enhanced antitumor effects by chemical modified igb3 analogues · therapeutic discovery enhanced...

Therapeutic Discovery

Enhanced Antitumor Effects by Chemical Modified IGb3Analogues

Zhixia Zhou1, Cai Zhang1, Chengfeng Xia3, Wenlan Chen4, Huawei Zhu2, Pingping Shang1, Fang Ma1,Peng George Wang4, Jian Zhang1, Wenfang Xu2, and Zhigang Tian1,5

AbstractCertain glycolipid antigens for natural killer T (NKT) cells can direct the overall cytokine balance of the

immune response. However, the molecular mechanism of Th1- or Th2-biased cytokine secretion by NKT

cells is still unknown. Previously, we synthesized isoglobotrihexosylceramide (iGb3) analogues by

introducing a hydroxyl group at C4 on the ceramide portion of iGb3 to produce 4-HO-iGb3 or to further

deoxylation on the terminal galactose to produce 40 0 0-dh-iGb3. Both modified iGb3, especially 40 0 0-dh-iGb3,

stimulated more IFN-g production by hepatic NKT cells, and thus elicited preferential Th1 responses. Here,

we found that 40 0 0-dh-iGb3–loaded bone marrow–derived dendritic cells (DC) could significantly inhibit

growth of subcutaneous melanoma and suppress lung metastasis in C57BL/6 mice compared with

unmodified iGb3-loaded DCs. In investigating the mechanisms of this improved activity, we found that

40 0 0-dh-iGb3 stimulation increased STAT1 signaling by NKT cells, whereas the phosphorylation of Th2 type

cytokine–associated transcription factor STAT6 signaling was not affected. Analysis of the structures of

iGb3 and 40 0 0-dh-iGb3 revealed that 40 0 0-dh-iGb3 provides greater stability and affinity between glycolipid

and CD1d or NKT TCR complex than iGb3. Thus, 40 0 0-dh-iGb3 can improve the antitumor effects of a DC-

based vaccine possibly by stabilizing the CD1d/glycolipid/TCR complex and stimulating IFN-g signalingof NKT cells. Furthermore, chemical modification of iGb3 can elicit Th1-biased responses by NKT cells, and

40 0 0-dh-iGb3 combined with a DC vaccine may serve as a potent new NKT-based therapy against tumors

and infectious diseases. Mol Cancer Ther; 10(8); 1–10. �2011 AACR.

Introduction

Natural killer T (NKT) cells are specialized immunecells that express NK markers along with a semiinvar-iant T-cell antigen receptor (TCR) and display uniquecharacteristics of innate rather than adaptive lympho-cytes (1). In mice, the TCR of most NKT cells consists ofan invariant Va chain encoded by the Va14 and Ja18gene segments paired with TCRb chains that belong toa restricted set of Vb families (2,3). Th1 responses are

associated with cell-mediated immunity by Th1 cyto-kines, which tend to produce the proinflammatoryresponses responsible for killing pathogens and forperpetuating autoimmune responses, whereas Th2responses are associated with humoral immunitymediated by Th2 cytokines. Two responses cross-reg-ulate each other: a shift in favor of Th2 may lessenautoimmune damage but also may render viral infec-tion; and shifting from Th2 to Th1 might benefit forclearance of infections and prevention of cancer butalso might lead to inflammation and tissue damage (4).NKT cells are implicated in the control of autoimmu-nity, resistance to tumors, and protection againstinfectious agents through prompt secretion of largeamounts of both T helper 1 (Th1) cytokines (IFN-g)and T helper 2 (Th2) cytokines [interleukin (IL) 4, IL-5and IL-13] after activation via TCR engagement (5).Furthermore, promotion of IFN-g–producing NK cellsalso occurs after NKT cell activation, accompaniedwith bystander activation of dendritic cells (DC),conventional T cells and B cells to strengthen Th1responses.

The first ligand identified for NKT cells was a-galac-tosylceramide (a-GalCer), a glycolipid derived from amarine sponge. It has been reported that a-GalCer exhi-bits multiple immunotherapy roles in autoimmune

Authors' Affiliations: 1Institute of Immunopharmacology & Immunother-apy,2Institute of Medicinal Chemistry, School of Pharmaceutical Sciences,Shandong University, Jinan; 3State Key Laboratory of Phytochemistry andPlant Resources in West China, Kunming Institute of Botany, ChineseAcademy of Sciences, Kunming, China; 4Departments of Biochemistryand Chemistry, The Ohio State University, Columbus, Ohio; and 5Depart-ment of Microbiology and Immunology, School of Life Sciences, Universityof Science and Technology of China, Hefei, China

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Cai Zhang or Zhigang Tian, Institute of Immu-nopharmacology & Immunotherapy, School of Pharmaceutical Sciences,Shandong University, 44 Wenhua, West Road, Jinan 250012,China. Phone: 86-531-8838-1980; Fax: 86-531-8838-3782; E-mail:[email protected] or [email protected]

doi: 10.1158/1535-7163.MCT-11-0030

�2011 American Association for Cancer Research.

