International Journal of Carbohydrate ChemistryVolume 2011 (2011), Article ID 749591, 13 pagesdoi:10.1155/2011/749591

Review Article

Endogenous and Exogenous CD1-Binding Glycolipids

Janice M. H. Cheng,1,2 Ashna A. Khan,1,2 Mattie S. M. Timmer,¹ and Bridget L. Stocker²

¹School of Chemical and Physical Sciences, Victoria University of Wellington, P.O. Box 600, Wellington 6012, New Zealand²Malaghan Institute of Medical Research, P.O. Box 7060, Wellington 6242, New Zealand

Received 15 August 2010; Revised 24 December 2010; Accepted 9 February 2011

Academic Editor: Yuriy A. Knirel

Copyright © 2011 Janice M. H. Cheng et al. Janice M. H. Cheng et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

In the same way that peptide antigens are presented by major histocompatibility complex (MHC) molecules, glycolipid antigens can also activate the immune response via binding to CD1 proteins on antigen-presenting cells (APCs) and stimulate CD1-restricted T cells. In humans, there are five members of the CD1 family, termed CD1a–e, of which CD1a–d are involved in glycolipid presentation at the cell surface, while CD1e is involved in the intracellular trafficking of glycolipid antigens. Both endogenous (self-derived) and exogenous (non-self-derived) glycolipids have been shown to bind to members of the CD1 family with varying degrees of specificity. In this paper we focus on the key glycolipids that bind to the different members of the CD1 family.

1. Introduction

Carbohydrates play an important role in various molecular recognition events. They are found on the surfaces of cells, bacteria, and viruses as glycoconjugates (such as glycoproteins and glycolipids), and it is therefore not surprising that their interaction with cells of the immune system forms an integral part of the body’s immune response. The innate immune system operates largely via receptor-ligand interactions, and glycoconjugates are capable of inducing the strongest immune responses known. Classic examples include the landmark discovery of endotoxin by Pfeiffer in the late 19th century [1] and the discovery in 1928 by Landsteiner and Levine that blood group determinants were responsible for the blood cell rejection (also known as transfusion reaction) observed during blood transfusion with incompatible donors [2]. Only later was it revealed that endotoxin is in fact a glycolipid [3] and that the A, B, and O antigens responsible for transfusion reaction are glycolipids and glycoproteins found on the surface of red blood cells [4].

Recently, cell-mediated immunity to glycolipid antigens has been discovered, and a glycolipid presentation pathway which parallels that of peptide antigen presentation has emerged. The glycolipid pathway involves CD1 proteins, major histocompatibility complex- (MHC-) like molecules that are capable of presenting glycolipids to a specialised subset of CD1-restricted T cells. Like MHC molecules, CD1 proteins are present on antigen-presenting cells (APCs), specialised immune cells that perform a crucial role in the immune system. After encountering an antigen, the APC sends messages to other immune cells, subsequently activating the full immune response. This paper will focus on CD1-binding glycolipids that are capable of activating and modulating the immune system via the activation of CD1-restricted T cells.

2. CD1 Molecules

The CD1 protein is a transmembrane glycoprotein consisting of a heavy chain with three extracellular domains (α1, α2, and α3) noncovalently linked to a β ₂-microglobulin (β ₂m) chain (Figure 1) [5]. The nonpolar amino acids in the α1 and α2 chains form the hydrophobic pockets that can accommodate the long lipid tails of a glycolipid. The CD1 family contains five members, CD1a, CD1b, CD1c, CD1d, and CD1e, which, depending on their sequence homology, can be further classified into three groups: group 1 (CD1a, CD1b, and CD1c), group 2 (CD1d), and group 3 (CD1e). In a CD1-glycolipid complex (Figure 1), the polar carbohydrate group of the glycolipid protrudes from the CD1 binding groove, allowing it to be recognised by CD1-restricted T cells via the T cell receptor (TCR). The trimolecular interaction between CD1, glycolipid, and TCR initiates a cascade of intracellular signalling that activates the T cells to produce signalling molecules called cytokines. The type of immune response generated is dependent on the cytokine profile produced.

