of March 15, 2018.This information is current as

ProductionγThat Preferentially Induces IFN-

A Novel Glycolipid Antigen for NKT Cells

and Mitchell KronenbergYuan, Steven A. Porcelli, Gurdyal S. Besra, Dirk M. Zajonc Petr A. Illarionov, Xiangshu Wen, Michelle Li, WeimingArchana Khurana, Jing Wang, Meng Zhao, Sonja Zahner, Alysia M. Birkholz, Enrico Girardi, Gerhard Wingender,

http://www.jimmunol.org/content/195/3/924doi: 10.4049/jimmunol.15000702015;

2015; 195:924-933; Prepublished online 15 JuneJ Immunol 

MaterialSupplementary

0.DCSupplementalhttp://www.jimmunol.org/content/suppl/2015/06/13/jimmunol.150007

Referenceshttp://www.jimmunol.org/content/195/3/924.full#ref-list-1

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The Journal of Immunology

A Novel Glycolipid Antigen for NKT Cells That PreferentiallyInduces IFN-g Production

Alysia M. Birkholz,*,†,‡ Enrico Girardi,†,1 Gerhard Wingender,* Archana Khurana,*

Jing Wang,† Meng Zhao,* Sonja Zahner,* Petr A. Illarionov,x Xiangshu Wen,{

Michelle Li,{ Weiming Yuan,{ Steven A. Porcelli,‖,# Gurdyal S. Besra,x Dirk M. Zajonc,†

and Mitchell Kronenberg*,‡

In this article, we characterize a novel Ag for invariant NKT (iNKT) cells capable of producing an especially robust Th1 response.

This glycosphingolipid, DB06-1, is similar in chemical structure to the well-studied a-galactosylceramide (aGalCer), with the only

change being a single atom: the substitution of a carbonyl oxygen with a sulfur atom. Although DB06-1 is not a more effective Ag

in vitro, the small chemical change has a marked impact on the ability of this lipid Ag to stimulate iNKT cells in vivo, with

increased IFN-g production at 24 h compared with aGalCer, increased IL-12, and increased activation of NK cells to produce

IFN-g. These changes are correlated with an enhanced ability of DB06-1 to load in the CD1d molecules expressed by dendritic

cells in vivo. Moreover, structural studies suggest a tighter fit into the CD1d binding groove by DB06-1 compared with aGalCer.

Surprisingly, when iNKT cells previously exposed to DB06-1 are restimulated weeks later, they have greatly increased IL-10

production. Therefore, our data are consistent with a model whereby augmented and or prolonged presentation of a glycolipid Ag

leads to increased activation of NK cells and a Th1-skewed immune response, which may result, in part, from enhanced loading

into CD1d. Furthermore, our data suggest that strong antigenic stimulation in vivo may lead to the expansion of IL-10–producing

iNKT cells, which could counteract the benefits of increased early IFN-g production. The Journal of Immunology, 2015, 195: 924–933.

Type 1 or invariant NKT (iNKT) cells are a lymphocytepopulation that is characterized by features of both theinnate and adaptive immune responses. The multiple

functions of these cells are remarkable in that they have beenimplicated in allergy, cancer, infection, autoimmunity, and a varietyof other conditions (1). Similar to other T lymphocytes, iNKT cellsarise from a CD4+, CD8+ double-positive thymocyte precursor (2).Unlike mainstream T cells, which recognize peptide moietiespresented by MHC-encoded molecules, iNKT cells recognizelipid Ags that are often glycosphingolipids (GSLs). Lipid Ags are

recognized when they are presented by CD1d, an MHC class I–like molecule (3). The CD1d binding groove is composed of twohydrophobic pockets labeled A9 and F9 (4). GSLs bind with thefatty acid localizing into the A9 groove and the sphingoid base intothe F9 groove. This binding mode allows the carbohydrate headgroup to protrude out of the CD1d molecule, such that it is ex-posed to be recognized by the iNKT cell TCR (5).The iNKT cell TCR contains a highly restricted, invariant TCR

a-chain that is formed by a Va14-Ja18 rearrangement in mice anda homologous Va24-Ja18 (TRAV10-TRAJ18) in humans (6).Although the b-chain of these TCRs is not invariant, it is biased toVb8.2, Vb7, or Vb2 in mice and Vb11 (TRBV25-1) in humans,with diverse CDR3 regions.The prototypical GSL recognized by iNKT cells is a-gal-

actosylceramide (aGalCer) (7, 8). When stimulated by a strongagonist, such as aGalCer, iNKT cells secrete both Th1 and Th2cytokines (e.g., IFN-g and IL-4) (9). Stimulation with aGalCercauses long-term changes in the iNKT cell population that origi-nally were likened to anergy (10, 11). We recently showed thatstimulation with aGalCer leads to an expansion of an iNKT cellpopulation capable of secreting IL-10, referred to as NKT10 cells(12). Interestingly, subtle chemical/structural alterations in aGalCerwere shown to alter the downstream cytokine response, skewing ittoward either a Th1 or a Th2 phenotype (13). Generation of an Agcapable of stimulating a strong Th1 cytokine profile has been anarea of great interest, because such an Ag would be beneficial forstimulating anticancer responses and for use as a vaccine adjuvant.Based on prior work, a heightened IFN-g response is due, in largepart, to the so-called “trans-activation” of NK cells occurringdownstream of iNKT cell stimulation (14–17). Therefore, a strongTh1 response induced by this and some other GSL Ags representsnot so much the tendency of an iNKT cell to produce more IFN-gwith reduced IL-4, but the output of a cellular network thatinvolves dendritic cells (DCs) expressing CD1d, iNKT cells to

*Division of Developmental Immunology, La Jolla Institute for Allergy and Immu-nology, La Jolla, CA 92037; †Division of Cell Biology, La Jolla Institute for Allergyand Immunology, La Jolla, CA 92037; ‡Division of Biological Sciences, Universityof California, San Diego, La Jolla, CA 92037; xSchool of Biosciences, University ofBirmingham, Edgbaston, Birmingham B15 2TT, United Kingdom; {Department ofMolecular Microbiology and Immunology, Keck School of Medicine, Universityof Southern California, Los Angeles, CA 90033; ‖Department of Microbiologyand Immunology, Albert Einstein College of Medicine, Bronx, NY 10461; and#Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461

1Current address: Research Center for Molecular Medicine of the Austrian Academyof Sciences, Vienna, Austria.

ORCID: 0000-0002-4780-7157 (W.Y.).

Received for publication January 13, 2015. Accepted for publication May 18, 2015.

