Journal of Immunological Methods 312 (2006) 34–39www.elsevier.com/locate/jim

Research paper

A modified α-galactosyl ceramide for staining and stimulatingnatural killer T cells

Yang Liu a, Randal D. Goff a, Dapeng Zhou b, Jochen Mattner b, Barbara A. Sullivan c,Archana Khurana c, Carlos Cantu III d, Eugene V. Ravkov e, Chris C. Ibegbu e,

John D. Altman e, Luc Teyton d, Albert Bendelac b, Paul B. Savage a,⁎

a Brigham Young University, Provo, UT 84602, United Statesb University of Chicago, Chicago, IL 60637, United States

c The La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, United Statesd Scripps Research Institute, La Jolla, CA 92037, United States

e Emory Vaccine Research Center, Atlanta, GA 30329, United States

Received 14 March 2005; received in revised form 3 October 2005; accepted 7 February 2006Available online 6 March 2006

Abstract

CD1d presentation of α-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells.KRN7000, an α-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine spongeglycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffersfrom poor solubility and availability. We have developed an α-galactosyl ceramide with improved solubility over KRN7000 thateffectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.© 2006 Elsevier B.V. All rights reserved.

Keywords: Natural killer T cell; T cell receptor; α-galactosyl ceramide; Cytokine

1. Introduction

The emerging role of natural killer T cells (NKTcells) in immune responses has prompted intense in-vestigation of their influences on various disease statesand their interactions with glycolipid antigens (Brigl andBrenner, 2004; Godfrey and Kronenberg, 2004; Kro-nenberg, 2005; Van Kaer, 2005). NKT cells display a

⁎ Corresponding author. Department of Chemistry and Biochemis-try, Brigham Young University, C100 BNSN, Provo, UT 84602,United States. Tel.: +1 801 422 4020; fax: +1 801 422 0153.

E-mail address: paul_savage@byu.edu (P.B. Savage).

0022-1759/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.jim.2006.02.009

limited repertoire of T cells receptors (TCRs) and re-spond to presentation of specific glycolipids via arelease of a variety of cytokines, including those as-sociated with both Th1 and Th2 responses. NKT cellshave been studied primarily in the context of CD1dpresentation of an α-galactosyl ceramide (αGC), termedKRN7000 (Fig. 1) (Morita et al., 1995), a glycolipid notconsidered to be a natural antigen for NKT cells. Animportant means of isolating and quantifying CD1dresponsive NKT cells via flow cytometry involves useof fluorophore-tagged CD1d tetramers loaded with αGC(Benlagha et al., 2000; Matusda et al., 2000). In addi-tion, αGC is used in studies of the influences of NKTcell stimulation on specific disease states. However,

O

OHOHO

HO

HN

OH

O

NHO

OH

O

OHOHO

HO

HN

OH

O

KRN7000

OH

OH

PBS-57

Fig. 1. Structures of αGC and PBS-57.

35Y. Liu et al. / Journal of Immunological Methods 312 (2006) 34–39

there have been some difficulties in procuring sufficientKRN7000, and this glycolipid has relatively poor solu-bility in either organic or aqueous solvents.

We have synthesized a series of modified αGCs in aneffort to determine the sites at which substitution can bemade without negatively influencing the interactions ofglycolipid-loaded CD1d with NKT cell receptors (Zhouet al., 2002). We found that replacement of the hydroxylgroup at the C6 position of galactose in αGC with anamide linked to a small molecule yields compounds thatretain the ability to stimulate cytokine release by NKTcells at levels comparable to KRN7000. Initial studiesinvolved incorporation of fluorophores at the C6 posi-tion of galactose; however, for NKT cell sorting by flowcytometry, a minimized structure was desirable. Conse-quently, an acetamide group was generated at C6. Fur-ther studies indicated that a cis-double bond in the acylchain in the ceramide portion of αGC resulted in anincrease in solubility over fully saturated compoundsand that the double bond facilitated loading into CD1dtetramers. The optimized glycolipid, PBS-57 in Fig. 1,stains mouse and human NKTcells as well as KRN7000and displays relatively high solubility. In vitro and invivo studies of the NKT cell stimulating properties ofPBS-57 indicated that this glycolipid stimulates NKTcells more effectively than KRN7000.

