracil (5-A-RU), an early intermediate in bacterial riboflavin synthesis. Although 5-A-RU does not bind MR1 or activate MAIT cells directly, it does form potent MAIT-activating antigens via non-enzymatic reactions with small molecules, such as glyoxal and methylglyoxal, which are derived from other metabolic pathways. The MAIT antigens formed by the reactions between 5-A-RU and glyoxal/methylglyoxal were simple adducts, 5-(2-oxoethylideneamino)-6-d-ribitylaminouracil (5-OE-RU) and 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), respectively, which bound to MR1 as shown by crystal structures of MAIT TCR ternary complexes. Although 5-OP-RU and 5-OE-RU are unstable intermediates, they became trapped by MR1 as reversible covalent Schiff base complexes. Mass spectra supported the capture by MR1 of 5-OP-RU and 5-OE-RU from bacterial cultures that activate MAIT cells, but not from non-activating bacteria, indicating that these MAIT antigens are present in a range of microbes. Thus, MR1 is able to capture, stabilize and present chemically unstable pyrimidine intermediates, which otherwise convert to lumazines, as potent antigens to MAIT cells. These pyrimidine adducts are microbial signatures for MAIT-cell immunosurveillance.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: The riboflavin pathway furnishes ligands that activate MAIT cells.
Figure 2: Chemical formation of pyrimidines and lumazines from condensation of small metabolites with 5-A-RU.
Figure 3: Structural basis of MR1-binding and recognition of transitory MAIT-cell antigens.
Figure 4: MR1–antigen tetramers and MAIT activation.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The atomic coordinates and structure factors for the TCR–MR1–antigen complexes have been deposited in the Protein Data Bank under accession numbers 4NQC, 4NQD and 4NQE.

References

  1. 1

    Brigl, M. & Brenner, M. B. CD1: antigen presentation and T cell function. Annu. Rev. Immunol. 22, 817–890 (2004)

  2. 2

    Yewdell, J. W. & Haeryfar, S. M. Understanding presentation of viral antigens to CD8+ T cells in vivo: the key to rational vaccine design. Annu. Rev. Immunol. 23, 651–682 (2005)

  3. 3

    Eckle, S. B. G., Turner, S. J., Rossjohn, J. & McCluskey, J. Predisposed αβ T cell antigen receptor recognition of MHC and MHC-I like molecules? Curr. Opin. Immunol. 25, 653–659 (2013)

  4. 4

    Rossjohn, J., Pellicci, D. G., Patel, O., Gapin, L. & Godfrey, D. I. Recognition of CD1d-restricted antigens by natural killer T cells. Nature Rev. Immunol. 12, 845–857 (2012)

  5. 5

    Kjer-Nielsen, L. et al. MR1 presents microbial vitamin B metabolites to MAIT cells. Nature 491, 717–723 (2012)

  6. 6

    Reantragoon, R. et al. Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells. J. Exp. Med. 210, 2305–2320 (2013)

  7. 7

    Gold, M. C. & Lewinsohn, D. M. Co-dependents: MR1-restricted MAIT cells and their antimicrobial function. Nature Rev. Microbiol. 11, 14–19 (2013)

  8. 8

    Treiner, E. et al. Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature 422, 164–169 (2003)

  9. 9

    Birkinshaw, R. W., Kjer-Nielsen, L., Eckle, S. B. G., McCluskey, J. & Rossjohn, J. MAITs, MR1 and vitamin B metabolites. Curr. Opin. Immunol. 26, 7–13 (2014)

  10. 10

    Vitreschak, A. G., Rodionov, D. A., Mironov, A. A. & Gelfand, M. S. Riboswitches: the oldest mechanism for the regulation of gene expression? Trends Genet. 20, 44–50 (2004)

  11. 11

    Burgess, C., O’Connell-Motherway, M., Sybesma, W., Hugenholtz, J. & van Sinderen, D. Riboflavin production in Lactococcus lactis: potential for in situ production of vitamin-enriched foods. Appl. Environ. Microbiol. 70, 5769–5777 (2004)

  12. 12

    Cushman, M. et al. Design, synthesis, and evaluation of 6-carboxyalkyl and 6-phosphonoxyalkyl derivatives of 7-oxo-8-ribitylaminolumazines as inhibitors of riboflavin synthase and lumazine synthase. J. Org. Chem. 67, 5807–5816 (2002)

  13. 13

    Bacher, A., Eberhardt, S., Fischer, M., Kis, K. & Richter, G. Biosynthesis of vitamin B2 (riboflavin). Annu. Rev. Nutr. 20, 153–167 (2000)

  14. 14

    Kis, K., Kugelbrey, K. & Bacher, A. Biosynthesis of riboflavin. The reaction catalyzed by 6,7-dimethyl-8-ribityllumazine synthase can proceed without enzymatic catalysis under physiological conditions. J. Org. Chem. 66, 2555–2559 (2001)

