MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens

Thierry Schmidlin; Enas Behiry; Hannah Thomas; Garry Dolton; Fabio Marino; Samiul Hasan; Magdalena von Essen; Rose M. Gathungu; Barbara A. Steigenberger; Hayden Selvadurai; Joseph Dukes; Paul E. Brennan; Owen B. Spiller; Jonathan D. Silk; Andrew K. Sewell; Nicola Ternette

doi:10.1016/j.crmeth.2025.101120

MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens

Thierry Schmidlin (Lead / Corresponding author)
, Enas Behiry
, Hannah Thomas
, Garry Dolton
, Fabio Marino
, Samiul Hasan
, Magdalena von Essen
, Rose M. Gathungu
, Barbara A. Steigenberger
, Hayden Selvadurai
, Joseph Dukes
, Paul E. Brennan
, Owen B. Spiller
, Jonathan D. Silk
, Andrew K. Sewell (Lead / Corresponding author)
, Nicola Ternette (Lead / Corresponding author)

Research output: Contribution to journal › Article › peer-review

5 Citations (Scopus)

89 Downloads (Pure)

Abstract

Major histocompatibility complex class I-related protein 1 (MR1) plays a central role in the immune recognition of infected cells and can mediate T cell detection of cancer. Knowledge of the nature of the ligands presented by MR1 is still sparse and has been limited by a lack of efficient approaches for MR1 ligand discovery. Here, we present a cross-linking strategy to investigate Schiff base-bound MR1 ligands. Our methodology employs reductive amination to stabilize the labile Schiff base bond between MR1 and its ligand, allowing for the detection of ligands as covalent MR1 adducts by mass spectrometry-based proteomics. We apply our approach to identifying vitamin B6 vitamers pyridoxal and pyridoxal 5′-phosphate (PLP) as MR1 ligands and show that both compounds are recognized by T cells expressing either A-F7, a mucosal-associated invariant T (MAIT) cell T cell receptor (TCR), or MC.7.G5, an MR1-restricted TCR reported to recognize cancer cells, highlighting them as immunogenic MR1 ligands.

Original language	English
Article number	101120
Journal	Cell Reports Methods
Volume	5
Issue number	8
Early online date	4 Aug 2025
DOIs	https://doi.org/10.1016/j.crmeth.2025.101120
Publication status	Published - 18 Aug 2025

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

antigen presentation
CP: Immunology
cross-linking
MAIT
mass spectrometry
metabolite antigens
MR1
pyridoxal
T cell
TCR
vitamin B6

ASJC Scopus subject areas

Biotechnology
Biochemistry
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Genetics
Radiology Nuclear Medicine and imaging
Computer Science Applications

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10.1016/j.crmeth.2025.101120Licence: CC BY

Final published version
Cell Reports Methods 5, 101120, August18, 2025© 2025 The Author(s). Published by Elsevier Inc. This is an open access article under the CCBY license (http://creativecommons.org/licenses/by/4.0/).
Final published version, 5.34 MBLicence: CC BY

Cite this

Schmidlin, T., Behiry, E., Thomas, H., Dolton, G., Marino, F., Hasan, S., von Essen, M., Gathungu, R. M., Steigenberger, B. A., Selvadurai, H., Dukes, J., Brennan, P. E., Spiller, O. B., Silk, J. D., Sewell, A. K., & Ternette, N. (2025). MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens. Cell Reports Methods, 5(8), Article 101120. https://doi.org/10.1016/j.crmeth.2025.101120

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title = "MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens",

abstract = "Major histocompatibility complex class I-related protein 1 (MR1) plays a central role in the immune recognition of infected cells and can mediate T cell detection of cancer. Knowledge of the nature of the ligands presented by MR1 is still sparse and has been limited by a lack of efficient approaches for MR1 ligand discovery. Here, we present a cross-linking strategy to investigate Schiff base-bound MR1 ligands. Our methodology employs reductive amination to stabilize the labile Schiff base bond between MR1 and its ligand, allowing for the detection of ligands as covalent MR1 adducts by mass spectrometry-based proteomics. We apply our approach to identifying vitamin B6 vitamers pyridoxal and pyridoxal 5′-phosphate (PLP) as MR1 ligands and show that both compounds are recognized by T cells expressing either A-F7, a mucosal-associated invariant T (MAIT) cell T cell receptor (TCR), or MC.7.G5, an MR1-restricted TCR reported to recognize cancer cells, highlighting them as immunogenic MR1 ligands.",

keywords = "antigen presentation, CP: Immunology, cross-linking, MAIT, mass spectrometry, metabolite antigens, MR1, pyridoxal, T cell, TCR, vitamin B6",

author = "Thierry Schmidlin and Enas Behiry and Hannah Thomas and Garry Dolton and Fabio Marino and Samiul Hasan and \{von Essen\}, Magdalena and Gathungu, \{Rose M.\} and Steigenberger, \{Barbara A.\} and Hayden Selvadurai and Joseph Dukes and Brennan, \{Paul E.\} and Spiller, \{Owen B.\} and Silk, \{Jonathan D.\} and Sewell, \{Andrew K.\} and Nicola Ternette",

