Isolation and Characterization of Antibodies Induced by Immunization with TNF-α Inducible Globotetraosylceramide.

Glycosphingolipids containing very-long-chain fatty acids (VLCFAs) regulate several immune responses, such as cytokine production, immune signaling, and antibody induction. We previously reported that stimulation with an inflammatory mediator, TNF-α, promotes the expression of glycosphingolipids in vascular endothelial cells. The major component is globotetraosylceramide containing VLCFAs (Gb4Cer-VLCFAs), but its role in inflammatory responses has not been fully investigated. In this study, the antibody-inducing properties of Gb4Cer-VLCFAs were analyzed using serum and hybridoma cells generated from Gb4Cer-VLCFA-immunized mice. The reactivity of serum antibodies against Gb4Cer indicated that immunization with Gb4Cer-VLCFAs immediately induced the production of anti-Gb4Cer antibodies. Over 81% of hybridomas generated from the splenocytes of an immunized mouse produced anti-Gb4Cer antibodies, a subset of which recognized an epitope shared by Gb4Cer and its precursor globotriaosylceramide (Gb3Cer). Further biochemical analyses of established monoclonal antibodies revealed that these antibodies included IgM and IgG3, which specifically react with Gb4Cer and Gb3Cer. These results indicate that immunization with Gb4Cer-VLCFAs can efficiently induce the production of anti-Gb4Cer and -Gb3Cer antibodies by B cells.

Asymmetrical and Isolated Hypoglossal Nerve Palsy Accompanied by a New Subset of Anti-ganglioside Antibodies in a Patient with Diffuse Large B Cell Lymphoma.

Malignant lymphoma sometimes involves peripheral nerves due to paraneoplastic syndrome associated with anti-ganglioside antibodies. We report a very rare case of malignant lymphoma accompanied by an asymmetrical and isolated hypoglossal nerve palsy associated with a new subset of anti-ganglioside antibodies. Magnetic resonance imaging and 18F-2-deoxy-2-fluoro-D-glucose position emission tomography showed no abnormalities of the hypoglossal nerve nucleus; however, the patient' s serum was positive for anti-sulfated glucuronyl paragloboside IgM antibodies as well as anti-GM1 IgM and anti-GQ1b IgM antibodies. The present case might suggest a paraneoplastic asymmetrical and isolated hypoglossal nerve palsy associated with a new subset of anti-ganglioside antibodies.

Antigen Specificity of Type I NKT Cells Is Governed by TCR ß-Chain Diversity.

NKT cells recognize lipid-based Ags presented by CD1d. Type I NKT cells are often referred to as invariant owing to their mostly invariant TCR α-chain usage (Vα14-Jα18 in mice, Vα24-Jα18 in humans). However, these cells have diverse TCR ß-chains, including Vß8, Vß7, and Vß2 in mice and Vß11 in humans, joined to a range of TCR Dß and Jß genes. In this study, we demonstrate that TCR ß-chain composition can dramatically influence lipid Ag recognition in an Ag-dependent manner. Namely, the glycolipids α-glucosylceramide and isoglobotrihexosylceramide were preferentially recognized by Vß7(+) NKT cells from mice, whereas the α-galactosylceramide analog OCH, with a truncated sphingosine chain, was preferentially recognized by Vß8(+) NKT cells from mice. We show that the influence of the TCR ß-chain is due to a combination of Vß-, Jß-, and CDR3ß-encoded residues and that these TCRs can recapitulate the selective Ag reactivity in TCR-transduced cell lines. Similar observations were made with human NKT cells where different CDR3ß-encoded residues determined Ag preference. These findings indicate that NKT TCR ß-chain diversity results in differential and nonhierarchical Ag recognition by these cells, which implies that some Ags can preferentially activate type I NKT cell subsets.

Protection against Shiga-Toxigenic Escherichia coli by Non-Genetically Modified Organism Receptor Mimic Bacterial Ghosts.

Shiga-toxigenic Escherichia coli (STEC) causes severe gastrointestinal infections in humans that may lead to life-threatening systemic sequelae, such as the hemolytic uremic syndrome (HUS). Rapid diagnosis of STEC infection early in the course of disease opens a window of opportunity for therapeutic intervention, for example, by administration of agents that neutralize Shiga toxin (Stx) in the gut lumen. We previously developed a recombinant bacterium that expresses a mimic of the Stx receptor globotriaosyl ceramide (Gb3) on its surface through modification of the lipopolysaccharide (A. W. Paton, R. Morona, and J. C. Paton, Nat Med 6:265-270, 2000, http://dx.doi.org/10.1038/73111). This construct was highly efficacious in vivo, protecting mice from otherwise fatal STEC disease, but the fact that it is a genetically modified organism (GMO) has been a barrier to clinical development. In the present study, we have overcome this issue by development of Gb3 receptor mimic bacterial ghosts (BGs) that are not classified as GMOs. Gb3-BGs neutralized Stx1 and Stx2 in vitro with high efficiency, whereas alternative Gb3-expressing non-GMO subbacterial particles (minicells and outer membrane blebs) were ineffective. Gb3-BGs were highly efficacious in a murine model of STEC disease. All mice (10/10) treated with Gb3-BGs survived challenge with a highly virulent O113:H21 STEC strain and showed no pathological signs of renal injury. In contrast, 6/10 mice treated with control BGs succumbed to STEC challenge, and survivors exhibited significant weight loss, neutrophilia, and histopathological evidence of renal damage. Thus, Gb3-BGs offer a non-GMO approach to treatment of STEC infection in humans, particularly in an outbreak setting.

A globotetraosylceramide (Gb4) receptor-based ELISA for quantitative detection of Shiga toxin 2e.

Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb4) receptor by ELISA (Gb4-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb4-ELISA increased linearly with Stx2eB concentration in the range of 20-2,500 ng/ml. The Gb4-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.

A hemolytic anti-LKE associated with a rare LKE-negative, "weak P" red blood cell phenotype: alloanti-LKE and alloanti-P recognize galactosylgloboside and monosialogalactosylgloboside (LKE) antigens.

BACKGROUND: "Weak P" is a rare red blood cell (RBC) phenotype, characterized by a global decrease in P(k) and P antigens. We now describe a second weak P individual who also typed LKE-negative (LKE-N) and possessed a clinically significant anti-LKE. STUDY DESIGN AND METHODS: Patient RBCs and plasma were examined by standard serology and flow cytometry. Glycosphingolipids (GSLs) from patient, P(k) , and LKE-strong (LKE-S) RBCs were isolated and analyzed by high-performance thin-layer chromatography (HPTLC). To confirm antibody specificity, patient serum and 30 human polyclonal controls, including alloanti-P and anti-PP1 P(k) , were tested against a panel of GSLs by HPTLC immunostaining. RESULTS: The patient typed P1 +, P+, and LKE-N and possessed a "P-like" panagglutinin. In a two-stage indirect antiglobulin test, the patient's plasma caused hemolysis of LKE-S cells but not p, P(k) , or LKE-N cells. Clinically, transfusion of P+ RBCs compatible by a prewarmed technique had shortened RBC survival with laboratory evidence of hemolysis. Analysis of the patient's isolated RBC GSLs showed a 30% relative decrease in Gb3 (P(k) ) and Gb4 (P) and a 90% decrease in monosialogalactosylgloboside (MSGG, LKE), accompanied by increased lactosylceramide (CDH), paragloboside, and GM3. On HPTLC immunostaining, the patient's plasma strongly bound MSSG with weak binding to galactosylgloboside (Gb5). Binding to MSGG, Gb5, and Gb4 was also observed with some examples of alloanti-P from P(k) individuals, but not anti-PP1 P(k) , autoanti-P, or normal controls. CONCLUSIONS: We describe the first example of a clinically significant anti-LKE in the setting of a rare weak P background. Human alloanti-LKE and some alloanti-P recognized Gb5 and MSGG.

Monoclonal anti-A activity against the FORS1 (Forssman) antigen.

BACKGROUND: The FORS blood group system (originally recognized as the Apae phenotype) was discovered by sporadic activity against polyclonal anti-A reagents and activity against the lectin Helix pomatia. The extent of monoclonal anti-A reagent activity against the FORS1 antigen is serologically and immunochemically incomplete. STUDY DESIGN AND METHODS: In the absence of natural FORS1-positive red blood cells (RBCs), kodecytes were created with synthetic disaccharide and pentasaccharide Forssman function-spacer-lipid (FSL) constructs, Fsdi -kodecytes, and FORS1-kodecytes, respectively. FSL constructs were also applied to solid surfaces and used in solid-phase enzyme immunoassays. A range of characterized monoclonal anti-A and anti-B reagents were then serologically and immunochemically characterized against these Forssman antigens. Polyclonal human anti-A, anti-B, the lectin H. pomatia serologic reagents; and canine RBCs were used as serologic controls. RESULTS: None of 19 different monoclonal anti-A reagents were able to detect the pentasaccharide Forssman on FORS1-kodecytes, while three reagents were able to detect disaccharide Forssman on Fsdi -kodecytes. Most anti-A reagents were immunochemically reactive with both the di- and the pentasaccharide Forssman antigens in the solid-phase assays. Historic polyclonal human anti-A and the lectin H. pomatia reacted strongly with the FORS1-kodecytes, correlating with the discovery of the Apae phenotype and supporting the use of FORS1-kodecytes as FORS1 surrogates. CONCLUSIONS: Monoclonal anti-A reagents, despite showing reactivity against the FORS1 antigen in solid-phase assays are unlikely to cause the agglutination of FORS1 antigen-positive RBCs.

[A case of IgM paraproteinemic neuropathy associated with anti-sulfated glucuronic paragloboside (SGPG) IgG antibody without anti-myelin-associated glycoprotein (MAG) activity].

We report a case of IgM paraproteinemic neuropathy associated with anti-sulfated glucuronic paragloboside (SGPG) IgG antibody. An 84-year old man complained of numbness on the left side of the face and in the distal portions of the limbs. Neurological examination showed mild sensory ataxia. The laboratory tests revealed the presence of IgM lambda paraproteinemia and anti-SGPG IgG antibody without anti-myelin-associated glycoprotein (MAG) activity and anti-MAG/SGPG IgM antibody. Results of nerve conduction study showed decreased sensory nerve action potential (SNAP) amplitude, indicating the presence of sensory-dominant axonal polyneuropathy, and the prolongation of distal latency was not observed. Treatment with corticosteroids resulted in a rapid improvement in neurological abnormalities. In IgM paraproteinemic neuropathy associated with anti-MAG/SGPG antibody, distal acquired demyelinating sensory neuropathy and resistance to immunological treatments are the characteristic pathologic and clinical features, respectively. On the other hand our rare case of IgM paraproteinemic neuropathy positive for anti-SGPG IgG antibody presented with axonal sensory polyneuropathy and a good responsiveness to corticosteroids.

P1PK: the blood group system that changed its name and expanded.

The antigens in the P1PK blood group system are carried on glycosphingolipids. The system currently includes three different antigens, P1, Pk, and NOR. The P1 antigen was disovered in 1927 by Landsteiner and Levine, and Pk and NOR were described in 1951 and 1982, respectively. As in the ABO system, naturally occurring antibodies of the immunoglobulin (Ig) M or IgG class, against the missing carbohydrate structures, can be present in the sera of people lacking the corresponding antigen. Anti-P1 is generally a weak and cold-reactive antibody not implicated in hemolytic transfusion reaction (HTR) or hemolytic disease of the fetus and newborn while Pk antibodies can cause HTR, and anti-NOR is regarded as a polyagglutinin. A higher frequency of miscarriage is seen in women with the rare phenotypes p, P1k, and P2k. Furthermore, the Pk and P1 antigens have wide tissue distributions and can act as host receptors for various pathogens and toxins. Why p individuals lack not only Pk and P expression but also P1 has been a longstanding enigma. Recently, it was shown that the same A4GALT-encoded galactosyltransferase synthesizes both the P1 and Pk antigens and that a polymorphism in a new exon in this gene predicts the P1 and P2 phenotypes.

P1/P2 genotyping of known and novel null alleles in the P1PK and GLOB histo-blood group systems.

BACKGROUND: The rare but clinically important null phenotypes of the P1PK and GLOB blood group systems are due to alterations in A4GALT and B3GALNT1, respectively. A recently identified single-nucleotide polymorphism in Exon 2a of A4GALT predicts the common P1 and P2 phenotypes but rare variants have not been tested. STUDY DESIGN AND METHODS: The aim of this study was to analyze 84 p, P1 (k) , and P2 (k) samples, with special emphasis on unknown alleles and the P(1) /P(2) marker. Of these, 27 samples came from individuals not previously investigated genetically and were therefore subjected to sequencing of A4GALT or B3GALNT1, and a subset was tested by flow cytometry. RESULTS: The P(1) /P(2) genotyping linked 20 p-inducing mutations in A4GALT to P(1) or P(2) allelic background. Eight p alleles remain unlinked due to compound heterozygosity. For 23 of 25 P(k) samples, concordant results were observed: P1 (k) samples had at least one P(1) allele while P2 (k) had P(2) only. The two remaining samples typed as P1+ and P1+(w) but were genetically P(2) /P(2) . A tendency toward higher P(k) antigen expression was observed on P1 (k) cells compared to P2 (k) . In total, six previously unknown null mutations were found and characterized in A4GALT while four new changes were revealed in B3GALNT1. CONCLUSION: For the first time, p alleles were shown to occur on both P(1) and P(2) allelic backgrounds. Furthermore, P(1) /P(2) genotyping predicted the P1 (k) versus P2 (k) phenotype in more than 90% of globoside-deficient samples. The number of GLOB-null alleles was increased by 50% and several P1PK-null alleles were identified.

