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αβ T cell receptors (αβTCRs) co-recognise antigens when bound to Major Histocompatibility Complex (MHC) or MHC class I-like molecules. Additionally, some αβTCRs can bind non-MHC molecules, but how much intact antigen reactivities are achieved remains unknown. Here, we identify an αβ T cell clone that directly recognises the intact foreign protein, R-phycoerythrin (PE), a multimeric (αβ) 6 γ protein complex. This direct αβTCR-PE interaction occurs in an MHC-independent manner, yet triggers T cell activation and bound PE with an affinity comparable to αβTCR-peptide-MHC interactions. The crystal structure reveals how six αβTCR molecules simultaneously engage the PE hexamer, mediated by the complementarity-determining regions (CDRs) of the αβTCR. Here, the αβTCR mainly binds to two α-helices of the globin fold in the PE α-subunit, which is analogous to the antigen-binding platform of the MHC molecule. Using retrogenic mice expressing this TCR, we show that it supports intrathymic T cell development, maturation, and exit into the periphery as mature CD4/CD8 double negative (DN) T cells with TCR-mediated functional capacity. Accordingly, we show how an αβTCR can recognise an intact foreign protein in an antibody-like manner., (© 2024. The Author(s).)

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., (© 2022 The University of Melbourne.)

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRβ-variable (TRBV) 17 + TCRs from the naïve mouse CD8 + T cell repertoire that recognizes the H-2D b -NP 366 epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., Competing Interests: Conflict of interest—J. R., J. M., L. L., and D. P. F. are named inventors on patent applications (PCT/AU2013/000742, WO2014005194) (PCT/AU2015/050148, WO2015149130) involving MR1 ligands for MR1-restricted MAIT cells owned by University of Queensland, Monash University, and University of Melbourne.

Mucosal-associated invariant T (MAIT) cells are important for immune responses against microbial infections. Although known to undergo marked numerical changes with age in humans, our understanding of how MAIT cells are altered during different phases across the human life span is largely unknown. Although also abundant in the tissues, our study focuses on MAIT cell analyses in blood. Across the human life span, we show that naive-like MAIT cells in umbilical cord blood switch to a central/effector memory-like profile that is sustained into older age. Whereas low-grade levels of plasma cytokine/chemokine were apparent in older donors (>65 y old), surprisingly, they did not correlate with the ex vivo MAIT hyperinflammatory cytokine profile observed in older adults. Removal of MAIT cells from older individuals and an aged environment resulted in the reversal of the baseline effector molecule profile comparable with MAIT cells from younger adults. An upregulated basal inflammatory profile accounted for reduced Escherichia coli -specific responses in aged MAIT cells compared with their young adult counterparts when fold change in expression levels of GzmB, CD107a, IFN-γ, and TNF was examined. However, the magnitude of antimicrobial MR1-dependent activation remained as potent and polyfunctional as with younger adults. Paired TCRαβ analyses of MAIT cells revealed large clonal expansions in older adults and tissues that rivalled, remarkably, the TCRαβ repertoire diversity of virus-specific CD8 + T cells. These data suggest that MAIT cells in older individuals, although associated with large clonal TCRαβ expansions and increased baseline inflammatory potential, demonstrate plasticity and provide potent antimicrobial immunity., (Copyright © 2020 by The American Association of Immunologists, Inc.)

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., (Copyright © 2019 by The American Association of Immunologists, Inc.)

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.

During αβ T cell development, T cell antigen receptor (TCR) engagement transduces biochemical signals through a protein-protein interaction (PPI) network that dictates dichotomous cell fate decisions. It remains unclear how signal specificity is communicated, instructing either positive selection to advance cell differentiation or death by negative selection. Early signal discrimination might occur by PPI signatures differing qualitatively (customized, unique PPI combinations for each signal), quantitatively (graded amounts of a single PPI series), or kinetically (speed of PPI pathway progression). Using a novel PPI network analysis, we found that early TCR-proximal signals distinguishing positive from negative selection appeared to be primarily quantitative in nature. Furthermore, the signal intensity of this PPI network was used to find an antigen dose that caused a classic negative selection ligand to induce positive selection of conventional αβ T cells, suggesting that the quantity of TCR triggering was sufficient to program selection outcome. Because previous work had suggested that positive selection might involve a qualitatively unique signal through CD3δ, we reexamined the block in positive selection observed in CD3δ 0 mice. We found that CD3δ 0 thymocytes were inhibited but capable of signaling positive selection, generating low numbers of MHC-dependent αβ T cells that expressed diverse TCR repertoires and participated in immune responses against infection. We conclude that the major role for CD3δ in positive selection is to quantitatively boost the signal for maximal generation of αβ T cells. Together, these data indicate that a quantitative network signaling mechanism through the early proximal TCR signalosome determines thymic selection outcome., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (© 2018 Australasian Society for Immunology Inc.)

