To investigate whether the difference in colonic iNKT cells between BFWT and BFΔSPT mice has biological significance, we subjected these mice to an oxazolone colitis challenge, in which intestinal inflammation characteristic of human ulcerative colitis is induced and is dependent on iNKT cell-produced interleukin 13 (IL-13) (). As expected, BFΔSPT mice had more severe weight loss ( Figure 2 A), higher cumulative histopathology scores ( Figure 2 B), and higher levels of IL-13 and IL-4 release than BFWT mice, although the difference in IL-1β production was not significant ( Figures 2 C–2E). Furthermore, we confirmed that the BFΔSPT mouse phenotype was CD1d dependent: blocking of CD1d with a monoclonal antibody (19G11) during the neonatal period reduced iNKT cell numbers and prevented the colitis phenotype when these mice were challenged in adulthood ( Figure S2 A–S2C). It is possible that different functional characteristics (e.g., cytokine production during colitis stimulation) of the iNKT cells from these two monoassociated mice may also partially contribute to the colitis results. Nonetheless, our results showed a direct link between symbiotic bacterial sphingolipids and both host iNKT cell homeostasis and disease susceptibility.

(A–C) BFΔSPT mice treated with CD1d antibody beginning in the neonatal period had reduced iNKT cell counts and were protected from colitis challenge. Total colonic LP iNKT cell numbers (A; n ≥ 3) were lower in α-CD1d-treated mice than in isotype-treated mice at 8 weeks. The α-CD1d-treated mice also lost less weight (B, P value compares data between the two groups at day 4) and had lower cumulative histopathology scores (C) after oxazolone colitis challenge (n = 7). Data in (A) and (C) were analyzed by the Mann Whitney test and are plotted with box plots. Whiskers are minima and maxima of the data. Data in (B) were confirmed to have normal distribution by the KS normality test with α = 0.05, analyzed by the Student’s t test and are presented as mean ± SEM.

Data in (A) were confirmed to have normal distribution by the KS normality test with α = 0.05, analyzed by Student’s t test and are presented as mean ± SEM. Data in (B)–(E) were analyzed by the Mann-Whitney test and are plotted with box plots. Whiskers are minima and maxima of the data; n ≥ 3 for each group; representative of three experiments. See also Figure S2

(C–E) In tissue explant cultures, levels of IL-13 (C) and IL-4 (D) production in colonic tissue after oxazolone challenge were higher in BFΔSPT mice than in BFWT mice, although IL-1β production was not (E).

In previous comparisons of iNKT cell numbers in SPF and GF mice, we found that SPF animals had higher numbers in the thymus, spleen, and liver but lower counts in the colon and lung (). In the current studies, we found that, despite differences in the numbers of iNKT cells in the colon, BFWT mice did not differ from BFΔSPT or GF mice in terms of iNKT cell numbers in the lung, liver, small intestine, thymus, spleen, or Peyer’s patches ( Figures 1 D–1F and S1 E–S1G). These results indicate that B. fragilis sphingolipids exert effects on iNKT cells only in the colon, where this bacterium is most abundant. A possible reason for the local effect of the B. fragilis glycosphingolipids in these mice is that their quantity, stability, and/or potency are not high enough to have an effect outside the large intestine for iNKT cell regulation.

After monocolonizing GF mice with either BFWT bacteria (termed BFWT mice) or BFΔSPT bacteria (termed BFΔSPT mice), we monitored absolute and relative numbers of iNKT cells in their pups’ colonic LP from birth to 9 weeks of age, as well as in age-matched GF and SPF mice ( Figures 1 A–1C). We found that iNKT cells were absent from the colon in all mice at birth but then were present in numbers that gradually increased until they reached steady state at the age of 6 weeks. However, the relative (to CD3T cells) and absolute numbers of iNKT cells were significantly higher in GF and BFΔSPT mice than in SPF and BFWT mice, despite lower cell numbers in BFΔSPT mice than in GF mice. We also found that colonic LP CD3T cell numbers were similar among GF, BFWT, and BFΔSPT mice ( Figure S1 C). These results suggest that bacterial sphingolipids from a single microbe, B. fragilis, negatively regulate iNKT cell numbers in the colon, although we do not know whether colonic iNKT cells from the BFWT and BFΔSPT mice are functionally similar (e.g., whether they have the same capacity for cytokine production upon activation). In addition, C-delta-monoassociated mice had colonic iNKT cell numbers similar to those observed in BFWT mice ( Figure S1 D).