Figure 1: Representation of CD1-glycolipid-TCR interaction during glycolipid antigen presentation.

The exact effect that the glycolipid molecular structure has on the immune response is only known in a very general sense. Unlike MHCs, which specifically bind peptide antigens, CD1 molecules accommodate structurally diverse glycolipids. Extensive crystal structure analysis of CD1 molecules with bound ligands suggests that CD1 binding is less specific, which supports the observation that the length of a lipid tail does not greatly affect the immunomodulatory profile of a given glycolipid [6]. Instead, the highly specific interaction of the TCR with the CD1-glycolipid complex plays the principal role in dictating the outcome of the immune response [7, 8]. Other processes such as CD1 and glycolipid trafficking into the cell and glycolipid processing prior to TCR recognition have also been found to have a profound effect on the immune response [9–11]. The ability of glycolipids to modulate the immune response prompted researchers to develop specific CD1-binding glycolipids that can be used in the investigation and treatment of many diseases, including cancer, bacterial infections, and autoimmune diseases such as multiple sclerosis and systemic lupus.

3. Endogenous and Exogenous Glycolipids

Production of antigen-specific T cells requires positive selection in the thymus. For the glycolipid reactive CD1-restricted T cells, this means that endogenous glycolipids, originating from within the host organism, must be presented by CD1 proteins and interact with the TCRs of developing CD1-restricted T cells. Isoglobotrihexosylceramide (iGb3) is the most studied endogenous glycolipid and has been suggested to be the pivotal ligand for the selection of CD1d-restricted T cells in the thymus [12], though further investigations are required to support this hypothesis [13]. Other endogenous glycolipids such as gangliosides and sulfatide, as well as phosphoglycerolipids and sphingomyelin, have also been found to stimulate group 1 CD1-restricted T cells. The exact role that endogenous lipids have in the activation of CD1-restricted T cells, however, remains unclear.

Of the exogenous glycolipids, numerous bacterial cell wall glycolipids have been shown to play an important role in the immune response. In particular, the discovery that mycolic acid, a nonpeptide lipid antigen from Mycobacterium tuberculosis (M. Tb), stimulates T cells via CD1 molecules paved the way towards the discovery of a host of glycolipids that activate the immune system in a similar manner [14]. Subsequently, other glycolipid cell wall components of M. Tb, such as glucose monomycolate (GMM) [14], phosphatidylinositolmannoside (PIM) [15], and lipoarabinomannan [16], have also been found to be immunogenic [17]. However, not all bacterial glycolipids are responsible for the activation of the immune system during infection. Interestingly, Mattner et al. discovered that the antigen responsible for activating CD1-restricted T cells in mice during an infection with Gram-negative, LPS-positive Salmonella typhimurium, was in fact the endogenous glycolipid, iGb3 [18].

When comparing endogenous and exogenous CD1-binding glycolipids, perhaps the most obvious structural difference is the linkage variation between the carbohydrate and the lipid backbone. Endogenous glycolipids such as iGb3 have β-linkages, while exogenous/pathogenic glycolipids (i.e., most bacterial glycolipids) typically have α-linkages. In this paper, endogenous and exogenous CD1-binding glycolipids will be described in the context of the CD1 isotype with which they interact. The origin/discovery, synthesis, and effect on the immune response of each glycolipid will also be discussed.

3.1. CD1a

The human CD1a molecule possesses two hydrophobic pockets (A′ and F′) that can bind aliphatic carbon chains of glycolipids via nonpolar Van der Waals interactions. The A′ pocket of CD1a is narrow with only one entry point and hence has been proposed to be a “molecular ruler” that allows CD1a to bind C₂₀ lipids or shorter [19]. The F′ pocket of CD1a is also unique in that it lies close to the plane of TCR recognition and hence directly participates in antigen presentation [6].