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on April 8, 2020. © 2011 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst June 8, 2011; DOI: 10.1158/1535-7163.MCT-11-0030

http://mct.aacrjournals.org/

diseases, cancer, atherosclerosis, and infectious diseases(6–8). However, the Th1 and Th2 cytokines that can bothbe produced by NKT cells after a-GalCer treatmentantagonize each other’s biological actions. Moreover,a-GalCer is a strong NKT agonist that can cause down-regulation of TCR expression and NKT function, leadingto anergy or unresponsiveness in mice (9). Such effectslargely limit the clinical use of a-GalCer. Isoglobotrihex-osylceramide (iGb3) is another glycolipid ligand for NKTcells that has been found to be stimulatory for bothmouseand human NKT cells and is assumed to be naturalantigenic ligand for NKT cells. The most distinctivedifference between a-GalCer and iGb3 is that the latteris a b-linked ceramide and displays 3 sugars. In addition,in terms of the preferential usage of Vb7>Vb8>Vb2chains, a-GalCer does not have such a bias, whereasiGb3 mimics closely the stimulatory properties of endo-genous ligands (10,11). However, iGb3 is a very weakagonist compared with a-GalCer. Recently, there havebeen reports that structure modifications of a-GalCerinduce polarization of cytokine production by NKT cells.For example, OCH, a synthetic analogue of a-GalCer,possessing a truncated sphingosine chain, was shown tobe Th2 polarizing (12,13); whereas a-C-GalCer, the C-glycoside of a-GalCer, was found to have a Th1-polariz-ing effect (14).

In our former study, we synthesized iGb3 analogues byintroducing a hydroxyl group at C4 on the ceramideportion of iGb3 to produce 4-HO-iGb3, and further deox-idation at the terminal galactose yielded 40 00-dh-iGb3. Wefound that both modified iGb3 molecules, particularly40 0 0-dh-iGb3, stimulated greater IFN-g production byhepatic NKT cells and thus elicited preferential Th1responses compared with the unmodified iGb3 (15).Here, we found that bone marrow–derived DCs (BM-DC) loaded with 40 0 0-dh-iGb3 exerted a significant greaterability to inhibit growth of subcutaneous melanoma andto suppress lung metastasis in C57BL/6 mice comparedwith the unmodified iGb3-loadedDC vaccine.We furtherinvestigated the underlyingmechanisms of this improve-ment in antitumor effect and the Th1 cytokine polariza-tion driven by 40 0 0-dh-iGb3 by analysis of the crystalstructures of 40 0 0-dh-iGb3 and iGb3 and expression ofkey transcription factors by NKT cells.

Materials and Methods

Mice and cell linesC57BL/6 mice were purchased from the Experimental

Animal Center of Beijing University (Beijing, China).All animals used in these experiments were between6 and 10 weeks of age and were maintained underspecific pathogen-free conditions. All animal studies wereapproved by the Institute Animal Care and Use Commit-tee of Shandong University. The Va14þ mouse CD1d-specific NKT cell hybridoma, N38-2C12 (2C12), waskindly provided by Dr. Mitchell Kronenberg (La JollaInstitute for Allergy and Immunology, San Diego, CA;

ref. 16) in 2008. The hybridoma cells were grown in RPMI1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FrontBiomedicals), 2mmol/LL-glutamine (LifeTechnologies), and b-mercaptoethanol(1� 10�5mol/L,LifeTechnologies) at 37�C inahumidifiedatmosphere containing 5% CO2 and were tested andauthenticated by flow cytometry to determine the TCRbexpression every time before being used in experiments.

ReagentsThe glycolipids iGb3 and 40 0 0-dh-iGb3 were synthe-

sized as previously described (the chemical structuresare shown in Supplementary Fig. S1; refs. 15,17,18). Theglycolipids were dissolved in dimethyl sulfoxide at1 mg/mL, and the working amount of glycolipids wasthen dissolved in PBS buffer. For in vivo experiments, themice were intraperitoneally injected with 100 mg/kg ofglycolipids or with vehicle. For in vitro experiments, 100ng/mL iGb3, 400 0-dh-iGb3, or vehicle was added to themedium. Alexa Fluor 488–labeled tetrameric CD1dmole-cules loaded with a-GalCer (CD1d tetramer) were kindlyprovided by theNIHTetramer Facility (Atlanta, GA). Thefollowing monoclonal antibodies (mAb) were from BDPharmingen: PE-Cy5.5-conjugated anti-CD3 mAb, PE-conjugated anti-IFN-g mAb, APC-conjugated anti-IL-4mAb, PE-conjugated anti-Stat1 (pY701)mAb, Alexa Fluor647–conjugated Stat6 (pY641) mAb, and isotype controlAbs. To identify the phenotypes of mature BM-DCs,phycoerythrin (PE)-conjugated anti-MHC class II anti-body, fluorescein isothiocyanate-conjugated CD11C andCD80 antibodies (eBioscience) were used.