2. Materials and methods

2.1. Sources of reagents

PBS-57 was synthesized as reported for related com-pounds with small molecules appended at C6 on galac-tose via amides (Zhou et al., 2002), except that aceticanhydride was used to form the acetamide group, andnervonic acid was used in the synthesis of the ceramideportion of the molecule. The structure of PBS-57 was

confirmed by 1H and 13C NMR and mass spectrome-try. sCD1d was obtained from the Teyton laboratory(Benlagha et al., 2000) and Kronenberg laboratory(Sidobre and Kronenberg, 2002), and streptavidin-APCand streptavidin-PE were from Pharmingen (San Diego,CA). The NKT hybridomas were established in theBendelac and Hayakawa laboratories as described (Zhouet al., 2004; Gui et al., 2001).

2.2. Loading of CD1d tetramers

For staining of the NKT cell hybridomas, the fol-lowing series of stock reagents were prepared: sCD1d(1 mg/mL in phospate-buffered saline (PBS)); PBS-57(1 mg/mL in DMSO); Tween 20 (0.5% in PBS); andstreptavidin-APC (80 μg/mL in PBS). The stock solu-tion of sCD1d (10 μL), PBS-57 (1 μL), and Tween 20(10 μL) were mixed. PBS was added (79 μL) to bringthe final volume to 100 μL, and the resulting solutionwas incubated at 37 °C for 3 h. To separate unboundglycolipid, the mixture was applied to a MicroconYM30 filter (Millipore) that had been previously wettedwith PBS (400 μL). The loaded membrane was centri-fuged until only ∼ 10 μL of solution remained then thevolume was increased to 100 μL by addition of PBS.The solution was agitated to aid in freeing the proteinfrom the filter. The Microcon unit was inverted in a freshEppendorf tube and the contents were centrifuged intothe tube. A 10 μL aliquot of the solution was removedand the streptavidin-APC solution (5 μL) was added.The resulting solution was incubated at 37 °C for 2 h.

For staining of thymoctyes and splenocytes, biotiny-lated mouse sCD1d (in PBS) was mixed with glycoli-pids (in PBS with 0.1% Tween 20, pH 7.4) at a molarratio of 1:3 (protein:lipid) and incubated at room tem-perature overnight. The following day, 80 μg ofstreptavidin-PE (Pharmingen) was added to 200 μg of

36 Y. Liu et al. / Journal of Immunological Methods 312 (2006) 34–39

the CD1–glycolipid mix and incubated at room tem-perature for 4 h. Tetramers were stored at 4 °C until use.

2.3. NKT cell staining

NKT cell hybridomas were suspended in PBS andstreptavidin (1 μg/mL) was used to block surface bio-tin of cells (20 min at room temperature). UnloadedsCD1d–streptavidin–cychrome was used to assess thenon-specific binding of unloaded empty CD1d tetramers(20 min at room temperature). Staining with the glyco-lipid–sCD1d–streptavidin–APC complex was per-formed for 40 min at 37 °C. The cells were washed byPBS and assayed via flow cytometry as described (Zhouet al., 2004).

Single cell suspensions of thymocytes and spleno-cytes from C57BL/6J mice (Jackson Laboratories, BarHarbor, Maine) were analyzed for cell surface expres-sion of the invariant Vα14i NKT TCR by flow cytom-etry. Briefly, 106 cells were incubated in 200 μL stainingmedia (2% BSA, 1% NaN3, 10 mM EDTA in PBS) with2.4G2 (1:100; ATCC, Manassas, VA) and Neutravidin(5 μg/200 μL; Molecular Probes, Eugene, OR) for20 min on ice. Cells were pelleted and resuspended instaining media with anti-TCRβ FITC (1:100; H57-597,BD-Pharmingen, San Diego, CA) and CD1/glycolipidor vehicle loaded tetramers conjugated with streptavi-

Fig. 2. Comparative analysis of Vα14i NKT cells from mouse thymus (A–C)B, E: KRN7000; C, F; PBS-57.

din-PE (1:400) and incubated on ice for 45 min. CD1–glycolipid tetramers were produced as previously des-cribed (Sidobre and Kronenberg, 2002). Cells werewashed twice in staining media, fixed with 1% para-formaldehyde in PBS and analyzed by flow cytometry.

