The following purified glycolipids were purchased from Fluka: sulfatide, galactosylceramide (GalCer), glucosylceramide (GlcCer), and sphingomyelin. Sulfatide, GM1, and lactosylceramide (LacCer) were purchased from Matreya. Semisynthetic sulfatide containing stearic acid or ¹⁴C-stearic acid as acyl moiety was purchased from Anawa. Purified ganglioside GM4 was kindly provided by S. Sonnino (University of Milano, Milano, Italy). All the glycolipids were 98–99% pure according to TLC analysis.

The sulfate group of sulfatide was hydrolyzed essentially as described previously (18). In brief, 1 mg sulfatide was incubated in 1 ml of methanol with 50 mM of HCl at room temperature. After 16 h, 3 ml of chloroform, 0.5 ml of methanol, and 1.1 ml of 0.2% Na₂CO₃ were added. The lower phase was washed with 2 ml of chloroform:methanol:0.1 M KCl (3:48:47, vol/vol) and then with 2 ml of chloroform:methanol:H₂O (3:48:47, vol/vol). Efficiency of sulfatide desulfation was tested by TLC on Silica gel high performance thin layer chromatography plates (Merck) using chloroform:methanol:0.25% KCl (50:40:10, vol/vol) as run-in solvent. Glycolipids were visualized by orcinol staining.

Sulfatide-specific T cell lines and clones were derived as described previously (19). DC were isolated from peripheral blood mononuclear cells by culturing in the presence of IL-4 and recombinant GM-CSF, as described previously (13). Each preparation of DC was tested for the expression of CD1 molecules using mAbs specific for CD1a (OKT6, ATCC CRL8019; American Type Culture Collection), CD1b (WM-25; Immunokontakt), and CD1c (L161; Instrumentation Laboratory). CD1a-, CD1b-, CD1c-, CD1d-, or mock-transfected C1R lymphoblastoma cells (provided by S. Porcelli, Albert Einstein College of Medicine, New York, NY) were used as APC in some experiments.

DC (5 × 10⁴/well) or CD1-transfected C1R cells (5 × 10⁴/well) in RPMI-1640 medium containing 10% FCS were preincubated for 2 h at 37°C with sonicated antigen (0.01–20 μM) before the addition of T cells (6 × 10⁴/well in triplicate). Supernatants were harvested after 36 h and released cytokines were measured by ELISA. TNF-α and IFN-γ were detected using sandwich ELISA kits according to manufacturer's instruction (Instrumentation Laboratory). IL-4 was detected using anti–IL-4 mAbs (BD PharMingen). Data are expressed as mean pg/ml ± SD of triplicates. All experiments were repeated at least two times.

For antibody blocking experiments, DC were preincubated with 3 μM of sulfatide and then with OKT6, WM25, or L161 mAbs, or with Fab fragments of TR66 mAbs (anti-CD3ε) for 20 min before the addition of T cells. Anti-TCRVγ9 mAbs were used as the isotype-matched control.

DC were washed and suspended in PBS (2–3 × 10⁶/ml) containing 0.05% glutaraldehyde for 30 s at 37°C. Additional fixation was blocked with 0.2 M of lysine. The efficiency of fixation was controlled by the [³H]thymidine incorporation of fixed cells and also by the stimulation of an MHC class II–restricted purified protein derivative (PPD)-specific T cell clone. In some experiments, APC were first fixed and then washed and pulsed with 10 μM of sulfatide for 1 h before the addition of T cells.

To investigate the requirements for antigen internalization, DC (10⁶) were preincubated for 1 h at 4°C, or at 37°C, in the presence or absence of 80 μM of chloroquine, or 20 μM of monensin, and then pulsed with 10 μM of sulfatide for an additional 1 h. After washing, DC were fixed and used to stimulate T cells.

T cell lines established for 12 d were used in these assays. Although the number of sulfatide-specific T cells was small, this time point was chosen to reduce in vitro bias for cytokine production. Intracellular staining was performed on T cells activated by CD1a-, CD1b-, CD1c-, or mock-transfected C1R cells pulsed with 10 μM of sulfatide. After 3 h, Brefeldin A (5 μg/ml; Sigma-Aldrich) was added and incubated for an additional 12 h. After washing, the cells were fixed with 2% of paraformaldehyde and permeabilized with 0.1% of saponin. Four color immunofluorescence analysis was performed using anti–CD3-ECD (Immunotech), anti–CD25-FITC, anti–IL-4–PE, and anti–IFN-γ–allophycocyanin mAbs (all from BD PharMingen). Cells were analyzed on a FACSVantage^® SE (Becton Dickinson), gated according to forward scatter and side scatter, and then on the CD3⁺CD25⁺ double-positive population.

Displacement studies were performed by using fixed DC pulsed with 60 μM of sulfatide for 1 h at 37°C, and then extensively washed and plated (3 × 10⁴/well) with various doses of other glycolipids (6–60 μM) for 1 h before the addition of T cells (5 × 10⁴/well). Isopentenyl-pyrophosphate–specific Vγ9Vδ2 T cell clone G2B9 was used as the negative control.