Preparation of glycolipid-pulsed BM-DCs andcoculture with NKT cells

BM-DCs were generated by culturing bone marrow–derived adherent cells with 40 ng/mL of recombinantmurine granulocyte macrophage colony-stimulating fac-tor (rmGM-CSF) and 20 ng/mL ofmIL-4 (PeproTech Inc.)for 6 days as described (19). Tomature theDCs, 50 ng/mLof lipopolysaccharide was added in the mediumfor 16 hours on day 7. The cells that became nonadherentwere recovered and contained more than 60% of matureDCs (CD11chigh, CD80high, and MHC class IIhigh) as iden-tified by flow cytometry. Mature DCs were pulsed withglycolipids (100 ng/mL) for 12 hours, and NKT cellhybridomas were added at 1 � 105 cells per well in thepresence of glycolipid- or PBS-pulsed DCs (1 � 105 cellsper well) for 6, 12, 24, or 48 hours, separately.

ELISAThe levels of IFN-g and IL-4 in cell culture supernatants

and serum were detected using standard sandwichELISA kits (BD Pharmingen) according to the man-ufacturer’s instructions.

Flow cytometryFor cell surface staining, DC cells were harvested,

blocked with anti-FcgR mAb, and stained with the

Zhou et al.

Mol Cancer Ther; 10(8) August 2011 Molecular Cancer TherapeuticsOF2




labeled mAbs at 4�C for 45 minutes. For intracellularcytokine staining, hepatic and splenic lymphocytes werecultured in RPMI 1640 containing 10% fetal calf serumand treated with monensin (Sigma) for 4 hours to inhibitextracellular secretion of cytokines. After blocking withanti-FcgR mAbs and staining for surface markers at 4�Cfor 1 hour, the cells were fixed for 30 minutes with 0.4%paraformaldehyde and permeabilized in buffer contain-ing 0.01% saponin (Sigma) and 0.09% NaN3 (Sigma),followed by incubation for 1 hour in the permeabilizationbuffer with PE- or adenomatous polyposis coli (APC)-conjugated Ab. For intracellular phospho-STAT analysis,NKT cells were treated with glycolipid-pulsed DCs for 10to 90 minutes at 37�C, fixed with 1.5% formaldehyde,permeabilized by resuspending in ice-cold MeOH, andthen incubated with anti-phospho-STAT1, anti-phospho-STAT6, or isotype Ab for 1 hour at 4�C. All stained cellswere analyzed using a flow cytometer (FACScalibur),and the data were processed with WinMDI 2.9 software(Scripps Research Institute, La Jolla, CA).

Real-time PCR analysisTotal RNA of splenic lymphocytes was isolated using

TRIzol reagent according to the standard manufacturer’sguide (Invitrogen). cDNA was generated with randomprimers using M-MLV reverse transcriptase (Promega)according to the manufacturer’s protocol. The primersequences were as follows: GATA-3 forward: 50-AGA-ACCGGCCCCTTATCAA-30, reverse: AGTTCGCGCAG-GATGTCC; T-bet forward: 50-CAACAACCCCTTTG-CCAAAG-30, reverse: TCCCCCAAGCAGCCAAAG; andb-actin forward: 50-AGAGGGAAATCGTGCGTGAC-30,reverse: CAATAGTGATGACCTGGCCGT. Triplicate20 mL PCR reactions were carried out using SYBR GreenSupermix (BioRad). The levels of mRNAwere normalizedto that of b-actin.

Western blotsHepatic and splenic lymphocytes were harvested and

lysed in ice-cold lysis buffer containing a dissolved pro-tease inhibitor tablet (Sigma) for 30 minutes. The whole-cell extracts were mixed in Laemmli loading buffer,boiled for 5 minutes, and then subjected to SDS-PAGE.After transferring to nitrocellulose membrane, immuno-blots were carried out with anti-GATA-3 mAb (cloneH48), anti-T-bet mAb (clone H-210), or anti-b-actinmAb (Santa Cruz Biotechnology) followed by incubationwith horseradish peroxidase–conjugated secondary anti-body and visualization by enhanced chemiluminescencesystem (Pierce). The results were analyzed by usingAlphaEaseFC software (version 4.0.0, Alpha InnotechCorporation) with normalization of each band to theircorresponding loading control.

Structural determinationThe docking model was applied using the Tripos

SYBYL 7.0 package (2009) on a Dell Precision 390 work-station. The glycolipids were constructed with the Sybyl/

Sketch module and geometry optimized using Powell’smethod with the Tripos force field with convergence

criterion set at 0.05 kcal/(A�mol) and assigned with the

Gasteiger–HUckel method. The docking studies wereconducted using a Sybyl/FlexX module, built with gly-colipids docked into the active site of mCD1d andmVa14TCRb, and the residues in a radius of 7.0 in thecocrystal structure (PDBcode:2DQM) were selected.