Data in (B) and (C) (days 21, 42, and 63) were confirmed to have normal distribution by the Kolmogorov-Smirnov (KS) normality test with α = 0.05, analyzed by Student’s t test, and are presented as mean ± SEM; n ≥ 3 for each group. Data in panels (D)–(F) were analyzed by the Mann-Whitney test. See also Figure S1

(B and C) The total numbers of colonic LP iNKT cells (B) and their percentages in CD3 + populations (C) were higher in GF and BFΔSPT than in SPF and BFWT mice.

In the model organism B. fragilis NCTC 9343, the enzyme encoded by gene BF2461 has a high degree of homology (E values ≤ −44 by standard BLASTP search) () with the eukaryotic enzyme serine palmitoyltransferase (SPT). SPT, the first committed enzyme in sphingolipid biosynthesis, produces 3-ketosphinganine from palmitoyl-coenzyme A and L-serine (). We knocked out gene BF2461 from wild-type B. fragilis NCTC 9343 (BFWT) to create a mutant strain BFΔSPT, and we complemented this mutant with a full copy of BF2461 in trans (C-delta). We found that the BFWT and BFΔSPT in vitro growth kinetics were generally comparable, although BFΔSPT had a slightly longer doubling time (64 ± 0 min versus 74 ± 1 min; Figure S1 A). Using thin-layer chromatography, we compared lipid extracts from BFWT and BFΔSPT strains and identified several spots that were present in the former but lacking in the latter. We further treated the two samples with mild alkaline hydrolysis to differentiate sphingolipids from phospholipids, the latter being the most common components of bacterial lipid membranes. The spots that were unique to the BFWT strain were indeed sphingolipids, as determined by their resistance to hydrolysis. In comparison, the spots that were present in both strains were hydrolyzed after treatment, a result suggesting that these spots were phospholipids. C-delta conferred the wild-type profile of sphingolipid generation ( Figure S1 B).

(D) At 6 weeks of age, numbers of colonic LP iNKT cells in C-delta mice were similar to those in BFWT mice; n ≥ 3. Data in (C) and (D) are plotted with box plots. Whiskers are minima and maxima of the data.

(B) B. fragilis mutant strain BFΔSPT did not produce sphingolipids, and the complemented strain C-delta restored sphingolipid production to the level seen in the BFWT strain. Sphingolipids were resistant to mild alkaline hydrolysis, while nonsphingolipids were not. Figure shows thin-layer chromatography of crude lipid extracts with or without 0.02 N NaOH treatment. The chromatography solvent used was chloroform–methanol–acetic acid–water at a ratio of 100:20:12:5. Data are representative of 3 experiments.