This work was supported by National Institutes of Health Grants AI045053 andAI071922 (to M.K.), AI074952 and AI107318 (to D.M.Z.), AI091987 (to W.Y.),and AI045889 (to S.A.P.). This work was also supported by University of California,San Diego Rheumatology T32 Grant AR064194 (to M.Z.).

Address correspondence and reprint requests to Dr. Mitchell Kronenberg, Division ofDevelopmental Immunology, La Jolla Institute for Allergy and Immunology, 9420Athena Circle, La Jolla, CA 92037. E-mail address: mitch@liai.org

The online version of this article contains supplemental material.

Abbreviations used in this article: 1.2, DN3A4-1.2; DC, dendritic cell; aGalCer,a-galactosylceramide; GGC, galactosyl (a1-2) galactosyl ceramide; GSL, glyco-sphingolipid; ICCS, intracellular cytokine staining; iNKT, invariant NKT; o/n, over-night; SPR, surface plasmon resonance; TD, tail deleted; WT, wild-type.

Copyright� 2015 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/15/$25.00

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activate the response, and NK cells that are stimulated down-stream of iNKT cells that are crucial for continued IFN-g release.Although aGalCer was shown to suppress tumor metastases in

mouse models (8), it has not been overwhelmingly successful inhuman trials; this may be due, in part, to the mixed Th1 and Th2response or the anergy that it induces (18, 19) or to the potentialinduction of NKT10 (12). Because of this, many analogs of aGalCerhave been generated in attempts to elicit a more pronouncedTh1-skewed response. C-glycoside, which differs from aGalCerby the replacement of the carbon–oxygen glycosidic linkagewith a carbon–carbon bond, was the first GSL Ag reported tohave a Th1-polarizing potential (20, 21). C-glycoside cannotstimulate human iNKT cells and, therefore, it is not useful fortherapeutic applications.In this study, we characterized, in detail, the biochemical

properties, immune responses, and mechanisms of action of a novelaGalCer analog, DB06-1. This compound is identical to aGalCerwith the exception of the replacement of the C2 carbonyl oxygenon the acyl chain with a sulfur atom. DB06-1 was identified ina screen of lipids that associated with detergent-resistant mem-brane domains, which is characteristic of other Th1-skewing Agstested (22). In this article, we provide evidence that DB06-1promoted a Th1-skewed response that was more prominent thanthat of aGalCer. This correlated with increased loading of this Aginto CD1d, which may be related to a tighter fit in the CD1dgroove. However, over the longer term, DB06-1 induced more IL-10–producing NKT10 cells than did aGalCer, suggesting that theTh1 effect of a strong antigenic stimulus may induce a counter-regulatory pathway in vivo.

Materials and MethodsStatistical tests

Unless otherwise noted, statistical comparisons were drawn using a two-tailed Student t test.

Cell-free Ag-presentation assay

Stimulation of iNKT cell hybridomas on MicroWell plates coated withsoluble mouse CD1d was carried out according to published protocols (23–25). The indicated amounts of compounds or vehicle were incubated for24 h in MicroWells that had been coated with 1 mg CD1d. After washing,5 3 104 DN3A4-1.2 (1.2) Va14 iNKT hybridoma cells were cultured inthe plate for 24 h, and IL-2 in the supernatant was measured by sandwichELISA (R&D Systems), following the manufacturer’s instructions. The 1.2Va14 iNKT cell hybridoma expresses a Va14-Vb8.2 TCR and was de-scribed previously (26).

Ag-presentation assays

The culture of bone marrow–derived DCs in GM-CSF was describedpreviously (27). Briefly, cells were isolated from mouse femurs and cul-tured in media containing GM-CSF for 7 d. The cells were then pulsedwith GSL Ags overnight (o/n) and incubated with 53 104 1.2 Va14 iNKThybridoma cells for 20–24 h. Similarly, the Ag-presentation assay usingA20 B lymphoma cells was described previously (28). Briefly, A20-CD1dtransfectants expressing wild-type (WT) CD1d or tail-deleted (TD) CD1dwere pulsed with a GSL Ag overnight. APCs (1 3 105/well) were incu-bated with 5 3 104 iNKT hybridomas for 20–24 h. IL-2 in the supernatantof hybridoma cultures was measured by sandwich ELISA (R&D Systems).Human Va24+ iNKT cells were purified by magnetic enrichment andexpanded according to a previously published protocol (29). Briefly,PBMCs were isolated by Percoll (Sigma-Aldrich) density-gradient cen-trifugation. Human donor PBMCs (1–1.5 3 106/ml) were cultured inRPMI 1640 (Invitrogen) supplemented with 10% (v/v) FBS and 1% (v/v)Pen-Strep-Glutamine (10,000 U/ml penicillin, 10,000 g/ml streptomycin,29.2 mg/ml L-glutamine; Invitrogen). Human iNKT cell cultures wereexpanded by weekly restimulation with aGalCer-pulsed irradiated PBMCsand recombinant human IL-2. Ag-pulsed PBMCs (1 3 105/ well) wereseeded in 96-well plates and cultured in the presence of 5 3 104 Va24+

human iNKT cells for 20–24 h. GM-CSF release, as a marker of iNKT cellactivation, was measured by sandwich ELISA (R&D Systems).

Mice

C57BL/6 mice were purchased from The Jackson Laboratory. CD1-TDmice were generously provided by the laboratory of Dr. Albert Bendelac(University of Chicago, Chicago, IL) (30). Cd1df/f mice were generated inthe laboratory using conventional strategies and crossed with a CD11c-Cre–transgenic mouse obtained from The Jackson Laboratory (31). IL-122/2

(strain B6.129S1-Il12atm1Jm/J) mice also were purchased from TheJackson Laboratory. All mice were housed in specific pathogen–freeconditions, and the experiments were approved by the InstitutionalAnimal Care and Use Committee of the La Jolla Institute for Allergyand Immunology. Humanized mice (hCD1d-Va24 Tg) (X. Wen, S. Kim,R. Xiong, M. Li, A. Lawrenczyk, X. Huang, S.-Y. Chen, P. Rao, G.S. Besra,P. Dellabona, G. Casorati, S.A. Porcelli, O. Akbari, M.A. Exley, andW. Yuan, submitted for publication) were generated by crossing a humanCD1d knockin mouse line (32) and a human Va24 TCR mouse line (33) andwere tested in the laboratory of W.Y. following the Animal Care and UseGuidelines of the University of Southern California. Mice were injected i.v.with 1–4 mg DB06-1 or aGalCer (positive control), and blood serum orspleens were harvested 2, 6, 22, or 24 h later or, in the case of NKT10staining, 4 wk later. Standard sandwich ELISAs for mouse IFN-g, IL-12p70,and IL-4 (R&D Systems) were performed to measure cytokines in the sera.