Tumor challenge and treatmentB16 melanoma cells (1 � 105) were injected into the

right flank of C57BL/6 mice subcutaneously or wereadministered in the tail vein. The mice were then ran-domly assigned to 3 groups with 3 mice in each group(PBS-loaded DC treatment control, iGb3-loaded DC treat-ment group, and 40 0 0-dh-iGb3–loaded DC treatmentgroup). Mature DCs (5 � 105) that had been exposed invitro to either 500 or 200 ng of iGb3 and 40 0 0-dh-iGb3 werevaccinated intravenously on days 3 and 5 respectively,and DCs with a corresponding volume of PBS wereinjected into the control mice. Four weeks later, the micewere sacrificed, and the tumors or lungs were removed.The tumorswereweighed, and the tumor volume (V) wasdetermined bymeasuring the length (l) and thewidth (w),then calculated by using the formula: V ¼ l � w2/2.

Pulmonary histopathologyThe superior lobe of the right lung from each tumor-

bearing mouse was harvested 4 weeks after the intrave-nous administration of B16 melanoma cells and fixed byintratracheal instillation of 1 mL buffered formalin (10%,pH 7.2) followed by immersion in 10% neutral-bufferedformalin for 24 hours. The lobe was then rinsed in waterfor 12 hours and subjected to routine histologic proces-sing and embedded in paraffin. Serial 5-mm thick cross-sections were obtained from paraffin-embedded blocksand stained with hematoxylin and eosin (H&E) using astandard protocol.

Statistical analysisStudent’s t test was used to compare the differences

between 2 different groups, and P < 0.05 was consideredstatistically significant.

Results

40 0 0-dh-iGb3–loaded DCs protect against melanomametastases

Treatmentwith glycolipids has been reported to induceprotection against metastatic tumors in mice, and bothNKT and NK cells contribute to the resistance (14, 20, 21).As it seemed that glycolipids, especially those that caninduce a Th1 cytokine bias, may be beneficial to anti-tumor immune responses, we tested whether DCs loadedwith the chemically modified 40 0 0-dh-iGb3 could primestrong NKT-mediated antitumor effects in mice chal-lenged with B16 murine melanoma cells. C57BL/6 micewere inoculated subcutaneously with 1 � 105 B16 cells

iGb3 Analogues Enhance NKT Cell–Mediated Antitumor Effects

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and then injected intravenously with iGb3, 400 0-dh-iGb3,or PBS-loaded DCs on days 3 and 5. The tumor volumesand weights were examined at day 28. As depicted inFig. 1A, 400 0-dh-iGb3–loaded DCs exerted a significantlygreater inhibitory effect and delayed the growth of B16melanoma compared with the iGb3-loaded DCs. Thetumor volume in PBS-loaded DC group increased to2,200.4 � 288.9 mm3, whereas it was only 156.3 � 150.0mm3 in the 40 0 0-dh-iGb3-loaded DC group, and 959.6 �249.8 mm3 in the iGb3-loaded DC group. Similarly,the tumor weights were 2.6 � 0.4, 1.8 � 0.1, and 0.7 �0.2 g in the PBS-loaded DC, iGb3-loaded DC, and 40 00-dh-iGb3-loaded DC group, respectively. These resultsindicated that both iGb3- and 400 0-dh-iGb3–loaded DCvaccine could inhibit and delay the growth of B16melanoma, with 400 0-dh-iGb3–loaded DC vaccine beingmore effective.

We further observed the effects of iGb3- and 40 00-dh-iGb3-loaded DCs in the B16 lung metastasis model.Metastases to the lungs were examined 2 weeks afterintravenous administration of DCs loaded with either ofthe 2 glycolipids. We found that 40 0 0-dh-iGb3–loaded DCswere more effective than iGb3-loaded DCs in reducingmetastases (Fig. 1B). H&E staining confirmed that themetastatic nodules in lungs of mice treated with 40 00-dh-

iGb3–loaded DCs were fewer than that in iGb3-loadedDCs treatment mice (Fig. 1B). These results indicated 40 00-dh-iGb3weremore potently inhibitory on the growth andmetastasis of B16 melanoma than iGb3 by DC-mediatedpresentation and activation of NKT cells.