αβ T cell receptors (TCRs) interact with peptides bound to the polymorphic major histocompatibility complex class Ia (MHC-Ia) and class II (MHC-II) molecules as well as the essentially monomorphic MHC class Ib (MHC-Ib) molecules. Although there is a large amount of information on how TCRs engage with MHC-Ia and MHC-II, our understanding of TCR/MHC-Ib interactions is very limited. Infection with cytomegalovirus (CMV) can elicit a CD8 + T cell response restricted by the human MHC-Ib molecule human leukocyte antigen (HLA)-E and specific for an epitope from UL40 (VMAPRTLIL), which is characterized by biased TRBV14 gene usage. Here we describe an HLA-E-restricted CD8 + T cell able to recognize an allotypic variant of the UL40 peptide with a modification at position 8 (P8) of the peptide (VMAPRTLVL) that uses the TRBV9 gene segment. We report the structures of a TRBV9 + TCR in complex with the HLA-E molecule presenting the two peptides. Our data revealed that the TRBV9 + TCR adopts a different docking mode and molecular footprint atop HLA-E when compared with the TRBV14 + TCR-HLA-E ternary complex. Additionally, despite their differing V gene segment usage and different docking mechanisms, mutational analyses showed that the TCRs shared a conserved energetic footprint on the HLA-E molecule, focused around the peptide-binding groove. Hence, we provide new insights into how monomorphic MHC molecules interact with T cells., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Adaptive immune responses protect against infection with dengue virus (DENV), yet cross-reactivity with distinct serotypes can precipitate life-threatening clinical disease. We found that clonotypes expressing the T cell antigen receptor (TCR) β-chain variable region 11 (TRBV11-2) were 'preferentially' activated and mobilized within immunodominant human-leukocyte-antigen-(HLA)-A*11:01-restricted CD8 + T cell populations specific for variants of the nonstructural protein epitope NS3 133 that characterize the serotypes DENV1, DENV3 and DENV4. In contrast, the NS3 133 -DENV2-specific repertoire was largely devoid of such TCRs. Structural analysis of a representative TRBV11-2 + TCR demonstrated that cross-serotype reactivity was governed by unique interplay between the variable antigenic determinant and germline-encoded residues in the second β-chain complementarity-determining region (CDR2β). Extensive mutagenesis studies of three distinct TRBV11-2 + TCRs further confirmed that antigen recognition was dependent on key contacts between the serotype-defined peptide and discrete residues in the CDR2β loop. Collectively, these data reveal an innate-like mode of epitope recognition with potential implications for the outcome of sequential exposure to heterologous DENVs.

Human T cell autoreactivity toward lipid antigens presented by CD1 proteins can manifest in numerous diseases, including psoriasis, contact hypersensitivities, and allergies. However, the molecular mechanisms for regulating T cell autoreactivity toward lipid antigens remain unclear. We determined the basis for T cell receptor (TCR) autoreactivity toward CD1b bound to self-phospholipids. The spectrum of self-antigens captured by CD1b skews toward abundant membrane phospholipids such as phosphatidylcholine and phosphatidylethanolamine. However, TCRs can specifically recognize rare phospholipids, including phosphatidylglycerol (PG). The structure of an autoreactive TCR bound to CD1b-PG shows that discrimination occurs through a marked induced fit movement of PG so that its polar head group fits snugly into the cationic cup of the TCR. Conversely, TCR binding toward ubiquitous self-phospholipids was sterically or electrostatically repelled. Accordingly, we describe a mechanism of TCR autoreactivity toward rare phospholipids and avoidance of autoreactivity to the most abundant self-phospholipids., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Glycolipid antigens recognized by αβ T-cell receptors (TCRs) drive the activation of invariant natural killer T (iNKT) cells, a specialized subset of innate T lymphocytes. Glycolipids with α-linked anomeric carbohydrates have been identified as potent microbial lipid antigens for iNKT cells, and their unusual α-anomeric linkage has been thought to define a "foreign" lipid antigen motif. However, mammals use endogenous lipids to select iNKT cells, and there is compelling evidence for iNKT cell responses in various types of sterile inflammation. The nature of endogenous or environmental lipid antigens encountered by iNKT cells is not well defined. Here, we sought to identify lipid antigens in cow's milk, a prominent part of the human diet. We developed a method to directly capture lipid antigens within CD1d-lipid-TCR complexes, while excluding CD1d bound to nonantigenic lipids, followed by direct biochemical analysis of the lipid antigens trapped at the TCR-CD1d interface. The specific antigens captured by this "TCR trap" method were identified as α-linked monohexosylceramides by mass spectrometry fragmentation patterns that distinguished α- from β-anomeric monohexosylceramides. These data provide direct biochemical evidence for α-linked lipid antigens from a common dietary source., Competing Interests: The authors declare no conflict of interest.

