Newborn and child-like molecular signatures in older adults stem from TCR shifts across human lifespan.

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)αß signatures. Suboptimal TCRαß 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.

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.

The intracellular pathway for the presentation of vitamin B-related antigens by the antigen-presenting molecule MR1.

The antigen-presenting molecule MR1 presents vitamin B-related antigens (VitB antigens) to mucosal-associated invariant T (MAIT) cells through an uncharacterized pathway. We show that MR1, unlike other antigen-presenting molecules, does not constitutively present self-ligands. In the steady state it accumulates in a ligand-receptive conformation within the endoplasmic reticulum. VitB antigens reach this location and form a Schiff base with MR1, triggering a 'molecular switch' that allows MR1-VitB antigen complexes to traffic to the plasma membrane. These complexes are endocytosed with kinetics independent of the affinity of the MR1-ligand interaction and are degraded intracellularly, although some MR1 molecules acquire new ligands during passage through endosomes and recycle back to the surface. MR1 antigen presentation is characterized by a rapid 'off-on-off' mechanism that is strictly dependent on antigen availability.

A three-stage intrathymic development pathway for the mucosal-associated invariant T cell lineage.

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.

T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex.

Central to adaptive immunity is the interaction between the αß T cell receptor (TCR) and peptide presented by the major histocompatibility complex (MHC) molecule. Presumably reflecting TCR-MHC bias and T cell signaling constraints, the TCR universally adopts a canonical polarity atop the MHC. We report the structures of two TCRs, derived from human induced T regulatory (iT(reg)) cells, complexed to an MHC class II molecule presenting a proinsulin-derived peptide. The ternary complexes revealed a 180° polarity reversal compared to all other TCR-peptide-MHC complex structures. Namely, the iT(reg) TCR α-chain and ß-chain are overlaid with the α-chain and ß-chain of MHC class II, respectively. Nevertheless, this TCR interaction elicited a peptide-reactive, MHC-restricted T cell signal. Thus TCRs are not 'hardwired' to interact with MHC molecules in a stereotypic manner to elicit a T cell signal, a finding that fundamentally challenges our understanding of TCR recognition.

Adjusting Microscale to Atomic-Scale Structural Order in PbS Nanocrystal Superlattice for Enhanced Photodetector Performance.

An investigation is presented into the effect of the long-range order on the optoelectronic properties of PbS quantum dot (QD) superlattices, which form mesocrystals, for potential use in photodetector applications. By self-assembly of QD nanocrystals on an Si/SiOx substrate, a highly ordered and densely packed PbS QD superlattice with a microscale size is obtained. The results demonstrate that annealing treatment induces mesocrystalline superlattices with preferred growth orientation, achieved by dislodging ligands. The improved orientation and electronic coupling of the mesocrystalline superlattices exhibit superior photodetector performance compared to disordered QD structures and closely packed superlattices. This improved performance is attributed to atomic alignment between QDs, leading to enhanced electronic coupling. The findings suggest that these mesocrystalline superlattices have promising potential for the next generation of QD optoelectronic devices.

The balance of interleukin-12 and interleukin-23 determines the bias of MAIT1 versus MAIT17 responses during bacterial infection.

Mucosal-associated invariant T (MAIT) cells are a major subset of innate-like T cells mediating protection against bacterial infection through recognition of microbial metabolites derived from riboflavin biosynthesis. Mouse MAIT cells egress from the thymus as two main subpopulations with distinct functions, namely, T-bet-expressing MAIT1 and RORγt-expressing MAIT17 cells. Previously, we reported that inducible T-cell costimulator and interleukin (IL)-23 provide essential signals for optimal MHC-related protein 1 (MR1)-dependent activation and expansion of MAIT17 cells in vivo. Here, in a model of tularemia, in which MAIT1 responses predominate, we demonstrate that IL-12 and IL-23 promote MAIT1 cell expansion during acute infection and that IL-12 is indispensable for MAIT1 phenotype and function. Furthermore, we showed that the bias toward MAIT1 or MAIT17 responses we observed during different bacterial infections was determined and modulated by the balance between IL-12 and IL-23 and that these responses could be recapitulated by cytokine coadministration with antigen. Our results indicate a potential for tailored immunotherapeutic interventions via MAIT cell manipulation.

