Stewart K. Richardson

A molecular basis for the exquisite CD1d-restricted antigen specificity and functional responses of natural killer T cells

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 t(1/2) 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.

T Cell Receptor CDR2β and CDR3β Loops Collaborate Functionally to Shape the iNKT Cell Repertoire

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.

A minimal binding footprint on CD1d-glycolipid is a basis for selection of the unique human NKT TCR

Although it has been established how CD1 binds a variety of lipid antigens (Ag), data are only now emerging that show how αβ T cell receptors (TCRs) interact with CD1-Ag. Using the structure of the human semiinvariant NKT TCR–CD1d–α-galactosylceramide (α-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 α-GalCer affected this interaction. The data revealed that an identical energetic footprint underpinned the human and mouse NKT TCR–CD1d–α-GalCer cross-reactivity. Some, but not all, of the contact residues within the Jα18-encoded invariant CDR3α loop and Vβ11-encoded CDR2β loop were critical for recognizing CD1d. The residues within the Vα24-encoded CDR1α and CDR3α 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–α-GalCer is small, the energetic footprint is smaller still, and reveals the minimal requirements for CD1d restriction.

Natural Sphingomonas Glycolipids Vary Greatly in Their Ability to Activate Natural Killer T Cells

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.

Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands

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.

Vβ2 natural killer T cell antigen receptor-mediated recognition of CD1d-glycolipid antigen

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.

Human and mouse type I natural killer T cell antigen receptors exhibit different fine specificities for CD1d-antigen complex

Background: Natural killer T cell antigen receptors (NKT TCRs) are restricted to lipid antigens presented by CD1d. Results: Fine specificity differences between human and mouse NKT TCRs toward CD1d-antigen complexes were observed. Conclusion: A structural basis underpins the fine specificity differences between human and mouse NKT TCRs. Significance: Understanding human NKT cell response to CD1d-restricted antigens has important therapeutic implications in developing NKT cell agonists. Human and mouse type I natural killer T (NKT) cells respond to a variety of CD1d-restricted glycolipid antigens (Ags), with their NKT cell antigen receptors (NKT TCRs) exhibiting reciprocal cross-species reactivity that is underpinned by a conserved NKT TCR-CD1d-Ag docking mode. Within this common docking footprint, the NKT TCR recognizes, to varying degrees of affinity, a range of Ags. Presently, it is unclear whether the human NKT TCRs will mirror the generalities underpinning the fine specificity of the mouse NKT TCR-CD1d-Ag interaction. Here, we assessed human NKT TCR recognition against altered glycolipid ligands of α-galactosylceramide (α-GalCer) and have determined the structures of a human NKT TCR in complex with CD1d-4′,4″-deoxy-α-GalCer and CD1d-α-GalCer with a shorter, di-unsaturated acyl chain (C20:2). Altered glycolipid ligands with acyl chain modifications did not affect the affinity of the human NKT TCR-CD1d-Ag interaction. Surprisingly, human NKT TCR recognition is more tolerant to modifications at the 4′-OH position in comparison with the 3′-OH position of α-GalCer, which contrasts the fine specificity of the mouse NKT TCR-CD1d-Ag recognition (4′-OH > 3′-OH). The fine specificity differences between human and mouse NKT TCRs was attributable to differing interactions between the respective complementarity-determining region 1α loops and the Ag. Accordingly, germline encoded fine-specificity differences underpin human and mouse type I NKT TCR interactions, which is an important consideration for therapeutic development and NKT cell physiology.

Atypical natural killer T-cell receptor recognition of CD1d-lipid antigens.

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.

Regulatory roles for NKT cell ligands in environmentally induced autoimmunity.

The development of autoimmune diseases is frequently linked to exposure to environmental factors such as chemicals, drugs, or infections. In the experimental model of metal-induced autoimmunity, administration of subtoxic doses of mercury (a common environmental pollutant) to genetically susceptible mice induces an autoimmune syndrome with rapid anti-nucleolar Ab production and immune system activation. Regulatory components of the innate immune system such as NKT cells and TLRs can also modulate the autoimmune process. We examined the interplay among environmental chemicals and NKT cells in the regulation of autoimmunity. Additionally, we studied NKT and TLR ligands in a tolerance model in which preadministration of a low dose of mercury in the steady state renders animals tolerant to metal-induced autoimmunity. We also studied the effect of Sphingomonas capsulata, a bacterial strain that carries both NKT cell and TLR ligands, on metal-induced autoimmunity. Overall, NKT cell activation by synthetic ligands enhanced the manifestations of metal-induced autoimmunity. Exposure to S. capsulata exacerbated autoimmunity elicited by mercury. Although the synthetic NKT cell ligands that we used are reportedly similar in their ability to activate NKT cells, they displayed pronounced differences when coinjected with environmental agents or TLR ligands. Individual NKT ligands differed in their ability to prevent or break tolerance induced by low-dose mercury treatment. Likewise, different NKT ligands either dramatically potentiated or inhibited the ability of TLR9 agonistic oligonucleotides to disrupt tolerance to mercury. Our data suggest that these differences could be mediated by the modification of cytokine profiles and regulatory T cell numbers.

Erratum: Adaptability of the semi-invariant natural killer T-cell receptor towards structurally diverse CD1d-restricted ligands (The EMBO Journal (2009) 28 (3579-3590) DOI: 10.1038/emboj.2009.286

Department of Microbiology and Immunology, Vanderbilt University, School of Medicine, Nashville, TN, USA, Department of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, National Jewish Centre for Allergy and Immunology Research, Denver, CO, USA, Division of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA, Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA, Department of Chemistry, University of Connecticut, Storrs, CT, USA, Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia, Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria, Australia, Fox Chase Cancer Centre, Philadelphia, PA, USA and Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA