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The Journal of Immunology, 2008, 180, 6452 -6456
Copyright © 2008 by The American Association of Immunologists, Inc.
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Cutting Edge: Nonglycosidic CD1d Lipid Ligands Activate Human and Murine Invariant NKT Cells1

Jonathan D. Silk2,*, Mariolina Salio2,*, B. Gopal Reddy2,{dagger}, Dawn Shepherd*, Uzi Gileadi*, James Brown{ddagger}, S. Hajar Masri*, Paolo Polzella*, Gerd Ritter§, Gurdyal S. Besra, E. Yvonne Jones{ddagger}, Richard R. Schmidt{dagger} and Vincenzo Cerundolo3,*

* Tumour Immunology Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; {dagger} Fachbereich Chemie, Universität Konstanz, Konstanz, Germany; {ddagger} Cancer Research U.K. Receptor Structure Research Group, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom; § Ludwig Institute for Cancer Research, New York Branch at Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than {alpha}-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/{alpha}-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Invariant natural killer T cells (iNKT cells)4 are CD1d-restricted T-lymphocytes expressing a semi-invariant {alpha}/β TCR encoded by V{alpha}14-J{alpha}18 segments in mice paired with restricted β-chains. Human iNKT cells express a V{alpha}24-J{alpha}18 {alpha}-chain often paired with Vβ11 (1). Although NKT cells expressing V{alpha}24-negative TCR were described (2), the CDR3 {alpha}-loops of V{alpha}24-negative and -positive TCR are almost superimposable (3). Activated iNKT cells rapidly produce IFN-{gamma} and IL-4 and induce dendritic cell (DC) maturation and IL-12 production (4, 5).

A number of bacterial, mammalian, and synthetic glycolipids that stimulate iNKT cells have been found (reviewed in Ref. 6). The best characterized, {alpha}-galactosylceramide ({alpha}-GalCer), comprises galactose attached via an {alpha}-linkage to ceramide (see Fig. 1A). The crystal structure of {alpha}-GalCer/CD1d showed that the ceramide is held in two hydrophobic A' and F' channels while the galactose head group protrudes from the binding groove (7, 8). The crystal structure of the human iNKT TCR/{alpha}-GalCer/CD1d complex (9) and mouse iNKT TCR mutagenesis (10) showed that the TCR has a different mode of docking onto {alpha}-GalCer/CD1d than TCR/peptide/MHC class I complexes, with ligand recognition being predominantly via the {alpha}-chain.


Figure 1
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  		FIGURE 1. Binding affinities of the iNKT TCR for hCD1d molecules loaded with nonglycosidic compounds. Equilibrium binding and kinetic measurements of a soluble human iNKT cell TCR were assessed for hCD1d molecules refolded with {alpha}-GalCer (A), ThrCer (B), GlyCer (C), and AraCer (D). Kd values (µM) were calculated from equilibrium binding. Kinetic and equilibrium binding values are the mean of at least two independent experiments. The structure of each ligand is indicated to the right.

 
Such structural studies enabled us to assess the effect of minimizing the head group of iNKT cell agonists on their ability to activate iNKT cells. We synthesized ligands in which galactose, the focal point of iNKT TCR recognition, is substituted with nonglycosidic variants. The ligands were used for functional and kinetic studies.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Mice, cell lines, and reagents

C57BL/6 and J{alpha}18 –/– mice (iNKT–/– mice) (11) were used. The tumor cell line E.G7-OVA (12) was used for in vivo experiments. Animal experiments were done under the authority of a U.K. Home Office Project License. Compounds were solubilized in 150 mM NaCl and 0.5% Tween 20 (vehicle). OVA was provided by T. Moran (Mount Sinai School of Medicine, New York, NY) and monophosphoryl lipid A (MPL) (Salmonella minnesota) from Sigma-Aldrich. Abs were from BD Biosciences or eBioscience. Flow cytometry was performed on a FACScalibur device with CellQuest software.

Soluble iNKT TCR and CD1d-ligand monomers

Production and use of soluble human V{alpha}24/Vβ11 TCR and human CD1d (hCD1d) monomers were described (13, 14).

Surface plasmon resonance

Experiments were performed with a Biacore 3000 (13).

Expansion of human iNKT cells and of DC maturation

Experiments were conducted as described (15).

Cytokine ELISA

Supernatants and sera were analyzed for IL-12p40, IFN-{gamma}, or IL-4 with ELISA (BD Biosciences and eBioscience).

Administration of Ags and adjuvants

All substances were diluted in PBS and administered i.v. Unless stated, doses used were 400 µg of OVA, 1 µg of compounds or vehicle, and 25 µg of MPL.

