Microbiology & Immunology - Research Publications
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ItemNo Preview AvailableNewborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan(Nature Research, 2023-11)CD8+ T cells provide robust antiviral immunity, but how epitope-specific T cells evolve across the human lifespan is unclear. Here we defined CD8+ T cell immunity directed at the prominent influenza epitope HLA-A*02:01-M158-66 (A2/M158) across four age groups at phenotypic, transcriptomic, clonal and functional levels. We identify a linear differentiation trajectory from newborns to children then adults, followed by divergence and a clonal reset in older adults. Gene profiles in older adults closely resemble those of newborns and children, despite being clonally distinct. Only child-derived and adult-derived A2/M158+CD8+ T cells had the potential to differentiate into highly cytotoxic epitope-specific CD8+ T cells, which was linked to highly functional public T cell receptor (TCR)αβ signatures. Suboptimal TCRαβ signatures in older adults led to less proliferation, polyfunctionality, avidity and recognition of peptide mutants, although displayed no signs of exhaustion. These data suggest that priming T cells at different stages of life might greatly affect CD8+ T cell responses toward viral infections.
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ItemCD8 coreceptor engagement of MR1 enhances antigen responsiveness by human MAIT and other MR1-reactive T cells(Rockefeller University Press, 2022)Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αβ coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αβ enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αβ act as functional coreceptors for MAIT and other MR1-reactive T cells.
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ItemNo Preview AvailableDrugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells(NATURE PUBLISHING GROUP, 2017-04)The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals.
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ItemCharacterization of Human Mucosal-associated Invariant T (MAIT) Cells(Wiley-Blackwell, 2019)Mucosal‐associated invariant T (MAIT) cells are a subset of unconventional T cells restricted by the major histocompatibility complex (MHC) class I–like molecule MHC‐related protein 1 (MR1). MAIT cells are found throughout the body, especially in human blood and liver. Unlike conventional T cells, which are stimulated by peptide antigens presented by MHC molecules, MAIT cells recognize metabolite antigens derived from an intermediate in the microbial biosynthesis of riboflavin. MAIT cells mediate protective immunity to infections by riboflavin‐producing microbes via the production of cytokines and cytotoxicity. The discovery of stimulating MAIT cell antigens allowed for the development of an analytical tool, the MR1 tetramer, that binds specifically to the MAIT T cell receptor (TCR) and is becoming the gold standard for identification of MAIT cells by flow cytometry. This article describes protocols to characterize the phenotype of human MAIT cells in blood and tissues by flow cytometry using fluorescently labeled human MR1 tetramers alongside antibodies specific for MAIT cell markers.
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ItemAntigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells(ROCKEFELLER UNIV PRESS, 2013-10-21)Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) α-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH₂OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8α(+)CD8β(-/lo), implying predominant expression of CD8αα homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH₂OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in Vα19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-β repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.
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ItemDiverse MR1-restricted T cells in mice and humans.(Nature Research (part of Springer Nature), 2019-05-21)Mucosal-associated invariant T (MAIT) cells express an invariant TRAV1/TRAJ33 TCR-α chain and are restricted to the MHC-I-like molecule, MR1. Whether MAIT cell development depends on this invariant TCR-α chain is unclear. Here we generate Traj33-deficient mice and show that they are highly depleted of MAIT cells; however, a residual population remains and can respond to exogenous antigen in vitro or pulmonary Legionella challenge in vivo. These residual cells include some that express Trav1+ TCRs with conservative Traj-gene substitutions, and others that express Trav1- TCRs with a broad range of Traj genes. We further report that human TRAV1-2- MR1-restricted T cells contain both MAIT-like and non-MAIT-like cells, as judged by their TCR repertoire, antigen reactivity and phenotypic features. These include a MAIT-like population that expresses a public, canonical TRAV36+ TRBV28+ TCR. Our findings highlight the TCR diversity and the resulting potential impact on antigen recognition by MR1-restricted T cells.
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ItemNo Preview AvailableA three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage(NATURE PUBLISHING GROUP, 2016-11)Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.
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ItemA molecular basis underpinning the T cell receptor heterogeneity of mucosal-associated invariant T cells(ROCKEFELLER UNIV PRESS, 2014-07-28)Mucosal-associated invariant T (MAIT) cells express an invariant T cell receptor (TCR) α-chain (TRAV1-2 joined to TRAJ33, TRAJ20, or TRAJ12 in humans), which pairs with an array of TCR β-chains. MAIT TCRs can bind folate- and riboflavin-based metabolites restricted by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. However, the impact of MAIT TCR and MR1-ligand heterogeneity on MAIT cell biology is unclear. We show how a previously uncharacterized MR1 ligand, acetyl-6-formylpterin (Ac-6-FP), markedly stabilized MR1, potently up-regulated MR1 cell surface expression, and inhibited MAIT cell activation. These enhanced properties of Ac-6-FP were attributable to structural alterations in MR1 that subsequently affected MAIT TCR recognition via conformational changes within the complementarity-determining region (CDR) 3β loop. Analysis of seven TRBV6-1(+) MAIT TCRs demonstrated how CDR3β hypervariability impacted on MAIT TCR recognition by altering TCR flexibility and contacts with MR1 and the Ag itself. Ternary structures of TRBV6-1, TRBV6-4, and TRBV20(+) MAIT TCRs in complex with MR1 bound to a potent riboflavin-based antigen (Ag) showed how variations in TRBV gene usage exclusively impacted on MR1 contacts within a consensus MAIT TCR-MR1 footprint. Moreover, differential TRAJ gene usage was readily accommodated within a conserved MAIT TCR-MR1-Ag docking mode. Collectively, MAIT TCR heterogeneity can fine-tune MR1 recognition in an Ag-dependent manner, thereby modulating MAIT cell recognition.
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ItemAntigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells(Rockefeller University Press, 2013)Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) alpha-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH2OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8 alpha(+)CD8 beta(-/lo), implying predominant expression of CD8 alpha alpha homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH2OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in V. 19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-beta repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.