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. Author manuscript; available in PMC: 2015 Oct 16.
Published in final edited form as: Immunity. 2014 Oct 16;41(4):543–554. doi: 10.1016/j.immuni.2014.08.017

The Identification of the Endogenous Ligands of Natural Killer T Cells Reveals the Presence of Mammalian α-Linked Glycosylceramides

Lisa Kain 1, Bill Webb 2, Brian L Anderson 3, Shenglou Deng 3, Marie Holt 1, Anne Constanzo 1, Meng Zhao 4, Kevin Self 1, Anais Teyton 1, Chris Everett 1, Mitchell Kronenberg 4, Dirk M Zajonc 4, Albert Bendelac 5, Paul B Savage 3, Luc Teyton 1,*
PMCID: PMC4220304  NIHMSID: NIHMS633508  PMID: 25367571

Summary

Glycosylceramides in mammalian species are thought to be present in the form of β-anomers. This conclusion was reinforced by the identification of only one glucosylceramide and one galactosylcerhamide synthase, both β-transferases, in mammalian genomes. Thus, the possibility that small amounts of α-anomers could be produced by an alternative enzymatic pathway, by an unfaithful enzyme, or spontaneously in unusual cellular compartments has not been examined in detail. We approached the question by taking advantage of the exquisite specificity of T and B lymphocytes and combined it with the specificity of catabolic enzymes of the sphingolipid pathway. Here, we demonstrate that mammalian immune cells produce constitutively very small quantities of α-glycosylceramides, which are the major endogenous ligands of natural killer T cells. Catabolic enzymes of the ceramide and glycolipid pathway tightly control the amount of these α-glycosylceramides. The exploitation of this pathway to manipulate the immune response will create new therapeutic opportunities.

Introduction

The biosynthetic pathways of glycolipids were described many decades ago in the context of enzymatic deficiencies that lead to inherited human diseases of the nervous system (Schulze and Sandhoff, 2011; Wennekes et al., 2009). A very good map of enzymes, their substrates, and their products was produced by classical biochemical methods and has not been challenged since. However, all lipid analytical methods lack sensitivity; it is nearly impossible to detect contaminations below 0.5%–1% in natural or synthetic preparations of lipids and glycolipids (Meisen et al., 2011). On the contrary, biological assays are exquisitely sensitive to low amounts of otherwise unmeasurable molecules. This particular situation has hampered the identification of immunologically relevant lipid species, a family of antigens that are presented to T cells by the family of major histocompatibility complex (MHC)-like molecules called CD1 (Bendelac et al., 2007).

In the current work, we have combined biological assays with immunological and enzymatic assays to interrogate glycolipid populations in order to elucidate the identity of glycolipids capable of triggering the activation of a regulatory T cell subset called type 1 natural killer T (NKT) cells; NKT cells make up a small population that sits at the interface between innate and adaptive immunities and is critical for the coordination of T and B cell responses (Bendelac et al., 2007). NKT cells are recruited very rapidly and transiently in the context of all microbial aggressions to allow the maturation of dendritic cells (DCs) and the recruitment of immune cells at the site of injury (Bendelac et al., 2007). The activation of NKT cells is believed in many cases to be dependent on the display of endogenous glycolipids by DCs in the context of CD1 MHC-like molecules. NKT cells are capable of almost immediate responses, leading to the hypothesis that endogenous ligands are either premade or quickly produced by an enzymatic modification that is tightly controlled to avoid persistent or overt activation leading to activation-induced cell death or stunning, such as when strong agonists are used (Wilson et al., 2003). A large number of potential self-antigens have been proposed over the years, and all are capable of activating NKT cells in vitro and/or in vivo (Brennan et al., 2011; Facciotti et al., 2012; Zhou et al., 2004b). It has proven difficult to study the chemistry of these potential candidates because of low sensitivity of the assays. To overcome the limitations of direct chemical methods, we have used the specificity of immunological and enzymatic assays to characterize and isolate the endogenous ligands of NKT cells in the thymus and in DCs. We found that these stimulatory NKT agonists are α-linked monoglycosylceramides, a class of glycolipids that were thought to be absent from mammalian cells given that the only two glycosylceramide synthases (glucosylceramide synthase [GCS] and ceramide galactosyltransferase [CGT]) were thought to be inverting glycosyltransferases; through a SN2-like ligation, these enzymes transfer α-glucose and α-galactose from uridine diphosphate (UDP)-sugar moieties in a β-anomeric linkage on a ceramide (Lairson et al., 2008). In addition, we demonstrate that catabolic enzymes tightly control the level of α-galactosylceramide (α-GalCer) in cells and tissues.

Results

β-Glucosylceramides Are Not the Natural Endogenous Ligands of NKT Cells

It has recently been proposed that β-linked monoglycosylceramides, such as β-glucosylceramides (β-GluCer), were natural endogenous ligands of NKT cells, and synthetic preparations of C12:0 and C24:1 β-GluCer have been shown to be strong activators of type 1 NKT cells (Brennan et al., 2011; Ortaldo et al., 2004; Parekh et al., 2004; Zigmond et al., 2007). However, because of the limitations of the analytical methods of lipids, the possibility that α-anomers could contaminate the synthetic preparations could not be easily ruled out. In addition, because β-GluCer is one of the most abundant glycosylceramides in all cell types, it remains very difficult to conceive the regulation of its presentation by CD1 molecules and recognition by NKT cells. Were purified by normal-phase chromatography a large quantity of commercial C24:1 β-GluCer and isolated seven fractions. Each was tested for the presence of β-GluCer by immunoblot using a polyclonal rabbit serum against β-GluCer, and only one fraction, fraction 3, tested positive (Figure S1, available online). However, in functional assays, six of the seven fractions were stimulatory.