Cell preparation

Single-cell suspensions of splenocytes were generated as described pre-viously (34). For DC isolation, the tissue was diced into 1-mm pieces anddigested using spleen dissociation media (Stem Cell Technologies), andDCs were enriched by positive selection using a CD11c+ isolation kit (StemCell Technologies) or MACS, according to the manufacturer’s protocols(Miltenyi Biotec). Isolated DCs were cocultured at various concentrationswith 5 3 104 1.2 Va14 iNKT hybridoma cells o/n, and activation wasmeasured by sandwich ELISA of culture supernatants for IL-2.

Flow cytometry and intracellular cytokine staining

For IFN-g intracellular cytokine staining (ICCS), the cells were cultured inmedia consisting of RPMI 1640 (Invitrogen) supplemented with 10% (v/v)FBS and 1% (v/v) Pen-Strep-Glutamine (Invitrogen) for 4 h at 37˚C in thepresence of GolgiPlug (BD Biosciences). To measure NKT10 cell IL-10,splenocytes were isolated from mice and restimulated with PMA (0.1 mg/ml)and ionomycin (0.9 mg/ml) (both from Sigma-Aldrich) for 4 h at 37˚C in thepresence of GolgiPlug and GolgiStop in media consisting of RPMI 1640(Invitrogen) supplemented with 10% (v/v) FBS and 1% (v/v) Pen-Strep-Glutamine (Invitrogen). Single-cell lymphocyte suspensions were purifiedusing Lymphoprep (Axis-Shield, Oslo, Norway) density gradient centri-fugation. Anti-mouse CD16/32 Ab (2.4 G2), purified in the Kronenberglaboratory, was used for FcR blocking. The following Abs were used inexperiments: CD1d-aGalCer tetramers labeled with the fluorochrome BV421(BD Biosciences) (generated in our laboratory) (35); CD1d, CD45R/B220,CD8, CD40, CD44, CD80, CD86, IL-10, and CD11b (all from BD Bio-sciences); NK1.1, IFN-g, and CD11c (all from eBioscience); CD4 (LifeTechnologies); and TCRb, CD3ε, CD19, and L363 (all from BioLegend).Dead cells were labeled with LIVE/DEAD yellow or aqua (Life Technologies).The cells were then fixed and permeabilized using Cytofix/Cytoperm buffer(BD Biosciences). The data were collected on an LSR II or Fortessa flowcytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).

NK cell depletion

C57BL/6 mice were depleted of NK cells by injecting 50 ml anti-asialoGM1 rabbit polyclonal Ab (Wako) 24 h prior to Ag challenge. NK celldepletion (NK1.1+ TCRb2 cells) was verified by flow cytometry.

Mouse CD1d expression, purification, and TCR refolding

Mouse CD1d–b2-microglobulin heterodimeric protein was expressedin a baculovirus-expression system, as reported previously (36). HumanCD1d–b2-microglobulin was prepared similarly to the mouse protein. TheVa14-Vb8.2 TCR construct design, refolding, and purification processeswere identical to the ones reported previously (37), whereas the autore-active human Va24-Vb11 TCR (auto-Va24) 4C1369 (38, 39) was gen-erously provided by Dr. Jamie Rossjohn (Monash University, Clayton,VIC, Australia) and prepared as reported (40).

Glycolipid loading and DB06-1–CD1d–TCR complexformation

The DB06-1 lipid, synthesized in the laboratory of G.S.B., was dissolved inDMSO at 1 mg/ml. Before loading, 25ml was diluted to 0.25 mg/ml with 25mlvehicle solution (50 mM Tris-HCl [pH 7], 4.8 mg/ml sucrose, 0.5 mg/ml

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sodium deoxycholate, and 0.022% Tween 20) and 50 ml 1% Tween 20 andincubated at 80˚C for 20 min. DB06-1 was loaded onto CD1d o/n (molarratio of protein/lipid of 1:3) in the presence of 10 mM Tris-HCl (pH 7).Refolded TCR was incubated at room temperature for 1 h with lipid-loadedCD1d at a 1:2 molar ratio, and the ternary CD1d–lipid–TCR complex wasisolated from uncomplexed CD1d and TCR by size-exclusion chromatog-raphy using a Superdex S200 10/300 GL (GE Healthcare).

Surface plasmon resonance binding analysis

Surface plasmon resonance (SPR) binding studies were conducted usinga Biacore 3000, as reported previously (37). Briefly, ∼300 response units ofbiotinylated CD1d (either human or mouse) loaded with DB06-1 wereimmobilized onto a streptavidin sensor chip (GE Healthcare) surface byinjecting the CD1d–DB06-1 mixture at 3 ml/min in HEPES buffered salinerunning buffer. A reference surface was generated in another flow channelwith unloaded CD1d. Mouse or autoreactive human TCRs were flowedover at a constant rate. Experiments were carried out at 25˚C with a flowrate of 30 ml/min and were performed at least twice. Kinetic parameters forthe mouse molecule interactions were calculated after subtracting the re-sponse to CD1d molecules in the reference channel, using a simpleLangmuir 1:1 model, in BIAevaluation software version 4.1. Human ki-netic parameters were obtained using steady-state solution graphs plottingTeq versus concentration and were fitted with binding response at equi-librium using BIAevaluation software version 4.1.

Crystallization and structure determination

The mouse CD1d–DB06-1–TCR complex was isolated by Superdex S20010/300 GL (GE Healthcare) column chromatography in 50 mM HEPES(pH 7.4), 150 mM NaCl and concentrated to 0.86 mg/ml. Crystals weregrown at 22.3˚C by sitting drop vapor diffusion while mixing 2 ml proteinwith 2 ml precipitate (0.2 M ammonium citrate dibasic [pH 4.98] 20% PEG4000). Crystals were flash-cooled at 100˚K in mother liquor containing20% glycerol. Diffraction data were collected at the Stanford SynchrotronRadiation Laboratory beamline 11-1 and processed with Mosflm software(41). The CD1d–DB06-1–TCR crystallized in space group C2221. Thestructure was solved by molecular replacement in Collaborative Compu-tational Project, Number 4 (1994) using the protein coordinates from theCD1d-iGb3 structure as the search model [PDB code 2Q7Y] (42), fol-lowed by the iNKT cell TCR [PDB code 3QUZ] (43). The model wasrebuilt into sA-weighted 2Fo – Fc and Fo – Fc difference electron-densitymaps using the COOT program (44). The lipid was built into a 2Fo – Fcmap and refined using REFMAC (1994). The final refinement steps wereperformed using the TLS procedure in REFMAC with five domains (a1–a2 domain including carbohydrates and glycolipid, a3-domain, b2-micro-globulin, variable domain, and constant domain of TCR). The CD1d–DB06-1–TCR structure was refined to 2.83 A to an Rcryst and Rfree of20.9 and 25.6%, respectively. The quality of the model was excellent, asassessed with MolProbity software (45) (Supplemental Table I).