On the basis of these findings, we hypothesized that only when mice are exposed to symbiotic sphingolipids very early in life are their iNKT cell numbers restricted in adulthood. We designed two cohousing experiments to test this hypothesis. In the first experiment, GF dams were mated with BFWT monocolonized mice. Ki-67 expression in offspring pups’ colonic LP iNKT cells was measured at 8 days of age ( Figure 3 C) and iNKT cell numbers were measured at 8 weeks ( Figure 3 D). As expected, the proliferation levels and total cell numbers in pups born to dams receiving BFWT bacteria (GF-WT(neo)) were similar to those in BFWT mice. In the second experiment, we cohoused GF pups at 10–14 days of age with BFWT mice (GF-WT(adu)), just after the cell proliferation window had closed. As expected, although GF-WT(adu) mice harbored numbers of BFWT bacteria equivalent to those in GF-WT(neo) mice, the GF-WT(adu) animals had much higher colonic LP iNKT cell numbers at 8 weeks of age. When we challenged these mice with oxazolone, GF-WT(neo) mice responded similarly to BFWT mice, with a significant reduction in the severity of the colitis phenotype and intestinal inflammation (as assessed by weight loss and colitis scores) from values obtained in GF-WT(adu) mice ( Figures 3 E and 3F). These studies established a critical time window for exposure to sphingolipid-producing symbionts to maintain host iNKT cell homeostasis and influence disease susceptibility. Interestingly, when we cohoused GF pregnant dams with BFWT mice 3 days before delivery, the pups’ colonic iNKT cell numbers were not normalized to the BFWT level but more resembled the numbers found in GF mice, although at time of delivery, the mother was heavily colonized with BFWT bacteria (data not shown). This finding indicates that either bacterial sphingolipids are important for iNKT cell development at the prenatal stage or as yet unidentified factors also control iNKT cell homeostasis.

One other possibility we considered was that some bacterial sphingolipids, including those of B. fragilis, inhibit the expansion of the iNKT cell population in the colon. To test this hypothesis, we measured the expression of Ki-67 (a nuclear protein marker for cellular proliferation) in iNKT cells within the colonic LP of mice from birth to 9 weeks of age. We found significantly higher mean fluorescence intensity (MFI) expression for this protein in both GF and BFΔSPT mice than in either SPF or BFWT mice during the neonatal period, particularly between days 5 and 12. At 8 days of age, the percentage of Ki-67iNKT cells was also higher in GF and BFΔSPT mice. Proliferation was reduced to similar low levels in all mice after 21 days ( Figures 3 A, S3 F, and S3G). To verify this observation, we used an alternative approach—the bromodeoxyuridine (BrdU) method—to measure DNA replication in colonic LP iNKT cells in 8-day-old mice. We confirmed that GF and BFΔSPT mice had higher levels of DNA replication in these cells than did SPF and BFWT mice, respectively ( Figures 3 B and S3 H). These studies showed that symbiotic bacterial sphingolipids can modulate the homeostasis of colonic iNKT cells by inhibiting cell proliferation, and thus their accumulation, during neonatal development.

Data in (A) (days 8 and 12) and (E) were confirmed to have normal distribution by the KS normality test with α = 0.05, analyzed by Student’s t test and are presented as mean ± SEM. Data in (B)–(D) and (F) were analyzed by the Mann-Whitney test and are plotted with box plots. Whiskers are minima and maxima of the data. See also Figure S3 and Table S1

We next investigated a number of possible causes for our findings that mice monocolonized with BFWT and BFΔSPT had different iNKT cell homeostasis in the colonic LP. We studied the relative numbers of BFWT and BFΔSPT bacteria contained within the colons, including microbes that were either in the lumen or tissue associated ( Figures S3 A and S3B), and we did not find appreciable differences in bacterial numbers between the two strains in any condition tested. We next studied the ability of B. fragilis to normalize the elevated colonic levels of CXCL16 observed in GF mice () and found that the two types of monocolonized mice had similar cxcl16 mRNA levels in the colon tissues, comparable to that of the GF mice ( Figure S3 C). Third, we analyzed the activation ( Figure S3 D) and apoptosis ( Table S1 ) of colonic iNKT cells in BFWT and BFΔSPT mice and found little difference. Lastly, we investigated whether PSA expression was quantitatively different between the BFWT and BFΔSPT strains, which could lead to differential activation of PSA-mediated pathways ( Figure S3 E). We found that PSA production levels were statistically identical in the two strains. Thus, we discovered no evidence to support any of the above mechanisms accounting for the differences in iNKT cell homeostasis in mice colonized with BFWT versus BFΔSPT bacteria.