40 0 0-dh-iGb3 promotes STAT1 activation and T-betexpression by NKT cells

To investigate the mechanisms underlying theimprovement of the antitumor effects by 40 00-dh-iGb3,we observed the effect of 400 0-dh-iGb3 on cytokine produc-tion byNKThybridoma in vitro. The freshly preparedDCswere loaded with iGb3, 40 0 0-dh-iGb3, or the vehicle for 12hours. The glycolipid-loaded DCs were cocultured withNKT cell hybridomas 2C12 for 6, 12, 24, or 48 hours, andsupernatants were collected to determine the secretedlevels of IFN-g and IL-4. As shown in Fig. 2A, the40 0 0-dh-iGb3–loadedDCs stimulatedmore IFN-g secretionby 2C12 hybridomas than the iGb3-loaded group. Therewere no differences in IL-4 production. Similar resultswere obtained with 2C2 cells by assaying intracellularIFN-g expression analyzed by flow cytometry (Fig. 2B). Inan in vivo experiment, the murine serum IFN-g wasenhanced after 400 0-dh-iGb3 stimulation (Fig. 2C),although the percentages of NKT cells were not increased

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esFigure 1. 40 0 0-dh-iGb3–loaded DC vaccine reduced the growth and pulmonary metastasis of B16 melanoma. A, a total of 1 � 105 B16 cells wereinjected subcutaneously into the right flank of B6mice to establish subcutaneous tumor-bearingmice. Then, 5� 105 DCs that had been exposed to 500 or 200ng/mL of iGb3, 40 0 0-dh-iGb3 were vaccinated intravenously on days 3 and 5. On day 28, the mice were sacrificed and the tumors were removed. Thetumor volumewas calculated (left) and weighed (right). B, B16 cells (1� 105) were administered intravenously by the tail vein. A total of 5� 105 DCs loadedwith500 or 200 ng/mL glycolipid were vaccinated intravenously on days 3 and 5. After 2 weeks, the tumor-bearing mice were sacrificed, and the lungs wereseparated and the tumor nodules were counted. H&E-stained histology showed the metastatic nodules in lungs. Representative photomicrographs arepresented at �100 magnification. The data are expressed as the mean � SD of 3 mice from at least 2 independent experiments. *, P < 0.01, compared withiGb3 group.

Zhou et al.





in both liver and spleen (Supplementary Fig. S2). Thesedata strongly suggested that 40 00-dh-iGb3 promoted IFN-gproduction and thus polarized the Th1 responses, whichmight be beneficial for antitumor immunity.During Th1/Th2 differentiation of conventional Th

precursor, signals through the TCR and cytokine recep-tors can lead to the initiation of a Th1 program viaSTAT1 or STAT4 activation and the induction ofT-bet, which promotes Th1 lineage commitment. Sig-nals that favor the activation of STAT6 induce GATA3leading to Th2 differentiation (22,23). To explorewhether these transcription factors are also involvedin the production of Th1/Th2 cytokines in NKT cellsand whether the changes of these transcription factorscontribute to the preferential induction of IFN-g by 40 0 0-dh-iGb3, we examined the phosphorylation of STAT1and STAT6 in 2C12 hybridomas upon stimulation withglycolipid-loaded DCs at various time points by intra-cellular staining. The results showed that 4-dh-iGb3–pulsed DCs treatment promoted the phosphorylation ofSTAT1 in NKT cells from 10 minutes, reaching themaximum at 30 minutes and returning to a lower levelat 60 and 90 minutes. The iGb3-pulsed DCs treatmentonly led to a slight increase at 20 and 30 minutes. The

capacity of 40 0 0-dh-iGb3 to promote STAT1 phosphory-tion was stronger than that of iGb3, especially at 30minutes (Fig. 3). Meanwhile, STAT6 was phosphory-lated after 20 minutes, peaked at 30 minutes, and thenregressed at 60 minutes in both the 40 00-dh-iGb3 andiGb3 treatment groups, with no significant differencesbetween the 2 groups (Fig. 3).

We next explored whether T-bet, a key downstreamtarget of STAT1 signaling, is involved in the mechanismof preferential induction of IFN-g by 400 0-dh-iGb3. Weused real-time PCR and Western blotting to assess themRNA and protein levels, respectively, of T-bet as well asGATA-3, a downstream target of STAT6, in splenic lym-phocytes from glycolipid and PBS-treated mice. Therewas an obvious increase in T-bet expression between theresting or stimulated groups at 24 hours, and the expres-sion of T-bet was significantly upregulated upon 40 00-dh-iGb3 treatment, compared with iGb3 treatment, both atthe mRNA (Fig. 4A) and protein (Fig. 4B) levels. Incontrast, GATA3 levels in splenocytes were comparablebetween the 2 groups (Fig. 4A and B). Similar results werealso obtained from hepatic lymphocytes (data notshown). These data suggested that 40 00-dh-iGb3 presentedby DCs upregulated the phosphorylation of STAT1 and

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Figure 2. 40 0 0-dh-iGb3 enhanced induction of IFN-g production from NKT cells. A, mature BM-DCs pulsed with glycolipid (100 ng/mL) were culturedat 1 � 105 cells per well with the 2C12 hybridoma (1 � 105 cells per well). The levels of IFN-g and IL-4 in supernatants were assessed by ELISA.B, 2C12 hybridoma cells were cultured with BM-DCs loaded with glycolipid for 48 hours. TCRbþ NKT cells were gated and analyzed for the presenceof intracellular IFN-g and IL-4 by fluorescence-activated cell sorter. The data are presented as the percentage of IFN-g- or IL-4–positive cells inthe gated population. The data shown are the mean � SD of 3 independent experiments. *, P < 0.05 versus iGb3-treated group. C, C57BL/6 micewere intraperitoneally injected with iGb3 and 40 0 0-dh-iGb3 (100 mg/kg). The serum levels of IFN-g and IL-4 were measured at different times byELISA. The results are expressed as the mean � SD of 3 mice from at least 3 independent experiments. *, P < 0.05; **, P < 0.01 compared with iGb3group.






expression of T-bet in NKT cells and thus resulted ingreater IFN-g secretion.