We synthesized C24:1 β-GluCer and tested it for presentation by DCs to the prototypic NKT cell hybridoma DN32.D3 and found no stimulation of DN32.D3 cells for this synthetic product, even at a high concentration (Figure 1A). We routinely used this test to assess the lack of α-anomer contamination in our β-anomer synthesis. The suspicion of contamination was confirmed by three experiments. First, we loaded CD1d-GT1b tetramers with commercial and in-house C24:1 β-GluCer in the presence of saposin B at acidic pH to allow exchange and completion of lipid loading. We used the C24:1-β-GluCer-CD1-loaded tetramers to stain DN32.D3 cells and polyclonal NKT populations from C57BL/6 mice and used C24:1-α-GalCer-loaded tetramers as positive controls. No staining of NKT cells could be observed, even at very high concentrations of tetramer (Figure 1B). Second, we produced recombinant acid glucosylceramidase (GBA), an enzyme specific to β-GluCer (Kacher et al., 2008), in fly cells and used it to digest fraction 3 to completion to produce glucose and ceramide (Figure 1C). This enzymatic treatment did not affect the stimulatory activity of the commercial C24:1 β-GluCer (Figure 1C), ruling out that β-GluCer could be an agonist of NKT cells. Finally, we confirmed the presence of α-linked contaminants by blocking the stimulatory activity with the anti-CD1-α-GalCer antibody L363 and by purifying stimulatory compounds with Pisum Sativum agglutinin, an anti-α-linked glucose lectin (Figure 1D and Figure S1). On the basis of the half maximal inhibitory concentration (IC50) in T cell assays of pure α-linked GluCer agonists, we could estimate that the contamination of commercial C24:1 β-GluCer with α-species was in the order of 0.2%–2%. Thus, concurrent evidence demonstrates that β-GluCer has no stimulatory properties toward NKT cells and that contamination of synthetic β-linked glycolipids with α-anomers is an important issue for biological studies.

Figure 1. β-GluCer Is Not the Endogenous Ligand of NKT Cells.

Figure 1

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(A) Comparison of the stimulatory capacity of commercial (closed circles) and in-house(open circles) C24:1 β-GluCer presented by DCs to DN32.D3 NKT cells.

(B) CD1d tetramers loaded with control lipid, C24:1 β-GluCer, or C24:1 α-GalCer were used to stain DN43.D3 NKT hybridoma cells and splenocytes and were examined by flow cytometry.

(C) β-GluCer was digested with recombinant GBA for 2 hr at 37°C, analyzed by TLC for confirmation of complete digestion (left panel), and tested functionally for its ability to stimulate DN32.D3 NKT cells when presented by WT splenocytes before (squares) and after (circles) digestion (right panel).

(D) The stimulatory activity of commercial β-GluCer was blocked by L363 (10 μg/ml; diamonds) and 20H2 (5 μg/ml; triangles), but not control (squares) antibodies (left panel), and could be recovered with Pisum Sativum lectin (right panel), an agglutinin specific to α-glucose (closed squares). Unbound fractions were used as a negative control (open circles). Seven two-fold dilutions of the purified material were used.

All experiments were repeated a minimum of three times with similar results. See also Figure S1.

“Autoreactivity” of NKT Cells toward Cellular Ligands

NKT cells have a memory phenotype and hallmarks of “preactivation” when analyzed ex vivo. In vitro, they have been described as being highly autoreactive given their propensity for being activated by syngeneic target cells expressing CD1d molecules (Bendelac et al., 2007; Park et al., 1998). This phenomenon is well illustrated by the activation of Vα14 NKT hybridoma cells, such as DN32.D3 cells, against CD1d-positive cell lines (such as rat basophilic leukemia [RBL]-CD1d cells; Figures 2A and 2B), primary cells (such as thymocytes; Figure 2C), and DCs that have been stimulated by Toll-like receptor (TLR) ligands (Figure 2D). The presentation of endogenous ligands requires a competent lysosome and lipid-transfer proteins (Zhou et al., 2004a), as illustrated in Figures 2E and 2F by the large decrease in stimulatory activity produced by the knockdown of prosaposin in RBL-CD1d cells. In these assays, as expected, anti-CD1 antibodies, such as 20H2, blocked the activation of DN32.D3 cells (Roark et al., 1998).

Figure 2. The Anti-CD1d-α-GalCer Antibody L363 Blocks the Autoreactivity of CD1-Expressing Cells toward NKT Cells.

Figure 2

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(A) IL-2 production by Vα14-expressing DN32.D3 NKT cells was tested after a 24 hr exposure to increasing numbers of RBL-CD1d cells in the presence of L363 (red dots) or control (black dots) antibody (10 μg/ml; left panel).

(B) Tested under similar conditions, the non-Vα14 NKT cell hybridoma, TBA7, was not affected by antibody treatment.

(C) The stimulatory activity of WT thymocytes toward DN32.D3 cells was tested in the presence of control (black dots) or L363 (red dots) antibody (20 μg/ml).

