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

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

Research paper

A modified α-galactosyl ceramide for staining and stimulating natural 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 States University of Chicago, Chicago, IL 60637, United States The La Jolla Institute for Allergy and Immunology, San Diego, CA 92121, United States d Scripps Research Institute, La Jolla, CA 92037, United States e Emory Vaccine Research Center, Atlanta, GA 30329, United States b

c

Received 14 March 2005; received in revised form 3 October 2005; accepted 7 February 2006 Available 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 sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an α-galactosyl ceramide with improved solubility over KRN7000 that effectively 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 (NKT cells) in immune responses has prompted intense investigation of their influences on various disease states and their interactions with glycolipid antigens (Brigl and Brenner, 2004; Godfrey and Kronenberg, 2004; Kronenberg, 2005; Van Kaer, 2005). NKT cells display a

⁎ Corresponding author. Department of Chemistry and Biochemistry, Brigham Young University, C100 BNSN, Provo, UT 84602, United States. Tel.: +1 801 422 4020; fax: +1 801 422 0153. E-mail address: [email protected] (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 respond to presentation of specific glycolipids via a release of a variety of cytokines, including those associated with both Th1 and Th2 responses. NKT cells have been studied primarily in the context of CD1d presentation of an α-galactosyl ceramide (αGC), termed KRN7000 (Fig. 1) (Morita et al., 1995), a glycolipid not considered to be a natural antigen for NKT cells. An important means of isolating and quantifying CD1d responsive NKT cells via flow cytometry involves use of fluorophore-tagged CD1d tetramers loaded with αGC (Benlagha et al., 2000; Matusda et al., 2000). In addition, αGC is used in studies of the influences of NKT cell stimulation on specific disease states. However,

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

HO

OH O

HO

35

O HN

HO

OH

O

KRN7000

OH

O HO

NH O

HO

HO

O HN

OH

O

PBS-57

OH

Fig. 1. Structures of αGC and PBS-57.

there have been some difficulties in procuring sufficient KRN7000, and this glycolipid has relatively poor solubility in either organic or aqueous solvents. We have synthesized a series of modified αGCs in an effort to determine the sites at which substitution can be made without negatively influencing the interactions of glycolipid-loaded CD1d with NKT cell receptors (Zhou et al., 2002). We found that replacement of the hydroxyl group at the C6 position of galactose in αGC with an amide linked to a small molecule yields compounds that retain the ability to stimulate cytokine release by NKT cells at levels comparable to KRN7000. Initial studies involved incorporation of fluorophores at the C6 position of galactose; however, for NKT cell sorting by flow cytometry, a minimized structure was desirable. Consequently, an acetamide group was generated at C6. Further studies indicated that a cis-double bond in the acyl chain in the ceramide portion of αGC resulted in an increase in solubility over fully saturated compounds and that the double bond facilitated loading into CD1d tetramers. The optimized glycolipid, PBS-57 in Fig. 1, stains mouse and human NKT cells as well as KRN7000 and displays relatively high solubility. In vitro and in vivo studies of the NKT cell stimulating properties of PBS-57 indicated that this glycolipid stimulates NKT cells more effectively than KRN7000. 2. Materials and methods 2.1. Sources of reagents PBS-57 was synthesized as reported for related compounds with small molecules appended at C6 on galactose via amides (Zhou et al., 2002), except that acetic anhydride was used to form the acetamide group, and nervonic acid was used in the synthesis of the ceramide portion of the molecule. The structure of PBS-57 was

confirmed by 1H and 13C NMR and mass spectrometry. sCD1d was obtained from the Teyton laboratory (Benlagha et al., 2000) and Kronenberg laboratory (Sidobre and Kronenberg, 2002), and streptavidin-APC and streptavidin-PE were from Pharmingen (San Diego, CA). The NKT hybridomas were established in the Bendelac and Hayakawa laboratories as described (Zhou et al., 2004; Gui et al., 2001). 2.2. Loading of CD1d tetramers For staining of the NKT cell hybridomas, the following 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); and streptavidin-APC (80 μg/mL in PBS). The stock solution of sCD1d (10 μL), PBS-57 (1 μL), and Tween 20 (10 μL) were mixed. PBS was added (79 μL) to bring the final volume to 100 μL, and the resulting solution was incubated at 37 °C for 3 h. To separate unbound glycolipid, the mixture was applied to a Microcon YM30 filter (Millipore) that had been previously wetted with PBS (400 μL). The loaded membrane was centrifuged until only ∼ 10 μL of solution remained then the volume was increased to 100 μL by addition of PBS. The solution was agitated to aid in freeing the protein from the filter. The Microcon unit was inverted in a fresh Eppendorf tube and the contents were centrifuged into the tube. A 10 μL aliquot of the solution was removed and 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, biotinylated mouse sCD1d (in PBS) was mixed with glycolipids (in PBS with 0.1% Tween 20, pH 7.4) at a molar ratio of 1:3 (protein:lipid) and incubated at room temperature overnight. The following day, 80 μg of streptavidin-PE (Pharmingen) was added to 200 μg of