Hypersensitivities following allergen antigen recognition by unconventional T cells.

Conventional T cells recognise protein-derived antigens in the context of major histocompatibility complex (MHC) class Ia and class II molecules and provide anti-microbial and anti-tumour immunity. Conventional T cells have also been implicated in type IV (also termed delayed-type or T cell-mediated) hypersensitivity reactions in response to protein-derived allergen antigens. In addition to conventional T cells, subsets of unconventional T cells exist, which recognise non-protein antigens in the context of monomorphic MHC class I-like molecules. These include T cells that are restricted to the cluster of differentiation 1 (CD1) family members, known as CD1-restricted T cells, and mucosal-associated invariant T cells (MAIT cells) that are restricted to the MHC-related protein 1 (MR1). Compared with conventional T cells, much less is known about the immune functions of unconventional T cells and their role in hypersensitivities. Here, we review allergen antigen presentation by MHC-I-like molecules, their recognition by unconventional T cells, and the potential role of unconventional T cells in hypersensitivities. We also speculate on possible scenarios of allergen antigen presentation by MHC-I-like molecules to unconventional T cells, the hallmarks of such responses, and the expected frequencies of hypersensitivities within the human population.

Mucosal-Associated Invariant T Cells Augment Immunopathology and Gastritis in Chronic Helicobacter pylori Infection.

Mucosal-associated invariant T (MAIT) cells produce inflammatory cytokines and cytotoxic granzymes in response to by-products of microbial riboflavin synthesis. Although MAIT cells are protective against some pathogens, we reasoned that they might contribute to pathology in chronic bacterial infection. We observed MAIT cells in proximity to Helicobacter pylori bacteria in human gastric tissue, and so, using MR1-tetramers, we examined whether MAIT cells contribute to chronic gastritis in a mouse H. pylori SS1 infection model. Following infection, MAIT cells accumulated to high numbers in the gastric mucosa of wild-type C57BL/6 mice, and this was even more pronounced in MAIT TCR transgenic mice or in C57BL/6 mice where MAIT cells were preprimed by Ag exposure or prior infection. Gastric MAIT cells possessed an effector memory Tc1/Tc17 phenotype, and were associated with accelerated gastritis characterized by augmented recruitment of neutrophils, macrophages, dendritic cells, eosinophils, and non-MAIT T cells and by marked gastric atrophy. Similarly treated MR1-/- mice, which lack MAIT cells, showed significantly less gastric pathology. Thus, we demonstrate the pathogenic potential of MAIT cells in Helicobacter-associated immunopathology, with implications for other chronic bacterial infections.

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

T cells discriminate between foreign and host molecules by recognizing distinct microbial molecules, predominantly peptides and lipids. Riboflavin precursors found in many bacteria and yeast also selectively activate mucosal-associated invariant T (MAIT) cells, an abundant population of innate-like T cells in humans. 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.

Chemical composition database establishment and metabolite profiling analysis of Yangyin qingfei decoction.

The chemical fingerprinting and metabolite profile in a rat plasma sample after intragastric administration of Yangyin qingfei decoction (YYQFD, 14 g/kg) were investigated. First, YYQFD was analyzed by UPLC/Q-TOF MS to establish the chemical composition database by comparing their retention behavior, accurate molecular mass and MS2 data with those of references or known compounds in the literature. In this database, 100 chemical constituents with information on retention time, molecular mass, molecular formula, MS2 data and compound name were identified, which can provide compound information for further metabolite profiling studies. Furthermore, 64 compounds including 37 prototypes and 27 metabolites were detected in the dosed rat plasma sample, and the metabolic pathways of YYQFD were hydrolyzation, hydroxylation, dehydrogenation, glucuronidation, glucosylation, sulfation and mixed modes. Among the five component herbs in the YYQFD, Glycyrrhizae Radix et Rhizome and Fritillariae Thunbergii bulbs were actively metabolized, contributing 16 and 7 metabolites, respectively. It is suggested that chemical characterization and metabolite profiling studies are valuable to elucidate the material basis of herbal preparations.