Data in (G) (days 8 and 12) were confirmed to have normal distribution by the KS normality test with α = 0.05, analyzed by the Student’s t test. Data in (A), (B), (C), and (E) were analyzed by the Mann Whitney test.

(H) Gating of BrdU staining on colonic LP iNKT cells in flow cytometry. Data were compiled from 3-5 mice for each group. Within each group, data were processed by subtracting PBS background from BrdU treatment, and then plotted in Figure 3 B. Data in (G) (days 8 and 12) were confirmed to have normal distribution by the KS normality test with α = 0.05, analyzed by the Student’s t test and are presented as mean ± SEM.

(G) The proportion of colonic LP iNKT cells expressing Ki-67 was lower in SPF and BFWT mice than in GF and BFΔSPT mice at 8 days of age; n ≥ 3. Data are presented as mean ± SEM.

(D) Colonic iNKT cells in BFWT mice (red) and BFΔSPT mice (blue) at 6–10 weeks of age expressed CD62L, CD69, and CD45RB at similar levels. Isotype control values (black) are also shown. Data were compiled from 3 independent experiments (2 or 3 mice per group for each experiment) and total of 4,000–9,000 colonic iNKT cells were analyzed for each group. Numbers indicate mean geometric fluorescence intensity.

Purified B. fragilis Glycosphingolipids Inhibit iNKT Cell Activation In Vitro and In Vivo

Figure 4 Chemical Analysis of B. fragilis Glycosphingolipid Peak GSL-Bf717 Show full caption (A) Three distinct sphingolipid clusters were identified in B. fragilis. (B) None of these clusters activated iNKT cells. The data are representative of two experiments (each done in triplicate) and presented as median ± range. (C) GL-Cers was inhibitory to iNKT cell activation by KRN7000 in vitro. The data were compiled from three experiments (n ≥ 6) and were confirmed to have normal distribution by the KS normality test with α = 0.05, analyzed by Student’s t test, and are presented as mean ± SEM. The p values compare KRN7000 + GSL-Bf717 (at ratios of 1:10 and 1:30) with KRN7000 alone (ratio 1:0). (D) GL-Cers had five chain-length variants (C32–C36), and C34 with MW = 717.6 (m/z 716) was predominant. (E–G) A peak from this variant, GSL-Bf717, was characterized by MS/MS (E), HPAEC (F), and 1H-NMR (G) and was found to have an α-galactosyl linkage. In (F), the hydrolyzed hexose head group coeluted with the spiked galactose standard (red) at 12.6 min. (H) The proposed molecular configuration of GSL-Bf717 is shown for comparison with that of the prototypical agonist KRN7000. In order to identify bioactive sphingolipids of B. fragilis, we performed comparative lipidomic profiling of BFWT and BFΔSPT bacteria by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). In BFWT lipid extracts, we identified three types of sphingolipids with characteristic MS/MS fragmentation patterns: ceramides (Cers), glycosylceramides (GL-Cers, signature fragments of 161,179), and phosphoethanolamine-ceramides (PE-Cers, signature fragment of 140) ( Figure 4 A; data not shown). None of these sphingolipids were detected in BFΔSPT lipid extracts. To determine whether any of these sphingolipids activated iNKT cells, we conducted coculture assays. Bone marrow dendritic cells (BMDCs) were pulsed with each sphingolipid type and then incubated with iNKT cell hybridoma 24.7. None of the lipids in the tested conditions activated iNKT cells to produce appreciable IL-2, although the prototypical ligand KRN7000 caused robust activation ( Figure 4 B). We next tested whether these lipids were inhibitory to iNKT cells. We pulsed BMDCs with each sphingolipid type in the presence of KRN7000 and then incubated the cells with hybridoma 24.7, measuring the production of IL-2 as an indicator of iNKT cell activation (100% activation was defined by the level of IL-2 production in the KRN7000 alone condition). In this assay, GL-Cers, but not the other two lipid types, significantly reduced iNKT cell activation by KRN7000 ( Figure 4 C). It is very likely that the difference in activity among the three classes of lipid molecules lies in the different head groups. It is possible that the hexose, in combination with certain unique features of bacterial ceramide chains, allows GL-Cers to interact more stably with CD1d and/or iNKT cells as compared with PE-Cers and Cers.