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the CD1–glycolipid mix and incubated at room temperature for 4 h. Tetramers were stored at 4 °C until use. 2.3. NKT cell staining NKT cell hybridomas were suspended in PBS and streptavidin (1 μg/mL) was used to block surface biotin of cells (20 min at room temperature). Unloaded sCD1d–streptavidin–cychrome was used to assess the non-specific binding of unloaded empty CD1d tetramers (20 min at room temperature). Staining with the glycolipid–sCD1d–streptavidin–APC complex was performed for 40 min at 37 °C. The cells were washed by PBS and assayed via flow cytometry as described (Zhou et al., 2004). Single cell suspensions of thymocytes and splenocytes from C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine) were analyzed for cell surface expression of the invariant Vα14i NKT TCR by flow cytometry. Briefly, 106 cells were incubated in 200 μL staining media (2% BSA, 1% NaN3, 10 mM EDTA in PBS) with 2.4G2 (1:100; ATCC, Manassas, VA) and Neutravidin (5 μg/200 μL; Molecular Probes, Eugene, OR) for 20 min on ice. Cells were pelleted and resuspended in staining media with anti-TCRβ FITC (1:100; H57-597, BD-Pharmingen, San Diego, CA) and CD1/glycolipid or vehicle loaded tetramers conjugated with streptavi-

din-PE (1:400) and incubated on ice for 45 min. CD1– glycolipid tetramers were produced as previously described (Sidobre and Kronenberg, 2002). Cells were washed twice in staining media, fixed with 1% paraformaldehyde in PBS and analyzed by flow cytometry. Whole blood samples (200 μL) (human, chimpanzee, rhesus macaques, pigtail macaques, and sooty mangabeys) were stained with both hCD1d-PBS-57 and mCD1d-PBS-57 tetramers, together with anti-CD3. These samples were processed, fixed, and analyzed by flow cytometry (Becton Dickinson FACS Lyse using the manufacturer's recommendations). 2.4. In vitro stimulation of splenocytes with KRN7000 and PBS-57 Splenocytes (5 × 105/well) from B6 mice were incubated with indicated doses of KRN7000 and PBS-57 in RPMI 1640 supplemented with 10% FCS, 50 μM 2-mercaptoethanol, 2 mM glutamine and antibiotics. After incubation for 48 h, cytokine concentrations were determined using ELISA (BD Pharmingen). 2.5. Administration of KRN7000 and PBS-57 in vivo Stock solutions of glycolipids in DMSO at 1 mg/mL were prepared. These solutions were added to PBS to

Fig. 2. Comparative analysis of Vα14i NKT cells from mouse thymus (A–C) and spleen (D–F) cell populations. A, D: Vehicle only (no glycolipid); B, E: KRN7000; C, F; PBS-57.

Y. Liu et al. / Journal of Immunological Methods 312 (2006) 34–39 DN32.D3(Vβ8.2)

N382C11(Vβ8.2)

DN3H1(Vβ2)

414A2(Vβ7)

37 431G11(Vβ8.1)

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.

give the glycolipid concentrations indicated. Aliquots of 100 μL of 1, 100, 10 000, or 1 000 000 pg/mL solutions of KRN7000 and PBS-57 were injected intravenously into 6 week-old B6 mice. Serum samples were isolated after 24 h and cytokine concentrations were determined using ELISA (BD Pharmingen). 3. Results and discussion While PBS-57 is slightly more complex than KRN7000, its synthesis is relatively straightforward. Consequently, it can be prepared in quantity and has been provided to the Tetramer Core Facility of the National Institutes of Health (USA) (http://www.yerkes. emory.edu/TETRAMER/CD1d_Tetramers.html). As with most glycolipids, solubility issues are central to handling the compounds. Methods of dissolving PBS57 are similar to those used with KRN7000, with the most convenient method involving dilution of concentrated DMSO solutions of the glycolipid into aqueous solutions. PBS-57 is soluble in DMSO above 20 mg/mL (22.4 mM), whereas in our hands the solubility of KRN7000 in DMSO is b5 mg/mL. Using high concenHuman

trations of the glycolipid in DMSO, followed by dilution into aqueous Tween 20 gives solutions with low residual concentrations of DMSO. For example, typical NKT cell staining protocols call for a final concentration of glycolipid of 10 μg/mL, which results in a final DMSO concentration of one percent or less (depending on the glycolipid concentration in the DMSO stock solution). Loading of CD1d tetramers with PBS-57 yields complexes that stain NKT cells comparably to KRN7000 (Fig. 2) using cells derived from mouse thymus and spleen. The TCR repertoire of NKT cells is limited with an invariable Vα subunit (Vα14 in mice and Vα24 in humans) and varied Vβ subunits that respond to glycolipid presentation by CD1d. The availability of NKT cell hybridomas that display distinct TCRs made it was possible to determine the effects of varied Vβ subunits on the ability of PBS-57 loaded tetramers to stain NKT cells. Flow cytometry results with a variety of mouse NKT cell hybridomas revealed that variations in Vβ subunits did not influence staining by PBS-57 loaded tetramers (Fig. 3). The influence of the Vβ subunits on cytokine production has not yet been fully elucidated; nevertheless, PBS-57 appears to be a “universal” ligand for NKT

Chimpanzee

Rhesus Macaque

0.1

0.44

0.18

0.091

0.46

mCD1d

hCD1d

0.14

CD3 Fig. 4. CD1d-responsive NKT cells observed using PBS-57 loaded into human and mouse CD1d tetramers.