ResultsDB06-1 activates mouse and human iNKT cells

DB06-1 is identical to aGalCer, with the exception of the re-placement of the C2 carbonyl oxygen on the acyl chain by a sulfuratom (Fig. 1A). We used several assays to measure the antigenicpotency of this compound. Initially, we tested DB06-1 in a cell-free Ag-presentation assay, whereby a soluble CD1d molecule wascoated on a plate, GSL Ags were added, and IL-2 release from aniNKT cell hybridoma was used to determine whether the lipidcould activate the iNKT cell TCR. DB06-1 only weakly stimu-lated the iNKT cell hybridoma compared with aGalCer (Fig. 1B).We also used a cell-based Ag-presentation assay, with bonemarrow–derived DCs as the APCs. This a more physiologicallyrelevant experimental set-up because it allows for the uptake byAPCs and endolysosomal loading of GSL Ags into CD1d. In thisexperimental set-up, DB06-1 was a more effective Ag, although itremained weaker in comparison with aGalCer (Fig. 1C).As noted in the Introduction, some lipids that activate mouse

iNKT cells do not stimulate their human counterparts; therefore,such compounds in this category are not relevant for the devel-opment of immune therapies. To address the possible usefulness ofDB06-1, we performed two studies. In the first, we determinedwhether DB06-1 could efficiently activate human iNKT cells derived

from human blood. We showed that DB06-1 activated iNKT cellsfrom two donors using human PBMCs as CD1d-expressing APCs(Fig. 2A). We also used a mouse strain in which the mouse Cd1d1gene was replaced with its human CD1d counterpart. These micealso contained a human iNKT cell Va24 TCRa transgene andwere crossed onto the Ca2/2 background. We refer to this strain asiNKT cell humanized mice. To assess their immune response, weinjected DB06-1 into these humanized mice and measured IFN-g at12 and 26 h postinjection (Fig. 2B), as well as IL-4 at 2 h postin-jection (Fig. 2C). IFN-g in the sera of humanized mice was higherwhen mice were injected with DB06-1 compared with aGalCer,whereas at the 2-h time point, IL-4 was decreased in the miceinjected with DB06-1. In summary, these two studies indicate thatDB06-1 is able to activate human iNKT cells and generate a Th1response in a humanized iNKT cell mouse model.

DB06-1 promotes increased IFN-g secretion

Th1 cytokine skewing following GSL stimulation is believed to bethe product of a cellular network that responds within the first 24 h(16). Therefore, we analyzed the in vivo response to DB06-1 bymeasuring the concentration of cytokines (IFN-g and IL-4) in thesera of mice 2 and 22 h after injection (Fig. 3A). Previous results(22) showed that DB06-1 can induce a robust serum IFN-gin vivo. The initial IFN-g response induced by DB06-1, measuredat 2 h, was similar to the response induced by aGalCer (Fig. 3A,Supplemental Fig. 1A) and is due to the rapid IFN-g secretionfrom iNKT cells. Although ICCS IFN-g at 2 h was higher in miceinjected with aGalCer compared with DB06-1, (SupplementalFig. 1C, 1D), this was not reflected in the sera data. The pro-duction of IFN-g at 22–24 h after GSL injection has been attrib-uted to the trans-activation of NK cells, due in part to IL-12production from APCs (46–49). Serum IL-12 levels from miceinjected with DB06-1 at 6 h postinjection were higher than in miceinjected with aGalCer (Fig. 3B). To measure NK cell trans-activation,we analyzed IFN-g production of splenic NK cells from miceinjected with DB06-1 24 h earlier. After a 4-h culture withGolgiPlug, splenic NK cells (NK1.1+ TCRb2) were identified byflow cytometry, and intracellular IFN-g was measured. NK cells ofmice injected with DB06-1 produced more IFN-g than NK cellsfrom aGalCer-injected mice (Fig. 3C), with 11–24% of NK cellsfrom mice injected with DB06-1 producing IFN-g comparedwith 1.5–6.5% of NK cells from mice injected with aGalCer(Supplemental Fig. 2A, 2B). To further determine that the seraIFN-g production at this 24-h time point was indeed due to NKcell trans-activation, we repeated the experiment in mice de-pleted specifically of NK cells using anti-asialo GM1 Abs (50)This treatment effectively depleted NK cells (Supplemental Fig.2C, 2D) but did not affect iNKT cells (Supplemental Fig. 2E). Theserum IFN-g levels 24 h after DB06-1 injection were significantlylower in these NK cell–depleted mice than in control mice (Fig.3D). We also injected Il122/2 mice and measured serum IFN-g at24 h by ELISA. In the absence of IL-12, the amount of IFN-g inthe serum from mice injected with DB06-1 was reduced ∼10-fold(Supplemental Fig. 2F). ICCS demonstrated that NK cells fromDB06-1–injected Il122/2 mice did not produce IFN-g (SupplementalFig. 2G). Based on these data, we conclude that DB06-1 causesa strongly Th1-skewed response in vivo, in both iNKT cell hu-manized and WT mice. Furthermore, it acts by causing increasedIL-12, which stimulates NK cells to produce IFN-g, with NK cellsproviding the majority of IFN-g in the serum.

