Syntheses of Gb3 and iGb3 have been reported,17,18
however, αGb3, and αiGb3 have not been reported and required a modified protecting group strategy, as compared to the syntheses of Gb3 and iGb3, to allow formation of the α-carbohydrate-ceramide bond. A key aspect in the syntheses of these glycolipids was generation of appropriately protected lactose derivatives. We found that 419
() was appropriate for our syntheses of αGb3 and αiGb3.
Synthesis of αiGb3 (yields in parentheses).
Synthesis of αiGb3 () began with coupling of 4 and acetobromogalactose (5) followed by protection of the remaining alcohol generating 6. Liberation of the C1 hydroxyl group gave 7 and coupling with 8 gave a mixture of anomers (predominately α). The anomers were difficult to separate at this stage, so the ester groups were removed and the anomers were separated effectively. Reductive deprotection gave αiGb3.
A closely related process was used in the synthesis of αGb3 (). Selective protection of the hydroxyl group at C3’ in 4 gave 10. Coupling with 5 provided the protected Gb3 trisaccharide (11). The anomeric position of the reducing sugar was revealed and the trisaccharide was coupled with 8 to give 12. Sequential deprotection yielded αGb3.
Measurement of the abilities of glycolipids to stimulate NKT cells requires a source of CD1d, typically expressed by antigen-presenting cells, and NKT cells. Dose-response experiments with these glycolipids were performed with dendritic cells as antigen-presenting cells in an assay measuring IL-2 release by a mouse DN32.D3 NKT cell hydridoma.20
As expected, results indicated that cytokine release (IL-2) is dependent on iGb3 concentration (). Notably, Gb3 did not stimulate, confirming that the CD1d-NKT cell recognition of glycolipids is specific for the substitution pattern offered by iGb3. In contrast, αiGb3 and αGb3 stimulated cytokine production with this NKT cell hybridoma. To confirm that this observation was not unique to mouse NKT cells, we determined stimulatory activity of αiGb3 with a human NKT cells line and found it to be a very potent stimulator (data not shown). However, from these results it was not clear if αiGb3 and αGb3 directly stimulated NKT cells or if they required truncation to α-glucosylceramide to become stimulatory.
Figure 3 Dose response (IL-2 production) of NKT cells (mouse hybridoma DN32.D3) to glycolipids: ○ iGb3, ● αiGb3, Gb3, ▲ αGb3.
Previous work demonstrated that α-galactosylceramides substituted at the C4 position on the carbohydrate with a second sugar required processing for stimulation of NKT cells.10,13
Both αiGb3 and αGb3 fit this pattern of substitution; therefore, it was expected that they would require truncation. Use of antigen-presenting cells that express tail-deleted CD1d (TD-CD1d) allows observation of influences of lysosomal processing and the influence of lipid transfer proteins on NKT cell stimulation. TD-CD1d does not cycle to lysosomal compartments and is therefore not loaded with processed glycolipids.21
Attempts to stimulate NKT cells using TD-CD1d and iGb3, αiGb3, and αGb3 resulted in minimal cytokine release (data not shown) indicating that protein-assisted loading and/or glycolipid processing was required for stimulation of NKT cells.
To determine the requirements for lipid transfer protein-assisted loading of CD1d, we used a gel-shift assay in which CD1d was first loaded with a charged glycolipid (trisialoganglioside GT1b).22
The complex was treated with αiGb3 or αGb3 in the presence or absence of saposin B, a lipid transfer protein.23
Displacement of GT1b from CD1d with either of these neutral glycolipids resulted in a decrease in electrophoretic mobility (). Without saposin B, no displacement of GT1b was observed, but efficient loading, as indicated by the shift in gel mobility, was observed with saposin B. This result indicated that without processing (i.e., loss of carbohydrate groups), these glycolipids were bound by CD1d and that a lipid transfer protein was required for loading.
Gel-shift assays of GT1b displacement by αiGb3 and αGb3. CD1d-GT1b complex- 2 µM, glycolipids-26 µM, saposin B- 2 µM.
The remaining issue was whether these glycolipids, loaded into CD1d, were capable of stimulating NKT cells without truncation of the oligosaccharide. To address this issue and to avoid any participation by glycosidases, we used CD1d immobilized in plastic plates rather than antigen presenting cells in observing NKT cell stimulation.22
As with the CD1d loading experiments, NKT cell stimulation was performed in the presence and absence of saposin B. Stimulation was observed as a function of IL-2 release from NKT cells (mouse DN32.D3 hybridomas), which was in turn measured by proliferation (incorporation of radiolabeled thymidine) of an IL-2 responsive cytotoxic T cell line (CTLL). As expected, in the absence of saposin B, no stimulation was observed (). However, in the presence of the lipid transfer protein, NKT cell stimulation was observed with both αiGb3 and αGb3. IL-2 production stimulated by these glycolipids was compared to that from an α-galactosylceramide closely related to 1
(). The α-galactosylceramide used contained ceramide derived from a C18
phytosphingosine chain and a nervonic acid acyl chain. We have shown that α-galactosylceramides based on this ceramide are highly stimulatory antigens of NKT cells.24
IL-2 release in the culture supernatant determined via tritiated thymidine incorporation by the IL-2 indicator cell line CTLL-2:.■: absence of saposin B. □: presence of saposin B.
Tetramer staining of NKT cells by glycolipid-loaded CD1d tetramers is a method commonly used to study association of glycolipids with T cell receptors on NKT cells.25,26
NKT cell receptors are comprised of Vα14 and Vα24 chains in mice and humans, respectively, and a limited repertoire of Vβ chains.1
It has been observed that iGb3 recognition is strongest with NKT cell receptors with Vβ7 chains,27
while α-galactosylceramides, such as PBS57,24
are recognized by NKT cell receptors with variety of Vβ chains (i.e., Vβ8.2, 8.1, 7, and 2). Staining of NKT cells with CD1d tetramers loaded with αiGb3 and αGb3 () indicated that only NKT cells with receptors with Vβ8.2 chains were significantly stained by tetramers loaded with these glycolipids, demonstrating the specific recognition of these trisaccharides.
NKT cell hybridomas (DN32.D3) expressing different Vβ chains in their T cell receptors were stained by CD1d tetramers loaded with the indicated glycolipids. The control used was alpha-galactosylcholesterol.