(D) A total of 2 × 104 DC3.2 cells treated for 16 hr with increasing concentrations of LPS were used to stimulate DN32.D3 cells in the presence of control (black dots) or L363 (red dots) antibody (10 μg/ml).

(E and F) Stimulation of DN32.D3 cells (E) and TBA7 cells (F) was tested against RBL-CD1d cells (black dots) or RBL-CD1d saposin-knockdown cells, in which saposin expression was reduced to undetectable levels with interfering RNAs. IL-2 production was measured with the NK reporter cell line from triplicate wells.

Experiments shown are representative of at least five independent experiments. See also Figure S2.

To probe the structure of the stimulatory CD1-lipid complexes, we used the antibodies L317 and L363 to block α-linked GluCer stimulation (Figure 1D). Both antibodies are specific to the complex CD1d-α-GalCer (Yu et al., 2007). The crystal structure of CD1d-α-GalCer in complex with L363 shows how the antibody can see both CD1 and the α-linked sugar (Yu et al., 2012). We used this structure to demonstrate by modeling that L363 could not bind β-linked glycolipids or diglycosylceramides because of obvious steric hindrances and that galactose was seen better than glucose as a result of a unique hydrogen bond between the C4 hydroxyl group of the sugar and the antibody (Figure 3A). As a control, we added L317 and L363 antibodies to the autoreactivity assays to determine their impact on endogenous antigen presentation. As shown for L363, both antibodies efficiently blocked the activation of DN32.D3 cells by RBL-CD1d cells, thymocytes, and TLR-activated DCs (Figures 2A, 2C, and 2D, respectively), whereas they did not affect the activation of non-Vα14 NKT cells, such as TBA7 cells (Figure 2B). The observation was expanded to the blocking of interferon-γ (IFN-γ) and interleukin-4 (IL-4) secretion by a polyclonal NKT cell line stimulated by in-vitro-matured bone-marrow-derived DCs (Figure S2). Because the specificity of antibodies is so exquisite, these results strongly suggest that the ligands for Vα14 NKT cells are α-linked monoglycosylceramides. In addition, the L363 antibody was also unable to block the stimulation of DN32.D3 T cells by DCs loaded with isoglobotrihexosylceramide (iGb3), a known agonist of NKT cells (Zhou et al., 2004b; data not shown).

Figure 3. The L363 Antibody Binds with Different Affinity to Both CD1-α-GalCer and CD1-α-GluCer Complexes but Does Not Recognize β-Glycosylceramide-CD1d Association.

Figure 3

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(A) Predicted L363 binding to glycosylceramides. In the crystal structure, L363 contacts α-GalCer with two H-bonds, G50 interacts with the axial 4″-OH, and R32 is specific to the sphingosine chain (Protein Data Bank [PDB] ID 3UBX, left panel). Modeling the interaction with α-GluCer illustrates the loss of the H-bond with G50, which as a result of equatorial rather than axial position of 4″-OH, leads to weaker L363 binding affinity (middle panel). However, N31 and R32 together form a cap over the sugar and bind through van der Waals interactions, predominantly through N31. The upright positioning of β-GalCer (modeled with the crystal structure of mCD1d-sulfatide [PDB ID 2AKR]) would prevent L363 binding as a result of steric clashes (right panel).

(B) The binding of the various α- and β-anomers of glycosylceramides mentioned and depicted in Table 1 was measured by SPR on a Biacore T200 instrument. Single-cycle analysis was performed on CM5 chips with 500–1,000 response units of immobilized antibody and increasing concentrations of the various CD1-lipid complexes. Analysis was performed with the Biacore T200 Biaevaluation global analysis software with subtracted sensorgrams (L363 antibody was used as a control antibody). Similar results were obtained in five independent experiments.

The Endogenous Ligands of NKT Cells Are Monoglycosylceramides

To unambiguously confirm these data, we examined the binding of the L363 antibody by surface plasmon resonance (SPR) against a large series of α- and β-GluCer and -GalCer. All α-linked monoglycosyl species demonstrated measurable binding and a strong preference for galactose over glucose (Figure 3B and Table 1). Binding of β-linked glycolipid-CD1 complexes could not be detected for either L317 or L363 antibody. Diglycosylceramides, such as α-Gal(α1-2)galactosylceramide, and trihexosylceramides, such as iGb3, loaded into CD1d also exhibited no ability to bind the same antibodies (data not shown). Interestingly, we also demonstrated the binding of L363 and L317 to CD1 loaded with α-lyso-galactosylceramide (α-psychosine) and α-lyso-glucosylceramide (α-glucopsychosine), two compounds that are potent stimulators of NKT cells in vitro and in vivo.

Table 1. Affinity Constants of L363 for a Series of α- and β-Monoglycosylceramide Anomers Bound to CD1d.