6 × 10⁶ of DC were pulsed with 60 μM of sulfatide at 37°C for 2 h, and then washed three times and plated (4 × 10⁵ cells/well) in triplicates. At different time points, DC were again washed twice before the addition of T cells (5 × 10⁵ cells/well). At each time point in control wells, 10 μM of sulfatide was added to assess the maximal presentation capacity of DC. Results are expressed as a percentage of control at each time point. As another control, CD1a, CD1b, and CD1c expression was tested by immunofluorescence, and median fluorescence values did not change during the culture periods.

Recombinant sCD1a and β2 microglobulin (β2m) were obtained as previously described for soluble CD1b (6). In vitro refolding of CD1a β2m was done by dilution at pH 7.2 in the presence of sulfatide. After concentration on Amicon 10K, the refolded sCD1a–sulfatide complexes were purified from aggregated proteins on a HiLoad 16/60 Superdex 200 column (Amersham Pharmacia Biotech). The purity of soluble protein was >90% as assessed by Coomassie staining.

To test the stability of sCD1a–sulfatide complexes, refolded sCD1a was loaded with 2 μM of [¹⁴C]sulfatide at room temperature for 2 h. The excess [¹⁴C]sulfatide was removed by size-exclusion chromatography using Superdex 200 10/30 column (Amersham Pharmacia Biotech), and purified sCD1a–sulfatide complexes were incubated for 1, 4, and 7 d at 18°C. At each time point, dissociated [¹⁴C]sulfatide was separated by size-exclusion chromatography, and radioactivity associated with sCD1a was measured by liquid scintillation. To assess proper refolding, sCD1a was tested in a sandwich ELISA using anti-β2m and anti-CD1a mAbs.

For activation assays, 30 μg/ml of sCD1a was preincubated with 10 μM of sulfatide and immobilized on 96-well plates. After washing away the excess protein and sulfatide, T cells were plated (1 × 10⁵/well) with 1 ng/ml of PMA. Released cytokines were measured after 24 h of incubation.

A panel of CD1-restricted T cell lines specific for sulfatide was established from peripheral blood of multiple sclerosis patients and normal donors. T cell clones were obtained by limiting dilution and scored for antigen specificity and CD1 restriction using CD1-transfected APC. A total of 58 sulfatide-specific T cell clones were isolated from two different lines. Reverse transcription-PCR analysis of the TCR BV genes of 26 clones showed that 9 out of 16 CD1a-restricted clones and 8 out of 10 CD1b-restricted clones present different rearrangements (unpublished data). It is reasonable to conceive that ∼50% of the 58 isolated clones are different cells. Most of the T cell clones only reacted to sulfatide-pulsed APC, whereas one CD1a-restricted clone was also weakly activated by DC in the absence of exogenous sulfatide (unpublished data). These latter findings could be attributed to pulsing with sulfatide present in the serum or produced by the APC themselves. Interestingly, only DC, and not B cells transfected with the CD1a gene, activated this clone.

Experiments conducted with CD1 transfectants showed that 19 clones were restricted by CD1a, 38 by CD1b, and 1 by CD1c. Fig. 1, A–C shows three examples of clones, each restricted by an individual CD1 molecule. The restriction by different CD1 molecules was confirmed by inhibition with specific relevant anti-CD1 mAbs, whereas control irrelevant mAbs had no effects (Fig. 1, D and F). Inhibition was also observed with anti-CD3ε mAb Fab fragments (Fig. 1, D and F), implying TCR-mediated activation. Most of the clones were CD4⁺, and only four were CD8⁺. All clones were TCR αβ⁺ and did not express the invariant Vα24/Vβ11 TCR present on NK T cells (unpublished data).

The above results show that sulfatide is a promiscuous ligand capable of forming immunogenic complexes with all group I CD1 molecules. As the bulk cell lines were established using a highly pure preparation of sulfatide from bovine brain, it was important to confirm that the stimulatory ligand is indeed sulfatide and not minor contaminating products. The following experiments confirmed that sulfatide is the active ligand. First, when sulfatide was desulfated (Fig. 2 D) it was no longer active (Fig. 2, A–C), thus confirming the importance of the sulfate group. Second, the purity of sulfatide was confirmed to be >98% by gas chromatography-mass spectrometry and its structure was validated by ¹H-nuclear magnetic resonance (unpublished data). Third, dose response studies showed that most of the clones were reactive even to doses of 1–10 nM (Fig. 2 E), thus making it unlikely that minor contaminants are the stimulatory ligands. The fine antigen specificity of T cell clones was also tested using a panel of highly pure glycolipids. Only four clones weakly cross reacted with β GalCer, which lacks the sulfate group present in sulfatide. None of the clones reacted to either GlcCer, LacCer, GM4 (Fig. 2 E), sphingomyelin, or GM1 ganglioside (unpublished data), all of which share the ceramide tail with sulfatide. These findings confirm the sulfatide specificity of the isolated clones and demonstrate that their recognition is highly sensitive to changes in the carbohydrate moiety of sulfatide.

