Total synthesis of Mycobacterium tuberculosis dideoxymycobactin-838 and stereoisomers: diverse CD1a-restricted T cells display a common hierarchy of lipopeptide recognition

Mycobacterium tuberculosis produces dideoxymycobactin-838 (DDM-838), a lipopeptide that potently activates T cells upon binding to the MHC-like antigen-presenting molecule CD1a. M. tuberculosis produces DDM-838 in only trace amounts and a previous solid-phase synthesis provided sub-milligram quantities. We describe a high-yielding solution-phase synthesis of DDM-838 that features a Mitsunobu substitution that avoids yield-limiting epimerization at lysine during esterification, and amidation conditions that prevent double-bond isomerization of the Z-C20:1 acyl chain, and provides material with equivalent antigenicity to natural DDM-838. Isomers of DDM-838 that varied in stereochemistry at the central lysine and the C20:1 acyl chain were compared for their ability to be recognised by CD1a-restricted T cell receptors (TCRs). These TCRs, derived from unrelated human donors, exhibited a similar spectrum of reactivity towards the panel of DDM-838 isomers, highlighting the exquisite sensitivity of lipopeptide-reactive T cells for the natural DDM stereochemistry

Alcohol Abstinence Does Not Fully Reverse Abnormalities of Mucosal-Associated Invariant T Cells in the Blood of Patients With Alcoholic Hepatitis

OBJECTIVES: Alcoholic hepatitis (AH) develops in approximately 30% of chronic heavy drinkers. The immune system of patients with AH is hyperactivated, yet ineffective against infectious diseases. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that are highly enriched in liver, mucosa, and peripheral blood and contribute to antimicrobial immunity. We aimed to determine whether MAIT cells were dysregulated in heavy drinkers with and without AH and the effects of alcohol abstinence on MAIT cell recovery. METHODS: MR1 tetramers loaded with a potent MAIT cell ligand 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil were used in multiparameter flow cytometry to analyze peripheral blood MAIT cells in 59 healthy controls (HC), 56 patients with AH, and 45 heavy drinkers without overt liver disease (HDC) at baseline and 6- and 12-month follow-ups. Multiplex immunoassays were used to quantify plasma levels of cytokines related to MAIT cell activation. Kinetic Turbidimetric Limulus Amebocyte Lysate Assay and ELISA were performed to measure circulating levels of 2 surrogate markers for bacterial translocation (lipopolysaccharide and CD14), respectively. RESULTS: At baseline, patients with AH had a significantly lower frequency of MAIT cells than HDC and HC. HDC also had less MAIT cells than HC (median 0.16% in AH, 0.56% in HDC, and 1.25% in HC). Further, the residual MAIT cells in patients with AH expressed higher levels of activation markers (CD69, CD38, and human leukocyte antigen [HLA]-DR), the effector molecule granzyme B, and the immune exhaustion molecule PD-1. Plasma levels of lipopolysaccharide and CD14 and several cytokines related to MAIT cell activation were elevated in patients with AH (interferon [IFN]-α, interleukin [IL]-7, IL-15, IL-17, IL-18, IL-23, IFN-γ, and tumor necrosis factor α). Decreased MAIT cell frequency and upregulated CD38, CD69, and HLA-DR correlated negatively and positively, respectively, with aspartate aminotransferase level. MAIT cell frequency negatively correlated with IL-18. HLA-DR and CD38 levels correlated with several cytokines. At follow-ups, abstinent patients with AH had increased MAIT cell frequency and decreased MAIT cell activation. However, MAIT cell frequency was not fully normalized in patients with AH (median 0.31%). DISCUSSION: We showed that HDC had a reduction of blood MAIT cells despite showing little evidence of immune activation, whereas patients with AH had a severe depletion of blood MAIT cells and the residual cells were highly activated. Alcohol abstinence partially reversed those abnormalities

Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells

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

Role of innate T cells in anti-bacterial immunity

Innate T cells are a heterogeneous group of αβ and γδ T cells that respond rapidly (<2 h) upon activation. These innate T cells also share a non MHC class I or II restriction requirement for antigen recognition. Three major populations within the innate T cell group are recognized, namely, invariant NKT cells, mucosal associated invariant T cells, and gamma delta T cells. These cells recognize foreign/self-lipid presented by non-classical MHC molecules, such as CD1d, MR1, and CD1a. They are activated during the early stages of bacterial infection and act as a bridge between the innate and adaptive immune systems. In this review, we focus on the functional properties of these three innate T cell populations and how they are purposed for antimicrobial defense. Furthermore, we address the mechanisms through which their effector functions are targeted for bacterial control and compare this in human and murine systems. Lastly, we speculate on future roles of these cell types in therapeutic settings such as vaccination

