Advertisement

MAIT Cells pp 191-207 | Cite as

Production of MR1 Tetramers Loaded with Microbial Ligands

  • Nicole A. Ladd
  • Cara A. Froyd
  • Erin J. AdamsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2098)

Abstract

In lieu of peptides, the monomorphic MHC-I-like molecule MR1 presents small molecule antigens to stimulate a subset of αβ T cells known as mucosal-associated (semi-) invariant T (MAIT) cells or, more broadly, MR1-restricted (MR1T) cells. The MR1 ligands identified to date are limited to derivatives and intermediates of the riboflavin and folate biosynthesis pathways and their presentation is therefore thought to be an indicator of infection by microbial species that can synthesize riboflavin. MAIT cells have, in recent years, been studied and isolated using a tetrameric reagent of recombinant MR1 loaded with the canonical ligand 5-OP-RU due to its potency toward MAIT clones. However, new evidence has shown that the repertoire of MR1 ligands is much more diverse than previously appreciated and, consistent with this, that the 5-OP-RU tetramer does not bind all MR1T cells. To study MR1-restricted T cell clones in the context of unique bacterial infection, we have generated a tetramer of MR1 loaded with diverse microbial antigens. The production of this reagent is detailed in this chapter.

Key words

MR1 MAIT Recombinant protein Microbes Mucosal immunity T Cell Receptor MR1T 

Notes

Acknowledgments

We would like to acknowledge the Bill and Melinda Gates Foundation for funding this protocol development, grant # OPP1131709, and the T32 Training Grant to the University of Chicago: GM007183. We thank members of the Lewinsohn Lab at Oregon Health and Science University, Dr. Melanie Harriff, Dr. Adrienne Luoma, Dr. Sobhan Roy, and Dr. Caitlin Castro for helpful discussions during protocol development.

References

  1. 1.
    Corbett AJ, Eckle SBD, Birkinshaw RW et al (2014) T-cell activation by transitory neo-antigens derived from distinct microbial pathways. Nature 509:361–365CrossRefGoogle Scholar
  2. 2.
    Harriff MJ, McMurtrey C, Froyd CA et al (2018) MR1 displays the microbial metabolome driving selective MR1-restricted T cell receptor usage. Sci Immunol 3:eaao2556.  https://doi.org/10.1126/sciimmunol.aao2556CrossRefPubMedGoogle Scholar
  3. 3.
    Treiner E, Duban L, Bahram S et al (2003) Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature 422:164–169CrossRefGoogle Scholar
  4. 4.
    Porcelli S, Yockey CE, Brenner MB et al (1993) Analysis of T cell antigen receptor (TCR) expression by human peripheral blood CD48 alpha/beta T cells demonstrates preferential use of several V beta genes and an invariant TCR alpha chain. J Exp Med 178:1–16CrossRefGoogle Scholar
  5. 5.
    Lopez-Sagaseta J, Dulberger CL, McFedries A et al (2013) MAIT recognition of a stimulatory bacterial antigen bound to MR1. J Immunol 191:5268–5277CrossRefGoogle Scholar
  6. 6.
    Lopez-Sagaseta J, Dulberger CL, Crooks JE et al (2013) The molecular basis for Mucosal-Associated Invariant T cell recognition of MR1 proteins. Proc Natl Acad Sci 110:E1771–E1778CrossRefGoogle Scholar
  7. 7.
    Gherardin NA, Keller AN, Woolley RE et al (2016) Diversity of T cells restricted by the MHC class I-related molecule MR1 facilitates differential antigen recognition. Immunity 44:32–45CrossRefGoogle Scholar
  8. 8.
    Murhammer DW (ed) (2007) Baculovirus and insect cell expression protocols, Methods in Mol Biol, vol 388. Springer, HeidelbergGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Nicole A. Ladd
    • 1
  • Cara A. Froyd
    • 1
  • Erin J. Adams
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of ChicagoChicagoUSA

Personalised recommendations