Phenotype of murine APCs

Expression of CD86 on CD11c+ and B220+ APCs from spleen was assessed by flow cytometry after 20 h. The VITAL assay was performed as described (16), staining with CD11c-allophycocyanin.

Induction of iNKT cell anergy

Mice were injected with 0.1 µg of either {alpha}-GalCer or threitolceramide (ThrCer) as described (17).

Monitoring OVA-specific immune responses

Mice were bled 6 days after immunization and PBL stained with H-2 Kb/SIINFEKL tetramers. The OVA-specific IgG ELISA for sera is described (18). To measure anti-tumor responses, immunized mice were injected with 1 x 106 E.G7-OVA cells s.c. and tumor size was calculated as the mean of the products of bisecting diameters (±SE).

Molecular modeling of ligands onto hCD1d/{alpha}-GalCer

Models were created using program O based on the TCR/{alpha}-GalCer/hCD1d structure (PDB Code 2PO6) (9). The CD1d, β2-microglobulin, and TCR structures were unmodified. Figures were generated with PyMol.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
ThrCer can activate both human and murine iNKT cells

The crystal structure of the iNKT TCR/{alpha}-GalCer/CD1d complex demonstrated that the 2', 3', and 4' hydroxyl groups of galactose make hydrogen bonds (H-bonds) with the iNKT TCR {alpha}-chain (9). We assessed whether iNKT cells could recognize clipped versions of the head group in which the alcohol moieties at C(5) and C(6) of the galactose were absent and the three key residues at C(2), C(3), and C(4) were retained. We synthesized a CD1d-binding, lipid-comprising ceramide, ether linked to sugar alcohols with four carbons (threitolceramide, referred to as ThrCer), and analyzed its ability to be recognized by human and murine iNKT cells.

Biacore affinity measurements were performed using a soluble iNKT TCR to compare the binding affinity of the human TCR for human CD1d molecules loaded with {alpha}-GalCer or ThrCer (Fig. 1, A and B). The iNKT TCR equilibrium binding constants (Kd) at 25°C were determined to be 1.3 and 5.78 µM for {alpha}-GalCer/CD1d or ThrCer/CD1d, respectively (Fig. 1, A and B). Good agreement was observed between the affinities determined kinetically (ratio of Koff to Kon) and by equilibrium measurements. The lower binding affinity was due to a combination of a faster off rate and a slower on rate for the iNKT TCR when bound to ThrCer/hCD1d than to {alpha}-GalCer/hCD1d.

Molecular modeling to assess the binding of the iNKT TCR/ThrCer/CD1d complex (Fig. 2B), based on the structure of iNKT TCR/{alpha}-GalCer/CD1d (9) (Fig. 2A), indicated that H-bonds between Phe29, Ser30, and Gly96 on the TCR {alpha}-chain and O4', O3' and O2' of the threitol head group are maintained, stabilizing the head group and allowing recognition by the iNKT TCR (Fig. 2B).


Figure 2
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  		FIGURE 2. Molecular modeling of ligands onto the structure of hCD1d/{alpha}-GalCer/TCR reveals potential conservation of H-bonds. Molecular models were superimposed onto the structure of the hCD1d/{alpha}-GalCer/TCR complex (A) (9 ). Models of ThrCer (B), GlyCer (C), and AraCer (D) have different numbers of stabilizing H-bonds with Phe29, Ser30, and Gly96 residues in the TCR, which may reflect their affinities. The hCD1d molecule is shown in green, the ligands in magenta, and the iNKT TCR CDR1{alpha} and CDR3{alpha} in yellow and cyan.

 
We confirmed the Biacore measurements by showing that ThrCer activated both human and mouse iNKT cells as defined by the maturation of human and mouse DCs (Fig. 3, A and B), the expansion of human iNKT cells in vitro (Fig. 3C), and in vivo secretion of IFN-{gamma} and IL-4 (Fig. 3D).


Figure 3
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  		FIGURE 3. Nonglycocosidic iNKT cell agonists activate human and murine iNKT cells. A, LPS or iNKT cell agonists were added to cocultures of human DCs and iNKT cells and DC maturation was assessed after 40 h. Histograms show the CD83 and CD86 profiles. Percentages indicate the percentage of DCs in the labeled gate. B, Twenty hours after injection, splenocytes from C57BL/6 or iNKT–/– mice were stained with anti-CD11c, B220, and CD86. Maturation was assessed by the expression of CD86 gating on DCs (CD11c+). Mean fluorescence intensity is shown. WT, Wild type. C, Monocyte-depleted fractions from PBL were cultured for 13 days in the presence of autologous DCs pulsed with 500 ng per ml of the indicated ligand to induce human iNKT cell expansion. Cells were stained with CD1d tetramer-PE and CD3-allophycocyanin, gated on live cells. The percentage of iNKT cells is shown (tetramer+/CD3+). The negative fraction from PBL in the absence of DCs was also tested. D, Mice were immunized and sera analyzed at different times for IFN-{gamma} or IL-4. E, Viability of human DCs was assessed after 40 h of coculture with iNKT cells and the indicated concentrations of ligands. Percentages of live DCs are shown. IL-12 release in the same experiment was measured with ELISA. F, Splenocytes from iNKT–/– mice were pulsed with {alpha}-GalCer or ThrCer, labeled, and injected into C57BL/6 or NKT–/– animals. After 24 h the mice were culled and splenocytes were stained with CD11c-allophycocyanin and the percentage of specific lysis of DCs was calculated.