2 O]), 680 ([M-H-2H 2 O]), 554 ([M-H-hexose+H 2 O]), 536 ([M-H-hexose]), and 490 ([M-H-C 15 H 29 O])—in addition to the above-mentioned hexose-derived fragments. Among these daughter ions, m/z 490 (loss of the hydroxyl aliphatic chain) assigned that sphingosine and the fatty acid chain have one hydroxyl group each (1H-NMR (1H-1H-NMR (correlated spectroscopy; data not shown) to verify the identity of the hexose and characterize its glycosidic linkage. NMR analysis revealed a galactose residue linked α-glycosidically to the sphingoid backbone. This α-galactosylceramide peak is referred to as GSL-Bf717, and a proposed molecular configuration is shown in Miyagawa et al., 1979 Miyagawa E.

Azuma R.

Suto T.

Yano I. Occurrence of free ceramides in Bacteroides fragilis NCTC 9343. To identify molecules inhibitory to iNKT cell activation, we further analyzed the GL-Cers cluster. We found that GL-Cers is composed of multiple molecular species, ranging from m/z 688 to m/z 744 ( Figure 4 D). The observation that all species had characteristic fragments at m/z 143, 161, and 179, which were from the hexose head group, implied that the source of the structural variability resided in the chain length of ceramide structures from C32 to C36 ( Figure 4 E; data not shown). MS/MS analysis of a prominent m/z 716 peak (C34: MW = 717.6) generated multiple daughter ions—assigned as m/z 698 ([M-H-HO]), 680 ([M-H-2HO]), 554 ([M-H-hexose+HO]), 536 ([M-H-hexose]), and 490 ([M-H-CO])—in addition to the above-mentioned hexose-derived fragments. Among these daughter ions, m/z 490 (loss of the hydroxyl aliphatic chain) assigned that sphingosine and the fatty acid chain have one hydroxyl group each ( Figure 4 E). The MW 717.6 peak was purified by HPLC and further analyzed. By high-pH anion-exchange chromatography (HPAEC), we identified the structure of the monosaccharide head group as galactose ( Figure 4 F). In addition, we usedH-NMR ( Figure 4 G) andH-H-NMR (correlated spectroscopy; data not shown) to verify the identity of the hexose and characterize its glycosidic linkage. NMR analysis revealed a galactose residue linked α-glycosidically to the sphingoid backbone. This α-galactosylceramide peak is referred to as GSL-Bf717, and a proposed molecular configuration is shown in Figure 4 H. In addition, we confirmed that GSL-Bf717 was detectable only in fecal samples from BFWT mice, but not in samples from BFΔSPT mice. The estimated yield of GSL-Bf717 was ∼1 ng per gram of fecal pellet. Although it shares key features with the known iNKT cell agonist KRN7000, GSL-Bf717 has unique structural characteristics, such as shorter chain lengths and different hydroxyl compositions in both chains. Ceramides produced by B. fragilis are known to have branched acyl chains in iso- or anteiso-positions (), which may present yet another distinction between GSL-Bf717 and KRN7000. As a result of these dissimilarities, GSL-Bf717 may possibly compete with KRN7000 for the limited space in CD1d grooves, but may be positioned differently so that the α-galactosyl head group does not interact with the iNKT cell receptor in the same way as KRN7000. It is unclear whether GSL-Bf717 and similar molecules are presented as antagonistic ligands or simply occupy the CD1d binding space due to high affinity. In either case, the end consequence is that these bacterial glycosphingolipids and KRN7000 have opposing immunological effects: GL-Cers restrains iNKT cell activation and KRN7000 activates iNKT cells.