The generation of T cell clones restricted by different CD1 molecules and with the same antigen specificity offered for the first time the possibility of comparing the antigen presentation requirements of CD1a, CD1b, and CD1c. DC, which express all of the CD1 molecules, were pulsed with sulfatide at 4°C or in the presence of monensin (to inhibit CD1 recycling) or chloroquine (to block endosomal acidification). These treatments did not inhibit sulfatide presentation by CD1a, CD1b, or CD1c isoforms (Fig. 3, A–C) , whereas they completely inhibited presentation to MHC class II–restricted T cells (Fig. 3 D). When DC were first fixed and then pulsed with sulfatide, the T cell clones were also efficiently activated (Fig. 3, A–C). Thus, neither internalization of sulfatide, the presence of acidified endosomes, nor CD1 recycling are necessary for the generation of immunogenic complexes between sulfatide and each of the three group I CD1 molecules.

Next, we investigated whether restriction by different CD1 molecules may influence lymphokine release. Evaluation of IL-4 and TNF-α release by each T cell clone after stimulation with sulfatide-pulsed DC showed no bias by CD1 restriction. Clones showed a Th1, Th2, or Th0 phenotype (Fig. 4) . To estimate the extent of in vitro–induced functional maturation, intracellular staining was performed on two freshly established sulfatide-specific bulk lines. Each line was challenged with CD1a-, CD1b-, CD1c-, or mock-transfected APC and then intracellular cytokines were studied. Anti-CD25 and anti-CD3 mAbs were used to gate activated T cells, and anti–IL-4 and anti–IFN-γ mAbs to detect these two intracellular cytokines (Table I). A very small number of T cells (1%) up-regulated CD25 in the absence of sulfatide, whereas a larger fraction (3.5–11%) became CD25⁺ after stimulation with sulfatide (unpublished data). The number of T cells up-regulating CD25 in the presence of sulfatide was similar for the three groups of CD1 transfectants. The analysis of intracellular cytokines showed that most of the cells produce IL-4 or IFN-γ. Interestingly, only a few cells produced both lymphokines after sulfatide recognition.

In conclusion, these experiments show that response to sulfatide can be either of a Th1 or Th2 type.

We have previously shown that self-glycosphingolipids bound to CD1b molecules are readily displaced by ligands that have an appropriate CD1b binding structure (6). Therefore, we asked whether sulfatide bound to each of the three CD1 molecules is displaceable. Fixed DC first pulsed with sulfatide and then incubated with increasing amounts of GM1 showed a reduced stimulation of all CD1-restricted T cells (Fig. 5, A–C) . The same concentration of GM1 did not influence the response of control TCR γδ T cells to their specific ligand (Fig. 5 D). Although GM1 at high doses almost completely eliminated T cell responsiveness, sphingomyelin was much less inhibitory even at high doses exceeding the critical micelle concentration (Fig. 5, A–C). Therefore, not all ligands containing a ceramide tail displace CD1-bound antigens.

It is not known whether CD1 molecules differ in their capacity to form long-lived complexes with glycolipids. To address this issue, DC were pulsed with sulfatide, washed extensively, and then chased for different times before the addition of T cell clones restricted by different CD1 molecules. As positive controls, DC treated as described above were also incubated with fresh sulfatide to restore maximal presentation capacity before the addition of T cells. In these experiments a high dose of sulfatide was used to avoid a possible influence due to different antigen sensitivity of individual T cell clones. Stimulation by CD1b– and CD1c–sulfatide complexes decreased to 50% within 24 h and remained at ∼30% of the control levels after 72 h. Surprisingly, CD1a continued to present sulfatide with >80% efficiency after a 72-h chase (Fig. 6 A). This was confirmed using two different CD1a-restricted sulfatide-specific T cell clones. In some experiments DC were also washed before the addition of T cells to avoid a possible reuptake of free sulfatide, and again similar results were obtained (unpublished data). Furthermore, DC did not change surface expression of CD1 molecules over 72 h (unpublished data), thus excluding CD1 downmodulation as a limiting condition. These findings indicate that CD1a–sulfatide complexes generated in living cells are immunogenic longer than complexes formed with CD1b and CD1c.

To study the stability of CD1a–sulfatide complexes, recombinant sCD1a molecules were generated. Radioactive sulfatide remained significantly associated with sCD1a for 7 d (71% of control values at time 0), even when the samples were maintained at room temperature, which shows a very stable interaction (Fig. 6 B). In similar experiments soluble recombinant mouse CD1d-α–GalCer complexes lost T cell stimulatory capacity after a 24-h chase (20).

Next, we studied whether the T cell stimulatory capacity of recombinant CD1a–sulfatide complexes might also persist for a long period as observed with living cells. The sCD1a molecules appeared properly refolded, as demonstrated by sandwich ELISA (unpublished data), because they stimulated CD1a-restricted sulfatide-specific T cells, but not T cells specific for another antigen (Fig. 6 C). When sulfatide-loaded sCD1a molecules were immobilized to plastic and chased for different times, they continued to stimulate specific T cells with an efficiency of 60% even after a 72-h chase (Fig. 6 D). These results confirm the data obtained with living cells, and clearly show that CD1a–sulfatide complexes are very stable and maintain T cell stimulatory capacity for a long time.