Stability and affinity of the CD1d/40 0 0-dh-iGb3/Va14TCRb complexes are augmented

It has been reported that the structural variants ofglycolipids, especially the differences in the affinity ofTCR binding to the glycolipid/CD1d complex and thestability of glycolipid ligands bound to CD1d molecules,may induce systemic polarization of cytokine productionby NKT cells (24). To investigate the mechanisms bywhich 400 0-dh-iGb3 preferentially induces IFN-g secretioncompared with iGb3, we compared the crystal structuresof 400 0-dh-iGb3 and iGb3 to analyze the stability andaffinity of the CD1d/glycolipid/Va14TCRb complex.40 0 0-dh-iGb3 was docked into the active sites of mCD1dand Va14TCRb using SYBYL 7.0 software. We encoun-tered the same problems as other researchers as weobserved that the iGb3 glycolipid ligand was very dif-ferent in size and structure with the linear trisaccharidehead group (Gal1-3-Gal1-4-Glc), and the ligands inCD1d-iGb3 and CD1d-40 0 0-dh-iGb3 complexes were lesswell ordered (10). Therefore, the a-1,3–linked galactose ofthe iGb3 or 40 00-dh-iGb3 was not visible in the crystalstructure. Consistent with the previous reports (25,26),the docking model showed that the acyl chain of 40 00-dh-iGb3 could insert to the pocket A0 by forming hydro-phobic interactions with this subsite, and the sphingosinechain could insert into the F0 pocket by forming hydro-phobic interactions. Meanwhile, the hydrophilic phenylgroup close to the 2 chains could interact with the rip

compartment of the CD1d binding groove (Fig. 5A). Tofurther understand the binding mode of 40 00-dh-iGb3with mCD1d in detail, a two-dimensional picture wascreated using the LIGPLOT program. As shown inFig. 5B, similar to iGb3, 40 0 0-dh-iGb3 could form hydrogenbondswith 3 residues of CD1d: Asp80, Asp153, and Thr156.In addition, the sphingosine chain of 40 00-dh-iGb3 couldform hydrophobic contacts with the Trp153, Phe120, Val118,He98, Leu84, and Leu143 residues, and the acyl chain couldform hydrophobic contacts with Val160, Ser76, Plm702, andTyr73. In contrast, iGb3 forms hydrophobic contacts withArg79, Phe70, Cys12, Leu100, Val98, Leu84, Phe77, and Tyr73

as reported by Zajonc and colleagues (10). We proposethat the differences in hydrophobic contacts by the sphin-gosine chain and acyl chain might increase the bindingstrength of 40 0 0-dh-iGb3 with CD1d. Although thehydroxy group introduced at C4 in 40 0 0-dh-iGb3 doesnot participate in binding with CD1d in the computeddocking model, we speculated that the hydroxyl groupmay strengthen the interaction with Asp80.

For the interaction of the glycolipid and TCR, we onlyobserved the interaction between the head groups ofiGb3/400 0-dh-iGb3 and Va14TCRb, as the tail parts ofthe glycolipid ligands were reported not to take part inthe binding (10). The docking results showed that thehead groups of 40 0 0-dh-iGb3 could insert into the "notch"of the TCR pocket as with iGb3 (Fig. 6A). 40 0 0-dh-iGb3could also form hydrophobic contacts with Asn23, Tyr110,and Lys167 of the TCR. However, the Lys167 from one sideof iGb3 is rotated to the other side of 40 0 0-dh-iGb3 in thetwo-dimensional image of the structures displayed by

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DC + iGb3DC + 4'-dh-iGb3

Figure 3. BM-DCs loaded with40 0 0-dh-iGb3 promotesphosphorylation of STAT1 by NKTcells. A, 2C12 cells were treatedwith BM-DCs that had beenexposed to glycolipids or controlfor 10 to 90 minutes. The treatedcells were then fixed and stainedwith TCRb, pSTAT1, pSTAT6, orisotype Ab. TCRb positive NKTcells were gated and analyzed forthe presence of pSTAT1 andpSTAT6 by fluorescence-activated cell sorter. Data arepresented as the percentage ofpSTAT1 or pSTAT6 positive cellsat different time points andexpressed as the mean � SD of 3individual experiments. *, P < 0.05,compared with iGb3 group.