Figure 5 B. fragilis GSL-Bf717 Inhibits iNKT Cell Activation Show full caption (A–D) GSL-Bf717 did not activate iNKT cells (A and C; data are representative of two independent experiments, each done in triplicate) but did inhibit iNKT cell activation by agonist KRN7000 (100 nM) in both hybridomas 24.7 and DN32.D3 (B, left columns, and D, data are representative of two independent experiments, each done in triplicate; p value compares KRN7000 + GSL-Bf717 at a 1:30 ratio with KRN7000 alone, which is at a ratio of 1:0). GSL-Bf717 also inhibited activation by agonist β-GlcCer (20 μM; right columns in B; p values compare β-GlcCer + GSL-Bf717 at ratios of 40:1 and 20:1 with β-GlcCer alone, which is at a ratio of 1:0). (E) The loading efficiency of PBS-44 to empty CD1d tetramer (phycoerythrin-labeled) was decreased significantly in the presence of GSL-Bf717, as evidenced by the reduced MFI of CD1d tetramer-stained iNKT cells; tetramers: PBS-44: GSL-Bf717=1:1:3; n = 4. (F and G) GSL-Bf717 reduced serum levels of IFN-γ (F) and IL-4 (G) released by KRN7000-stimulated iNKT cells in vivo; 100% production was defined as the level of cytokines produced in KRN7000-only-treated mice (n ≥ 4). Mice were intraperitoneally injected with 1,000 ng of bacterial lipids and/or 100 ng of KRN7000. Data in (A)–(D) and (F) are presented as median ± range. Data in (E) are plotted with box plots. Whiskers are minima and maxima of the data. Data in (B) and (D)–(F) were analyzed by the Mann-Whitney test. Data in (G) were confirmed to have normal distribution by the KS normality test with α = 0.05, analyzed by Student’s t test, and are presented as mean ± SEM. See also Figure S4 Figure S4 GSL-Bf717 Does Not Activate iNKT Cells, Related to Figure 5 Show full caption (A–H) CD1d tetramers added with bacterial sphingolipids did not bind to iNKT cell line 24.7. (I–K) CD1d tetramers added with GSL-Bf717 did not stain liver lymphocytes. Representative FACS plots are shown from 2 independent experiments. To confirm that GSL-Bf717 has inhibitory activity, we performed several coculture assays. In cocultures of BMDCs and iNKT cell hybridoma 24.7, GSL-Bf717 did not activate iNKT cells ( Figure 5 A). Moreover, GSL-Bf717 did not activate noninvariant NKT cell line 14S6 (data not shown). We next conducted a CD1d-loading experiment using phycoerythrin-stained CD1d empty tetramers and the iNKT cell hybridoma 24.7. When the empty CD1d tetramers are loaded with lipid antigens, they can bind to the iNKT cell receptor and the complex can be detected by flow cytometry, as shown in Figures S4 A–S4D for tetramers either preloaded at the NIH facility ( Figure S4 B) or lab loaded ( Figure S4 D) with PBS-57 (a lipid variant of KRN7000 that also stimulates iNKT cells). Previously empty tetramers added with either one of the three identified lipid fractions ( Figure 4 A) or GSL-Bf717 did not bind to iNKT cells ( Figures S4 E–S4H). In addition, GSL-Bf717-added, previously empty CD1d tetramers did not effectively stain liver lymphocytes, in contrast to the control tetramers loaded with PBS-57 ( Figures S4 I–S4K). These CD1d-loading experiments provide supporting evidence that the purified bacterial lipids do not activate 24.7 iNKT hybridoma cells in the tested conditions. The likely mechanisms could be that the lipids do not sit or do not sit correctly in the CD1d-binding grooves and/or that the iNKT TCRs do not recognize these CD1d-lipid complexes.

