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Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host–microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d–BfaGC–NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system. The symbiotic gut bacterium Bacteroides fragilis produces unique α-galactosylceramides from host dietary branched-chain amino acids, which are presented as CD1d ligands and immunomodulate natural killer T cells.

The Major Histocompatibility Complex class I-related protein 1 (MR1) presents small molecule metabolites, drugs, and drug-like molecules that are recognized by MR1-reactive T cells. While we have an understanding of how antigens bind to MR1 and upregulate MR1 cell surface expression, a quantitative, cell-free, assessment of MR1 ligand-binding affinity was lacking. Here, we developed a fluorescence polarization-based assay in which fluorescent MR1 ligand was loaded into MR1 protein in vitro and competitively displaced by candidate ligands over a range of concentrations. Using this assay, ligand affinity for MR1 could be differentiated as strong (IC

The two main T cell lineages, αβ and γδ T cells, play a central role in immunity. Unlike αβ T cells that recognize antigens bound to the Major Histocompatibility Complex (MHC) or MHC class I-like antigen-presenting molecules, the ligands for γδ T cell receptors (TCRs) are much more diverse. However, it is now clear that γδ TCRs can also recognize MHC class I-like molecules, including CD1b, CD1c, CD1d and the MHC class I-related protein 1 (MR1). Yet, our understanding at the molecular level of γδ T cell immunity to CD1 and MR1 is still very limited. Here, we discuss new molecular paradigms underpinning γδ TCRs recognition of antigens, antigen-presenting molecules or both. The recent discovery of recognition of MR1 by a γδ TCR at a position located underneath the antigen display platform reinforces the view that γδ TCRs can approach their ligands from many directions, unlike αβ TCRs that bind MHC, CD1 and MR1 targets in an aligned, end to end fashion.

MR1 presents vitamin B-related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2+ αβ T cell receptor (TCR). In addition, a more diverse TRAV1-2- MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2- TCRs interact with MR1-antigen complexes remains unclear. Here, we describe how a TRAV12-2+ TCR (termed D462-E4) recognizes an MR1-antigen complex. We report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. Here, the TRBV29-1 β-chain of the D462-E4 TCR binds over the F'-pocket of MR1, whereby the complementarity-determining region (CDR) 3β loop surrounded and projected into the F'-pocket. Nevertheless, the CDR3β loop anchored proximal to the MR1 A'-pocket and mediated direct contact with the 5-OP-RU antigen. The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2+ and TRAV36+ TCRs' docking topologies on MR1. Accordingly, diverse MR1-restricted T cell repertoire reveals differential docking modalities on MR1, thus providing greater scope for differing antigen specificities.

Mucosal-associated invariant T (MAIT) cells are activated by microbial riboflavin-based metabolite antigens when presented by MR1. How modifications to the potent antigen 5-OP-RU affect presentation by MR1 and MAIT cell activation remains unclear. Here we design 20 derivatives, termed altered metabolite ligands (AMLs), to dissect the impact of different antigen components on the human MAIT-MR1 axis. Analysis of 11 crystal structures of MAIT T cell antigen receptor (TCR)-MR1-AML ternary complexes, along with biochemical and functional assays, shows that MR1 cell-surface upregulation is influenced by ribityl and non-ribityl components of the ligand and the hydrophobicity of the MR1-AML interface. The polar ribityl chain of the AML strongly influences MAIT cell activation potency through dynamic compensatory interactions within a MAIT TCR-MR1-AML interaction triad. We define the basis by which the MAIT TCR can differentially recognize AMLs, thereby providing insight into MAIT cell antigen specificity and potency.

Type I and type II natural killer T (NKT) cells are restricted to the lipid antigen-presenting molecule CD1d. While we have an understanding of the antigen reactivity and function of type I NKT cells, our knowledge of type II NKT cells in health and disease remains unclear. Here we describe a population of type II NKT cells that recognise and respond to the microbial antigen, α-glucuronosyl-diacylglycerol (α-GlcADAG) presented by CD1d, but not the prototypical type I NKT cell agonist, α-galactosylceramide. Surprisingly, the crystal structure of a type II NKT TCR-CD1d-α-GlcADAG complex reveals a CD1d F’-pocket-docking mode that contrasts sharply with the previously determined A’-roof positioning of a sulfatide-reactive type II NKT TCR. Our data also suggest that diverse type II NKT TCRs directed against distinct microbial or mammalian lipid antigens adopt multiple recognition strategies on CD1d, thereby maximising the potential for type II NKT cells to detect different lipid antigens., Natural killer T (NKT) cells include type I that express semi-invariant T cell receptor (TCR), and type II that cover a broader repertoire. Here the authors describe the crystal structure of a type II NKT TCR complexed with CD1d/antigen to propose that type II NKT TCRs may adapt multiple CD1d docking modes to maximise antigen recognition efficacy.