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20000

cells with TCRs of varied structures among these hybridomas. We also observed the abilities of PBS-57 loaded CD1d tetramers (both mouse and human) to stain NKT cells in blood samples from humans and a variety of non-human primates (Karadimitris et al., 2001; Gumperz et al., 2002). Examples are shown in Fig. 4. A majority of human blood samples contained sufficient NKT cells (N0.08% of CD3-positive cells) to observe staining (14 out of 17 samples), while some samples may have contained too few NKT cells to allow detection of staining (Lee et al., 2002). Among non-human primates, significant NKT cell staining was observed with a majority of chimpanzee blood samples (6 out of 10 samples) and one quarter of samples from rhesus macaques (12 samples). NKT cell staining was not observed with samples from pigtail macaques (six samples) and sooty mangabeys (six samples). Due to the limited populations of NKT cells in circulating blood and the small sample sizes, it is possible that staining would be observed in pigtail macaques and sooty mangabeys if samples from more individuals were analyzed. A high affinity interaction between NKT cell receptors and CD1d facilitated by a specific glycolipid would be expected to result in cytokine release from NKT cells. To determine if PBS-57 stimulates cytokine release, we examined NKT cell responses to CD1d presentation of this glycolipid in vitro and in vivo. For in vitro testing, mouse splenocytes were harvested and subjected to the concentrations of glycolipids indicated in Fig. 5. To determine if the cytokine release profiles were modified by the structural differences of PBS-57 as compared to KRN7000, release of both IFN-γ and IL-4 were quantified. Responses to PBS-57 plateaued at approximately 100 pg/mL, as compared to ca. 1000 pg/mL for KRN7000, and both glycolipids effectively induce Th1 and Th2 cytokine secretion. To verify that PBS-57

17500

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15000

10000 7500 5000 2500 0

0

0

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00

00

1,

, 00

1

1

0.

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▪

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

simulates NKT cells in vivo, mice were injected with varied amounts of PBS-57 and KRN7000 (100 μL injections of varied concentrations of glycolipids). Serum IFN-γ concentrations are shown in Fig. 6. As observed in vitro, PBS-57 appeared slightly more active, although the difference between PBS-57 and KRN7000 was attenuated in vivo. Due to the improved solubility properties of PBS-57 over KRN7000, it may be better available for loading into CD1d resulting in stimulation of NKT cells at lower concentrations. 4. Conclusions Modification of the functionality at the C6 position of αGC and incorporation of a double bond in the acyl chain gives a glycolipid that stains NKT cells, via CD1d tetramers, comparably to KRN7000, and NKT cells 7500

100

IFN-γ (pg/mL)

75

IL-4 (pg/mL)

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25

5000

2500

0

0 1

10

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1,000 10,000 100,000

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Fig. 5. Cytokine release from B6 mouse splenocytes stimulated with PBS-57 and □ KRN7000. 105 spleen cells were incubated with the indicated concentrations of glycolipids and cytokine concentrations were measured after 48 h.

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presenting TCRs with a variety of Vβ chains are effectively stained. Presumably due to better solubility, PBS57 stimulates NKT cells in vitro at lower concentrations than KRN7000, and in vivo it is at least as effective in eliciting cytokine release. It is anticipated that the availability of this glycolipid (PBS-57 loaded CD1d tetramers are available through the NIH Tetramer Core Facility) will facilitate research involving these important regulatory T cells. Acknowledgement Financial support from the National Institutes of Health (P01 AI053725 to A. B., L. T., and P. B. S.) is gratefully acknowledged. D. Z. and J. M. were supported by Cancer Research Institute (New York) fellowship grants, and B. A. S. was supported by an NRSA Fellowship from the NIH (F32 AI62015). The authors gratefully acknowledge Mitchell Kronenberg for providing critical reagents. References Benlagha, K., Weiss, A., Beavis, A., Teyton, L., Bendelac, A., 2000. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191, 1895. Brigl, M., Brenner, M.B., 2004. CD1: T cell function and antigen presentation. Annu. Rev. Immunol. 22, 817. Gumperz, J.E., Miyake, S., Yamamura, T., Brenner, M.B., 2002. Functional distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195, 625. Godfrey, D.I., Kronenberg, M., 2004. J. Clin. Invest. 114, 1379.

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