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doi = "10.1016/j.crmeth.2025.101120",

language = "English",

volume = "5",

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Schmidlin, T, Behiry, E, Thomas, H, Dolton, G, Marino, F, Hasan, S, von Essen, M, Gathungu, RM, Steigenberger, BA, Selvadurai, H, Dukes, J, Brennan, PE, Spiller, OB, Silk, JD, Sewell, AK & Ternette, N 2025, 'MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens', Cell Reports Methods, vol. 5, no. 8, 101120. https://doi.org/10.1016/j.crmeth.2025.101120

TY - JOUR

T1 - MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens

AU - Schmidlin, Thierry

AU - Behiry, Enas

AU - Thomas, Hannah

AU - Dolton, Garry

AU - Marino, Fabio

AU - Hasan, Samiul

AU - von Essen, Magdalena

AU - Gathungu, Rose M.

AU - Steigenberger, Barbara A.

AU - Selvadurai, Hayden

AU - Dukes, Joseph

AU - Brennan, Paul E.

AU - Spiller, Owen B.

AU - Silk, Jonathan D.

AU - Sewell, Andrew K.

AU - Ternette, Nicola

PY - 2025/8/18

Y1 - 2025/8/18

N2 - Major histocompatibility complex class I-related protein 1 (MR1) plays a central role in the immune recognition of infected cells and can mediate T cell detection of cancer. Knowledge of the nature of the ligands presented by MR1 is still sparse and has been limited by a lack of efficient approaches for MR1 ligand discovery. Here, we present a cross-linking strategy to investigate Schiff base-bound MR1 ligands. Our methodology employs reductive amination to stabilize the labile Schiff base bond between MR1 and its ligand, allowing for the detection of ligands as covalent MR1 adducts by mass spectrometry-based proteomics. We apply our approach to identifying vitamin B6 vitamers pyridoxal and pyridoxal 5′-phosphate (PLP) as MR1 ligands and show that both compounds are recognized by T cells expressing either A-F7, a mucosal-associated invariant T (MAIT) cell T cell receptor (TCR), or MC.7.G5, an MR1-restricted TCR reported to recognize cancer cells, highlighting them as immunogenic MR1 ligands.

AB - Major histocompatibility complex class I-related protein 1 (MR1) plays a central role in the immune recognition of infected cells and can mediate T cell detection of cancer. Knowledge of the nature of the ligands presented by MR1 is still sparse and has been limited by a lack of efficient approaches for MR1 ligand discovery. Here, we present a cross-linking strategy to investigate Schiff base-bound MR1 ligands. Our methodology employs reductive amination to stabilize the labile Schiff base bond between MR1 and its ligand, allowing for the detection of ligands as covalent MR1 adducts by mass spectrometry-based proteomics. We apply our approach to identifying vitamin B6 vitamers pyridoxal and pyridoxal 5′-phosphate (PLP) as MR1 ligands and show that both compounds are recognized by T cells expressing either A-F7, a mucosal-associated invariant T (MAIT) cell T cell receptor (TCR), or MC.7.G5, an MR1-restricted TCR reported to recognize cancer cells, highlighting them as immunogenic MR1 ligands.

KW - antigen presentation

KW - CP: Immunology

KW - cross-linking

KW - MAIT

KW - mass spectrometry

KW - metabolite antigens

KW - MR1

KW - pyridoxal

KW - T cell

KW - TCR

KW - vitamin B6

UR - https://www.scopus.com/pages/publications/105013246866

U2 - 10.1016/j.crmeth.2025.101120

DO - 10.1016/j.crmeth.2025.101120

M3 - Article

C2 - 40763744

AN - SCOPUS:105013246866

VL - 5

JO - Cell Reports Methods

JF - Cell Reports Methods

IS - 8

M1 - 101120

ER -

MR1-ligand cross-linking identifies vitamin B6 metabolites as TCR-reactive antigens

Abstract

UN SDGs

Keywords

ASJC Scopus subject areas

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