Lipid Name Affinity (Kdiss / Kass = Kd) s−1 / M.s−1 = M Structure
α-GalCer 0.0083 / 8.57 × 104 = 9.67 × 10−8 graphic file with name nihms633508t1.jpg
C24:1 α-GalCer 0.05 / 1.42 × 105 = 3.50 × 10−7 graphic file with name nihms633508t2.jpg
C24:1 β-GalCer NM graphic file with name nihms633508t3.jpg
α-psychosine 0.021 / 9.43 × 105 = 2.23 × 10−6 graphic file with name nihms633508t4.jpg
β-psychosine NM graphic file with name nihms633508t5.jpg
α-GluCer 0.4711 /9.37 × 104 = 5.02 × 10−6 graphic file with name nihms633508t6.jpg
C24:1 β-GluCer NM graphic file with name nihms633508t7.jpg
α-glucopsychosine 0.1152 /4.59 × 103 = 8.14 × 10−6 graphic file with name nihms633508t8.jpg
β-glucopsychosine NM graphic file with name nihms633508t9.jpg
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The structure of each ceramide is depicted in the right column. Measurements were carried out on a Biacore T200 at 25°C. The same experiment was repeated three times with similar results. NM indicates a nonmeasurable interaction.

Because it is likely that the endogenous ligands are made in very small amounts, we decided to isolate them from cell lines that we could grow in large quantities. For enrichment and purification, we used L363 immunoprecipitation in the presence of a detergent that was compatible with mass spectrometry (MS) analysis and unlikely to compete with CD1-bound lipids efficiently. Sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl) methoxy]-1-propanesulfonate satisfied most of these criteria and was shown in vitro to not bind CD1d to any measurable extent. Because both L317 and L363 antibodies have low affinity for their antigens and because these antigens are present at low density on cell surfaces (no staining by flow cytometry), we decided to use multiple reaction monitoring (MRM)-MS (Kitteringham et al., 2009) to increase specificity and sensitivity of detection in the immunoprecipitates. Transition profiles of all glycosylceramides presented in Table 1 were determined and used as standards (see Experimental Procedures). Using the RBL-CD1d cell line as starting material, we demonstrated the presence of a C24:1 glycosylceramide (mass of 809.67 Da) in six independent experiments, whereas only traces of lyso-glycosylceramide (mass of 462 Da) could be found in two experiments. Similar compounds were isolated from the DC line DC3.2. Both L363 and L317 antibodies isolated the same molecular species (Figure 4A).

Figure 4. Endogenous NKT Ligands Recognized by L363 and L317 Antibodies Are Monoglycosylceramides and α-Linked.

Figure 4

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(A) Liquid chromatography-MRM-MS identified an 810 Da monoglycosylceramide in DC3.2 and RBL-CD1d cells. The lipid content of L363 and L317 antibody immunoprecipitations from DC3.2 and RBL-CD1d cells (2 × 109 cells) was analyzed by MRM-MS. Ionization-transition profiles were defined for α-GalCer, C24:1 α-GalCer (shown in the figure), psychosines, and phytopsychosines. Untransfected RBL cells (5 × 109 cells) were used as a negative control.

(B) The α-linkage of the anomer was demonstrated by analysis of control antibody (ctl, normal rabbit IgG), anti-α-GalCer (āα, left), and anti-L363 (right) immunoprecipitations of C14-labeled RBL cells by TLC. C14UDP-galactose was used to label 5 × 107 RBL cells for 48 hr. In separate experiments, protein extracts were immunoprecipitated with L363, whereas lipid extracts were immunoprecipitated with a purified anti-α-GalCer rabbit antibody. Immunoprecipitates were extracted with a 2:1 chloroform-methanol mixture and analyzed by high-performance TLC next to standards (std.) for α-GalCer and β-GalCer on plates that had been pretreated with borate. Standards were cut and stained with cerium-ammonium-molybdate stain and charring. The experimental part of the TLCs was fluorographed on Biomax MS films for 4–8 weeks.

Each experiment was repeated a minimum of three times. See also Figures S3 and S4.

This direct demonstration of a C24:1 monoglycosylceramide species by MRM-MS did not allow us to eliminate that other stimulatory species were also associated with CD1d. Indeed, because we tested in vitro and in vivo, all α-glycosylceramides with or without acyl chains (psychosines) had stimulatory activity but much lower affinity for CD1d molecules and the L363 antibody (Table 1); therefore, it is most likely that glucosylceramides and lysoceramides would have been lost during the immunopurification procedure. The stimulatory nature of the isolated glycolipids could not be tested directly because of the very limited quantities that we could isolate from 2 × 109 cells. Also, the anomeric identity of the isolated compound could not be probed directly by MS given that α- and β-anomers are isobaric species or by NMR becuase quantities were so limiting. In conclusion, we could isolate from cells molecular species corresponding to C24:1 monoglycosylceramides by using two antibodies for which we confirmed the specificity to CD1d-α-linked monoglycosylceramide complexes.

α-GalCer Is One of the Endogenous Ligands of NKT Cells

On the basis of mass, we knew that the ligand we isolated from RBL cells and DCs was a monoglycosylceramide with a C24:1 fatty acid. To demonstrate the α-linkage, we produced anti-α-GalCer antibodies in rabbits by repeated injections and purification after ammonium sulfate precipitation on a streptavidin-biotinylated α-GalCer column. Specificity of the purified antibodies was tested by SPR on liposomes retaining either α- or β-GalCer (Figure S3). After developing a thin-layer chromatography (TLC) protocol to separate α- and β-GalCer anomers (see Experimental Procedures), we radiolabeled RBL-CD1d cells with C14-UDP-galactose and used them for either immunoprecipitation with L363 antibodies or lipid extraction and immunoprecipitation with the rabbit anti-α-GalCer antibodies. Each immunoprecipitation was extracted with a 2:1 chloroform-methanol mixture and analyzed by TLC next to α- and β-GalCer standards that we stained to locate the migration pattern of each species. In both cases, the isolated monoglycosylceramides migrated as α-anomers, confirming the previous results (Figure 4B). Three independent experiments yielded the same results and allowed us to estimate that the abundance of α-GalCer in RBL-CD1d cells was 0.02% of all galactosylceramides and that ∼25% of these were associated with CD1d.