Brennan et al., 2011 Brennan P.J.

Tatituri R.V.

Brigl M.

Kim E.Y.

Tuli A.

Sanderson J.P.

Gadola S.D.

Hsu F.-F.

Besra G.S.

Brenner M.B. Invariant natural killer T cells recognize lipid self antigen induced by microbial danger signals. When in competition with KRN7000 at a ratio of 1:30 in the coculture assay using BMDCs and 24.7 cells, GSL-Bf717 in excess moderately (i.e., by 40%) inhibited KRN7000-induced activation of iNKT cells ( Figure 5 B, left group of columns). This limited suppressive activity of GSL-Bf717 suggested that it likely acts as a competitive inhibitor to agonists in binding to CD1d, instead of as a true antagonist. However, when we tested another iNKT cell agonist, β-glucosylceramide (β-GlcCer, d18:1/24:1(15Z)), we found that GSL-Bf717 was more effective in inhibiting its function. β-GlcCer is an endogenous agonist that has much weaker activity than KRN7000 (). Indeed, in our assay, to induce comparable IL-2 production in the 24.7 hybridoma, it took micromolar concentrations of β-GlcCer as compared with nanomolar concentrations of KRN7000 ( Figure 5 A). Remarkably, GSL-Bf717 not only could inhibit β-GlcCer function in a dose-dependent fashion but also could achieve ∼40% inhibition with as little as 2.5% of the β-GlcCer concentration ( Figure 5 B, right group of columns, 40:1). We next used the hybridoma DN32.D3 to test the inhibitory activity of GSL-Bf717 on KRN7000 function. We found that GSL-Bf717 was not able to activate this iNKT cell line when BMDC was used as the CD1d-expressing antigen-presenting cell, but could inhibit iNKT cell activation by KRN7000 to a similar extent as in 24.7 ( Figures 5 C and 5D). As seen with hybridoma 24.7, Cers could not inhibit DN32.D3 iNKT hybridoma cell activation by KRN7000. A comparison of Figures 4 C and 5 B shows that the GL-Cers fraction was a more potent inhibitor of iNKT cell activation than was GSL-Bf717. It is highly likely that many B. fragilis glycosphingolipid species can additively or synergistically inhibit iNKT cells just as GSL-Bf717 does. Nonetheless, GSL-Bf717 is representative of these glycosphingolipids and can be used to further explore the inhibitory activity of GL-Cers.

To understand at the molecular level the inhibitory activity of GSL-Bf717, we performed a CD1d tetramer competitive loading experiment. We incubated PBS-44 (another variant of KRN7000 that is also agonistic to iNKT cells and can be efficiently loaded to empty CD1d tetramers) with phycoerythrin-labeled unloaded CD1d tetramers in the presence and absence of GSL-Bf717 in a 1:1:3 ratio (tetramers/PBS-44/GSL-Bf717). We then used the CD1d-lipid complex (in excess) to stain iNKT hybridoma 24.7 cells and measured the MFI. We found that, in comparison with the PBS-44+vehicle control, addition of GSL-Bf717 to PBS-44 was able to significantly reduce the MFI of the stained iNKT cells to ∼57% of that of the vehicle control, reflecting the decreased staining efficiency of the tetramers, as well as the poor loading capacity of PBS-44 to CD1d in the presence of GSL-Bf717 ( Figure 5 E). This experiment strongly suggested that the inhibitory function of GSL-Bf717 was likely mediated at the molecular level during CD1d-lipid interactions in which GSL-Bf717 could occupy the CD1d grooves and prevent PBS-44 loading, and/or that the GSL-Bf717-loaded CD1d complex was not recognized properly by the iNKT cells (leading to a decreased MFI).