  15. 15

    Wang, Y. & Ho, C.-T. Flavour chemistry of methylglyoxal and glyoxal. Chem. Soc. Rev. 41, 4140–4149 (2012)

  16. 16

    Patel, O. et al. Recognition of vitamin B metabolites by mucosal-associated invariant T cells. Nature Commun. 4, 2142 (2013)

  17. 17

    Moody, D. B. et al. Cd1b-mediated T cell recognition of a glycolipid antigen generated from mycobacterial lipid and host carbohydrate during infection. J. Exp. Med. 192, 965–976 (2000)

  18. 18

    Hoiseth, S. K. & Stocker, B. A. D. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature 291, 238–239 (1981)

  19. 19

    Strugnell, R. et al. Characterization of a Salmonella typhimurium aro vaccine strain expressing the P.69 antigen of Bordetella pertussis. Infect. Immun. 60, 3994–4002 (1992)

  20. 20

    Plaut, G. W. E. & Harvey, R. A. in Methods in Enzymology Vol. 18, Part B (eds McCormick, D. B. & Wright, L. D. ) 515–538 (Academic, 1971)

  21. 21

    Huang, S. et al. Evidence for MR1 antigen presentation to mucosal-associated invariant T cells. J. Biol. Chem. 280, 21183–21193 (2005)

  22. 22

    Reantragoon, R. et al. Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor. J. Exp. Med. 209, 761–774 (2012)

  23. 23

    CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  24. 24

    McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007)

  25. 25

    Zwart, P. H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008)

  26. 26

    Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2004)

  27. 27

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

Download references

Acknowledgements

We thank the staff of the Monash crystallization facility and the Australian Synchrotron for assistance with crystallization and data collection, respectively, and M. J. McConville, P. O’Donnell and C.-S. Ang from the University of Melbourne Bio21 Institute mass spectrometry platform for assistance with mass spectrometry experiments. The Australian Research Council (ARC), a Program Grant and Project Grant of the National Health and Medical Research Council of Australia (NHMRC) supported this research; D.V.S. is a recipient of a Science Foundation Ireland (SFI) Principal Investigator award (ref. no. 08/IN.1/B1909); O.P. was supported by an ARC Future Fellowship; D.P.F. was supported by an NHMRC Senior Principal Research Fellowship (1027369); J.R. was supported by an NHMRC Australia Fellowship.

Author information

A.J.C., S.B.G.E., R.W.B. and L.L. are joint first authors. O.P., J.Ma., H.C., N.A.W., R.A.S., Z.C., R.R., B.M., D.V.S. and J.Y.W.M. either performed experiments, provided key reagents, and/or analysed data, and/or provided intellectual input or helped write the manuscript. D.P.F., L.K.N., J.Mc. and J.R. co-led the investigation and contributed to design and interpretation of data, project management, and writing of the manuscript. D.P.F., L.K.N., J.Mc. and J.R. are joint senior authors.

Correspondence to David P. Fairlie or Lars Kjer-Nielsen or Jamie Rossjohn or James McCluskey.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 MR1 ligand identification from different bacterial strains.

a, Detection of m/z 329.11 species in MR1 refolded with 5-A-RU and methylglyoxal (control), and supernatants from wild-type (CB013) and CB013-derivative (that is, ΔRibA, ΔRibB, ΔRibG or ΔRibH) L. lactis bacteria. Shown are counts on the y-axis versus retention time on the x-axis. b, Lack of activation of Jurkat.MAIT cells by supernatant from mutant ΔRibD/H S. typhimurium (strain SL1344) but not wild-type (WT), or ΔRibD/H plus RibD/H bacteria. Shown is MFI of CD69-APC on the y-axis. c, Detection of m/z 329.11 species in MR1 refolded with supernatants from wild-type, ΔRibD/H or ΔRibD/H plus RibD/H S. typhimurium bacteria, or control media. Shown are counts on the y-axis versus retention time on the x-axis. d, Detection of m/z 329.11 species in MR1 refolded with 5-A-RU and methylglyoxal (control), or bacterial supernatants from L. lactis (CB013) or E. faecalis bacteria, or control media. Shown are counts on the y-axis versus retention time on the x-axis. e, Detection of m/z 329.11 species in MR1 refolded with 5-A-RU and methylglyoxal (control), or supernatant from E. coli bacteria, or media. Shown are counts on the y-axis versus retention time on the x-axis. Experiments ae were performed three, three, three, two and three times, respectively.