Degradation Controls the Amount of Natural Endogenous NKT Ligands

As one would predict for an innate-like lymphocyte, NKT cells are activated rapidly and briefly so that stunning and anergy can be avoided (Wilson et al., 2003). The time scale of the de novo synthesis of glycosylceramides would not be the most appropriate to address such a requirement for rapid stimulation (Hayes and Jungalwala, 1976). An alternative mechanism would be to mobilize a pre-existing pool or to limit the degradation of a compound produced in very limited quantities and degraded efficiently. Chemists and biochemists have ruled out the synthesis of α-linked glycosylceramides by mammalian cells on the basis of theoretical arguments favoring a SN2-like reaction over a SN1 reaction in the ligation of UDP-α glucose or galactose to ceramide by glycosyltransferases (Lairson et al., 2008); this prediction has been largely confirmed experimentally. However, the possibility that small amounts of α-linked glycosylceramides could be produced by these enzymes or that under certain physiologic conditions, such as the acidic environment of the lysosome, α-linked species could be produced with the help of anomerases will have to be examined; we already know that the prolonged exposure of α-GalCer and psychosine to pH 4.0 did not lead to any measurable conversion to α-anomers (Figure S4). The role that catabolism might play in the availability of endogenous NKT ligands was suggested by the phenotype of NKT cells in α-galactosidase (GLA) deficiency, also known as Fabry disease, in mice and humans (Darmoise et al., 2010; Pereira et al., 2013). Indeed, the absence of GLA resulted in a profile of NKT cell hyperstimulation that correlated with the presence of an increased amount of self-ligands at the surface of selecting thymocytes and peripheral antigen-presenting cells (Darmoise et al., 2010; Pereira et al., 2013). We confirmed that compared to DCs from littermate control animals, GLA-deficient DCs were hyperstimulatory toward DN32.D3 T cells and that this activity was inhibited by L363 antibody (data not shown). GLA has been described as being exclusively specific to terminal α-galactoses (Guce et al., 2010), and in vitro digestion of α-GalCer confirmed that the proximal sugar was indeed inaccessible when displayed by a ceramide. However, when we tested α-psychosine as a substrate, GLA was capable of removing the α-linked galactose and eliminating the stimulatory activity of this compound (Figures 5A and 5B). In vivo, so far, only two lysosomal enzymes have been shown to remove fatty acids from glycosylceramides: acid ceramidase (ASAH1; Park and Schuchman, 2006), the most studied but whose deficiency is embryonically lethal in mice (Eliyahu et al., 2007), and N-acylamidehydrolase (ASHL), a poorly characterized enzyme that is homologous to ASAH1 (Tsuboi et al., 2005). In the absence of gene-deficient animal models, we used enzyme inhibitors to assess the role of ceramidases in the control of NKT ligands. We used carmofur, a known commercial drug with high specificity for ASAH1 (Realini et al., 2013), to demonstrate that ASAH1 was upstream of GLA and necessary for its action; indeed, the inhibition of ASAH1 induced an important increase in stimulatory activity toward NKT cells, whereas the inhibition of ASHL with a specific inhibitor (Li et al., 2012) resulted in a very limited effect on the stimulatory activity of untreated or lipopolysaccharide (LPS)-treated DCs. Similarly, the inhibition of α-GLA in DCs with 1-deoxygalactonojirimycin reproduced in vitro the Fabry phenotype of increased stimulation, whereas 1-deoxygluconojirimycin, an inhibitor of acid α-glucosidase (GAA; Ishii et al., 2009; Khanna et al., 2012; Wennekes et al., 2009), did not influence stimulation (Figure 5C); however, in the context of stimulation by a cytokine, such as tumor necrosis factor (TNF), which induced endogenous NKT ligands in DCs, both inhibitors increased stimulation of NKT cells similarly (Figure S5). The accumulation of α-GalCer during GLA and ASAH1 inhibition was confirmed by indirect immunofluorescence using the purified rabbit anti-α-GalCer antibodies in RBL-CD1d cells treated with the same inhibitors (Figure 5D). The specificity of staining was confirmed by competing antibody binding with either free β-GalCer (Figures 5Da–5Dd), which showed no inhibition, or free α-GalCer (Figures 5De–5Dg), which completely blocked staining. α-GalCer was found in small vesicles, some of which colocalized with CD1d-containing vesicles (Figures 5Db–5Dd). Treatment of the RBL-CD1d cells for 24 hr with carmofur and 1-deoxygalactonojirimycin (Figures 5Dh–5Dk) increased the number and size of the vesicles but never reached levels comparable to what the addition of exogenous α-GalCer produced (Figure 5Dk).

Figure 5. Catabolic Enzymes Control the Availability of α-Glycosylceramides.

Figure 5

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(A) High-performance TLC was used to separate glycosylceramides (left panel) and lyso-glycosylceramides (right panel) before and after digestion with recombinant GLA. Lipids were visualized with a cerium ammonium molybdate stain. Abbreviations are as follows: Gal, GalCer; Glu, GluCer.