3.1.1. Endogenous Glycolipids

Sulfatide, illustrated by 1 (Scheme 1), is a β-D-galactosylceramide sulfated at C-3 of D-galactose. Ceramides are long chain dihydroxy or trihydroxy bases which are linked to a fatty acid via an amide linkage at C-2. The sphingosine base, a dihydroxylated base with mono-unsaturation across C-4 and C-5, is commonly found in endogenous glycolipids and forms the lipid backbone of sulfatides. Sulfatide is a potent glycolipid produced by mammalian cells and is found mainly in the myelin sheath but is also present in small amounts in other tissues [20]. The ability of sulfatide to bind to all group 1 and group 2 CD1 proteins (CD1a–d) gives it the just reputation of being the most promiscuous CD1-binding glycolipid [20, 21]. Nonetheless, its binding affinity differs with each CD1 isotype, with the CD1a-sulfatide complex seeming to be the most stable [20]. Here the narrow A′ pocket allows CD1a to selectively bind to the C₁₈ sphingosine backbone while the acyl lipid protrudes along the surface of the CD1a molecule and extends into the F′ pocket. The 3-O-sulfogalactose protrudes from the CD1 pocket and interacts with the TCR of the T cell.

Figure 2: Gangliosides GM1 and GQ1b.

GM1 (6, Figure 2) is the most abundant ganglioside found in the human myelin sheath and consists of a ceramide with a core tetrasaccharide to which a single sialic acid residue is attached. GM1 binds to CD1b on the cell surface at neutral pH and is recognised without internalisation and processing [27]. These CD1b-GM1 complexes can stimulate T cells to induce a T-helper 1- (Th1-) type response measured by the release of cytokines such as TNF-α and IFN-γ, which are important cytokines for the generation of a cytotoxic immune response [27]. Ganglioside-specific T cells can discriminate small differences in the carbohydrate portion of the glycolipid as revealed by a study whereby analogues of GM1 that lack the terminal galactose residue (GM2), sialic acid residue (asialo-GM1), or both were unable to stimulate production of IFN-γ by T cells [27]. This suggests that the GM1 pentasaccharide represents the minimum epitope for recognition by TCRs. The structure of the lipid tail and the fatty acid is also important for the specificity of CD1b binding, with stearic acid having the highest T cell stimulatory capacity [27]. GM1 binding to CD1b is highly reversible, and other ceramide-containing glycosphingolipids can displace GM1 in the CD1b complex. The first total synthesis of GM1 occurred in 1986 by Sugimoto et al. and took an impressive twelve linear steps [28]. From the retrosynthesis (Scheme 2), it can be seen that the main steps involved the coupling of the peracetylated pentasaccharide imidate 8 to sphingosine acceptor 9, which was accomplished in 33% yield using BF₃·Et₂O as the activator. Donor 8 in turn was accessible via coupling of galactosyl galactosamine 10 and sialylated lactoside 11.

Figure 3: Building blocks for the synthesis of GQ1b.

3.2.2. Exogenous Glycolipids

Glucose-monomycolate (GMM, 14, Figure 4) is the 6-O-mycolate ester of D-glucose and is found in bacterial species including Mycobacteria, Rhodococcus, and Nocardia. Mycolic acids themselves are α-alkyl, β-hydroxy fatty acids that are present in most mycobacterium species and also in related taxa. Mycolic acids from different species of bacteria may vary to give rise to alpha-, keto-, methoxy-, or epoxy-mycolic acids. The GMM (14) illustrated contains the alpha-mycolic acid backbone. It should also be noted that mycolic acids were the first lipid antigens revealed to activate CD1-restricted T cells via binding to CD1b molecules [14].

Figure 4: Representative glucose monomycolate (GMM).