DB06-1 is presented more effectively by DCs

A previous study indicated that CD8a+ CD11c+ DCs are thedominant APC type essential for activation of iNKT cells by

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injected GSL Ags (51), although, in some circumstances, macro-phages were shown to be important too, especially for Ags that arein particulate form (52). To determine whether DCs were essentialfor the presentation of DB06-1, we generated a mouse strain withfloxed CD1d alleles (Cd1df/f mice) and crossed this line witha CD11c-Cre–transgenic mouse strain (Cd1df/f Cre+ mice), therebydeleting CD1d expression on CD11c+ cells, including most DCs(Fig. 4A). When Cd1df/f Cre+ mice were injected with DB06-1, weobserved a significant decrease in the amount of IFN-g in mousesera at 24 h (Fig. 4B). However, because IFN-g production was notcompletely absent, these data suggest that CD11c+ DCs may not bethe sole population capable of presenting DB06-1 to iNKT cellsin vivo. IL-4 in the sera at 2 h also was determined and was de-creased in Cd1df/f Cre+ mice as well (Supplemental Fig. 3A).Therefore, we conclude that CD11c+ DCs likely were important forDB06-1 presentation in vivo, although the participation of other celltypes was not excluded. To further investigate how aGalCer andDB06-1 might affect DCs, we analyzed surface markers on CD8a+

DCs, comparing the 2- and 24-h time points. Surface levels ofCD1d did not change in mice injected with either GSL Ag; how-ever, CD80 and CD86 were increased at 24 h postinjection ofaGalCer or DB06-1. There was a trend toward higher expression ofboth CD80 and CD86 with injection of DB06-1 at 24 h, but thedifference was not significant (Supplemental Fig. 3B)We previously found that a common feature of several Th1

cytokine–skewing aGalCer analogs is that they persist longer ascomplexes with CD1d on the surface of APCs in vivo (53). Toaddress this, we injected lipid Ags and we used an Ab that bindsspecifically to aGalCer–CD1d complexes (L363) to measuresurface GSL–CD1d complexes on DCs using flow cytometry.After injection of either aGalCer or DB06-1, complexes withCD1d were barely detectable on the surface of DCs by flowcytometry at 2 h postinjection compared with control uninjectedmice. However, at 24 h, DB06-1–CD1d complex staining washigher and increased compared with the aGalCer–CD1d complex(Supplemental Fig. 3C).

FIGURE 1. DB06-1 is recognized by mouse iNKT cells. (A) Chemical structures of aGalCer and DB06-1, with the sulfur atom that differentiates DB06-

1 shown in red. (B) CD1d-coated plate assay whereby soluble CD1d bound to a plate was incubated with the indicated Ags o/n. The 1.2 Va14 iNKT cell

hybridoma was added, and IL-2 was measured after 16 h by ELISA to assess TCR engagement. Data are representative of one of two independent

experiments. Error bars represent 6 SEM of three wells/condition. (C) Bone marrow DCs were incubated with 100 ng/ml lipid Ag o/n and then cocultured

with 1.2 Va14 iNKT hybridoma cells o/n. Secreted IL-2 was measured the following day by ELISA to assess hybridoma stimulation. Data are from one

representative of two independent experiments. Error bars represent 6 SEM of three wells/condition. *p # 0.05, ***p # 0.001.

FIGURE 2. DB06-1 is recognized by hu-

man iNKT cells. (A) Two human iNKT cell

lines were stimulated o/n with PBMCs that

had been pulsed for 16 h with the indicated

Ag concentrations. ELISA for GM-CSF was

used as a readout of iNKT cell activation.

Data are representative from one of two inde-

pendent experiments. Error bars represent 6SEM of three wells/condition. (B and C) iNKT

cell humanized mice (hCD1d 3 Va24 Tg)

were injected with 1 mg of GSL Ag. Serum

IFN-g cytokine levels were assessed by ELISA

at 12 and 26 h postinjection (B), and serum IL-4

was assessed by ELISA at 2 h (C). ***p #

0.001, ****p # 0.0001.

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We analyzed the presence of these complexes using a T cellfunctional assay, which is more sensitive than flow cytometry,because it is likely that very few Ag–CD1d complexes are requiredto activate an iNKT cell. We injected mice with one of the GSLAgs, isolated a splenic fraction enriched for DCs at 2 or 24 h, andused these APCs to stimulate iNKT cell hybridomas in vitro (13,53). The Th1-skewing lipids that had been analyzed in this waypreviously showed an increased ability to activate iNKT cellhybridomas at 24 h compared with aGalCer (53). In accordancewith these studies, we observed that APCs purified 24 h after Aginjection were better able to activate iNKT cell hybridomasin vitro when they had been exposed in vivo to DB06-1 than whenexposed to aGalCer (Fig. 4D). However, unlike the previousstudies, the presentation of DB06-1 by APCs loaded in vivo in-duced a clearly stronger iNKT cell response ex vivo comparedwith aGalCer, even at 2 h after Ag injection (Fig. 4C). Althoughwe did not detect surface Ag–CD1d complexes by flow cytometryon DCs of mice injected 2 h earlier, it is likely that an amount ofcomplexes below the detection limit of flow cytometry was able togive an optimal stimulation of iNKT cell hybridomas in vitro.However, it is also possible that some lipid Ag taken up by DCsin vivo was able to load into CD1d during the culture period with theiNKT cell hybridoma cells. Regardless, our data demonstrate thatDB06-1 is taken up and presented more effectively by DCs in vivo.

CD1d recycling enhances DB06-1 presentation

Previous data indicated that presentation of Th1 cytokine–skewing lipid Ags was increased when CD1d cycled throughendosomal compartments. Furthermore, CD1d molecules presenting

Th1-skewing Ags were preferentially associated with lipid rafts(22). CD1d contains a tyrosine motif in its cytoplasmic tail thatallows trafficking to endosomal compartments. To study therole of endosome trafficking in the presentation of DB06-1, weused transfected cell lines that either expressed WT CD1d or TDCD1d lacking the cytoplasmic tail, including the tyrosine motifimportant for endosomal location. As a reference Ag, we usedgalactosyl (a1-2) galactosyl ceramide (GGC), which is known torequire lysosomal processing to cleave the terminal sugar to yieldthe active compound, aGalCer. Without recycling through endo-lysosomal compartments, GGC is not recognized by iNKT cellhybridomas (54). Like GGC, DB06-1 presentation to an iNKT cellhybridoma also was greatly reduced when CD1d lacked the abilityto recycle through endosomal compartments compared with theWT CD1d cells (Fig. 5A). To confirm that this recycling was alsoimportant in vivo, we used a mouse strain that lacked the tyrosinemotif of the Cd1d gene (CD1-TD). Although surface expression ofCD1d is significantly higher on APCs from CD1-TD mice com-pared with control mice, iNKT cells do not develop to WT numbersin CD1d-TD mice, likely due to the inability of CD1d to acquire theligands appropriate for iNKT cell positive selection (30). To studythe capability of APCs from these mice, we injected CD1-TD micewith DB06-1 or aGalCer, isolated splenic CD11c+ cells at 24 h, andused different concentrations of the DCs to stimulate an iNKT cellhybridoma ex vivo as a readout of Ag presentation. aGalCer wasused as the control Ag because, although it shows a dependenceon CD1d recycling in some experiments, in our experience it canload effectively into CD1d molecules on the cell surface (54).Although an iNKT cell response could be elicited by DCs derived

