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Mycobacterial cell wall lipids bind the conserved CD1 family of antigen-presenting molecules and activate T cells via their T cell receptors (TCRs). Sulfoglycolipids (SGLs) are uniquely synthesized by Mycobacterium tuberculosis, but tools to study SGL-specific T cells in humans are lacking. We designed a novel hybrid synthesis of a naturally occurring SGL, generated CD1b tetramers loaded with natural or synthetic SGL analogs, and studied the molecular requirements for TCR binding and T cell activation. Two T cell lines derived using natural SGLs are activated by synthetic analogs independently of lipid chain length and hydroxylation, but differentially by saturation status. By contrast, two T cell lines derived using an unsaturated SGL synthetic analog were not activated by the natural antigen. Our data provide a bioequivalence hierarchy of synthetic SGL analogs and SGL-loaded CD1b tetramers. These reagents can now be applied to large-scale translational studies investigating the diagnostic potential of SGL-specific T cell responses or SGL-based vaccines., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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.

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.

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 anti- gens 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. [ABSTRACT FROM AUTHOR]

The most potent foreign antigens for natural killer T cells (NKT cells) are ?-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct ?-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 ?-linked agonists ?-galactosylceramide (?-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 ?-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 ?-linked self ligands to resemble the conformation of foreign ?-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to ?-linked antigens. [ABSTRACT FROM AUTHOR]

Type I natural killer T cells (NKT cells) are characterized by an invariant variable region 14-joining region 18 (V?14-J?18) T cell antigen receptor (TCR) ?-chain and recognition of the glycolipid ?-galactosylceramide (?-GalCer) restricted to the antigen-presenting molecule CD1d. Here we describe a population of ?-GalCer-reactive NKT cells that expressed a canonical V?10-J?50 TCR ?-chain, which showed a preference for ?-glucosylceramide (?-GlcCer) and bacterial ?-glucuronic acid-containing glycolipid antigens. Structurally, despite very limited TCR? sequence identity, the V?10 TCR-CD1d-?-GlcCer complex had a docking mode similar to that of type I TCR-CD1d-?-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. [ABSTRACT FROM AUTHOR]

Abstract: αβ T cell receptors (TCRs) have traditionally been viewed as receptors for peptide antigens presented by either Major Histocompatibility Complex (MHC) class I (for CD8 T cells) or MHC class II (for CD4 T cells) antigen-presenting molecules. However, it is now clear that some T cell lineages express TCRs that are specialized for recognition of lipid-based antigens presented by the MHC class I-like CD1 family. Recently, the molecular basis for the TCR recognition of glycolipid antigens presented by CD1d has revealed an evolutionarily conserved-docking mode that is distinct from that of peptide-based recognition. T cells carrying these receptors follow a unique developmental pathway that results not only in unconventional antigen specificity, but also seemingly exaggerated functional capabilities, which makes these cells and their antigens highly attractive targets for immunotherapeutic manipulation. [Copyright &y& Elsevier]

The semi-invariant natural killer (NK) T-cell receptor (NKTcr) recognises structurally diverse glycolipid antigens presented by the monomorphic CD1d molecule. While the α-chain of the NKTcr is invariant, the β-chain is more diverse, but how this diversity enables the NKTcr to recognise diverse antigens, such as an α-linked monosaccharide (α-galactosylceramide and α-galactosyldiacylglycerol) and the β-linked trisaccharide (isoglobotriaosylceramide), is unclear. We demonstrate here that NKTcrs, which varied in their β-chain usage, recognised diverse glycolipid antigens with a similar binding mode on CD1d. Nevertheless, the NKTcrs recognised distinct epitopic sites within these antigens, including α-galactosylceramide, the structurally similar α-galactosyldiacylglycerol and the very distinct isoglobotriaosylceramide. We also show that the relative roles of the CDR loops within the NKTcr β-chain varied as a function of the antigen. Thus, while NKTcrs characteristically use a conserved docking mode, the NKTcr β-chain allows these cells to recognise unique aspects of structurally diverse CD1d-restricted ligands. [ABSTRACT FROM AUTHOR]