αβT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized. To provide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR cross-reactivity remain unclear, with conflicting results on anti-viral T cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M1 58 , respectively) or with the hepatitis C and influenza viruses (NS3 1073 and NA 231 , respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell cross-reactivity. We combined ex vivo and in vitro functional assays, single-cell αβTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αβTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell cross-reactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mucosal-associated invariant T (MAIT) cells are thought to detect microbial antigens presented by the HLA-Ib molecule MR1 through the exclusive use of a TRAV1-2-containing TCRα. Here we use MR1 tetramer staining and ex vivo analysis with mycobacteria-infected MR1-deficient cells to demonstrate the presence of functional human MR1-restricted T cells that lack TRAV1-2. We characterize an MR1-restricted clone that expresses the TRAV12-2 TCRα, which lacks residues previously shown to be critical for MR1-antigen recognition. In contrast to TRAV1-2(+) MAIT cells, this TRAV12-2-expressing clone displays a distinct pattern of microbial recognition by detecting infection with the riboflavin auxotroph Streptococcus pyogenes. As known MAIT antigens are derived from riboflavin metabolites, this suggests that TRAV12-2(+) clone recognizes unique antigens. Thus, MR1-restricted T cells can discriminate between microbes in a TCR-dependent manner. We postulate that additional MR1-restricted T-cell subsets may play a unique role in defence against infection by broadening the recognition of microbial metabolites.

Memory CD8(+)T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158and the hypervariable HLA-B*3501-NP418antigens. The TCRαβs for HLA-B*3501-NP418 (+)CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19(+)TCRαβ was selected in HLA-A*0201(+)donors responding to M158 This public TCR cross-recognized naturally occurring M158variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19(+)TCR specificity for the WT and mutant M158peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201(+)individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158 Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs.

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.

NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR β-chains, including Vβ8, Vβ7, and Vβ2 in mice and Vβ11 in humans, joined to a range of TCR Dβ and Jβ genes. In this study, we demonstrate that TCR β-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vβ7(+) NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vβ8(+) NKT cells from mice. We show that the influence of the TCR β-chain is due to a combination of Vβ-, Jβ-, and CDR3β-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3β-encoded residues determined Ag preference. These findings indicate that NKT TCR β-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Mucosal-associated invariant T (MAIT) cells have a semi-invariant TCR Vα-chain, and their optimal development is dependent upon commensal flora and expression of the nonpolymorphic MHC class I-like molecule MR1. MAIT cells are activated in an MR1-restricted manner by diverse strains of bacteria and yeast, suggesting a widely shared Ag. Recently, human and mouse MR1 were found to bind bacterial riboflavin metabolites (ribityllumazine [RL] Ags) capable of activating MAIT cells. In this study, we used MR1/RL tetramers to study MR1 dependency, subset heterogeneity, and protective effector functions important for tuberculosis immunity. Although tetramer(+) cells were detected in both MR1(+/+) and MR1(-/-) TCR Vα19i-transgenic (Tg) mice, MR1 expression resulted in significantly increased tetramer(+) cells coexpressing TCR Vβ6/8, NK1.1, CD44, and CD69 that displayed more robust in vitro responses to IL-12 plus IL-18 and RL Ag, indicating that MR1 is necessary for the optimal development of the classic murine MAIT cell memory/effector subset. In addition, tetramer(+) MAIT cells expressing CD4, CD8, or neither developing in MR1(+/+) Vα19i-Tg mice had disparate cytokine profiles in response to RL Ag. Therefore, murine MAIT cells are considerably more heterogeneous than previously thought. Most notably, after mycobacterial pulmonary infection, heterogeneous subsets of tetramer(+) Vα19i-Tg MAIT cells expressing CXCR3 and α4β1 were recruited into the lungs and afforded early protection. In addition, Vα19iCα(-/-)MR(+/+) mice were significantly better protected than were Vα19iCα(-/-)MR1(-/-), wild-type, and MR1(-/-) non-Tg mice. Overall, we demonstrate considerable functional diversity of MAIT cell responses, as well as that MR1-restricted MAIT cells are important for tuberculosis protective immunity., (Copyright © 2015 by The American Association of Immunologists, Inc.)

A central paradigm in αβ T cell-mediated immunity is the simultaneous co-recognition of antigens and antigen-presenting molecules by the αβ T cell antigen receptor (TCR). CD1a presents a broad repertoire of lipid-based antigens. We found that a prototypical autoreactive TCR bound CD1a when it was presenting a series of permissive endogenous ligands, while other lipid ligands were nonpermissive to TCR binding. The structures of two TCR-CD1a-lipid complexes showed that the TCR docked over the A' roof of CD1a in a manner that precluded direct contact with permissive ligands. Nonpermissive ligands indirectly inhibited TCR binding by disrupting the TCR-CD1a contact zone. The exclusive recognition of CD1a by the TCR represents a previously unknown mechanism whereby αβ T cells indirectly sense self antigens that are bound to an antigen-presenting molecule.

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., (© 2014 Pellicci et al.)