Extended Data Figure 2 NMR characterization of 5-OP-RU.

ac, NMR characterization of 5-OP-RU (3d) in DMSO-d6 with internal solvent peak at 2.50 p.p.m. and 39.52 p.p.m. for 1H and 13C, respectively. a, 1H NMR (600 MHz); b, 13C NMR (150 MHz); c, HSQC. The compound 5-OP-RU (3d) was synthesized from the reaction of 5-A-RU and methylglyoxal in DMSO-d6, and then isolated from aqueous media by reversed-phase high-performance liquid chromatography (rpHPLC). Although it was less stable in water, it could still be identified and characterized at pH > 6.

Extended Data Figure 3 Stability of 5-OP-RU.

a, Reaction between 5-A-RU (0.5 mM) and methylglyoxal (3 equivalents (eq)) at pH 6.8, 37 °C in MilliQ water. b, Stability of purified 5-OP-RU (65 μM) at pH 6.8 and 37 °C. The half-life was 135 min. c, Stability of purified 5-OP-RU (65 μM) at variable pH in aqueous TBS buffer (10 mM Tris, 150 mM NaCl, pH 8.0), MilliQ water (pH 6.8) or ammonium acetate buffer (20 mM, pH 5.4) at 15 °C. The half-lives were 15 h at pH 8.0, 14.2 h at pH 6.8, 49 min at pH 5.4.

Extended Data Figure 4 Electron density for ligands, and contacts associated with MR1(K43A)–5-OP-RU MAIT TCR complex.

ah, Electron density of 5-OP-RU in MR1, 5-OE-RU in MR1 and 5-OP-RU in MR1(K43A). ac, Final 2Fo − Fc map, contoured at 1σ for 5-OP-RU (a) and 5-OE-RU (b) in the MAIT TCR–MR1–antigen complex, and 5-OP-RU (c) in the MAIT TCR–MR1(K43A)–antigen complex. df, Simulated annealing omit maps showing unbiased Fo − Fc electron density, contoured at 3σ, for 5-OP-RU (d) and 5-OE-RU (e) in MR1, and 5-OP-RU (f) in MR1(K43A). g, h, MR1(K43A)–5-OP-RU MAIT TCR complex showing contacts between MR1(K43A) and 5-OP-RU (g) and contacts between MAIT TCR and 5-OP-RU (h). MR1 is shown in grey, MAIT TCR CDR3α in yellow and CDR3β in orange with ribbon representation, and 5-OP-RU is shown in cyan with stick representation. Hydrogen bonds are indicated with black dashed lines with a water molecule mediating hydrogen bonding between the CDR3β 5-OP-RU shown in dark blue sphere representation.

Extended Data Figure 5 Chromatographic and mass spectrometry properties of MR1 ligands.

a, Ligand eluted from MR1 complexed with the product of (i) 5-A-RU and methylglyoxal condensation reaction (top); (ii) 5-A-RU and glyoxal condensation reaction (middle); or (iii) 5-A-RU and 13C-glycolaldehyde condensation reaction (bottom). Shown are extracted ion chromatograms (left); m/z spectrum (centre); and product ions from targeted fragmentation (right). Black diamonds indicate precursor ions. This experiment was performed three times. b, Mass spectrometry characterization of 5-OP-RU (top) and 5-OE-RU and 13C-5-OE-RU (bottom).

Extended Data Figure 6 Mass spectrometry of the 315.09 species.

Extracted ion chromatograms of m/z 315.09 species in MR1 refolded with 5-A-RU and glyoxal (control), or E. coli supernatant, or media. Shown are counts on the y-axis versus retention time on the x-axis. This experiment was performed three times.

Extended Data Figure 7 NMR characterization of RL.

ac, Spectra were recorded as a solution in D2O–CD3OD (9:1) with internal solvent peak at 3.31 p.p.m. and 49.0 p.p.m. for 1H and 13C, respectively. a, 1H NMR (600 MHz); b, 13C NMR (150 MHz); c, HMBC.

Extended Data Figure 8 NMR characterization of RL-7-Me.

ac, Spectra were recorded as a solution in D2O–CD3OD (9:1) with internal solvent peak at 3.31 p.p.m. and 49.0 p.p.m. for 1H and 13C, respectively. a, 1H NMR (600 MHz) and mechanism for deuterium exchange of CH3 at position 7. Identical exchange was also observed in pure D2O at slower rate (data not shown). b, 13C NMR (150 MHz) showing characteristic heptet from 7-CD3 after complete deuterium exchange. c, HMBC.

Extended Data Table 1 Data collection and refinement statistics

Supplementary information

Supplementary Information

This file contains a list of abbreviations and Supplementary Methods parts 1 and 2. (PDF 151 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Corbett, A., Eckle, S., Birkinshaw, R. et al. T-cell activation by transitory neo-antigens derived from distinct microbial pathways. Nature 509, 361–365 (2014). https://doi.org/10.1038/nature13160

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.