An overview on the identification of MAIT cell antigens.

Mucosal associated invariant T (MAIT) cells are restricted by the monomorphic MHC class I-like molecule, MHC-related protein-1 (MR1). Until 2012, the origin of the MAIT cell antigens (Ags) was unknown, although it was established that MAIT cells could be activated by a broad range of bacteria and yeasts, possibly suggesting a conserved Ag. Using a combination of protein chemistry, mass spectrometry, cellular biology, structural biology and small molecule chemistry, we discovered MR1 ligands derived from folic acid (vitamin B9) and from an intermediate in the microbial biosynthesis of riboflavin (vitamin B2). While the folate derivative 6-formylpterin generally inhibited MAIT cell activation, two riboflavin pathway derivatives, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil and 5-(2-oxoethylideneamino)-6-D-ribitylaminouracil, were potent MAIT cell agonists. Other intermediates and derivatives of riboflavin synthesis displayed weak or no MAIT cell activation. Collectively, these studies revealed that in addition to peptide and lipid-based Ags, small molecule natural product metabolites are also ligands that can activate T cells expressing αß T-cell receptors, and here we recount this discovery.

Antigen ligation triggers a conformational change within the constant domain of the alphabeta T cell receptor.

Ligation of the alphabeta T cell receptor (TCR) by a specific peptide-loaded major histocompatibility complex (pMHC) molecule initiates T cell signaling via the CD3 complex. However, the initial events that link antigen recognition to T cell signal transduction remain unclear. Here we show, via fluorescence-based experiments and structural analyses, that MHC-restricted antigen recognition by the alphabeta TCR results in a specific conformational change confined to the A-B loop within the alpha chain of the constant domain (Calpha). The apparent affinity constant of this A-B loop movement mirrored that of alphabeta TCR-pMHC ligation and was observed in two alphabeta TCRs with distinct pMHC specificities. The Ag-induced A-B loop conformational change could be inhibited by fixing the juxtapositioning of the constant domains and was shown to be reversible upon pMHC disassociation. Notably, the loop movement within the Calpha domain, although specific for an agonist pMHC ligand, was not observed with a pMHC antagonist. Moreover, mutagenesis of residues within the A-B loop impaired T cell signaling in an in vitro system of antigen-specific TCR stimulation. Collectively, our findings provide a basis for the earliest molecular events that underlie Ag-induced T cell triggering.

T cell allorecognition via molecular mimicry.

T cells often alloreact with foreign human leukocyte antigens (HLA). Here we showed the LC13 T cell receptor (TCR), selected for recognition on self-HLA-B( *)0801 bound to a viral peptide, alloreacts with B44 allotypes (HLA-B( *)4402 and HLA-B( *)4405) bound to two different allopeptides. Despite extensive polymorphism between HLA-B( *)0801, HLA-B( *)4402, and HLA-B( *)4405 and the disparate sequences of the viral and allopeptides, the LC13 TCR engaged these peptide-HLA (pHLA) complexes identically, accommodating mimicry of the viral peptide by the allopeptide. The viral and allopeptides adopted similar conformations only after TCR ligation, revealing an induced-fit mechanism of molecular mimicry. The LC13 T cells did not alloreact against HLA-B( *)4403, and the single residue polymorphism between HLA-B( *)4402 and HLA-B( *)4403 affected the plasticity of the allopeptide, revealing that molecular mimicry was associated with TCR specificity. Accordingly, molecular mimicry that is HLA and peptide dependent is a mechanism for human T cell alloreactivity between disparate cognate and allogeneic pHLA complexes.

Immune self-reactivity triggered by drug-modified HLA-peptide repertoire.