(B) Samples from α-GalCer (left) and α-psychosine (right) were tested for their stimulatory ability toward DN32.D3 NKT cells before (empty circles) and after (filled circles) digestion with recombinant GLA. DC3.2 cells (20,000 cells/well) were used as antigen-presenting cells. A representative experiment out of three independent experiments is shown.

(C) DC3.2 cells were differentiated with LPS, treated with inhibitors of α-glycosidases (GLAi and/or GAAi), 1-deoxygalactonojirimycin (0.5 μM), and 1-deoxy-gluconojirimycin (2.0 μM) or with ceramidase inhibitors ASAHLi (20 μM;Li et al., 2012) or ASAH1i (carmofur, 1.0 μM) for 24 hr, and used to stimulate DN32.D3 NKT cells. A control (empty circles) is included in each panel for comparison with inhibitors (filled circles).

(D) Confocal microscopy of α-GalCer in RBL-CD1d cells by indirect immunofluorescence. Flattened Z series are presented. Anti-α-GalCer antibody is in red, whereas anti-CD1d antibody is in green. Control stains include normal rabbit Ig, 14.4.4 antibody, and secondary antibodies (a and h). In (b), (c), and (d), cells were incubated in the presence of 10 μg/ml β-GalCer at the time of staining, whereas in (e), (f), and (g), cells were incubated in the presence of 10 μg/ml α-GalCer. An overlay of each group is presented in (d) and (g). In (h)–(k), cells were either untreated (i) or treated with 0.5 μM 1-deoxygalactonojirimycin and 1.0 μM carmofur (j) or 2.0 μg/ml α-GalCer (k) for 24 hr in 0.05% serum-free media containing BSA. All cells were permeabilized with 0.05% saponin. All scale bars represent 20 μm.

See also Figures S4–S6.

These results support a model in which the amount of endogenous α-glycosylceramides is directly controlled by catabolic enzymes in a two-step mechanism: removal of the acyl chain of α-ceramide and subsequent removal of the sugar by an α-glycosidase. All together, these experiments have revealed the existence of an important catabolic pathway that controls the availability of natural NKT ligands and utilizes a two-step enzymatic process to degrade α-glycosylceramides.

Ex Vivo and In Vivo Effect of CD1-α-GalCer Blockade

Using the same enzyme inhibitors, we examined the stimulatory activity of thymocytes toward DN32.D3 T cells. In contrast to the results of DCs, both glycosidase inhibitors modulated the amount of cell-surface NKT ligands only marginally, and only the inhibition of ASAH1 by carmofur had a substantial effect (Figure S6). The possibility that both α-GluCer and α-GalCer are produced in thymocytes is supported by an inhibition experiment with L363 antibody. RBL-CD1d cells and thymocytes were calibrated for stimulation toward DN32.D3 cells and compared side by side against increasing concentration of L363 antibody (Figure 6A). With increasing concentration of antibody, stimulatory activity of both cell types diminished, but the IC50 was ∼5 μg/ml for thymocytes and less than ∼0.325 μg/ml for RBL-CD1d cells. This difference could not be explained by antibody target density given that RBL-CD1d cells were more potent than thymocytes but could be explained by differential affinity of the antibody on the two cell types tested. Given the exclusive specificity of L363 for α-linked monoglycosylceramides, we could reasonably hypothesize that on thymocytes, in addition to α-GalCer, CD1d-α-GluCer complexes were likely to be present and would require higher concentration of antibody to be blocked, whereas RBL-CD1d cells expressed mainly the higher-affinity CD1-α-GalCer complexes. This information was critical for carrying out ex vivo experiments because we argued that the low affinity of L363 antibody for thymic ligands would most likely result in the absence of a detectable phenotype if concentrations of the antibody were not sufficient. Therefore, we tested L363 at high concentration on fetal thymic organ cultures and compared it to a negative control antibody (14.4.4 s) and a positive control anti-CD1 antibody (20H2). At day 18 of culture, day 14.5 thymi treated with L363 antibody did not contain detectable NKT cells, as measured with CD1d tetramers loaded with PBS-57 (Figure 6B). Then, we performed the same experiment in vivo by injecting 8-week-old female C57BL/6 mice with either 14.4.4 or L363 antibody over a 4-week period and examining thymic NKT numbers. Compared to control thymi, thymi in the L363-treated group were almost completely depleted of NKT cells (Figure 6C); it is likely that the few remaining NKT cells represented the long-lived thymic resident NKT cells that have been described (Berzins et al., 2006). Finally, we used the anti-α-GalCer polyclonal antibody to try to visualize α-GalCer-expressing cells in the thymi of wild-type (WT) and GLA-deficient mice. In normal thymi, few islands (five to ten cells) were labeled in the cortex and costained with anti-CD3 antibodies, whereas GLA-deficient thymi were intensely stained throughout the cortex and medulla, indicating a large increase in the quantities of α-GalCer (Figure 6D). Thus, we confirmed ex and in vivo that thymic selection of NKT cells could be inhibited by L363 antibody blocking of CD1d-α-monoglycosylceramide complexes. In addition, it is likely that both α-GalCer and α-GluCer are selecting ligands of NKT cells.

Figure 6. Thymocytes Present More Than One α-Glycosylceramide.