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.

T cells represent a critical arm of our immune defense against pathogens. Over the past two decades, considerable inroads have been made in understanding the fundamental principles underpinning the molecular presentation of peptide-based antigens by the Major Histocompatibility Complex molecules (MHC-I and II), and their molecular recognition by specialized subsets of T cells. However, some T cells can recognize lipid-based antigens presented by MHC-I-like molecules that belong to the Cluster of Differentiation 1 (CD1) family. Here, we will review the advances that have been made in the last five years to understand the molecular mechanisms orchestrating the presentation of novel endogenous and exogenous lipid-based antigens by the CD1 glycoproteins and their recognition by specific populations of CD1-reactive T cells.

CD1a-autoreactive T cells contribute to skin disease, but the identity of immunodominant self-lipid antigens and their mode of recognition are not yet solved. In most models, MHC and CD1 proteins serve as display platforms for smaller antigens. Here, we showed that CD1a tetramers without added antigen stained large T cell pools in every subject tested, accounting for approximately 1% of skin T cells. The mechanism of tetramer binding to T cells did not require any defined antigen. Binding occurred with approximately 100 lipid ligands carried by CD1a proteins, but could be tuned upward or downward with certain natural self-lipids. TCR recognition mapped to the outer A' roof of CD1a at sites remote from the antigen exit portal, explaining how TCRs can bind CD1a rather than carried lipids. Thus, a major antigenic target of CD1a T cell autoreactivity in vivo is CD1a itself. Based on their high frequency and prevalence among donors, we conclude that CD1a-specific, lipid-independent T cells are a normal component of the human skin T cell repertoire. Bypassing the need to select antigens and effector molecules, CD1a tetramers represent a simple method to track such CD1a-specific T cells from tissues and in any clinical disease.

CD1a molecules capture lipid classes that can prevent the binding of autoreactive T cell antigen receptors., CD1a-autoreactive T cells represent a significant proportion of circulating αβ T cells in humans and appear to be enriched in the skin. How their autoreactivity is regulated remains unclear. In this issue of JEM, Cotton et al. (2021. J. Exp. Med. https://doi.org/10.1084/jem.20202699) show that CD1a molecules do not randomly survey cellular lipids but instead capture certain lipid classes that broadly interfere with the binding of autoreactive T cell antigen receptors to the target CD1a. These findings provide new potential therapeutic avenues for manipulating CD1a autoreactive T cell responses.

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.

In non-eutherians, a third type of T cell The two established T cell lineages found in jawed vertebrates use either an αβ or a γδ T cell receptor (TCR) to detect antigens. Recently, another type of TCR chain (TCRµ) was found in marsupials and monotremes. Morrissey et al. analyzed T cells from the gray short-tailed opossum and uncovered a third lineage resident in the spleen that uses a γµ TCR (see the Perspective by Criscitiello). The authors then characterized the crystal structures of two different γµ TCRs, which exhibited an architecture distinct from αβ or γδ TCRs in which a highly diverse, unpaired immunoglobulin-like variable domain was predicted to be the major antigen recognition determinant. Like camelid VHH and shark IgNAR antibodies, γµ TCRs could potentially inform future nanobody development. Science , this issue p. 1383 ; see also p. 1308

The role unconventional T cells play in protective immunity in humans is unclear. Mucosal-associated invariant T (MAIT) cells are an unconventional T cell subset restricted to the antigen-presenting molecule MR1. Here, we report the discovery of a patient homozygous for a rare Arg31His (R9H in the mature protein) mutation in MR1 who has a history of difficult-to-treat viral and bacterial infections. MR1R9H was unable to present the potent microbially derived MAIT cell stimulatory ligand. The MR1R9H crystal structure revealed that the stimulatory ligand cannot bind due to the mutation lying within, and causing structural perturbation to, the ligand-binding domain of MR1. While MR1R9H could bind and be up-regulated by a MAIT cell inhibitory ligand, the patient lacked circulating MAIT cells. This shows the importance of the stimulatory ligand for MAIT cell selection in humans. The patient had an expanded γδ T cell population, indicating a compensatory interplay between these unconventional T cell subsets.