Human leukocyte antigens (HLAs) are highly polymorphic proteins that initiate immunity by presenting pathogen-derived peptides to T cells. HLA polymorphisms mostly map to the antigen-binding cleft, thereby diversifying the repertoire of self-derived and pathogen-derived peptide antigens selected by different HLA allotypes. A growing number of immunologically based drug reactions, including abacavir hypersensitivity syndrome (AHS) and carbamazepine-induced Stevens-Johnson syndrome (SJS), are associated with specific HLA alleles. However, little is known about the underlying mechanisms of these associations, including AHS, a prototypical HLA-associated drug reaction occurring exclusively in individuals with the common histocompatibility allele HLA-B*57:01, and with a relative risk of more than 1,000 (refs 6, 7). We show that unmodified abacavir binds non-covalently to HLA-B*57:01, lying across the bottom of the antigen-binding cleft and reaching into the F-pocket, where a carboxy-terminal tryptophan typically anchors peptides bound to HLA-B*57:01. Abacavir binds with exquisite specificity to HLA-B*57:01, changing the shape and chemistry of the antigen-binding cleft, thereby altering the repertoire of endogenous peptides that can bind HLA-B*57:01. In this way, abacavir guides the selection of new endogenous peptides, inducing a marked alteration in 'immunological self'. The resultant peptide-centric 'altered self' activates abacavir-specific T-cells, thereby driving polyclonal CD8 T-cell activation and a systemic reaction manifesting as AHS. We also show that carbamazepine, a widely used anti-epileptic drug associated with hypersensitivity reactions in HLA-B*15:02 individuals, binds to this allotype, producing alterations in the repertoire of presented self peptides. Our findings simultaneously highlight the importance of HLA polymorphism in the evolution of pharmacogenomics and provide a general mechanism for some of the growing number of HLA-linked hypersensitivities that involve small-molecule drugs.

MR1 presents microbial vitamin B metabolites to MAIT cells.

Antigen-presenting molecules, encoded by the major histocompatibility complex (MHC) and CD1 family, bind peptide- and lipid-based antigens, respectively, for recognition by T cells. Mucosal-associated invariant T (MAIT) cells are an abundant population of innate-like T cells in humans that are activated by an antigen(s) bound to the MHC class I-like molecule MR1. Although the identity of MR1-restricted antigen(s) is unknown, it is present in numerous bacteria and yeast. Here we show that the structure and chemistry within the antigen-binding cleft of MR1 is distinct from the MHC and CD1 families. MR1 is ideally suited to bind ligands originating from vitamin metabolites. The structure of MR1 in complex with 6-formyl pterin, a folic acid (vitamin B9) metabolite, shows the pterin ring sequestered within MR1. Furthermore, we characterize related MR1-restricted vitamin derivatives, originating from the bacterial riboflavin (vitamin B2) biosynthetic pathway, which specifically and potently activate MAIT cells. Accordingly, we show that metabolites of vitamin B represent a class of antigen that are presented by MR1 for MAIT-cell immunosurveillance. As many vitamin biosynthetic pathways are unique to bacteria and yeast, our data suggest that MAIT cells use these metabolites to detect microbial infection.

Human leukocyte antigen class I-restricted activation of CD8+ T cells provides the immunogenetic basis of a systemic drug hypersensitivity.

The basis for strong immunogenetic associations between particular human leukocyte antigen (HLA) class I allotypes and inflammatory conditions like Behçet's disease (HLA-B51) and ankylosing spondylitis (HLA-B27) remain mysterious. Recently, however, even stronger HLA associations are reported in drug hypersensitivities to the reverse-transcriptase inhibitor abacavir (HLA-B57), the gout prophylactic allopurinol (HLA-B58), and the antiepileptic carbamazepine (HLA-B*1502), providing a defined disease trigger and suggesting a general mechanism for these associations. We show that systemic reactions to abacavir were driven by drug-specific activation of cytokine-producing, cytotoxic CD8+ T cells. Recognition of abacavir required the transporter associated with antigen presentation and tapasin, was fixation sensitive, and was uniquely restricted by HLA-B*5701 and not closely related HLA allotypes with polymorphisms in the antigen-binding cleft. Hence, the strong association of HLA-B*5701 with abacavir hypersensitivity reflects specificity through creation of a unique ligand as well as HLA-restricted antigen presentation, suggesting a basis for the strong HLA class I-association with certain inflammatory disorders.

Molecular architecture of the αß T cell receptor-CD3 complex.