Figure 6

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(A) Titration of DN32.D3-cell-stimulation inhibition by L363 antibody with the use of thymocytes (left panel) or RBL-CD1 d cells as antigen-presenting cells. Seven two-fold dilutions were tested from a 20 μg/ml stock. In the lower panel, the percentage of inhibition was plotted as the percentage of maximal response (100%) for RBL-CD1d cells (black symbols) and thymocytes (empty circles).

(B) Day 14.5 thymic lobes were cultured for 18 days in the presence of 40–60 μg of antibody. 14.4.4 s was the negative control antibody, and 20H2 was the positive control antibody. In this particular experiment, percentages of CD1-PBS57-positive cells were 0.27%, 8.02%, 1.14%, and 0.27% for adult thymus, 14.4.4 s, L363, and 20H2, respectively. Similar results were obtained on four other thymic lobes. The experiment was repeated three times. FTOC, fetal thymic organ culture.

(C) Mice were treated with either 14.4.4 control or L363 antibody for 4 weeks and examined for thymic NKT cell numbers. The lowering of NKT cell numbers in the L363-treated group was significant (p = 0.0147).

(D) Staining of WT and Gla−/− thymic sections with purified anti-α-GalCer rabbit antibody (green) and anti-CD3 antibody (red). Control staining was with secondary antibodies only. Sections (a), (b), and (c) were magnified 20×. Section (d), from a WT thymus, was magnified 40×. Similar images were obtained in three independent experiments on WT and Gla−/− thymi. Scale bars represent 100 μm in (a)–(c) and 50 μm in (d).

See also Figure S6.

Discussion

The biochemical analysis of lipids has been limited by the lack of sensitivity of available analytical techniques. We have addressed these limitations by combining the exquisite sensitivity of T cells in biological assays with the specificity of immunoglobulins, the ability to immunopurify lipids with antibodies, the stereospecificity of catabolic enzymes, and new techniques such as MRM-MS to identify and characterize the natural endogenous ligands of NKT cells. This approach identified several types of α-glycosylceramides as the main endogenous ligands of NKT cells in the thymus and the periphery if we consider in a reductionist approach that DCs are the most relevant cell type for NKT functions in the periphery (Arora et al., 2014). The current discovery sheds light on a number of unexplained observations in the field of NKT biology. First, the likely presence of both α-GluCer and α-GalCer in the thymus explains why the removal of a single pathway, such as performed in the acid-galactosylceramidase-deficient animal, did not eliminate NKT cells (Bendelac et al., 2007; Stanic et al., 2003). Indeed, the reciprocal compensation of glucosylceramide versus galactosylceramide species has been well documented in various lysosomal-storage diseases (Coetzee et al., 1996; Jennemann et al., 2005; Tadano-Aritomi et al., 2000). Second, the presence of the two species of α-glycosylceramide in the thymus would explain the differential reactivity of thymic NKT cells to α-GluCer and α-GalCer CD1d tetramers and their preferential reactivity to Vβ7 and Vβ8 NKT cells, respectively (Wun et al., 2011). Third, our results explain the phenotype of the Fabry mouse that was recently reported (Darmoise et al., 2010) and support the idea that catabolism is an important factor controlling the amount of NKT agonists. In the Fabry mouse, even though the lysosome is dysfunctional, NKT cells are hyperstimulated in the periphery and exhibit signs of permanent activation. The idea that degradation is an important factor controlling the amount of NKT cell ligands is also supported by proteomics studies of the secretome of monocytes; these demonstrated that all the essential enzymes needed to degrade α-ceramides (GLA, GAA, and ASAH1) are rapidly produced upon activation and allow a fast control of NKT cell activation in the context of inflammation and infection (Meissner et al., 2013). It is also important to note that a two-step degradation process is required for the complete removal of stimulatory activity given that α-glycosidase, as shown for GLA, only cleaves the sugar from lysoceramides. In this respect, we will have to evaluate the respective roles that ASAH1 and ASHL play in the process of producing α-psychosines, a task that will be made difficult by the fact that the ASAH1 deficiency is embryonically lethal in mice (Eliyahu et al., 2007). The fourth conclusion of our study is that the dual nature of the endogenous ligands of NKT cells, as mentioned for thymic NKT cells, most likely is the basis for the tissue specificity of NKT cell subpopulations (Park et al., 1998). It is reasonable to hypothesize that the balance between glucosyl and galactosyl species, as seen between thymocytes and DCs, is a general mechanism that favors the local expansion of NKT cells tuned for the recognition of one or the other ligand. The possibility that both α-GluCer and α-galactosylceramides could be modified at positions 3 and 6 by either a sulfate or an acetyl group will also have to be explored, given that we know that the synthetic forms of these modified α-glycosylceramides are powerful agonists of NKT cells in vitro. The possibility that α-lyso-glycosylceramide and α-glycosylceramide recognition is finely tuned by the nature of the Vβ chain should also be examined. Finally, follow-up studies will have to address the mechanisms leading to the production of α-anomers of glycolipids. It could be as simple as the noninversion of the sugar by GCS or CGT during synthesis or the synthetic function of ASAH1 (Okino et al., 2003) and glycosidases (Yamashita et al., 2005) after alteration of substrate availability. More complex situations requiring specific anomerases will also have to be considered, given that none is exclusive.