The T-cell antigen receptor is a heterodimeric αβ protein (TCR) expressed on the surface of T-lymphocytes, with each chain of the TCR comprising three complementarity-determining regions (CDRs) that collectively form the antigen-binding site. Unlike antibodies, which are closely related proteins that recognize intact protein antigens, TCRs classically bind, via their CDR loops, to peptides (p) that are presented by molecules of the major histocompatibility complex (MHC). This TCR-pMHC interaction is crucially important in cell-mediated immunity, with the specificity in the cellular immune response being attributable to MHC polymorphism, an extensive TCR repertoire and a variable peptide cargo. The ensuing structural and biophysical studies within the TCR-pMHC axis have been highly informative in understanding the fundamental events that underpin protective immunity and dysfunctional T-cell responses that occur during autoimmunity. In addition, TCRs can recognize the CD1 family, a family of MHC-related molecules that instead of presenting peptides are ideally suited to bind lipid-based antigens. Structural studies within the CD1-lipid antigen system are beginning to inform us how lipid antigens are specifically presented by CD1, and how such CD1-lipid antigen complexes are recognized by the TCR. Moreover, it has recently been shown that certain TCRs can bind to vitamin B based metabolites that are bound to an MHC-like molecule termed MR1. Thus, TCRs can recognize peptides, lipids, and small molecule metabolites, and here we review the basic principles underpinning this versatile and fascinating receptor recognition system that is vital to a host's survival., (© 2013 The Protein Society.)

The self-reactivity of their T-cell antigen receptor (TCR) is thought to contribute to the development of immune regulatory cells, such as invariant NK T cells (iNKT). In the mouse, iNKT cells express TCRs composed of a unique Vα14-Jα18 rearrangement and recognize lipid antigens presented by CD1d molecules. We created mice expressing a transgenic TCR-β chain that confers high affinity for self-lipid/CD1d complexes when randomly paired with the mouse iNKT Vα14-Jα18 rearrangement to study their development. We show that although iNKT cells undergo agonist selection, their development is also shaped by negative selection in vivo. In addition, iNKT cells that avoid negative selection in these mice express natural sequence variants of the canonical TCR-α and decreased affinity for self/CD1d. However, limiting the affinity of the iNKT TCRs for "self" leads to inefficient Egr2 induction, poor expression of the iNKT lineage-specific zinc-finger transcription factor PLZF, inadequate proliferation of iNKT cell precursors, defects in trafficking, and impaired effector functions. Thus, proper development of fully functional iNKT cells is constrained by a limited range of TCR affinity that plays a key role in triggering the iNKT cell-differentiation pathway. These results provide a direct link between the affinity of the TCR expressed by T-cell precursors for self-antigens and the proper development of a unique population of lymphocytes essential to immune responses.

The diverse αβ T cell receptor (TCR) repertoire exhibits versatility in its ability to generate antigen (Ag) receptors capable of interacting with polymorphic Major Histocompatibility Complex (MHC) molecules and monomorphic MHC-I like molecules, including the CD1 and MR1 families. Collectively, these evolutionarily related Ag-presenting molecules present peptides, lipids and vitamin B metabolites for T cell surveillance. Interestingly, whilst common TCR gene usage can underpin recognition of these distinct classes of Ags, it is unclear whether the 'rules' that govern αβTCR-Ag MHC interactions are shared. We highlight recent observations in the context of TCR biases towards MHC and MHC-I like molecules., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Human T cell antigen receptors (TCRs) pair in millions of combinations to create complex and unique T cell repertoires for each person. Through the use of tetramers to analyze TCRs reactive to the antigen-presenting molecule CD1b, we detected T cells with highly stereotyped TCR α-chains present among genetically unrelated patients with tuberculosis. The germline-encoded, mycolyl lipid-reactive (GEM) TCRs had an α-chain bearing the variable (V) region TRAV1-2 rearranged to the joining (J) region TRAJ9 with few nontemplated (N)-region additions. Analysis of TCRs by high-throughput sequencing, binding and crystallography showed linkage of TCRα sequence motifs to high-affinity recognition of antigen. Thus, the CD1-reactive TCR repertoire is composed of at least two compartments: high-affinity GEM TCRs, and more-diverse TCRs with low affinity for CD1b-lipid complexes. We found high interdonor conservation of TCRs that probably resulted from selection by a nonpolymorphic antigen-presenting molecule and an immunodominant antigen.

αβ-TCRs expressed at the CD8(+) T-cell surface interact with short peptide fragments (p) bound to MHC class I molecules (pMHCI). The TCR/pMHCI interaction is pivotal in all aspects of CD8(+) T-cell immunity. However, the rules that govern the outcome of TCR/pMHCI engagement are not entirely understood, and this is a major barrier to understanding the requirements for both effective immunity and vaccination. In the present study, we discovered an unexpected feature of the TCR/pMHCI interaction by showing that any given TCR exhibits an explicit preference for a single MHCI-peptide length. Agonists of nonpreferred length were extremely rare, suboptimal, and often entirely distinct in sequence. Structural analysis indicated that alterations in peptide length have a major impact on antigenic complexity, to which individual TCRs are unable to adapt. This novel finding demonstrates that the outcome of TCR/pMHCI engagement is determined by peptide length in addition to the sequence identity of the MHCI-bound peptide. Accordingly, the effective recognition of pMHCI Ag, which is a prerequisite for successful CD8(+) T-cell immunity and protective vaccination, can only be achieved by length-matched Ag-specific CD8(+) T-cell clonotypes.