GMM is a potent ligand for CD1-restricted T cells when presented by CD1 molecules [7]. For example, studies have shown that the CD1b-restricted T-cell recognition of GMM is highly specific for the glucose moiety since naturally occurring mycolate esters of glycerol and arabinose are not recognised by CD1-restricted T cells. In addition, the R,R configuration of the hydroxy acid and the position of the linkage of the mycolate to the glucose unit are also important for CD1 binding [7]. Structural and mass-spectroscopy analysis has shown that high-affinity binding also depends on the exact structure of the acyl side chains of the antigen [38]. Crystal structure studies of the CD1b-GMM complex indicate that both acyl chains are buried in the antigen binding groove of CD1b, thus leaving the glucose unit exposed to the surface for recognition by TCRs. This explains the observation that one TCR can recognise GMMs with varying chain lengths derived from different bacterial sources [39].

Diacylated trehalose sulphates (Acyl₂SGL, 15, Figure 5) are lipid antigens produced by virulent M. Tb and consist of a trehalose core that is acylated at the 2-position by palmitic (or stearic) acid and acylated at the 3-position by hydroxyphthioceranoic acid [40]. While the methyl branches were assigned to be of the L-series, the stereochemistry of the hydroxy-substituted carbon has not been assigned [41].

Figure 5: Diacylated trehalose sulphate (Acyl₂SGL).

Sulfoglycolipids bind to human CD1b, stimulate CD1b-restricted T cells to release the cytotoxic cytokine IFN-γ, and recognise M. Tb-infected cells in a host, leading to killing of intracellular bacteria [40]. The influence that the lipid chains have on T cell activation in relation to the length, position, and structure of the fatty acid residues has been studied, and it was observed that the presence and stereochemistry of a multimethyl-branched fatty acid appendage on the 3-position in Acyl₂SGL were crucial for T cell stimulation [42]. Naturally occurring Acyl₂SGL, illustrated in 15, was seen to be most potent in T-cell activation as revealed in a study by Guiard and coworkers, who synthesised a range of sulfoglycolipid analogues in which the hydroxyphthioceranoic acid has been replaced by less complex acids (Scheme 3) [42]. The synthesis started with the selective acylation of the readily available 4,6:4′,6′-di-O-benzylidene protected α,α′-trehalose 16, which gave the 2-O-acyl product 17 without the need for stannylene chemistry or elaborate protecting group manipulations. Subsequent protection of the diol in 17 with dichloro-tetraisopropyldisiloxane gave alcohol 18 which was then acylated at the 3-position with various long chain branched fatty acids. Finally, the siloxane protection in 19 was removed using TBAF and the resulting diol 20 selectively sulphated at the 2′-position to gain sulphate 21. Final deprotection using catalytic acid provided the SGL analogues (e.g., 22). Although none of the synthetic compounds were as potent as the natural sulfoglycolipid 15, some of the sulfoglycolipid analogues showed promising T-lymphocyte-activation properties [42]. It was later shown that the presence of T cells that can recognise Acyl₂SGL, in patients with active or latent tuberculosis, demonstrates the potential of sulfoglycolipids as subunit vaccine against tuberculosis [43].

Figure 6: Mannosyl-β-1-phosphodolichol (MPD).

MPD consists of a mannoside β-linked to a phosphopolyprenoid lipid via a phosphate group. Except for the proximal isoprene, the lipid contains unsaturated isoprene units linked in trans and cis configurations. The ability of CD1c, but not other CD1 proteins, to accommodate methyl branching within a single lipid indicates the specificity of CD1c for this branched motif [45].

Through the testing of various isolated and semisynthetic MPD analogues, a correlation between dolichol chain length and T-cell response has been observed. MPDs with a chain length similar to those found in Mycobacteria (C30–35) gave the strongest T-cell responses, while no activation was observed for lipids with longer chains (C55–95) [45]. This is probably because long lipid chains resemble the endogenously expressed MPD, which should not generate any immune response in the absence of infection and illustrates the capability of CD1c to distinguish between self-like and foreign-like glycolipids.