 
Importantly, activation of human iNKT cells by ThrCer-pulsed DCs, while inducing DC maturation as defined by IL-12 production (Fig. 3E) and IFN-{gamma} secretion (data not shown), ensured a greater proportion of live DCs as compared with the DC survival after the activation of human iNKT cells by {alpha}-GalCer- or {alpha}-GalCer analog C20:2-pulsed DCs (19) (Fig. 3E). To confirm and extend these results, mouse splenocytes were fluorescently labeled, pulsed with {alpha}-GalCer or ThrCer, and injected into syngeneic mice. Ninety percent of {alpha}-GalCer-pulsed DCs were killed while 50% of ThrCer-pulsed DCs survived (Fig. 3F), showing that the lifespan of DCs pulsed with ThrCer is longer than that of DCs pulsed with {alpha}-GalCer.

Differential ability of glycerolceramide and arabinitolceramide to activate human and murine iNKT cells

Having shown that ThrCer can activate both human and murine iNKT cells, we compared the activity of two other compounds with head groups of three (glycerolceramide, referred to as GlyCer) or five (arabinitolceramide, referred to as AraCer) hydroxymethylene residues linked to ceramide. These studies revealed an unexpected dichotomy in the ability of the two compounds to be recognized by iNKT cells. The human iNKT TCR bound to GlyCer/CD1d with an affinity of 2.96 µM (Fig. 1C). GlyCer efficiently activated human iNKT cells (Fig. 3C), resulting in DC maturation (Fig. 3A). In contrast, GlyCer failed to stimulate murine iNKT cells (Fig. 3D) or induce DC maturation (Fig. 3B). The iNKT TCR had a weaker affinity for AraCer/CD1d (25 µM) (Fig. 1D) and AraCer had a significantly reduced ability to activate human iNKT cells (Fig. 3C) and induce DC maturation (Fig. 3A) compared with GlyCer and ThrCer. Surprisingly, when AraCer was injected into mice it induced secretion of IFN-{gamma} and IL-4 (Fig. 3D) and maturation of DCs (Fig. 3B). The dichotomy in the ability of GlyCer and AraCer to be recognized by human and murine iNKT cells was confirmed by pulsing C1R cells, transfected with either hCD1d or murine CD1d, with GlyCer or AraCer and using them to stimulate either human or murine iNKT cells (data not shown).

Modeling GlyCer (Fig. 2C) onto the CD1d/{alpha}-GalCer/TCR structure suggested that although GlyCer could only maintain three of the H-bonds formed by ThrCer (Fig. 2B) given its shorter length, three H-bonds may be sufficient to lock the head group into position. This reasoning is consistent with the observation that the human iNKT TCR had a similar binding affinity for GlyCer and ThrCer (Fig. 1, B and C). The weaker affinity of the iNKT TCR for AraCer/CD1d (Fig. 1D) could possibly be accounted for by an increased flexibility of the head group due to the additional hydroxymethylene group (C5), which may attain a different conformation and inhibit the interaction with the TCR (Fig. 2D).

Nonglycosidic iNKT cell agonists assist Ag specific T and B cell responses

We and others have shown that coinjection of {alpha}-GalCer with Ag induces enhanced T and B cell responses (4, 5, 18, 20, 21). ThrCer, GlyCer, or AraCer with OVA were injected into mice and the magnitude of OVA-specific CD8+ T and B cell responses was assessed. Immune responses in mice injected with ThrCer were comparable to those seen with {alpha}-GalCer (Fig. 4, A and C) and were further enhanced by coinjecting the TLR ligand MPL (data not shown). Injection with AraCer induced OVA-specific B cell responses similar to those in {alpha}-GalCer treated mice, whereas there were fewer OVA-specific T cells (Fig. 4, A and B). OVA-specific T cells in mice injected with OVA and ThrCer or AraCer rejected E.G7-OVA tumor cells compared with control groups (Fig. 4C). Mice injected with GlyCer/OVA had reduced tumor growth but were not protected.