CD1d-restricted natural killer T (NKT) cells include two major subgroups. The most widely studied are Vα14Jα18(+) invariant NKT (iNKT) cells that recognize the prototypical α-galactosylceramide antigen, whereas the other major group uses diverse T-cell receptor (TCR) α-and β-chains, does not recognize α-galactosylceramide, and is referred to as diverse NKT (dNKT) cells. dNKT cells play important roles during infection and autoimmunity, but the antigens they recognize remain poorly understood. Here, we identified phosphatidylglycerol (PG), diphosphatidylglycerol (DPG, or cardiolipin), and phosphatidylinositol from Mycobacterium tuberculosis or Corynebacterium glutamicum as microbial antigens that stimulated various dNKT, but not iNKT, hybridomas. dNKT hybridomas showed distinct reactivities for diverse antigens. Stimulation of dNKT hybridomas by microbial PG was independent of Toll-like receptor-mediated signaling by antigen-presenting cells and required lipid uptake and/or processing. Furthermore, microbial PG bound to CD1d molecules and plate-bound PG/CD1d complexes stimulated dNKT hybridomas, indicating direct recognition by the dNKT cell TCR. Interestingly, despite structural differences in acyl chain composition between microbial and mammalian PG and DPG, lipids from both sources stimulated dNKT hybridomas, suggesting that presentation of microbial lipids and enhanced availability of stimulatory self-lipids may both contribute to dNKT cell activation during infection.

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.

T cells that express clonally distributed αβ T-cell receptors (TCRs) corecognize antigenic peptides (p) bound to major histocompatibility complex class I (MHC-I) and class II molecules (MHC-II). Extensive human leukocyte antigen (HLA) polymorphism enables HLA molecules from different haplotypes to capture an array of self- and microbe-derived peptide antigens that is fundamental to adaptive immunity. T cells developing in the thymus are selected for weak binding to self-peptide-HLA complexes generating a vast repertoire of clonally distinct T cells in the periphery. Indeed, diversity within germline loci and the finally assembled TCR genes, coupled with inherent TCR cross-reactivity, enables CD8(+) T cells to survey the multitude of pHLA-I landscapes. Precisely how does the TCR ligate to pHLA-I, and how does knowledge of the detailed structural interactions inform immunobiology? A recent number of our structural studies concerning the TCR-pMHC-I axis, alongside others in the field, have provided insight into HLA-I polymorphism, pMHC-I flexibility, TCR bias, TCR polymorphism, maintenance of self-tolerance, T-cell cross-reactivity, and alloreactivity. Collectively, the data also provide an opportunity to address the structural correlates of MHC-I restriction. Here, we provide our perspective concerning these advances in the field. Although much key information has been gleaned, the structural data show that some of the key concepts surrounding the TCR-pMHC-I interaction remain controversial and unresolved., (© 2012 John Wiley & Sons A/S.)

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 TCR plays a critical role in recognizing intracellular pathogens and initiating pathways leading to the destruction of infected cells by the immune system. Although genetic variability is known to greatly impact on the human immune system and the outcome of infection, the influence of sequence variation leading to the inactivation or deletion of TCR gene segments is unknown. To investigate this issue, we examined the CD8(+) T cell response to an HLA-B7-restricted epitope ((265)RPHERNGFTVL(275)) from the pp65 Ag of human CMV that was highly biased and frequently dominated by a public TCR β-chain encoded by the variable gene segment TRBV4-3. Approximately 40% of humans lack T cells expressing TRBV4-3 because of a 21.5-kb insertion/deletion polymorphism, but these individuals remain responsive to this epitope, using a diverse T cell repertoire characterized by private TCR usage. Although most residues within the bulged 11-mer peptide were accessible for TCR contact, the public and private TCRs showed distinct patterns of sensitivity to amino acid substitution at different positions within the peptide, thereby suggesting that the repertoire diversity generated in the absence of the dominant public TRBV4-3(+) TCR could lead to better protection from viral escape mutation. Thus, variation in the size of the TRBV repertoire clearly contributes toward interindividual variability in immune responses and is presumably maintained in many ethnic groups to enhance the diversity of Ag-specific T cell responses.

EBV is a ubiquitous and persistent human pathogen, kept in check by the cytotoxic T cell response. In this study, we investigated how three TCRs, which differ in their T cell immunodominance hierarchies and gene usage, interact with the same EBV determinant (FLRGRAYGL), bound to the same Ag-presenting molecule, HLA-B8. We found that the three TCRs exhibit differing fine specificities for the viral Ag. Further, via structural and biophysical approaches, we demonstrated that the viral Ag provides the greatest energetic contribution to the TCR-peptide-HLA interaction, while focusing on a few adjacent HLA-based interactions to further tune fine-specificity requirements. Thus, the TCR engages the peptide-HLA with the viral Ag as the main glue, such that neighboring TCR-MHC interactions are recruited as a supportive adhesive. Collectively, we provide a portrait of how the host's adaptive immune response differentially engages a common viral Ag.