FIGURE 3. Th1 cytokine skewing depends on IL-12 and NK cells. (A) Ratio (DB06-1/aGalCer) of 2- and 22-h cytokine concentrations in sera obtained

from C57BL/6 mice after i.v. injection of 1 mg lipid (DB06-1 or aGalCer), as measured by ELISA. Data are a combination from four independent

experiments. Error bars represent 6 SEM. (B) IL-12p70 measured by ELISA in sera from C57BL/6 mice injected i.v. with 1 mg of aGalCer or DB06-1

compared with uninjected control mice at 6 h postinjection. Data are representative of mouse triplicates for each group for one of two independent

experiments. Error bars represent6 SEM. (C) NK cell IFN-g production from C57BL/6 mice injected iv with 1 mg DB06-1. Splenocytes from DB06-1– or

aGalCer-injected mice and controls were isolated and cultured in complete media containing GolgiPlug (BD Biosciences) for 4 h. NK cells were gated as

live B2202NK1.1+ CD3ε2 cells. The total ICCS IFN-g mean fluorescence intensity (MFI) of NK cells is plotted. Data are representative of mouse triplicates for

each group from one of three independent experiments. Error bars represent 6 SEM. (D) IFN-g in the sera, as measured by ELISA, at 24 h postinjection of

DB06-1 into C57BL/6 mice depleted of NK cells with anti-asialo GM1 Ab (NK block) was compared with controls. Data are representative of mouse triplicates

for each group for one of two independent experiments. Error bars represent 6 SEM. *p # 0.05, **p # 0.01, ***p # 0.001, ****p # 0.0001.

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from CD1-TD mice that had been injected with DB06-1, this re-sponse was reduced compared with the one stimulated by DCsfrom mice injected with aGalCer (Fig. 5B). This profile is op-posite from the one obtained when these two Ags were injectedinto WT mice (Fig. 4D). Together, these data demonstrate thatDB06-1 is presented when CD1d traffics normally through lateendosomal compartments, and it is more dependent on CD1drecycling than is aGalCer.

The iNKT cell TCR has a high affinity for the CD1d–DB06-1complex

One hypothesis for the ability of Ags to induce a Th1 cytokine–skewed response in vivo is that those Ags have an increased af-finity for the iNKT cell TCR when bound to CD1d (55).Equilibrium-binding analysis using SPR demonstrated a bindingaffinity (KD) of the mouse Va14-Vb8.2 iNKT cell TCR for mouseCD1d–DB06-1 complexes of 56 6 6 nM (Fig. 6A), ∼2-foldweaker than the binding affinity observed with aGalCer-loaded

CD1d (24 nM; data not shown). Ka (5.2 6 0.7 104 M21 s21) and

Kd (2.9 6 0.7 1023 s21) were comparable to the rates for aGalCer

(Ka = 7.84 104 M21 s21 and Kd = 1.61 1023 s21; data not shown).We also analyzed the affinity of a human autoreactive iNKT cell

TCR for the human CD1d–DB06-1 complex (Fig. 6B). Using

steady-state kinetic modeling, whereby we plotted the residual

units of the sensorgram against the concentration of the TCR and

calculated a line of best fit (Fig. 6C), we determined that the af-

finity of the autoreactive human TCR for the CD1d–DB06-1

complex (0.485 6 0.265 mM) was again ∼2-fold weaker than

for the aGalCer–CD1d complex (0.22 6 0.11 mM). Therefore, we

conclude that, although the iNKT cell TCR has a high affinity for

the CD1d–DB06-1 complex, the interaction with the aGalCer–

CD1d complexes is even stronger. These results are consistent

with other data demonstrating that increased TCR affinity cannot

explain increased Th1 cytokine release resulting from iNKT cellstimulation.

FIGURE 4. Enhanced presentation of DB06-

1 by DCs. (A) CD1d deletion in CD11c+ cells in

Cd1dfl/fl 3 Cd11c-Cre mice. Analysis of gated

live B2202, TCRb2, CD11c+ cells from WT,

Cd1dfl/fl3 Cd11c-Cre (CD11c Cre), and Cd1d2/2

(CD1d2/2) mice. (B) Cd1dfl/fl 3 Cd11c-Cre (Cre+)

mice and littermate Cd1dfl/fl controls (Cre2) were

injected with 1 mg DB06-1 and bled at 2 and 22 h,

and serum IFN-g was measured by ELISA. Data

are representative from one of two independent

experiments. Error bars represent 6 SEM of at

least two mice per condition. (C and D) Ex vivo

Ag-presentation assay. C57BL/6 mice were

injected with 1 mg of the indicated glycolipid,

and CD11c+ splenic DCs were enriched using

magnetic bead isolation at 2 h (C) and 24 h (D)

postinjection. Indicated numbers of enriched

DCs were cultured with 1.2 Va14 iNKT hy-

bridoma cells o/n, and IL-2 in the supernatant

was measured by ELISA. Data are plotted as the

number of enriched DCs/well versus IL-2 (pg/ml).

Data are representative of mouse triplicates for

each group for one of three independent experi-

ments. Error bars represent SEM. *p # 0.05. ns,

not significant (p . 0.05).

FIGURE 5. Presentation of DB06-1 is enhanced by endolysosomal localization. (A) Ag-presenting A20 cells expressing WT or TD CD1d were pulsed

with 0.1 ng DB06-1 or 100 ng GGC o/n. Cells were then cultured with the 1.2 Va14 iNKT hybridoma o/n, and IL-2 in the supernatant was quantified by

ELISA. Data are representative of two independent experiments. Error bars represent 6 SEM of three wells/condition. (B) Ex vivo Ag presentation assay.

Mice with the WT CD1d gene replaced with a TD CD1d gene were injected i.v. with 1 mg of the indicated lipid Ags. At 22 h, splenic CD11c+ DCs were

enriched by magnetic bead isolation, and various numbers were cocultured with 1.2 Va14 iNKT hybridoma cells. Data are plotted as the number of

enriched DCs/well versus IL-2 (U/ml) and are representative of mouse triplicates for each group for one of two independent experiments. Error bars

represent SEM. *p # 0.05, ****p # 0.0001.