Zhou et al.





LIGPLOT. This change in spatial configuration led to theincrease in index of stability and affinity of glycolipids/TCR from 3.3 to 4.9 points, perhaps due to the removal ofthe 40 0 0hydroxyl group in the terminal galactose of iGb3(Fig. 6B). Together, these results indicated that the struc-tural changes of 400 0-dh-iGb3 may increase the stability ofbinding to CD1d and the affinity to the NKT TCR com-pared with that of the unmodified iGb3.

Discussion

The highly conserved CD1d-restricted NKT cells, iden-tified as a bridge between innate and adaptive immuneresponses, exert potent immune regulatory functions byreleasing a variety immunomodulatory cytokines. Up tonow, the response of NKT cells has been studied exten-sively by multiple groups with a-GalCer that has beenproven to be a unique type of adjuvant for vaccinedevelopment (7). New analogues of a-GalCer are beingsynthesized to search for newNKT cell agonists that mayhave superior properties for the treatment of autoim-mune and inflammatory diseases. One of these, a-C-GalCer was found to be more potent in helping miceto defend against mouse malaria and B16 melanoma byinducing a more prolonged IL-12 and IFN-g response(14). Moreover, a-C-GalCer was reported bind morestably to DCs than a-GalCer, and a-C-GalCer–loaded

DCs induced higher levels and longer lasting IFN-g–producing NKT cell responses and more effectiveadaptive protective T-cell–mediated immunity (21).

iGb3, the first found natural ligand of NKT cells, is alsodescribed as a candidate for mediating the developmentand function of NKT cells in infections, malignancy, andautoimmunity (27). A recent report showed that iGb3-primed DCs exerted a significant NKT cell–mediatedantitumor activity in mice challenged with melanomacells (28), which supported that iGb3 can display anti-tumor activities similar to a-GalCer. In our previouswork, we found that 2 chemical modification of iGb3analogues, especially 400 0-dh-iGb3, were much moreactive than iGb3 for increasing IFN-g production byhepatic NKT cells (15). In the present study, we foundthat 400 0-dh-iGb3–loaded BM-DCs exerted a more signifi-cant inhibitory effect on the growth of subcutaneous B16melanoma and better suppression of lung metastasis inC57BL/6 mice when comparing with the unmodifiediGb3-loaded DC vaccine. We further confirmed the effectof 400 0-dh-iGb3 on Th1-biased cytokine production byhepatic and splenic NKT cells (data not shown) as wellas NKT hybridoma cell lines both in vivo and in vitro. Wehypothesize that this Th1 bias status of NKT cells drivenby 40 00-dh-iGb3 may be beneficial for antitumor therapy.

The differentiation process of Th1/Th2 cells by con-ventional T cells is critical for their function in immune

Figure 4. T-bet, but not GATA-3,is upregulated in splenocytes after40 0 0-dh-iGb3 stimulation. A,splenic lymphocytes fromC57BL/6 mice treated withglycolipids intraperitoneally wereseparated, and themRNA levels ofGATA-3 and T-bet were assayedby real-time PCR. Each gene wasnormalized to that of b-actin. Thedata are expressed as themean � SD of three mice from atleast 3 independent experiments.*, P < 0.05, compared with iGb3group. B, Western blot analysis forprotein levels of GATA-3 andT-bet in splenocytes. The datais representative of at least3 independent experiments.

iGb3PBS

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responses. Th1 differentiation is promoted by IL-12 sig-naling, which is mediated by STAT1/4, whereas Th2differentiation is promoted by IL-4 signals by means of

STAT6. In addition, the downstream targets of STATsignaling are important for the differentiation. T-bethas been identified as a key transcription factor for the

Ligand bond

LP

Nonligand bond

Hydrogen bond and its lengthNonligand residues involved in hydrophobiccontact(s)

Corresponding atoms involved inhydrophobic contact(s)

His53

C1

C1

Asn23(A)Asn23(A)

Tyr110(C)Tyr110(C)

Lys167(C)

Lys167(C)

A

B

Figure 6. Structural overview ofthemV14TCR-glycolipid complex.A, head groups of glycolipidsinsert into the notch TCR pocket.Presentation of the head group ofiGb3 (left) and 40 0 0-dh-iGb3 (right)in the V14TCR binding groove. B,the docking model of iGb3 and40 0 0-dh-iGb3 with mV14TCRproduced by LIGPLOT.Glycolipids are shown in violet.The red dots by the arrow indicatethe 40 0 0 hydroxyl group removedfrom the terminal galactose ofiGb3 (left), resulting in themodifiedsugar head group of glycolipid40 0 0-dh-iGb3 (right).