Natural killer T cell antigen receptors (NKT TCRs) recognize lipid-based antigens (Ags) presented by CD1d. Although the TCR α-chain is invariant, NKT TCR Vβ exhibits greater diversity, with one (Vβ11) and three (Vβ8, Vβ7, and Vβ2) Vβ chains in humans and mice, respectively. With the exception of the Vβ2 NKT TCR, NKT TCRs possess canonical tyrosine residues within complementarity determining region (CDR) 2β that are critical for CD1d binding. Thus, how Vβ2 NKT TCR docks with CD1d-Ag was unclear. Despite the absence of the CDR2β-encoded tyrosine residues, we show that the Vβ2 NKT TCR engaged CD1d-Ag in a similar manner and with a comparable affinity and energetic footprint to the manner observed for the Vβ8.2 and Vβ7 NKT TCRs. Accordingly, the germline-encoded regions of the TCR β-chain do not exclusively dictate the innate NKT TCR-CD1d-Ag docking mode. Nevertheless, clear fine specificity differences for the CD1d-Ag existed between the Vβ2 NKT TCR and the Vβ8.2 and Vβ7 NKT TCRs, with the Vβ2 NKT TCR exhibiting greater sensitivity to modifications to the glycolipid Ag. Furthermore, within the Vβ2 NKT TCR-CD1d-αGalCer complex, the CDR2β loop mediated fewer contacts with CD1d, whereas the CDR1β and CDR3β loops contacted CD1d to a much greater extent compared with most Vβ11, Vβ8.2, and Vβ7 NKT TCRs. Accordingly, there is a greater interplay between the germline- and nongermline-encoded loops within the TCR β-chain of the Vβ2 NKT TCR that enables CD1d-Ag ligation.

NKT cells respond to a variety of CD1d-restricted glycolipid Ags that are structurally related to the prototypic Ag α-galactosylceramide (α-GalCer). A modified analog of α-GalCer with a carbon-based glycosidic linkage (α-C-GalCer) has generated great interest because of its apparent ability to promote prolonged, Th1-biased immune responses. In this study, we report the activation of spleen NKT cells to α-C-GalCer, and related C-glycoside ligands, is weaker than that of α-GalCer. Furthermore, the Vβ8.2 and Vβ7 NKT TCR affinity for CD1d-α-C-GalCer, and some related analogs, is ∼10-fold lower than that for the NKT TCR-CD1d-α-GalCer interaction. Nevertheless, the crystal structure of the Vβ8.2 NKT TCR-CD1d-α-C-GalCer complex is similar to that of the corresponding NKT TCR-CD1d-α-GalCer complex, although subtle differences at the interface provide a basis for understanding the lower affinity of the NKT TCR-CD1d-α-C-GalCer interaction. Our findings support the concept that for CD1d-restricted NKT cells, altered glycolipid ligands can promote markedly different responses while adopting similar TCR-docking topologies.

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.

Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V(α)14-J(α)18) T cell antigen receptor (TCR) α-chain and recognition of the glycolipid α-galactosylceramide (α-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of α-GalCer-reactive NKT cells that expressed a canonical V(α)10-J(α)50 TCR α-chain, which showed a preference for α-glucosylceramide (α-GlcCer) and bacterial α-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCRα sequence identity, the V(α)10 TCR-CD1d-α-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-α-GalCer complexes, although differences at the antigen-binding interface accounted for the altered antigen specificity. Our findings provide new insight into the structural basis and evolution of glycolipid antigen recognition and have notable implications for the scope and immunological role of glycolipid-specific T cell responses.

Mouse type I natural killer T cell receptors (iNKT TCRs) use a single V alpha 14-J alpha 18 sequence and V beta s that are almost always V beta 8.2, V beta 7, or V beta 2, although the basis of this differential usage is unclear. We showed that the V beta bias occurred as a consequence of the CDR2 beta loops determining the affinity of the iNKT TCR for CD1d-glycolipids, thus controlling positive selection. Within a conserved iNKT-TCR-CD1d docking framework, these inherent V beta-CD1d affinities are further modulated by the hypervariable CDR3 beta loop, thereby defining a functional interplay between the two iNKT TCR CDR beta loops. These V beta biases revealed a broadly hierarchical response in which V beta 8.2 > V beta 7 > V beta 2 in the recognition of diverse CD1d ligands. This restriction of the iNKT TCR repertoire during thymic selection paradoxically ensures that each peripheral iNKT cell recognizes a similar spectrum of antigens.

The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.

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.