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Structure of the mouse TCR–DB06-1–CD1d ternary complex

To characterize the biochemical features of the binding of theDB06-1 GSL to CD1d and the iNKT cell TCR, we determined thestructure of the ternary complex by x-ray crystallography (Fig. 7A,Supplemental Table I). The complex crystallized in the spacegroup C2221, with one complex in the asymmetric unit. Thebinding orientation of the iNKT cell TCR is consistent with theconserved parallel docking mode described previously (56).The TCR docks over the CD1d Ag binding groove in an orien-tation that is markedly different from the diagonal bindingfootprint seen in MHC–peptide–TCR complexes. As shownpreviously with models using the same TCR construct (53), theTCR interacts with CD1d using amino acids in the TCR CDR3aand CDR2b loops, as well as a CDR3b-dependent contact (Fig.7B). The same previously seen polar contacts are also formedbetween the a-chain (N30, R95, G96) of the iNKT cell TCR andthe DB06-1 Ag (Fig. 7B). There is well-defined density for theDB06-1 ligand (Fig. 7C), with the galactose head group exposedto recognition by the iNKT cell TCR. Polar contacts between theGSL Ag and amino acids E80, E153, and T156 of CD1d are

virtually identical to the interactions of CD1d with aGalCer. Thebinding orientation of DB06-1 within the hydrophobic A9 and F9

grooves of CD1d is also conserved, with the acyl chain localizing to

the A9 groove and the sphingoid base to the F9 groove, with min-

imal to no differences when superimposed on the aGalCer–CD1d

structure (data not shown). Because the two molecules are chemi-

cally similar in these regions, this similarity was expected. DB06-1

differs from aGalCer by the replacement of an oxygen with a sulfur.

The sulfur atom, which is 50% bigger than the oxygen atom present

in aGalCer (Fig. 7D), took up more space in CD1d (Fig. 7E),

possibly forming more intimate contacts with the surrounding

CD1d residues.

DB06-1 induces IL-10

Recent studies identified an iNKT cell subset, which we calledNKT10 cells, that produces IL-10 and may have regulatory

function (12, 57). Previous experiments demonstrated that

iNKT cells exposed to aGalCer in vivo were more capable of

producing IL-10 when restimulated weeks to months later. To

compare a strongly Th-1–biasing GSL Ag with aGalCer for the

FIGURE 6. TCR binding to

CD1d–DB06-1 complexes. (A) Bia-

core SPR sensorgram showing the

binding of increasing concentrations

of a mouse Va14-Vb8.2 iNKT cell

TCR (0.004–2 mM) to complexes of

mouse CD1d–DB06-1 (upper panel).

Data are representative of three in-

dependent experiments. Plot of the

residuals, which is the deviation in

the actual data from the fitted model

(lower panel). (B) Binding of an au-

toreactive human Va24 iNKT cell

TCR (0.2–5 mM) to human CD1d–

DB06-1 complexes, as assessed by

SPR. Data are representative of three

independent experiments. (C) Satu-

ration plot demonstrating equilibrium

binding of the human iNKT cell TCR

to immobilized CD1d—DB06-1.

FIGURE 7. Crystal structure of

the mouse CD1d–DB06-1–Va14-

Vb8.2 TCR ternary complex. (A)

Overview of the ternary complex

(PDB ID 4ZAK). (B) TCR/glycolipid

Ag contacts. (C) Final 2F0 2 Fc elec-

tron-density map, shown as blue mesh,

contoured at 1.0 s level for the DB06-1

glycolipid. Note that the a2-helix is

removed for clarity. CD1d surface

showing fit of aGalCer with oxygen

highlighted (D) and DB06-1 with the

sulfur and local amino acids high-

lighted (E).

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induction of NKT10 cells, we injected mice with DB06-1 oraGalCer; 4 wk later, we measured the capacity for splenic iNKTcells to produce IL-10 following a brief stimulation in vitro withPMA and ionomycin, followed by ICCS. Remarkably, the fre-quency of IL-10+ iNKT cells 1 mo after DB06-1 immunization wassignificantly greater than after aGalCer immunization (Fig. 8). Asimilar enhancement of IL-10 production was observed after im-munization with DB06-1 when the iNKT cells were restimulatedwith Ag 1 mo later (data not shown). These data suggest a possiblelink between a strong Th1 cytokine response and the induction ofIL-10–producing iNKT cells.

DiscussionUnderstanding how structural changes in GSL Ags can differen-tially modulate the immune response is important for under-standing how iNKT cells influence immunity, as well as fordeveloping therapeutic GSLs. For example, Ags that preferentiallyskew the immune response toward Th1 cytokine production couldbe useful as vaccine adjuvants and anticancer therapeutics (58, 59).In this study, we characterized DB06-1, a GSL Ag that differsfrom the well-studied aGalCer by only a single atom. Despite thissubtle change, we show that DB06-1 leads to changes in the im-mune system of mice that are more pronounced than those in-duced by aGalCer. Within 1 d after immunization, DB06-1 causedan increased Th1-cytokine response, and in the long-term it in-duced more NKT10 cells than aGalCer. Moreover, becauseDB06-1 can activate human iNKT cells, it, or related Ags withthioamide groups, could be therapeutically relevant. It is note-worthy that DB06-1 was not better or markedly different fromaGalCer using in vitro assays, considering TCR binding to theGSL–CD1d complex, as well as activation in vitro using CD1d-coated plates or APCs. However, in almost all of the in vivo assaysit was superior, including increased loading onto DCs in vivo,increased IFN-g in the serum, augmented IL-12 in the serum, andincreased NK cell IFN-g production. These findings emphasizethe complexity of assaying GSL Ag efficacy and the need to studythese compounds in vivo.A number of theories have been proposed for Th1 cytokine

skewing following GSL Ag immunization; however, despite mucheffort and investigation, this process remains incompletely un-derstood. Selective uptake and presentation by an APC type, forexample DCs versus B lymphocytes, could be important (60),although recent evidence indicates that CD8a+ DCs are the mostessential APCs for the presentation of injected lipid Ags, re-gardless of whether they are Th1 or Th2 skewing (51). The effectof a GSL Ag on the APC could be critical, perhaps as a result ofits trafficking in the cell and the site where it is loaded into CD1d.In fact, Th1 cytokine skewing has been associated with a requirement

for Ag internalization for loading into CD1d, as opposed to loadinginto CD1d on the cell surface (61). This type of immune response isalso correlated with the appearance of GSL–CD1d complexes on thecell surface in detergent-resistant domains (22). Consistent with this,DB06-1 originally was identified in a screen of lipid Ags that wereassociated with detergent-resistant domains when bound to CD1d.Furthermore, this Ag was shown previously to stimulate robust IFN-gproduction in vivo; however, cytokine production in response to itwas not compared with aGalCer, which could be classified as a Th0Ag, considering the ratio of IL-4/IFN-g that it induces (22). Forunknown reasons, the different trafficking of GSLs in APCs is linkedto changes in the DCs, such as increased expression of CD86, whichcould help to stimulate Th1 responses.Another theory for Th1 cytokine skewing proposes that IFN-g