Ligand bond

Nonligand bond

A

BHydrogen bond and its length

Nonligand residues involved in hydrophobiccontact(s)

His53

Corresponding atoms involved in hydrophobic contact(s)

Trp142(C)

Val118(C)

Val160(C)

Phe120(C)

Leu84(C)

Leu143(C)

Ile98(C)Tyr73(C)Thr156(C)

Asp153(C)

Asp80(C)

Igc602

Plm702(C)

Ser76(C)

Figure 5. The docking mode ofglycolipid compounds withmCD1d. A, visualization of themodels of mCD1d–glycolipidcomplexes generated by theSYBYL 7.3 software. View of thepresentation of iGb3 (left) and 40 0 0-dh-iGb3 (right) by CD1d along thebinding groove. Lipophilicitypotential (LP) of mCD1d isrepresented as surfaces color-coded according to the degree ofpositional displacement. Lightblue residues represent very lowaffinity, gray to green isintermediate, and yellow redresidues show significantly highaffinity. B, the docking model of40 0 0-dh-iGb3 with mCD1dproduced by LIGPLOT. 40 0 0-dh-iGb3 is shown in violet. The reddots by the arrow indicate thehydroxyl group introduced at C4of iGb3 (071).

Zhou et al.





development of Th1 cells and the induction of IFN-gproduction, whereas GATA-3 is crucial for Th2 develop-ment (29–31). However, the transcriptional control of thedevelopment and differentiation of Th1/Th2 cytokines inNKT cells remains poorly understood. There is evidencethat STAT6 signaling pathway acts as an important factorfor the expression of many Th2 cytokines by NKT cells(32). GATA-3 is also intrinsically critical in regulating thedevelopment, survival, activation, and effector functionof NKT cells (33). Moreover, T-bet was also identified as amajor regulator necessary for thematuration of NKT cells(34). In this study, we found that, compared with iGb3,400 0-dh-iGb3 presented by DCs more significantly upre-gulated the phosphorylation of STAT1 in NKT cells,whereas there were no differences in STAT6 phosphor-ylation between the 2 glycolipid-loaded DC treatmentgroups. Our data suggest that STAT1/T-bet signalingmay be an important pathway to regulate IFN-g tran-scription in NKT cells, leading to preferential Th1-biasedresponses. These findings are consistent with other stu-dies supporting the idea that T-bet might be responsiblefor the remodeling of the IFN-g locus in NKT cells (35).The spatial configurations of glycolipids apparently

affect the differentiation and development of NKT cells.Subtle alterations in a glycolipid antigen, especially differ-ences in the affinity of TCRbinding to the glycolipid-CD1dcomplex and the stability of glycolipid ligands bound toCD1dmolecules, can alter the immune responses initiatedby NKT cells, possibly by impairing cytokine polarization(24). Here, by docking the 2 glycolipids into the active sitesof CD1d and Va14TCRb, we proposed that 40 0 0-dh-iGb3,made by introduction of a hydroxyl group at C4 of iGb3and removing the 40 00 hydroxyl group of the terminalgalactose, possibly stabilizes the CD1d-40 00-dh-iGb3 com-plexes, augments the interactionwith TCR ofNKT. Unfor-tunately, introducing a hydroxy group (O71) at C4 of 40 0 0-

dh-iGb3doesnot cause it tobindwithany residueofCD1d.We presumed that O71 in the sphingosine chain maystabilize the lipid backbone, together with hydrophobiccontacts between chains and additional residues of CD1dthat may change the affinity of the mCD1d-glycolipidcomplexes to TCR. In addition, the change in spatial con-figuration by removal of the 400 0hydroxyl group in theterminal galactose of iGb3 increases the stability and affi-nity of glycolipids/TCR, and thus eventually promotesIFN-g secretion ofNKT cells.Wehave attempted but failedto dock the CD1d-40 0 0-dh-iGb3/TCR complexes, perhapsdue to the high complexity of the glycolipid ligands.

In summary, we provided evidence that 40 0 0-dh-iGb3can improve the antitumor effects of a DC-based vaccine,possibly by stabilizing the CD1d/glycolipid/TCR com-plex and stimulating IFN-g signaling of NKT cells. Ourfindings highlight that owing to the privilege of prefer-ential induction of Th1 responses, the 40 00-dh-iGb3–loaded DC vaccine or 40 0 0-dh-iGb3 as an adjuvant mayachieve more effective therapy against tumors and infec-tious diseases when translated into clinical trials.

Disclosure of Potential Conflict of Interest

No potential conflicts of interest were disclosed.

Grant Support

This work was supported by the Natural Science Foundation of China(90713033) and the National 973 Basic Research Program of China(2007CB815803) and the Important National Science & Technology Spe-cific Projects (2008ZX10002-008).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 13, 2011; revised March 27, 2011; accepted April 18,2011; published OnlineFirst June 8, 2011.

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Published OnlineFirst June 8, 2011.Mol Cancer Ther Zhixia Zhou, Cai Zhang, Chengfeng Xia, et al. Enhanced Antitumor Effects by Chemical Modified IGb3 Analogues

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