MR1 presents vitamin B–related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2(+) αβ T cell receptor (TCR). In addition, a more diverse TRAV1-2(−) MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2(−) TCRs interact with MR1–antigen complexes remains unclear. Here, we describe how a TRAV12-2(+) TCR (termed D462-E4) recognizes an MR1–antigen complex. We report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. Here, the TRBV29-1 β-chain of the D462-E4 TCR binds over the F′-pocket of MR1, whereby the complementarity-determining region (CDR) 3β loop surrounded and projected into the F′-pocket. Nevertheless, the CDR3β loop anchored proximal to the MR1 A′-pocket and mediated direct contact with the 5-OP-RU antigen. The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2(+) and TRAV36(+) TCRs' docking topologies on MR1. Accordingly, diverse MR1-restricted T cell repertoire reveals differential docking modalities on MR1, thus providing greater scope for differing antigen specificities.

The major histocompatibility complex (MHC) class-I related molecule MR1 is a monomorphic and evolutionary conserved antigen (Ag)-presenting molecule that shares the overall architecture of MHC-I and CD1 proteins. However, in contrast to MHC-I and the CD1 family that present peptides and lipids, respectively, MR1 specifically presents small organic molecules. During microbial infection of mammalian cells, MR1 captures and presents vitamin B precursors, derived from the microbial biosynthesis of riboflavin, on the surface of antigen-presenting cells. These MR1-Ag complexes are recognized by the mucosal-associated invariant T cell receptor (MAIT TCR), which subsequently leads to MAIT cell activation. Recently, MR1 was shown to trap chemical scaffolds including drug and drug-like molecules. Here, we review this metabolite Ag-presenting molecule and further define the key molecular interactions underlying the recognition and reactivity of MAIT TCRs to MR1 in an Ag-dependent manner.

During industrialization, humans have been exposed to increasing numbers of foreign chemicals. Failure of the immune system to tolerate drugs, cosmetics, and other skin products causes allergic contact dermatitis, a T cell-mediated disease with rising prevalence. Models of αβ T cell response emphasize T cell receptor (TCR) contact with peptide-MHC complexes, but this model cannot readily explain activation by most contact dermatitis allergens, which are nonpeptidic molecules. We tested whether CD1a, an abundant MHC I-like protein in human skin, mediates contact allergen recognition. Using CD1a-autoreactive human αβ T cell clones to screen clinically important allergens present in skin patch testing kits, we identified responses to balsam of Peru, a tree oil widely used in cosmetics and toothpaste. Additional purification identified benzyl benzoate and benzyl cinnamate as antigenic compounds within balsam of Peru. Screening of structurally related compounds revealed additional stimulants of CD1a-restricted T cells, including farnesol and coenzyme Q2. Certain general chemical features controlled response: small size, extreme hydrophobicity, and chemical constraint from rings and unsaturations. Unlike lipid antigens that protrude to form epitopes and contact TCRs, the small size of farnesol allows sequestration deeply within CD1a, where it displaces self-lipids and unmasks the CD1a surface. These studies identify molecular connections between CD1a and hypersensitivity to consumer products, defining a mechanism that could plausibly explain the many known T cell responses to oily substances.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1+ TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1–encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag.

Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d–α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1+ type I NKT cell repertoire. These cells express a range of TCR α- and β-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7–8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A′-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d–α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition., The invariant αβTCR of type I NKT cells recognizes a lipid α-GalCer presented by CD1d. Here the authors describe atypical α-GalCer-reactive NKT cells with diverse TCRs, which bind to CD1d-α-GalCer in a manner distinct from type I NKT cells, thus unveiling greater diversity in lipid antigen recognition.

A different way for γδ T cells to bind The ligands bound by γδ T cell receptors (TCRs) are less well characterized than those of their αβ TCR cousins, which are antigens presented by major histocompatibility complex (MHC) and related proteins. Le Nours et al. identified a phenotypically diverse γδ T cell subset in human tissues that reacts to MHC-related protein 1 (MR1), which presents vitamin B derivatives. A crystal structure of a γδ TCR–MR1–antigen complex revealed that some of these TCRs can bind underneath the MR1 antigen-binding cleft instead of recognizing the presented antigen. This work thus uncovers an additional ligand for γδ T cells and reconceptualizes the nature of T cell antigen recognition. Science , this issue p. 1522

Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α‐galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly pro-inflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α‐galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings, and suggested basic principles for capturing useful properties of sphinganine analogues of synthetic iNKT cell activators in the design of immunotherapeutic agents.