production is a result of prolonged stimulation by GSL–CD1dcomplexes, which could be due to increased TCR affinity, in-creased stability of GSL–CD1d complexes, or pharmacokineticproperties. For example, a decreased rate of compound degrada-tion, as originally proposed for C-glycoside, could contribute toprolonged iNKT cell stimulation. In fact, our data indicate thatseveral Th1-skewing GSL Ags have an increased half-life in vivoas Ag–CD1d complexes on the surface of APCs (13, 53). Fur-thermore, the results from structural studies indicate that someTh1-skewing glycolipids may have increased contacts with CD1d,and this enhanced interaction with CD1d may contribute to theprolonged antigenic stimulation (53, 62). Although these differenttheories have merit, one class of theory alone probably does notaccount for all compounds that cause Th1-biasing cytokine re-lease, and these mechanisms are not mutually exclusive.The novel compound characterized in this study fits with several

of the theories and mechanisms described above for the activity ofAgs that cause Th1-biased cytokine responses. For example, DB06-1is preferentially presented by CD11c+ DCs to iNKT cells. Aspreviously mentioned, it was known that DB06-1 bound to CD1dlocalized to detergent-resistant domains (22). Moreover, DB06-1also shows a strong preference for internalization by APCs for ef-fective presentation. Although DB06-1 can be loaded into CD1din vitro in a cell-free assay, optimal presentation of this lipid isachieved by recycling through endosomal compartments. AlthoughDB06-1 has a slightly weaker TCR affinity when presented byCD1d than aGalCer, a weaker TCR affinity also has been observedwith other Th1-biasing GSL Ags, including the most well-studiedAg, C-glycoside (13). Similar to other Th1-biasing iNKT cell GSLAgs (53), DB06-1 persists as GSL–CD1d complexes on DCsin vivo, as measured by the ability of ex vivo DCs to activate aniNKT cell hybridoma. When DCs from mice injected with DB06-1were analyzed ex vivo, they were more effective at stimulating theiNKT cell hybridoma at both the early (2 h) and late (24 h) time

FIGURE 8. Increased IL-10 induced by DB06-1. (A and B) IL-10 production, as measured by intracellular staining of splenic iNKT cells from C57BL/6

mice. iNKT cells were gated as live cells that were B2202, CD8a2, CD3ε+, CD1d-tet+. Mice were injected with 4 mg of the indicated Ags, DB06-1 or

aGalCer 1 mo earlier. Cells were restimulated with PMA and ionomycin in vitro and intracellular IL-10 produced by CD4+ and double-negative

iNKT cells. (A) Representative flow cytometry plots showing IL-10 production. (B) Summary graph. Data are representative of two independent

experiments. Error bars represent SEM of at least three mice/condition. *p # 0.05.

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points, demonstrating increased loading into the CD1d groovein vivo. Consistent with this, we also observed increased Ag–CD1dcomplexes by flow cytometry but only at 24 h, likely because theincreased epitope density at the earlier times was not sufficient to bedetected by flow cytometry. Therefore, we propose that, althoughthe larger sulfur atom may make CD1d loading more difficultin vitro in a cell-free assay, this may be overcome in the presence oflipid-transfer proteins in the lysosome. Conversely, the sulfur atomalso may allow for better locking within the CD1d groove, inhib-iting the GSL Ag from replacement. Furthermore, sulfur is also lesselectronegative than oxygen, and this may allow DB06-1 to bemaintained in the CD1d hydrophobic pocket longer. Therefore, wespeculate that, as for other Th1-biasing iNKT cell Ags, the en-hanced molecular interaction of DB06-1 with CD1d permits anincreased and prolonged iNKT cell stimulation that leads to in-creased IFN-g production by trans-activated NK cells.A population of iNKT cells that produces IL-10, called NKT10

cells, was described recently (12, 57). These cells are induced orexpand greatly after a strong antigenic stimulation, for exampleafter aGalCer immunization, they are long-lived, and they pref-erentially localize to adipose tissue. An intriguing property ofDB06-1 described in this article is the robust IL-10 production byiNKT cells from mice injected with this compound 1 mo earliercompared with those injected with aGalCer. We made a similarobservation of increased IL-10 production when iNKT cells wererestimulated weeks after injection with several other Th1-biasingGSL Ags (G. Wingender, A. Birkholz, D. Sag, A.R. Howell, andM. Kronenberg, manuscript in preparation), suggesting that thisproperty could be a more general one. This effect, extending farbeyond the initial IFN-g burst, could have profound implicationsregarding the development of iNKT cell GSL Ags as therapeutics.Previous studies showed that mice pretreated with aGalCer havea decreased ability to reject tumors due to the IL-10 production byNKT10 cells (12). If we extrapolate, it is possible that a GSL thatleads to a more pronounced IL-10 response would theoreticallylead to less tumor rejection and, thus, would be a poor target fora cancer therapeutic. However, other diseases may benefit fromactivation of NKT10 cells. Repetitive aGalCer injections wereshown to lead to a reduced disease score in a mouse model ofmultiple sclerosis. This protection was suggested to be correlatedwith the ability of aGalCer to induce NKT10 cell IL-10 produc-tion, because IL-102/2 mice are not protected (63). Single injec-tions of aGalCer are unable to lead to this protection (64);however, a strong IL-10–inducing GSL like DB06-1 may prove tobe more effective, and IFN-g is known to be necessary to promotethis response (65). In addition to this, aGalCer was shown to beprotective in a mouse rheumatoid arthritis model through an IL-10–mediated response (66). Although humans have NKT10 cells,it is unknown whether the profound response seen in mice will befound in humans; more studies are needed to address the impli-cations of IL-10–producing NKT10 cells on human therapeutics.In summary, DB06-1 is a powerful activating GSL Ag capable of

impacting the mouse immune system days and weeks after im-munization. Its chemical properties allow for the stable formationof complexes with CD1d when it can be internalized within DCsin vivo. The characterization of this GSL, along with previousiNKT cell GSL Ags, contributes to our understanding of themechanisms for diverse iNKT cell influences on the immune re-sponse and will aid in the logical design of potential futureiNKT cell GSL Ag therapeutics.

AcknowledgmentsWe thank Dr. Jamie Rossjohn for the autoreactive human TCR plasmid, Dr.

Albert Bendelac for the CD1-TD mice, and Kyowa Hakko Kirin for

aGalCer. We also thank the Stanford Synchrotron Radiation Laboratory,

the Flow Cytometry Core Facility, and the Department of Laboratory Animal

Care at the La Jolla Institute for Allergy and Immunology for excellent

technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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