In conclusion, the immune system and immune probes have allowed us to reveal the existence of a metabolic and degradative pathway that produces α-linked glycolipids. This pathway is most likely critical for the generation and control of NKT cell activation in the thymus and the periphery. Given the close identity of the murine and human NKT cell systems, it is most likely that the same α-glycolipids will be identified in man. The understanding of the transcriptional control of this pathway should allow new approaches in the utilization of NKT cells in immunotherapy. Finally, potential nonimmunological functions of these new glycolipids will have to be examined.

Experimental Procedures

Cells, Cell Lines, and DC Maturation

DN32.D3 and TBA7 cells have been described extensively in other publications and are commonly used as representatives of type 1 semi-invariant Vα14 NKT cells and type 2 non-Vα14 NKT cells, respectively. DC3.2 cells are a DC line expressing CD1d and are susceptible to differentiation induced by TLR ligands and cytokines, such as LPS and TNF (Shen et al., 1997).

Polyclonal primary NKT cell lines were established by fluorescence-activated cell sorting of fresh NKT cells from thymi with CD1d tetramers for staining. After sorting, cells were expanded in vitro by stimulation with anti-TCRβ and -CD28 antibodies for 2 days and the subsequent addition of IL-7 for 5 days. Bone-marrow-derived DCs were cultured in the presence of GM-CSF for 7 days.

Mice were purchased from the Jackson Laboratory and kept in the care of the Scripps Research Institute Immunology Vivarium according to the Institutional Animal Care and Use Committee guidelines.

Antibodies and Antibody Production and Purification

L363 (IgG2a) and L317 (IgG1) antibodies were a generous gift from Steve Porcelli. In most experiments, the anti-MHC class II antibodies MKD6 (anti-I-Ad, IgG2a) and 14.4.4 s (anti-I-Ek, IgG2a) were used as controls. Rabbit polyclonal anti-α-Galcer antibodies were produced over a 6-month period by repeated subcutaneous immunizations of New Zealand rabbits with 20 μg α-GalCer in incomplete Freund's adjuvant. See also Supplemental Experimental Procedures.

Radiolabeling of RBL Cells

RBL-CD1d cells at 50% confluency were labeled with 10 μM C14-UDP galactose per flask. After washing with PBS, protein extraction was carried out for L363 immunoprecipitation, whereas lipids were extracted for immunoprecipitation with anti-α-GalCer antibodies. See also Supplemental Experimental Procedures.

T Cell Activation Assay

Antigen-presentation assays were carried out with 5 × 103 to 20 × 103 DC3.2 cells or 1 × 105 splenocytes and 4 × 104 T cells per well in 96-well tissue-culture plates in triplicates. Cell-culture supernatants were collected 24 hr later for determination of IL-2 concentrations with an IL-2-dependent NK cell line reporter system.

Flow Cytometry

NKT cells were quantified with CD1d tetramers empty or loaded with PBS-57. Samples were acquired on a MACSQuant analyzer with MACSQuantify software (both Miltenyi Biotec) and analyzed with FlowJo software (Tree Star). See also Supplemental Experimental Procedures.

Fetal Thymic Organ Culture

Embryonic day 14.5 fetal thymic lobes were harvested from timed pregnant C57BL/6J mice and cultured on nitrocellulose filters (Whatman) placed on a sponge (Gelfoam size 4; Upjohn Pharmacia). Lobes were cultured for 18 days in Dulbecco's modified Eagle's medium before being analyzed. See also Supplemental Experimental Procedures.

In Vivo Treatment with Antibodies

Eight-week-old C57BL/6 mice were injected intraperiteonally every 5 days with 1 mg of either 14.4.4 or L363 antibody for a period of 4 weeks. Thymi were harvested and processed individually at week 5.

TLC and TLC Blot

TLC analysis was carried out with aluminum back plates (EMD Bioscience). Running solutions and visualization are detailed in the Supplemental Experimental Procedures.

Liquid Chromatography and MS

An Agilent 1200 UPLC system was coupled to a 6490 Triple Quadrupole mass spectrometer for use in C24:1 monoglycosylceramide determination using MRM for enhanced sensitivity and selectivity. See also Supplemental Experimental Procedures.

Histology and Indirect Immunofluorescence

Tissues were embedded in optimum cutting temperature compound and flash frozen (Tissue-Tek) before being sectioned and processed. For immunofluorescence on cells, cells were cultured for 24 hr in serum-free media containing 0.05% BSA on coated coverslips. Visualization was by confocal microscopy. See also Supplemental Experimental Procedures.

SPR

A Biacore T200 instrument (GE Healthcare) was used for SPR measurements. Measurements were performed according to single-cycle protocols for avoiding the repeated use of regeneration buffer on the immobilized ligands. See also Supplemental Experimental Procedures.

Supplementary Material

Supplement

Acknowledgments

The making of L317 and L363 by Steve Porcelli (Albert Einstein College of Medicine, New York) and his generosity in making these reagents available to us were critical for the present work. Ken Rock (University of Massachusetts– Worcester) provided us with the CD1d-positive dendritic cell line DC3.2. Deborah Wittherton (the Scripps Research Institute) helped us establish fetal thymus organ culture. Ajit Varki (University California, San Diego) gave us critical advice for the radioactive labeling of glycolipids. This work was supported by NIH grants AI053725 (to P.B.S., A.B., and L.T.), AI102892 (to L.T.), and AI71922 (to M.K.). This is manuscript 23088 from the Scripps Research Institute.

Footnotes

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