MHC class I-like CD1 molecules have evolved to present lipid-based antigens to T cells. Differences in the antigen-binding clefts of the CD1 family members determine the conformation and size of the lipids that are presented, although the factors that shape CD1 diversity remain unclear. In mice, two homologous genes, CD1D1 and CD1D2, encode the CD1d protein, which is essential to the development and function of natural killer T (NKT) cells. However, it remains unclear whether both CD1d isoforms are equivalent in their antigen presentation capacity and functions. Here, we report that CD1d2 molecules are expressed in the thymus of some mouse strains, where they select functional type I NKT cells. Intriguingly, the T cell antigen receptor repertoire and phenotype of CD1d2-selected type I NKT cells in CD1D1 −/− mice differed from CD1d1-selected type I NKT cells. The structures of CD1d2 in complex with endogenous lipids and a truncated acyl-chain analog of α-galactosylceramide revealed that its A′-pocket was restricted in size compared with CD1d1. Accordingly, CD1d2 molecules could not present glycolipid antigens with long acyl chains efficiently, favoring the presentation of short acyl chain antigens. These results indicate that the two CD1d molecules present different sets of self-antigen(s) in the mouse thymus, thereby impacting the development of invariant NKT cells.

Most studies on the immunotherapeutic potential of T cells have focused on CD8 and CD4 T cells that recognize peptide antigens (Ag) presented by polymorphic major histocompatibility complex (MHC) class I and MHC class II molecules, respectively. However, unconventional T cells, which interact with MHC class Ib and MHC-I like molecules, are also implicated in tumor immunity, although their role therein is unclear. These include unconventional T cells targeting MHC class Ib molecules such as HLA-E and its murine ortholog Qa-1b, natural killer T (NKT) cells, mucosal associated invariant T (MAIT) cells, and γδ T cells. Here, we review the current understanding of the roles of these unconventional T cells in tumor immunity and discuss why further studies into the immunotherapeutic potential of these cells is warranted.

The hallmark function of αβ T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

Lipids are now widely considered to play a variety of important roles in T-cell mediated immunity, including serving as antigens. Lipid-based antigens are presented by a specialised group of glycoproteins termed CD1. In humans, three classes of CD1 molecules exist: group 1 (CD1a, CD1b, CD1c), group 2 (CD1d), and group 3 (CD1e). While CD1d-mediated T-cell immunity has been extensively investigated, we have only recently gained insights into the structure and function of group 1 CD1 molecules. Structural studies have revealed how lipid-based antigens are presented by group 1 CD1 molecules, as well as shedding light on the molecular requirements for T-cell recognition. Here, we provide an overview of our current understanding of lipid presentation by group 1 CD1 molecules in humans and their recognition by T-cells, as well as examining the potential differences in lipid presentation that may occur across different species.

Godfrey, Rossjohn, and colleagues define a population of T cells in healthy humans that express T cell receptors (TCRs) comprised of δ variable gene segments fused to α joining and constant domains and paired with a variety of TCR-β chains. Functional and structural analyses reveal how components of αβ and γδ TCR gene loci combine to create T cells with unique patterns of antigen recognition., αβ and γδ T cells are disparate T cell lineages that can respond to distinct antigens (Ags) via the use of the αβ and γδ T cell Ag receptors (TCRs), respectively. Here we characterize a population of human T cells, which we term δ/αβ T cells, expressing TCRs comprised of a TCR-δ variable gene (Vδ1) fused to joining α and constant α domains, paired with an array of TCR-β chains. We demonstrate that these cells, which represent ∼50% of all Vδ1+ human T cells, can recognize peptide- and lipid-based Ags presented by human leukocyte antigen (HLA) and CD1d, respectively. Similar to type I natural killer T (NKT) cells, CD1d-lipid Ag-reactive δ/αβ T cells recognized α-galactosylceramide (α-GalCer); however, their fine specificity for other lipid Ags presented by CD1d, such as α-glucosylceramide, was distinct from type I NKT cells. Thus, δ/αβTCRs contribute new patterns of Ag specificity to the human immune system. Furthermore, we provide the molecular bases of how δ/αβTCRs bind to their targets, with the Vδ1-encoded region providing a major contribution to δ/αβTCR binding. Our findings highlight how components from αβ and γδTCR gene loci can recombine to confer Ag specificity, thus expanding our understanding of T cell biology and TCR diversity.