3.3.2. Exogenous Glycolipids

Mannosyl-β-1-phosphomy-coketides (MPMs, 24, Figure 7) are potent mycobacterial antigens recently isolated from Mycobacterium avium (24a) and Mycobacterium tuberculosis (24b) [44]. These phospholipids consist of a mannosyl-β-phosphate moiety similar to that in the endogenous MPD (23) but possess an unusual saturated isoprenoid backbone. The antigenicity of the MPM is largely attributed to the saturation of the lipid chain and the shorter length of the backbone (C₃₀) as compared to their endogenous dolichol counterparts [48]. Like MPDs, the methyl branches (S-configuration) of this mannosyl-β-1-phosphomycoketide help retain the lipid in the F′ pocket, thus allowing for T-cell recognition and activation [49]. The synthesis of MPM 24a was first reported by Crich and Dudkin in 2002 [50], and, more recently, MPM 24b and analogues thereof were synthesised by Van Summeren and coworkers [51]. Crich and Dudkin (Scheme 4(a)) used a strategy whereby triflate donor 25 was coupled to the isoprenoid phosphate 26 to give mannosyl phosphate 27 which could be deprotected in a single Birch reduction to provide MPM 24a. Van Summeren and coworkers, however, phosphorylated tetraacetylmannose 28 to give phosphate triester 29 (Scheme 4(b)). After deprotection of 29, phosphate 30 was coupled to isoprenoid alcohol 31 to give phosphodiester 32. Final deacetylation then gave MPM 24b. These syntheses not only confirmed the structural identification of these naturally occurring MPMs lipids but also assist in the development of a subunit vaccine against tuberculosis [52].

Figure 7: Mannosyl-β-1-phosphomycoketide (MPM).

3.4. CD1d

CD1d is perhaps the most widely studied CD1 protein due to its structural homology across mice and humans [6] and its importance in cancer immunotherapy. The heavy α chains form the hydrophobic binding pockets A′ and F′ that are able to accommodate lipid tails of glycolipids, as illustrated by the crystal structure of CD1d bound to α-galactosyl ceramide [53]. An extensive hydrogen bonding network holds the sugar head group in place for recognition by the T cell receptor.

3.4.1. Endogenous Glycolipids

Isoglobotrihexosylceramide (iGb3, 33, Figure 8) is a triglycosylceramide with a lactose residue β-linked to a ceramide with a sphingosine backbone and a terminal galactose residue, bound via an α(1–3) linkage to the nonreducing end of the lactosylceramide. iGb3 can be isolated from cat intestines but has also been chemically [54] and enzymatically [10] synthesised. Interestingly, iGb3 has been shown to play an important role in the clearing of bacterial infections [12]. This was illustrated in a study whereby mice, lacking in an enzyme that converts the biological precursor iGb4 to iGb3, were found to be deficient in CD1d-restricted T cells and subsequently incapable of clearing infection by Salmonella typhimurium. Also of note is that the globoside Gb3, which contains an α(1–4)-linked terminal galactose instead of the α(1–3) linkage in iGb3, does not stimulate CD1-restricted T cells [55]. This indicates that there is a high level of specificity for the interaction between CD1d-glycolipid complexes with the TCRs of CD1-restricted T cells.

Figure 8: iGb3 and Gb3.

Qiu and Schmidt provided convenient routes for the synthesis of globosides (Scheme 5) [54]. Here, glycosylation of lactose acceptor 34 with imidate 35 provided the isogloboside trisaccharide 36, while regioselective glycosylation of lactose acceptor 37 with the same donor provided the isomeric trisaccharide 38. The total syntheses of iGb3 and Gb3 were finalised by transforming 36 and 38 into their respective imidates, coupling to a sphingosine acceptor (cf. 2, Scheme 1) and deprotection/acylation.

Figure 9: Phosphatidylinositolmannosides (PIMs).