Antigen-loaded MR1 tetramers define T cell receptor heterogeneity in mucosal-associated invariant T cells

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

Recognition of vitamin B metabolites by mucosal-associated invariant T cells

The mucosal-associated invariant T-cell antigen receptor (MAIT TCR) recognizes MR1 presenting vitamin B metabolites. Here we describe the structures of a human MAIT TCR in complex with human MR1 presenting a non-stimulatory ligand derived from folic acid and an agonist ligand derived from a riboflavin metabolite. For both vitamin B antigens, the MAIT TCR docks in a conserved manner above MR1, thus acting as an innate-like pattern recognition receptor. The invariant MAIT TCR a-chain usage is attributable to MR1-mediated interactions that prise open the MR1 cleft to allow contact with the vitamin B metabolite. Although the non-stimulatory antigen does not contact the MAIT TCR, the stimulatory antigen does. This results in a higher affinity of the MAIT TCR for a stimulatory antigen in comparison with a non-stimulatory antigen. We formally demonstrate a structural basis for MAIT TCR recognition of vitamin B metabolites, while illuminating how TCRs recognize microbial metabolic signatures

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses

Structural insight into MR1-mediated recognition of the mucosal associated invariant T cell receptor

Mucosal-associated invariant T (MAIT) cells express a semiinvariant αβ T cell receptor (TCR) that binds MHC class I-like molecule (MR1). However, the molecular basis for MAIT TCR recognition by MR1 is unknown. In this study, we present the crystal structure of a human Vα7.2Jα33-Vβ2 MAIT TCR. Mutagenesis revealed highly conserved requirements for the MAIT TCR-MR1 interaction across different human MAIT TCRs stimulated by distinct microbial sources. Individual residues within the MAIT TCR β chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR α chain controlled specificity through a small number of residues, which are conserved across species and located within the Vα-Jα regions. Mutagenesis of MR1 showed that only two residues, which were centrally positioned and on opposing sides of the antigen-binding cleft of MR1, were essential for MAIT cell activation. The mutagenesis data are consistent with a centrally located MAIT TCR-MR1 docking that was dominated by the α chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR-CD1d-antigen interaction, in which both the α and β chain of the NKT TCR is required for ligation above the F\u27-pocket of CD1d

The allopurinol metabolite, oxypurinol, drives oligoclonal expansions of drug‐reactive T cells in resolved hypersensitivity cases and drug‐naïve healthy donors

Allopurinol (ALP) is a successful drug used in the treatment of gout. However, this drug has been implicated in hypersensitivity reactions that can cause severe to life‐threatening reactions such as Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Individuals who carry the human leukocyte antigen (HLA)‐B*58:01 allotype are at higher risk of experiencing a hypersensitivity reaction (odds ratios ranging from 5.62 to 580.3 for mild to severe reactions, respectively). In addition to the parent drug, the metabolite oxypurinol (OXP) is implicated in triggering T cell‐mediated immunopathology via a labile interaction with HLA‐B*58:01. To date, there has been limited information regarding the T‐cell receptor (TCR) repertoire usage of reactive T cells in patients with ALP‐induced SJS or TEN and, in particular, there are no reports examining paired αβTCRs. Here, using in vitro drug‐treated PBMCs isolated from both resolved ALP‐induced SJS/TEN cases and drug‐naïve healthy donors, we show that OXP is the driver of CD8+ T cell‐mediated responses and that drug‐exposed memory T cells can exhibit a proinflammatory immunophenotype similar to T cells described during active disease. Furthermore, this response supported the pharmacological interaction with immune receptors (p‐i) concept by showcasing (i) the labile metabolite interaction with peptide/HLA complexes, (ii) immunogenic complex formation at the cell surface, and (iii) lack of requirement for antigen processing to elicit drug‐induced T cell responsiveness. Examination of paired OXP‐induced αβTCR repertoires highlighted an oligoclonal and private clonotypic profile in both resolved ALP‐induced SJS/TEN cases and drug‐naïve healthy donors