The T cell repertoire comprises αβ and γδ T cell lineages. Although it is established how αβ T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for γδ TCRs. We describe a population of human V δ 1 + γδ T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a γδ TCR binds CD1d-α- galactosylceramide (α-GalCer). The γδ TCR docked orthogonally, over the A′ pocket of CD1d, in which the V δ 1-chain, and in particular the germ line-encoded CDR1δ loop, dominated interactions with CD1d. The TCR γ-chain sat peripherally to the interface, with the CDR3γ loop representing the principal determinant for α-GalCer specificity. Accordingly, we provide insight into how a γδ TCR binds specifically to a lipid-loaded antigen-presenting molecule.

AB5 toxins are key virulence factors found in a range of pathogenic bacteria. AB5 toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 A resolution are reported.

CD1a is a lipid-presenting molecule that is abundantly expressed on Langerhans cells. However, the in vivo role of CD1a has remained unclear, principally because CD1a is lacking in mice. Through the use of mice with transgenic expression of CD1a, we found that the plant-derived lipid urushiol triggered CD1a-dependent skin inflammation driven by CD4(+) helper T cells that produced the cytokines IL-17 and IL-22 (TH17 cells). Human subjects with poison-ivy dermatitis had a similar cytokine signature following CD1a-mediated recognition of urushiol. Among various urushiol congeners, we identified diunsaturated pentadecylcatechol (C15:2) as the dominant antigen for CD1a-restricted T cells. We determined the crystal structure of the CD1a-urushiol (C15:2) complex, demonstrating the molecular basis of urushiol interaction with the antigen-binding cleft of CD1a. In a mouse model and in patients with psoriasis, CD1a amplified inflammatory responses that were mediated by TH17 cells that reacted to self lipid antigens. Treatment with blocking antibodies to CD1a alleviated skin inflammation. Thus, we propose CD1a as a potential therapeutic target in inflammatory skin diseases.

CD1 proteins present microbial lipids to T cells. Germline-encoded mycolyl lipid-reactive (GEM) T cells with conserved αβ T cell receptors (TCRs) recognize CD1b presenting mycobacterial mycolates. As the molecular basis underpinning TCR recognition of CD1b remains unknown, here we determine the structure of a GEM TCR bound to CD1b presenting glucose-6-O-monomycolate (GMM). The GEM TCR docks centrally above CD1b, whereby the conserved TCR α-chain extensively contacts CD1b and GMM. Through mutagenesis and study of T cells from tuberculosis patients, we identify a consensus CD1b footprint of TCRs present among GEM T cells. Using both the TCR α- and β-chains as tweezers to surround and grip the glucose moiety of GMM, GEM TCRs create a highly specific mechanism for recognizing this mycobacterial glycolipid., Germline-encoded mycolyl lipid-reactive (GEM) T cells recognize CD1b proteins presenting mycobacterial mycolates via their T-cell receptors (TCRs). Here, the authors present the structure of this interaction and provide a molecular basis for the co-recognition of CD1b and a mycobacterial glycolipid.

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.

SummaryMany pathogenic bacteria have sophisticated mechanisms to interfere with the mammalian immune response. These include the disruption of host extracellular ATP levels that, in humans, is tightly regulated by the nucleoside triphosphate diphosphohydrolase family (NTPDases). NTPDases are found almost exclusively in eukaryotes, the notable exception being their presence in some pathogenic prokaryotes. To address the function of bacterial NTPDases, we describe the structures of an NTPDase from the pathogen Legionella pneumophila (Lpg1905/Lp1NTPDase) in its apo state and in complex with the ATP analog AMPPNP and the subtype-specific NTPDase inhibitor ARL 67156. Lp1NTPDase is structurally and catalytically related to eukaryotic NTPDases and the structure provides a basis for NTPDase-specific inhibition. Furthermore, we demonstrate that the activity of Lp1NTPDase correlates directly with intracellular replication of Legionella within macrophages. Collectively, these findings provide insight into the mechanism of this enzyme and highlight its role in host-pathogen interactions.