Although it has been established how CD1 binds a variety of lipid antigens (Ag), data are only now emerging that show how alphabeta T cell receptors (TCRs) interact with CD1-Ag. Using the structure of the human semiinvariant NKT TCR-CD1d-alpha-galactosylceramide (alpha-GalCer) complex as a guide, we undertook an alanine scanning mutagenesis approach to define the energetic basis of this interaction between the NKT TCR and CD1d. Moreover, we explored how analogues of alpha-GalCer affected this interaction. The data revealed that an identical energetic footprint underpinned the human and mouse NKT TCR-CD1d-alpha-GalCer cross-reactivity. Some, but not all, of the contact residues within the Jalpha18-encoded invariant CDR3alpha loop and Vbeta11-encoded CDR2beta loop were critical for recognizing CD1d. The residues within the Valpha24-encoded CDR1alpha and CDR3alpha loops that contacted the glycolipid Ag played a smaller energetic role compared with the NKT TCR residues that contacted CD1d. Collectively, our data reveal that the region distant to the protruding Ag and directly above the F' pocket of CD1d was the principal factor in the interaction with the NKT TCR. Accordingly, although the structural footprint at the NKT TCR-CD1d-alpha-GalCer is small, the energetic footprint is smaller still, and reveals the minimal requirements for CD1d restriction.

TCR repertoire diversity has been convincingly shown to facilitate responsiveness of CD8+ T cell populations to mutant virus peptides, thereby safeguarding against viral escape. However, the impact of repertoire diversity on the functionality of the CD8+ T cell response to cognate peptide-MHC class I complex (pMHC) recognition remains unclear. Here, we have compared TCRbeta chain repertoires of three influenza A epitope-specific CD8+ T cell responses in C57BL/6 (B6) mice: D(b)NP(366-374), D(b)PA(224-233), and a recently described epitope derived from the +1 reading frame of the influenza viral polymerase B subunit (residues 62-70) (D(b)PB1-F2(62)). Corresponding to the relative antigenicity of the respective pMHCs, and irrespective of the location of prominent residues, the D(b)PA(224)- and D(b)PB1-F2(62)-specific repertoires were similarly diverse, whereas the D(b)NP(366) population was substantially narrower. Importantly, parallel analysis of response magnitude, cytotoxicity, TCR avidity, and cytokine production for the three epitope-specific responses revealed no obvious functional advantage conferred by increased T cell repertoire diversity. Thus, whereas a diverse repertoire may be important for recognition of epitope variants, its effect on the response to cognate pMHC recognition appears minimal.

Natural killer T cells expressing 'invariant' T cell receptor alpha-chains (TCRalpha chains) containing variable (V) and joining (J) region V(alpha)14-J(alpha)18 (V(alpha)14i) rearrangements recognize both endogenous and microbial glycolipids in the context of CD1d. How cells expressing an invariant TCRalpha chain and a restricted set of TCRbeta chains recognize structurally diverse antigens is not clear. Here we show that a V(alpha)14i TCR recognized many alpha-linked glycolipids by means of a 'hot-spot' of germline-encoded amino acids in complementarity-determining regions 3alpha, 1alpha and 2beta. This hot-spot did not shift during the recognition of structurally distinct antigens, suggesting that the V(alpha)14i TCR functions as a pattern-recognition receptor, conferring on natural killer T cells the ability to sense and respond in an innate way to pathogens displaying antigenic alpha-linked glycolipids.

The CD1 family is a large cluster of non-polymorphic, major histocompatibility complex (MHC) class-I-like molecules that bind distinct lipid-based antigens that are recognized by T cells. The most studied group of T cells that interact with lipid antigens are natural killer T (NKT) cells, which characteristically express a semi-invariant T-cell receptor (NKT TCR) that specifically recognizes the CD1 family member, CD1d. NKT-cell-mediated recognition of the CD1d-antigen complex has been implicated in microbial immunity, tumour immunity, autoimmunity and allergy. Here we describe the structure of a human NKT TCR in complex with CD1d bound to the potent NKT-cell agonist alpha-galactosylceramide, the archetypal CD1d-restricted glycolipid. In contrast to T-cell receptor-peptide-antigen-MHC complexes, the NKT TCR docked parallel to, and at the extreme end of the CD1d-binding cleft, which enables a lock-and-key type interaction with the lipid antigen. The structure provides a basis for the interaction between the highly conserved NKT TCR alpha-chain and the CD1d-antigen complex that is typified in innate immunity, and also indicates how variability of the NKT TCR beta-chain can impact on recognition of other CD1d-antigen complexes. These findings provide direct insight into how a T-cell receptor recognizes a lipid-antigen-presenting molecule of the immune system.

The interaction between the alphabeta T cell receptor (TCR) and the peptide bound to the major histocompatibility complex class I molecule (pMHC-I) constitutes a central interaction in adaptive immunity. How these receptors interact with such low affinity while maintaining exquisite specificity for peptide antigen and host MHC (MHC-I restriction) remains a challenge to be explained by structural immunologists. Moreover, how this extracellular interaction is transmitted as an intracellular signal via the CD3 complex remains unresolved. Nevertheless, several structures of TCRs, non-liganded and ligated to a defined pMHC-I, combined with detailed biophysical analyses, have provided insight of the structural basis of MHC-I restriction. In addition, structures of isolated CD3 components have enabled T cell signalling mechanisms to be postulated. Recent findings in this area, which include seven distinct TCR/pMHC-I complexes, have fundamental implications in adaptive immunity as well as therapeutic applications to modulate the adaptive immune response.