Whole blood samples (200 μL) (human, chimpanzee,rhesus macaques, pigtail macaques, and sooty manga-beys) were stained with both hCD1d-PBS-57 andmCD1d-PBS-57 tetramers, together with anti-CD3.These samples were processed, fixed, and analyzed byflow cytometry (Becton Dickinson FACS Lyse using themanufacturer's recommendations).

2.4. In vitro stimulation of splenocytes with KRN7000and PBS-57

Splenocytes (5×105/well) from B6 mice were incu-bated with indicated doses of KRN7000 and PBS-57in RPMI 1640 supplemented with 10% FCS, 50 μM2-mercaptoethanol, 2 mM glutamine and antibiotics.After incubation for 48 h, cytokine concentrations weredetermined using ELISA (BD Pharmingen).

2.5. Administration of KRN7000 and PBS-57 in vivo

Stock solutions of glycolipids in DMSO at 1 mg/mLwere prepared. These solutions were added to PBS to

and spleen (D–F) cell populations. A, D: Vehicle only (no glycolipid);

DN32.D3(Vβ8.2) N382C11(Vβ8.2) 431G11(Vβ8.1)DN3H1(Vβ2) 414A2(Vβ7)

control

PBS-57

Fig. 3. NKT cell hybridomas expressing different Vβ TCRs were stained by CD1-tetramers loaded with PBS-57, or a non-stimulating glycolipid(α-galactosylcholesterol). PBS-57 stained all hybridomas regardless of Vβ usage.

37Y. Liu et al. / Journal of Immunological Methods 312 (2006) 34–39

give the glycolipid concentrations indicated. Aliquots of100 μL of 1, 100, 10000, or 1000000 pg/mL solutionsof KRN7000 and PBS-57 were injected intravenouslyinto 6 week-old B6 mice. Serum samples were isolatedafter 24 h and cytokine concentrations were determinedusing ELISA (BD Pharmingen).

3. Results and discussion

While PBS-57 is slightly more complex thanKRN7000, its synthesis is relatively straightforward.Consequently, it can be prepared in quantity and hasbeen provided to the Tetramer Core Facility of theNational Institutes of Health (USA) (http://www.yerkes.emory.edu/TETRAMER/CD1d_Tetramers.html). Aswith most glycolipids, solubility issues are central tohandling the compounds. Methods of dissolving PBS-57 are similar to those used with KRN7000, with themost convenient method involving dilution of concen-trated DMSO solutions of the glycolipid into aqueoussolutions. PBS-57 is soluble in DMSO above 20 mg/mL(22.4 mM), whereas in our hands the solubility ofKRN7000 in DMSO is b5 mg/mL. Using high concen-

Human Chimpa

hCD

1dm

CD

1d

CD

0.14

0.18

Fig. 4. CD1d-responsive NKT cells observed using PBS-

trations of the glycolipid in DMSO, followed by dilutioninto aqueous Tween 20 gives solutions with low residualconcentrations of DMSO. For example, typical NKTcell staining protocols call for a final concentration ofglycolipid of 10 μg/mL, which results in a final DMSOconcentration of one percent or less (depending on theglycolipid concentration in the DMSO stock solution).

Loading of CD1d tetramers with PBS-57 yieldscomplexes that stain NKTcells comparably to KRN7000(Fig. 2) using cells derived from mouse thymus andspleen. The TCR repertoire of NKT cells is limited withan invariable Vα subunit (Vα14 in mice and Vα24 inhumans) and varied Vβ subunits that respond to glyco-lipid presentation by CD1d. The availability of NKTcellhybridomas that display distinct TCRs made it was pos-sible to determine the effects of varied Vβ subunits on theability of PBS-57 loaded tetramers to stain NKT cells.Flow cytometry results with a variety of mouse NKTcellhybridomas revealed that variations in Vβ subunits didnot influence staining by PBS-57 loaded tetramers(Fig. 3). The influence of the Vβ subunits on cytokineproduction has not yet been fully elucidated; neverthe-less, PBS-57 appears to be a “universal” ligand for NKT

nzeeRhesus

Macaque

3

0.1 0.44

0.091 0.46

57 loaded into human and mouse CD1d tetramers.