A characteristic of mucosal-associated invariant T (MAIT) cells is the expression of TRAV1-2(+) T cell receptors (TCRs) that are activated by riboflavin metabolite-based antigens (Ag) presented by the MHC-I related molecule, MR1. Whether the MR1-restricted T cell repertoire and associated Ag responsiveness extends beyond these cells remains unclear. Here, we describe MR1 autoreactivity and folate-derivative reactivity in a discrete subset of TRAV1-2(+) MAIT cells. This recognition was attributable to CDR3β loop-mediated effects within a consensus TRAV1-2(+) TCR-MR1-Ag footprint. Furthermore, we have demonstrated differential folate- and riboflavin-derivative reactivity by a diverse population of "atypical" TRAV1-2(-) MR1-restricted T cells. We have shown that TRAV1-2(-) T cells are phenotypically heterogeneous and largely distinct from TRAV1-2(+) MAIT cells. A TRAV1-2(-) TCR docks more centrally on MR1, thereby adopting a markedly different molecular footprint to the TRAV1-2(+) TCR. Accordingly, diversity within the MR1-restricted T cell repertoire leads to differing MR1-restricted Ag specificity.

Semi-invariant/type I NKT cells are a well-characterized CD1d-restricted T cell subset. The availability of potent Ags and tetramers for semi-invariant/type I NKT cells allowed this population to be extensively studied and revealed their central roles in infection, autoimmunity, and tumor immunity. In contrast, diverse/type II NKT (dNKT) cells are poorly understood because the lipid Ags that they recognize are largely unknown. We sought to identify dNKT cell lipid Ag(s) by interrogating a panel of dNKT mouse cell hybridomas with lipid extracts from the pathogen Listeria monocytogenes. We identified Listeria phosphatidylglycerol as a microbial Ag that was significantly more potent than a previously characterized dNKT cell Ag, mammalian phosphatidylglycerol. Further, although mammalian phosphatidylglycerol-loaded CD1d tetramers did not stain dNKT cells, the Listeria-derived phosphatidylglycerol-loaded tetramers did. The structure of Listeria phosphatidylglycerol was distinct from mammalian phosphatidylglycerol because it contained shorter, fully-saturated anteiso fatty acid lipid tails. CD1d-binding lipid-displacement studies revealed that the microbial phosphatidylglycerol Ag binds significantly better to CD1d than do counterparts with the same headgroup. These data reveal a highly potent microbial lipid Ag for a subset of dNKT cells and provide an explanation for its increased Ag potency compared with the mammalian counterpart.