The underlying generic properties of alphabeta TCRs that control MHC restriction remain largely unresolved. To investigate MHC restriction, we have examined the CTL response to a viral epitope that binds promiscuously to two human leukocyte Ags (HLAs) that differ by a single amino acid at position 156. Individuals expressing either HLA-B*3501 (156Leucine) or HLA-B*3508 (156Arginine) showed a potent CTL response to the 407HPVGEADYFEY417 epitope from EBV. Interestingly, the response was characterized by highly restricted TCR beta-chain usage in both HLA-B*3501+ and HLA-B*3508+ individuals; however, this conserved TRBV9+ beta-chain was associated with distinct TCR alpha-chains depending upon the HLA-B*35 allele expressed by the virus-exposed host. Functional assays confirmed that TCR alpha-chain usage determined the HLA restriction of the CTLs. Structural studies revealed significant differences in the mobility of the peptide when bound to HLA-B*3501 or HLA-B*3508. In HLA-B*3501, the bulged section of the peptide was disordered, whereas in HLA-B*3508 the bulged epitope adopted an ordered conformation. Collectively, these data demonstrate not only that mobile MHC-bound peptides can be highly immunogenic but can also stimulate an extremely biased TCR repertoire. In addition, TCR alpha-chain usage is shown to play a critical role in controlling MHC restriction between closely related allomorphs.

The underlying basis of major histocompatibility complex (MHC) restriction is unclear. Nevertheless, current data suggest that a common thermodynamic signature dictates alphabeta T cell receptor (TcR) ligation. To evaluate whether this thermodynamic signature defines MHC restriction, we have examined the thermodynamic basis of a highly characterized immunodominant TcR interacting with its cognate peptide-MHC-I ligand. Surprisingly, we observed this interaction to be governed by favorable enthalpic and entropic forces, which is in contrast to the prevailing generality, namely, enthalpically driven interactions combined with markedly unfavorable entropic forces. We conclude that extrinsic molecular factors, such as coreceptor ligation, conformational adjustments involved in TcR signaling, or constraints dictated by higher-order arrangement of ligated TcRs, might play a greater role in guiding MHC restriction than appreciated previously.

Little is known regarding the basis for selection of the semi-invariant alphabeta T cell receptor (TCR) expressed by natural killer T (NKT) cells or how this mediates recognition of CD1d-glycolipid complexes. We have determined the structures of two human NKT TCRs that differ in their CDR3beta composition and length. Both TCRs contain a conserved, positively charged pocket at the ligand interface that is lined by residues from the invariant TCR alpha- and semi-invariant beta-chains. The cavity is centrally located and ideally suited to interact with the exposed glycosyl head group of glycolipid antigens. Sequences common to mouse and human invariant NKT TCRs reveal a contiguous conserved "hot spot" that provides a basis for the reactivity of NKT cells across species. Structural and functional data suggest that the CDR3beta loop provides a plasticity mechanism that accommodates recognition of a variety of glycolipid antigens presented by CD1d. We propose a model of NKT TCR-CD1d-glycolipid interaction in which the invariant CDR3alpha loop is predicted to play a major role in determining the inherent bias toward CD1d. The findings define a structural basis for the selection of the semi-invariant alphabeta TCR and the unique antigen specificity of NKT cells.

Unusually long major histocompatibility complex (MHC) class I-restricted epitopes are important in immunity, but their 'bulged' conformation represents a potential obstacle to alphabeta T cell receptor (TCR)-MHC class I docking. To elucidate how such recognition is achieved while still preserving MHC restriction, we have determined here the structure of a TCR in complex with HLA-B(*)3508 presenting a peptide 13 amino acids in length. This complex was atypical of TCR-peptide-MHC class I interactions, being dominated at the interface by peptide-mediated interactions. The TCR assumed two distinct orientations, swiveling on top of the centrally bulged, rigid peptide such that only limited contacts were made with MHC class I. Although the TCR-peptide recognition resembled an antibody-antigen interaction, the TCR-MHC class I contacts defined a minimal 'generic footprint' of MHC-restriction. Thus our findings simultaneously demonstrate the considerable adaptability of the TCR and the 'shape' of MHC restriction.

Alphabeta T-cell receptor (TcR) recognition of antigenic peptides bound to the major histocompatibility complex (pMHC), is integral to the cellular immune system. Crystallographic studies over the last decade have provided significant insight into this unique trimolecular recognition event. The TcR-pMHC structural information has been paralleled by biophysical studies that have further explored the emerging binding models in an attempt to answer fundamental immunological questions regarding MHC restriction, T-cell immunodominance and TcR cross-reactivity. However, despite the important data that has been generated regarding TcR-pMHC interactions, the scope of this information is still incomplete due to the limited range of TcRs that have been studied. These limitations are primarily due to difficulties in obtaining high yields of recombinant alphabeta TcR for crystallographic and biophysical analysis; here we will discuss some of the protein engineering strategies that have been employed to expand the pool of recombinant TcRs suitable for crystallographic studies and the subsequent studies that have utilized these proteins.