0

0.110

1,00

0

100,

000

2500

5000

7500

10000

12500

15000

17500

20000

IFN

-γ (p

g/m

L)

pg

Fig. 6. Serum IFN-γ concentrations from mice injected (i.v.) with theindicated quantities of ▪ PBS-57 and □ KRN7000. Cytokineconcentrations were determined 24 h after injection.

38 Y. Liu et al. / Journal of Immunological Methods 312 (2006) 34–39

cells with TCRs of varied structures among thesehybridomas.

We also observed the abilities of PBS-57 loadedCD1d tetramers (both mouse and human) to stain NKTcells in blood samples from humans and a variety ofnon-human primates (Karadimitris et al., 2001; Gum-perz et al., 2002). Examples are shown in Fig. 4. Amajority of human blood samples contained sufficientNKT cells (N0.08% of CD3-positive cells) to observestaining (14 out of 17 samples), while some samplesmay have contained too few NKT cells to allow detec-tion of staining (Lee et al., 2002). Among non-humanprimates, significant NKT cell staining was observedwith a majority of chimpanzee blood samples (6 out of10 samples) and one quarter of samples from rhesusmacaques (12 samples). NKT cell staining was not ob-served with samples from pigtail macaques (six sam-ples) and sooty mangabeys (six samples). Due to thelimited populations of NKT cells in circulating bloodand the small sample sizes, it is possible that stainingwould be observed in pigtail macaques and sooty man-gabeys if samples from more individuals were analyzed.

A high affinity interaction between NKT cell recep-tors and CD1d facilitated by a specific glycolipid wouldbe expected to result in cytokine release from NKTcells.To determine if PBS-57 stimulates cytokine release, weexamined NKT cell responses to CD1d presentation ofthis glycolipid in vitro and in vivo. For in vitro testing,mouse splenocytes were harvested and subjected to theconcentrations of glycolipids indicated in Fig. 5. Todetermine if the cytokine release profiles were modifiedby the structural differences of PBS-57 as compared toKRN7000, release of both IFN-γ and IL-4 were quan-tified. Responses to PBS-57 plateaued at approximate-ly 100 pg/mL, as compared to ca. 1000 pg/mL forKRN7000, and both glycolipids effectively induce Th1and Th2 cytokine secretion. To verify that PBS-57

0

25

50

75

100

IFN

-γ (p

g/m

L)

1 10 100 1,000 10,000 100,000

pg/mL

IL-4

(pg

/mL)

Fig. 5. Cytokine release from B6 mouse splenocytes stimulated with▪ PBS-concentrations of glycolipids and cytokine concentrations were measured af

simulates NKT cells in vivo, mice were injected withvaried amounts of PBS-57 and KRN7000 (100 μLinjections of varied concentrations of glycolipids).Serum IFN-γ concentrations are shown in Fig. 6. Asobserved in vitro, PBS-57 appeared slightly more active,although the difference between PBS-57 and KRN7000was attenuated in vivo. Due to the improved solubilityproperties of PBS-57 over KRN7000, it may be betteravailable for loading into CD1d resulting in stimulationof NKT cells at lower concentrations.

4. Conclusions

Modification of the functionality at the C6 position ofαGC and incorporation of a double bond in the acylchain gives a glycolipid that stains NKT cells, via CD1dtetramers, comparably to KRN7000, and NKT cells

0

2500

5000

7500

pg/mL1 10 100 1,000 10,000 100,000

57 and□ KRN7000. 105 spleen cells were incubated with the indicatedter 48 h.

39Y. Liu et al. / Journal of Immunological Methods 312 (2006) 34–39

presenting TCRs with a variety of Vβ chains are effec-tively stained. Presumably due to better solubility, PBS-57 stimulates NKT cells in vitro at lower concentrationsthan KRN7000, and in vivo it is at least as effective ineliciting cytokine release. It is anticipated that theavailability of this glycolipid (PBS-57 loaded CD1dtetramers are available through the NIH Tetramer CoreFacility) will facilitate research involving these impor-tant regulatory T cells.

Acknowledgement

Financial support from the National Institutes ofHealth (P01 AI053725 to A. B., L. T., and P. B. S.) isgratefully acknowledged. D. Z. and J. M. were sup-ported by Cancer Research Institute (New York) fellow-ship grants, and B. A. S. was supported by an NRSAFellowship from the NIH (F32 AI62015). The authorsgratefully acknowledge Mitchell Kronenberg for pro-viding critical reagents.

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