Dihydrorotate dehydrogenase (DHOD) (EC 1.3.99.11) catalyzes the fourth step and only redox reaction in the de novo pyrimidine biosynthesis, the stereospecific oxidation of (S)-DHO to orotate accompanied by the reduction of the prosthetic flavin (FMN) group (Fig. 1 ▶). A phylogenetic analysis of available DHOD sequences revealed that DHOD from different organisms can be assigned to two different major classes: class 1 and class 2 (Bjornberg et al. 1997). Class 1 DHODs originating mainly from gram-positive bacteria can furthermore be divided into subclasses 1A, 1B, and a new type 1S identified in Sulfolobus solfataricus (Sorensen and Dandanell 2002). The DHODs belonging to the different classes differ also in their location in the cell. The class 1A and 1B DHODs are found in the cytosol, whereas those from class 2 are membrane associated. Another distinct difference between the two classes of enzymes is their natural electron acceptor used to reoxidize the flavin group. Figure 1. The reaction catalyzed by class 2 DHODs and chemical structure of the DHOD inhibitors atovaquone, brequinar, and A771726. This figure is produced by ISIS draw 2.4 (MDL Information Systems, Inc.). Lactococcus lactis contains two genes encoding for DHODs representing subclass 1A and 1B, DHODA and DHODB, respectively. They differ in their structural organization and use of electron acceptor. The DHODA enzyme is a homodimer comprising two PyrDA subunits with an (αβ)8 barrel fold and the prosthetic FMN group located at the C-terminal ends of the β-strands at the top of the barrel (Rowland et al. 1997); it uses fumarate as its natural electron acceptor (Andersen et al. 1996). The DHODB is a heterotetramer composed of a central homodimer of PyrDB subunits resembling the DHODA structure and two PyrK subunits (Rowland et al. 2000). It is the presence of the PyrK subunits, which contain an FAD group and a [2Fe-2S] cluster, that enables the class 1B enzymes to use NAD+ as the natural electron acceptor (Nielsen et al. 1996). Class 1S DHOD can use Q0 and molecular oxygen as electron acceptors, together with the unphysiological substrates ferricyanide and DCIP used in in vitro measurements (Sorensen and Dandanell 2002). The membrane-associated class 2 DHODs found in gram-negative bacteria and in eukaryotes are monomeric enzymes that have the respiratory quinones as their physiological electron acceptors (Fig. 1 ▶; Bjornberg et al. 1999). A major structural difference between the class 1 and class 2 DHODs is their extended N terminus. The structure determinations for the DHODC and DHODH, truncated to be of the same length as DHODC, showed that the N terminus in the class 2 enzymes comprises a separate domain with two α-helices located on the top of the catalytic (αβ)8 barrel close to the FMN group (Liu et al. 2000; Norager et al. 2002). All eukaryotic enzymes from class 2 are located in the mitochondrial membrane attached by transmembrane α -helices, whereas the gram-negative bacterial enzymes are associated with the cytosolic side of the outer membrane. The extension of the N terminus in class 2 DHODs is thought to serve as a targeting signal guiding the enzyme to its location in the inner mitochondrial membrane (Rawls et al. 2000; Loffler et al. 2002) A basic residue in the active site mediates the stereospecific oxidation of (S)-DHO. It is a cysteine in the class 1 enzymes (Bjornberg et al. 1997) and a serine residue in the class 2 DHODs (Bjornberg et al. 1999). The basic residue is located in a loop in close contact to DHO bound on top of the FMN group. This position facilitates abstraction of a proton from the C5 atom of DHO in the enzymatic reaction, where a double bond between C5 and C6 is formed due to the transfer of a hydride ion from C6 to the N5 atom of FMN (Fig. 1 ▶). The second half reaction uses the respiratory quinones as electron acceptors. Their proposed binding site (Liu et al. 2000) is the N-terminal domain, where they are able to mediate the electron transfer to the FMNH2 group bound in the (αβ)8 barrel, as shown in Figure 1 ▶. The inhibition of DHODs causes a lowering of the intracellular pools of uracil, cytosine, and thymine nucleotides in cells, which makes DHODs attractive drug targets (Fairbanks et al. 1995). Most organisms are able to use a salvage pathway for pyrimidine nucleotide biosynthesis. It allows the pyrimidine bases or nucleosides formed from degradation of nucleotides and nucleic acids to be reused by salvage reactions. Some of the genes encoding for the enzymes in the pyrimidine salvage pathway were not identified in the genomes of two organisms affecting human health, the bacterium Helicobacter pylori causing stomach ulcers and stomach cancer and the malaria-causing parasite Plasmodium. They therefore depend exclusively on de novo synthesis of pyrimidine nucleotides, which explains why DHODs from these organisms are very attractive drug targets. Rapidly dividing human cells, like activated lymphocytes (Cutolo et al. 2003) and cancer cells (Shawver et al. 1997) require also a functional de novo nucleotide pathway to meet their requirement for nucleotides because recycling using salvage pathways of the already existing nucleotide pool through salvage pathways is not sufficient (Fairbanks et al. 1995). The immunomodulating drug leflunomide (Arava) has been approved for the treatment of rheumatoid arthritis (Goldenberg 1999). It has been shown that this drug inhibits DHODH and thereby inhibits the pyrimidine de novo biosynthetic pathway (Davis et al. 1996). The structure of DHODH is known in complex with A771726, the active metabolite of the prodrug leflunomide (DHODH-lefl) and brequinar (DHODH-breq) (Liu et al. 2000). From the analysis of the two structures of DHODH, it was concluded that the inhibitors could bind to the same site as the second natural substrate, the respiratory quinone. This feature was also deduced from enzyme kinetics studies of most of the class 2 enzyme inhibitors reported so far (Bader et al. 1998; Knecht et al. 2000). Considerable efforts have been put into structure activity analysis for DHODs from different organisms, among them rat and mouse (Knecht et al. 2000). An interesting feature of class 2 DHODs is the relatively small number of conserved residues located in their extended N termini. This explains why the N-terminal domains in the known structures of the class 2 DHODs display significant variations in the length and orientation of the helices that form this domain (Norager et al. 2002). The comparison of the sequences from DHODH, DHODC, and DHODR in Figure 2 ▶ reveals that, among the residues corresponding to the first 40 residues of DHODC, there are only six conserved. It is likely that this variation is the origin of the different behavior of inhibitors even for very closely related DHODs like the rat and human (Knecht and Loffler 1998). Thus, it seems possible to design inhibitors that are specific for a given organism, as demonstrated by structure-activity studies made on class 2 DHODs (Copeland et al. 2000). Figure 2. Structural alignment of DHODR, DHODH, and DHODC sequences. The structural elements correspond to the DHODR structures. α-Helixes in the central barrel are named α1–α8 and β-sheets in the barrel are named β1–β8. ... The work presented here addresses the differences between the class 2 DHODs. We have determined the crystal structures of the DHOD from rat, truncated like DHODH to be of the same length as DHODC, in complex with brequinar (DHODR-breq) and atovaquone (DHODR-ato). Atovaquone (Fig. 1 ▶) is a structural analog of ubiquinone. It is used as a broad-spectrum antiparasitic drug and has showed activity against various parasitic infections, such as malaria, toxoplasmosis (caused by Toxoplasma gondii), and pneumonia (Pneumocystis carinii) (Kaneshiro et al. 2000). Atovaquone has passed clinical trials and thereby received approval to combat Plasmodium falciparum. The primary mechanism of action in Plasmodium falciparum is the irreversible binding to the mitochondrial cytochrome bc1 complex, but it is also a potent inhibitor of DHOD activity (Ittarat et al. 1994). Atovaquone is marketed in the United States under the trade name Mepron. Atovaquone is one of the active compounds in Malarone (GlaxoSmithKline), used in the prophylaxis (prevention) and treatment of malaria. Brequinar is a quinoline carboxylic acid, which has been tested preclinically as a cytostatic agent. The three inhibitors atovaquone, A77126, and brequinar (known to inhibit different class 2 DHODs) are chemically different and do not mimic the natural electron acceptor, as shown in Figure 1 ▶. Our analysis of the two structures of DHODR-ato and DHODR-breq revealed a remarkable difference in the conformation of their small N-terminal domain. A comparison to the structures of inhibited DHODH have revealed subtle differences in the N-terminal domain that can explain why the DHOD inhibitors act differently on the two highly homologous enzymes. These results are valuable for the structural-based drug design of organism-specific inhibitors of DHOD, and have formed the basis for a modeling of quinone binding. Furthermore, we present an analysis of the differences in the N-terminal domain between the class 2 membrane-bound and membrane-associated DHOD, based on computational GRID modeling.