αß T-cell receptor (TCR) activation plays a crucial role for T-cell function. However, the TCR itself does not possess signaling domains. Instead, the TCR is noncovalently coupled to a conserved multisubunit signaling apparatus, the CD3 complex, that comprises the CD3εγ, CD3εδ, and CD3ζζ dimers. How antigen ligation by the TCR triggers CD3 activation and what structural role the CD3 extracellular domains (ECDs) play in the assembled TCR-CD3 complex remain unclear. Here, we use two complementary structural approaches to gain insight into the overall organization of the TCR-CD3 complex. Small-angle X-ray scattering of the soluble TCR-CD3εδ complex reveals the CD3εδ ECDs to sit underneath the TCR α-chain. The observed arrangement is consistent with EM images of the entire TCR-CD3 integral membrane complex, in which the CD3εδ and CD3εγ subunits were situated underneath the TCR α-chain and TCR ß-chain, respectively. Interestingly, the TCR-CD3 transmembrane complex bound to peptide-MHC is a dimer in which two TCRs project outward from a central core composed of the CD3 ECDs and the TCR and CD3 transmembrane domains. This arrangement suggests a potential ligand-dependent dimerization mechanism for TCR signaling. Collectively, our data advance our understanding of the molecular organization of the TCR-CD3 complex, and provides a conceptual framework for the TCR activation mechanism.

Recognition of Vitamin B Precursors and Byproducts by Mucosal Associated Invariant T Cells.

Vitamin B2 (riboflavin) is essential for metabolic functions and is synthesized by many bacteria, yeast, and plants, but not by mammals and other animals, which must acquire it from the diet. In mammals, modified pyrimidine intermediates from the microbial biosynthesis of riboflavin are recognized as signature biomarkers of microbial infection. This recognition occurs by specialized lymphocytes known as mucosal associated invariant T (MAIT) cells. The major histocompatibility class I-like antigen-presenting molecule, MR1, captures these pyrimidine intermediates, but only after their condensation with small molecules derived from glycolysis and other metabolic pathways to form short-lived antigens. The resulting MR1-Ag complexes are recognized by MAIT cell antigen receptors (αß T cell receptors (TCRs)), and the subsequent MAIT cell immune responses are thought to protect the host from pathogens at mucosal surfaces. Here, we review our understanding of how these novel antigens are generated and discuss their interactions with MR1 and MAIT TCRs.

The structural basis for autonomous dimerization of the pre-T-cell antigen receptor.

The pre-T-cell antigen receptor (pre-TCR), expressed by immature thymocytes, has a pivotal role in early T-cell development, including TCR ß-selection, survival and proliferation of CD4(-)CD8(-) double-negative thymocytes, and subsequent αß T-cell lineage differentiation. Whereas αßTCR ligation by the peptide-loaded major histocompatibility complex initiates T-cell signalling, pre-TCR-induced signalling occurs by means of a ligand-independent dimerization event. The pre-TCR comprises an invariant α-chain (pre-Tα) that pairs with any TCR ß-chain (TCRß) following successful TCR ß-gene rearrangement. Here we provide the basis of pre-Tα-TCRß assembly and pre-TCR dimerization. The pre-Tα chain comprised a single immunoglobulin-like domain that is structurally distinct from the constant (C) domain of the TCR α-chain; nevertheless, the mode of association between pre-Tα and TCRß mirrored that mediated by the Cα-Cß domains of the αßTCR. The pre-TCR had a propensity to dimerize in solution, and the molecular envelope of the pre-TCR dimer correlated well with the observed head-to-tail pre-TCR dimer. This mode of pre-TCR dimerization enabled the pre-Tα domain to interact with the variable (V) ß domain through residues that are highly conserved across the Vß and joining (J) ß gene families, thus mimicking the interactions at the core of the αßTCR's Vα-Vß interface. Disruption of this pre-Tα-Vß dimer interface abrogated pre-TCR dimerization in solution and impaired pre-TCR expression on the cell surface. Accordingly, we provide a mechanism of pre-TCR self-association that allows the pre-Tα chain to simultaneously 'sample' the correct folding of both the V and C domains of any TCR ß-chain, regardless of its ultimate specificity, which represents a critical checkpoint in T-cell development. This unusual dual-chaperone-like sensing function of pre-Tα represents a unique mechanism in nature whereby developmental quality control regulates the expression and signalling of an integral membrane receptor complex.