PIMs stimulate CD1-restricted T cells via CD1d binding and the subsequent triggering of antigen-specific IFN-γ and cell-mediated cytotoxicity [15]. The structural requirements for PIMs to bind to CD1d are the possession of two fatty acyl chains and a polar head group [58]. The increased complexity of the carbohydrate moiety does not affect CD1d binding with mycobacterial PI behaving in the same way as PIM₂ and PIM₆ [58]. Interestingly, the CD1e isoform has been shown to activate lysosomal mannosidases, which break down larger PIM structures, such as PIM₆, into PIM₂ that is then presented by CD1d [59]. PIM ether analogues are also effective in enhancing IL-12 production by immature bovine dendritic cells and thus have the potential to be used as an adjuvant [60]. The first total synthesis of the more complex PIM₆ was reported in 2006 by Liu and coworkers [61]. Pseudoheptasaccharide 40 (Scheme 6) was first acylated and ester 41 subsequently allyl-deprotected to give alcohol 42. Alcohol 42 was then phosphorylated, oxidised, and deprotected to obtain the triacyl PIM₆.

Figure 10: Agelasphin-9b (AGL-9b) and α-galactosyl ceramide (αGalCer).

αGalCer has been reported to have potential in the treatment of several diseases including cancer, malaria, type 1 diabetes, and multiple sclerosis [64]. The mechanism by which αGalCer exerts its therapeutic effect only became known with the discovery that it binds to CD1d and activates iNKT cells [65]. αGalCer is the first agonist found to activate the CD1d-restricted iNKT cells that express an invariant TCRα chain (Vα24Jα18 in humans and Vα14Jα18 in mice). The first chemical synthesis of αGalCer was reported by Morita et al. [63] in 1995 and subsequently optimised by others in representative syntheses such the use of thioglycoside donor using NIS/TfOH as the promoter system [66] and trichloroacetimidate galactosyl donor using boron trifluoride etherate as the activating agent [67] in the key glycosylation step. Much effort is currently being made to develop derivatives of αGalCer [68] with improved antitumour activity, particularly within the context of cancer immunotherapy.

3.5. CD1e

Unlike CD1a–d, which are expressed on the cell surface of antigen presenting cells, CD1e is primarily found in intracellular compartments (i.e., Golgi apparatus, endosome, and lysosome) and is believed to be crucial for glycolipid processing and loading onto CD1 molecules [59, 69]. In this context, CD1e has been shown to process PIM₆ to the smaller PIM₂ motif for presentation by CD1d [59].

4. Concluding Remarks

Like their protein counterparts, glycolipids can regulate the immune system in a very specific manner via binding to the CD1 family of proteins and subsequent activation of CD1-restricted T cells. Glycolipids that bind to CD1 include both endogenous glycolipids, found within the host, and exogenous glycolipids from foreign materials or pathogens (Table 1). The five members of the CD1 family (CD1a–e) show binding specificities to individual or families of glycolipids, and as such, the resulting immune response to each CD1-glycolipid complex can be used for therapies ranging from cancer vaccine adjuvants to the treatment of autoimmune diseases. As more complex biochemical tools are developed, it is almost certain that the role of CD1-binding glycolipids in the immune system becomes better understood and opens the gateway to more specific therapies and enhanced preventative vaccination protocols.

Table 1: Ligands of CD1 molecules.

CD1 isoform
Endogenous ligand
References
Exogenous ligand
References

CD1a
Sulfatide
[20, 22, 23]
—
—

CD1b
GM1
[27–29]
GMM
[14, 38, 39]

GQ1b
[31, 35, 37]
Acyl₂SGL
[40–43]

CD1c
MPD
[45–47]
MPM
[48–52]

CD1d
iGb3
[10, 54]
PIMs
[15, 56–61]

Sulfatide
[20–23]
α-GalCer
[62–68]

CD1e
Involved in glycolipid antigen presentation
[59, 69]

Acknowledgments

The authors would like to thank the Cancer Society of New Zealand (B. L. Stocker) and the Health Research Council of New Zealand (M. S. M. Timmer) for financial support.

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