Figure 4
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  		FIGURE 4. Adjuvant activity of nonglycosidic iNKT cell agonists. A, OVA-specific CD8+ T cell responses were analyzed and are shown as tetramer+ cells as percentage of CD8+ cells. B and C, Mice were bled 11–14 days after priming and the sera were tested by ELISA for the presence of OVA-specific IgGs. D, Mice were immunized and challenged 7 days later with EG7.OVA cells s.c. Tumor growth was monitored and the mean tumor size was calculated (±SE). E, iNKT cells stimulated with ThrCer recover from activation-induced anergy more quickly than with {alpha}-GalCer. Mice (n = 3) were immunized with 0.1 µg of {alpha}-GalCer or ThrCer i.v. on day –7 or –14. Splenocytes were cultured for 60 h in the presence of vehicle or {alpha}-GalCer and the supernatants were tested for IFN-{gamma}.

 
These results confirmed the ability of ThrCer and AraCer to induce murine iNKT cell-dependent DC maturation and demonstrated their ability to facilitate the crosstalk between iNKT and B cells.

Previous studies showed that {alpha}-GalCer induces iNKT cell anergy as defined by a stunted response to a second exposure to {alpha}-GalCer (17). Although iNKT cells stimulated with ThrCer in vivo were initially refractory to a subsequent challenge with {alpha}-GalCer in vitro, after 14 days they recovered the ability to produce IFN-{gamma} (Fig. 4E) and IL-4 (data not shown). In contrast, iNKT cells stimulated in vivo with {alpha}-GalCer failed to respond after 14 days to in vitro stimulation with {alpha}-GalCer. These data indicate that the unresponsiveness of iNKT cells after their in vivo stimulation with ThrCer is shorter lived than iNKT cell unresponsiveness caused by in vivo stimulation with {alpha}-GalCer, a property that may have advantages for the use of ThrCer in vaccination strategies.

Concluding Remarks

Our results identify a new class of iNKT cell agonists, broadening the range of potential compounds that can activate iNKT cells. Although it is unclear whether such nonglycosidic compounds can be naturally synthesized, processing of monogalactosyl diacylglycerol, derived from Borrelia burgdorferi (22), may result in the generation of diacylglycerol capable of activating iNKT cells.

The divergence in Ag recognition of GlyCer and AraCer by murine and human iNKT cells highlights important differences in the ability of iNKT cells to recognize CD1d-restricted ligands, which have previously been overlooked using {alpha}-GalCer analogues. Comparison of murine and human iNKT TCRs revealed a "hot spot" of conserved residues at the ligand-binding site conferring cross-species reactivity to ligands (10, 23). The 2', 3', and 4' OH groups of the galactose were found to be important for hydrogen bonding with the iNKT TCR {alpha}-chain, while the 6' OH group pointed toward the solvent (9). These structural arguments are consistent with the observation that human iNKT cells recognize ThrCer and GlyCer, possibly because H-bonds are likely to be maintained through the 2' and 3' OH groups.

Finally, these compounds should be considered as adjuvants capable of harnessing iNKT cells for vaccination strategies, as their weaker affinity for the human iNKT TCR minimizes overstimulation of iNKT cells without compromising DC maturation and expansion of Ag-specific T and B cells.

The ability of these nonglycosidic compounds to activate iNKT cells, together with the observation that an iNKT cell hybridoma was activated by phosphoethanolamine (24), underlines the degree of promiscuity of human and murine iNKT cells with regard to ligand recognition, suggesting that the search for natural iNKT cell ligands should not be limited to glycolipids but that other lipid structures should also be considered.


   Acknowledgments
 
We thank Andrea Tarlton, Ian Hermans, Michael Palmowski, and Michael Koch for discussions and assistance with the project.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Funding for this work was provided by the Ludwig Institute for Cancer Research and Cancer Research U.K. Grants C399/A2291 (to V.C.) and C375 (to E.Y.J.) and the Royal Society Wolfson Research Merit Award, a Personal Research Chair from James Bardrick, and a former Lister Institute-Jenner Research Fellowship (to G.S.B.). Back

2 J.D.S., M.S., and B.G.R. contributed equally to the work. Back

3 Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, Tumour Immunology Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom. E-mail address: vincenzo.cerundolo{at}imm.ox.ac.uk Back

4 Abbreviations used in this paper: iNKT cell, invariant NK T cell; {alpha}-GalCer, {alpha}-galactosylceramide; AraCer, arabinitolceramide; DC, dendritic cell; GlyCer, glycerolceramide; H-bond, hydrogen bond; hCD1d, human CD1d; MPL, monophosphoryl lipid A; ThrCer, threitolceramide. Back

Received for publication December 17, 2007. Accepted for publication March 19, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 

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