Natural killer T cells (NKT cells) are selected in the thymus by self-glycolipid antigens presented by CD1d molecules. It is currently thought that one specific component of the lysosomal processing pathway, which leads to the production of isoglobotrihexosylceramide (iGb3), is essential for normal NKT cell development. New evidence now shows that NKT cell development can be disrupted by a diverse range of mutations that interfere with different elements of the lysosomal processing and degradation of glycolipids. This suggests that lysosomal storage diseases (LSDs) in general, rather than one specific defect, can disrupt CD1d antigen presentation, leading to impaired development of NKT cells. [ABSTRACT FROM AUTHOR]

The article focuses on a study in mice which reported that Natural killer T (NKT) cells are crucial for progression of Aspergillus fumigatus–induced asthma. It informs that Aspergillus fumigatus is a fungus that is associated with severe forms of asthma. The study also reported that a glycolipid antigen from the fungus drives NKT cell–mediated inflammatory response.

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. [ABSTRACT FROM AUTHOR]

Current views emphasize TCR diversity as a key feature that differentiates the group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 systems. Whereas TCR sequence motifs define CD1d-reactive NKT cells, the available data do not allow a TCR-based organization of the group 1 CD1 repertoire. The observed TCR diversity might result from donor-to-donor differences in TCR repertoire, as seen for MHC-restricted T cells. Alternatively, diversity might result from differing CD1 isoforms, Ags, and methods used to identify TCRs. Using CD1b tetramers to isolate clones recognizing the same glycolipid, we identified a previously unknown pattern of V gene usage (TRAV17, TRBV4-1) among unrelated human subjects. These TCRs are distinct from those present on NKT cells and germline-encoded mycolyl lipid-reactive T cells. Instead, they resemble the TCR of LDN5, one of the first known CD1b-reactive clones that was previously thought to illustrate the diversity of the TCR repertoire. Interdonor TCR conservation was observed in vitro and ex vivo, identifying LDN5-like T cells as a distinct T cell type. These data support TCR-based organization of the CD1b repertoire, which consists of at least two compartments that differ in TCR sequence motifs, affinity, and coreceptor expression. [ABSTRACT FROM AUTHOR]

Summary: Natural killer T (NKT) cells respond to a variety of CD1d-restricted antigens (Ags), although the basis for Ag discrimination by the NKT cell receptor (TCR) is unclear. Here we have described NKT TCR fine specificity against several closely related Ags, termed altered glycolipid ligands (AGLs), which differentially stimulate NKT cells. The structures of five ternary complexes all revealed similar docking. Acyl chain modifications did not affect the interaction, but reduced NKT cell proliferation, indicating an affect on Ag processing or presentation. Conversely, truncation of the phytosphingosine chain caused an induced fit mode of TCR binding that affected TCR affinity. Modifications in the glycosyl head group had a direct impact on the TCR interaction and associated cellular response, with ligand potency reflecting the t1/2 life of the interaction. Accordingly, we have provided a molecular basis for understanding how modifications in AGLs can result in striking alterations in the cellular response of NKT cells. [ABSTRACT FROM AUTHOR]

Summary: Mouse type I natural killer T cell receptors (iNKT TCRs) use a single Vα14-Jα18 sequence and Vβs that are almost always Vβ8.2, Vβ7, or Vβ2, although the basis of this differential usage is unclear. We showed that the Vβ bias occurred as a consequence of the CDR2β 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 Vβ-CD1d affinities are further modulated by the hypervariable CDR3β loop, thereby defining a functional interplay between the two iNKT TCR CDRβ loops. These Vβ biases revealed a broadly hierarchical response in which Vβ8.2 > Vβ7 > Vβ2 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. [Copyright &y& Elsevier]