beta-1,4-Galactanases hydrolyze the galactan side chains that are part of the complex carbohydrate structure of the pectin. They are assigned to family 53 of the glycoside hydrolases and display significant variations in their pH and temperature optimum and stability. Two fungal beta-1,4-galactanases from Myceliophthora thermophila and Humicola insolens have been cloned and heterologously expressed, and the crystal structures of the gene products were determined. The structures are compared to the previously only known family 53 structure of the galactanase from Aspergillus aculeatus (AAGAL) showing approximately 56% identity. The M. thermophila and H. insolens galactanases are thermophilic enzymes and are most active at neutral to basic pH, whereas AAGAL is mesophilic and most active at acidic pH. The structure of the M. thermophila galactanase (MTGAL) was determined from crystals obtained with HEPES and TRIS buffers to 1.88 A and 2.14 A resolution, respectively. The structure of the H. insolens galactanase (HIGAL) was determined to 2.55 A resolution. The thermostability of MTGAL and HIGAL correlates with increase in the protein rigidity and electrostatic interactions, stabilization of the alpha-helices, and a tighter packing. An inspection of the active sites in the three enzymes identifies several amino acid substitutions that could explain the variation in pH optimum. Examination of the activity as a function of pH for the D182N mutant of AAGAL and the A90S/ H91D mutant of MTGAL showed that the difference in pH optimum between AAGAL and MTGAL is at least partially associated with differences in the nature of residues at positions 182, 90, and/or 91.

AB₅ toxins are key virulence factors found in a range of pathogenic bacteria. AB₅ toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB₅ toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB₅ toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB₅ toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 Å resolution are reported.

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 α-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.

Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRβ chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.

The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct β-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated β-linked agonists β-galactosylceramide (β-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the β-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the β-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to β-linked antigens.

β-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal β-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been kinetically characterized using the chromogenic substrate analog 4-nitrophenyl β-1,4-d-thiogalactobioside synthesized by the thioglycoligase approach. Values of k(cat)/K(m) for this substrate with A. aculeatus β-1,4-galactanase at pH 4.4 and for M. giganteus β-1,4-galactanase at pH 5.5 are 333M(-1)s(-1) and 62M(-1)s(-1), respectively. By contrast the B. licheniformis β-1,4-galactanase did not hydrolyze 4-nitrophenyl β-1,4-d-thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial β-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis β-1,4-galactanase. This loop contains substrate binding subsites -3 and -4, which presumably cause B. licheniformis β-1,4-galactanase to bind 4-nitrophenyl -1,4-β-d-thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-β-d-thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-β-d-galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4mM.