Esther Dawen Yu

Galactose-modified iNKT cell agonists stabilized by an induced fit of CD1d prevent tumour metastasis

Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic α‐galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1‐biasing glycolipid, α‐C‐GalCer, whose CD1d binding follows a conventional key‐lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose‐modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

The Vα14 invariant natural killer T cell TCR forces microbial glycolipids and CD1d into a conserved binding mode

The first crystal structures of iNKT cell TCRs bound to complexes of CD1d and microbe-derived glycolipids provide insight into the structural basis of iNKT cell microbial antigen recognition.

Cutting Edge: Structural Basis for the Recognition of β-Linked Glycolipid Antigens by Invariant NKT Cells

Invariant NKT (iNKT) cells expressing a semi-invariant Vα14 TCR recognize self and foreign lipid Ags when presented by the nonclassical MHCI homolog CD1d. Whereas the majority of known iNKT cell Ags are characterized by the presence of a single α-linked sugar, mammalian self Ags are β-linked glycosphingolipids, posing the interesting question of how the semi-invariant TCR can bind to such structurally distinct ligands. In this study, we show that the mouse iNKT TCR recognizes the complex β-linked Ag isoglobotrihexosylceramide (iGb3; Galα1-3-Galβ1-4-Glcβ1-1Cer) by forcing the proximal β-linked sugar of the trisaccharide head group to adopt the typical binding orientation of α-linked glycolipids. The squashed iGb3 orientation is stabilized by several interactions between the trisaccharide and CD1d residues. Finally, the formation of novel contacts between the proximal and second sugar of iGb3 and CDR2α residues of the TCR suggests an expanded recognition logic that can possibly distinguish foreign Ags from self Ags.

Unique Interplay between Sugar and Lipid in Determining the Antigenic Potency of Bacterial Antigens for NKT Cells.

Structural and biophysical studies reveal the induced-fit mechanism underlying the stringent specificity of invariant natural killer T cells for unique glycolipid antigens from the pathogen Streptococcus pneumoniae.

Structural and Functional Characterization of a Novel Nonglycosidic Type I NKT Agonist with Immunomodulatory Properties

Activation of type I NKT (iNKT) cells by CD1d-presented agonists is a potent immunotherapeutic tool. α-Galactosylceramide (α-GalCer) is the prototypic agonist, but its excessive potency with simultaneous production of both pro- and anti-inflammatory cytokines hampers its potential therapeutic use. In search for novel agonists, we have analyzed the structure and function of HS44, a synthetic aminocyclitolic ceramide analog designed to avoid unrestrained iNKT cell activation. HS44 is a weaker agonist compared with α-GalCer in vitro, although in vivo it induces robust IFN-γ production, and highly reduced but still functional Th2 response. The characteristic cytokine storm produced upon α-GalCer activation was not induced. Consequently, HS44 induced a very efficient iNKT cell-dependent antitumoral response in B16 animal model. In addition, intranasal administration showed the capacity to induce lung inflammation and airway hyperreactivity, a cardinal asthma feature. Thus, HS44 is able to elicit functional Th1 or Th2 responses. Structural studies show that HS44 binds to CD1d with the same conformation as α-GalCer. The TCR binds to HS44 similarly as α-GalCer, but forms less contacts, thus explaining its weaker TCR affinity and, consequently, its weaker recognition by iNKT cells. The ability of this compound to activate an efficient, but not massive, tailored functional immune response makes it an attractive reagent for immune manipulation.

Structural Basis for the Recognition of C20:2-αGalCer by the Invariant Natural Killer T Cell Receptor-like Antibody L363

Background: Antibodies that recognize glycolipids presented by the antigen-presenting molecule CD1d are useful tools in studying natural killer T cell biology. Results: The CD1d-α-galactosylceramide specific antibody L363 has TCR-like binding properties. Conclusion: Glycolipid-reactive antibodies are more antigen-specific than the TCR of iNKT cells. Significance: This is the first crystal structure of a glycolipid-reactive TCR-like antibody. Natural killer T (NKT) cells express a semi-invariant Vα14 T cell receptor (TCR) and recognize structurally diverse antigens presented by the antigen-presenting molecule CD1d that range from phosphoglycerolipids to α- and β-anomeric glycosphingolipids, as well as microbial α-glycosyl diacylglycerolipids. Recently developed antibodies that are specific for the complex of the prototypical invariant NKT (iNKT) cell antigen αGalCer (KRN7000) bound to mouse CD1d have become valuable tools in elucidating the mechanism of antigen loading and presentation. Here, we report the 3.1 Å resolution crystal structure of the Fab of one of these antibodies, L363, bound to mCD1d complexed with the αGalCer analog C20:2, revealing that L363 is an iNKT TCR-like antibody that binds CD1d-presented αGalCer in a manner similar to the TCR. The structure reveals that L363 depends on both the L and H chains for binding to the glycolipid-mCD1d complex, although only the L chain is involved in contacts with the glycolipid antigen. The H chain of L363 features residue Trp-104, which mimics the TCR CDR3α residue Leu-99, which is crucial for CD1d binding. We characterized the antigen-specificity of L363 toward several different glycolipids, demonstrating that whereas the TCR can induce structural changes in both antigen and CD1d to recognize disparate lipid antigens, the antibody L363 can only induce the F′ roof formation in CD1d but fails to reorient the glycolipid headgroup necessary for binding. In summary, L363 is a powerful tool to study mechanism of iNKT cell activation for structural analogs of KRN7000, and our study can aid in the design of antibodies with altered antigen specificity.

Microbial glycolipid antigen recognition by invariant natural killer T cells

Microbial glycolipid antigen recognition by invariant natural killer T cells Dirk M. Zajonc,a Yali Li,a Enrico Girardi,a Esther Dawen Yu,a Jing Wang,a Gavin F. Painter,b Petr Illarionov,c Yuki Kinjo,d Mitchell Kronenberg,e aDivision of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037 (USA). bCarbohydrate Chemistry Team, Industrial Research Limited, Lower Hutt (New Zealand). cSchool of Biosciences, University of Birmingham, Edgbaston (UK) dDepartment of Chemotherapy and Mycoses, National Institute for Infectious Diseases, Tokyo (Japan). eDivision of Developmental Immunology, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037 (USA). E-mail: dzajonc@liai.org