TLR4-MD-2 complex is negatively regulated by an endogenous ligand, globotetraosylceramide.

Although endogenous ligands for Toll-like receptor (TLR)4-myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4-MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4-MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4-MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4-MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4-MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.

CD1d protein structure determines species-selective antigenicity of isoglobotrihexosylceramide (iGb3) to invariant NKT cells.

Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d-presented self-antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3-dependent human iNKT-cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human-mouse cross-species analysis of iNKT-cell activation by iGb3-CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3-hCD1d was unable to support cognate interactions with the iNKT-cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine-to-tryptophan modification within the α2-helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3-hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT-cell biology.

Further structural characterization of the Echinococcus granulosus laminated layer carbohydrates: the blood-antigen P1-motif gives rise to branches at different points of the O-glycan chains.

The glycobiology of the cestodes, a class of parasitic flatworms, is still largely unexplored. An important cestode species is Echinococcus granulosus, the tissue-dwelling larval stage of which causes hydatid disease. The E. granulosus larva is protected from the host by a massive mucin-based extracellular matrix termed laminated layer (LL). We previously reported ( Díaz et al. 2009. Biochemistry 48:11678-11691) the molecular structure of the most abundant LL O-glycans, comprising up to six monosaccharide residues. These are based on Cores 1 and 2, in cases elongated by a chain of Galpß1-3 residues, which can be capped by Galpα1-4. In addition, the Core 2 GlcNAcp residue can be decorated with the Galpα1-4Galpß1-4 disaccharide. Larger glycans also detected contained additional HexNAc residues that could not be explained by the structural repertoire described above. In this work, we elucidate, by mass spectrometry (MS) and nuclear magnetic resonance (NMR), six additional glycans from the E. granulosus LL between six and eight residues in size. Their structures are related to those already described but in cases bear GlcNAcpß1-6 or Galpα1-4Galpß1-4GlcNAcpß1-6 as ramifications on the core Galpß1-3 residue. We also obtained evidence that noncore Galpß1-3 residues can be similarly ramified. Thus, the new motif together with the previous information may explain all the glycan compositions detected in the LL by MS. In addition, we show that the anti-Echinococcus monoclonal antibody E492 (Parasite Immunol 21:141, 1999) recognizes Galpα1-4Galpß1-4GlcNAcp (the blood P(1)-antigen motif). This explains the antibody's reactivity with a range of Echinococcus tissues, as the P(1)-motif is also carried on non-LL N-glycans and glycolipids from this genus.

Molecular genetic basis of the human Forssman glycolipid antigen negativity.

Forssman heterophilic glycolipid antigen has structural similarity to the histo-blood group A antigen, and the GBGT1 gene encoding the Forssman glycolipid synthetase (FS) is evolutionarily related to the ABO gene. The antigen is present in various species, but not in others including humans. We have elucidated the molecular genetic basis of the Forssman antigen negativity in humans. In the human GBGT1 gene, we identified two common inactivating missense mutations (c.688G>A [p.Gly230Ser] and c.887A>G [p.Gln296Arg]). The reversion of the two mutations fully restored the glycosyltransferase activity to synthesize the Forssman antigen in vitro. These glycine and glutamine residues are conserved among functional GBGT1 genes in Forssman-positive species. Furthermore, the glycine and serine residues represent those at the corresponding position of the human blood group A and B transferases with GalNAc and galactose specificity, respectively, implicating the crucial role the glycine residue may play in the FS α1,3-GalNAc transferase activity.

Complete absence of the αGal xenoantigen and isoglobotrihexosylceramide in α1,3galactosyltransferase knock-out pigs.

BACKGROUND: Anti-Galα1,3Galß-R natural antibodies are responsible for hyperacute rejection in pig-to-primate xenotransplantation. Although the generation of pigs lacking the α1,3galactosyltransferase (GalT) has overcome hyperacute rejection, antibody-mediated rejection is still a problem. It is possible that other enzymes synthesize antigens similar to Galα1,3Gal epitopes that are recognized by xenoreactive antibodies. The glycosphingolipid isoglobotrihexosylceramide (iGb3) represents such a candidate expressing an alternative Galα1,3Gal epitope. The present work determined whether the terminal Galα1,3Gal disaccharide is completely absent in Immerge pigs lacking the GalT using several different highly sensitive methods. METHODS: The expression of Galα1,3Gal was evaluated using a panel of antibodies and lectins by flow cytometry and fluorescent microscopy; GalT activity was detected by an enzymatic assay; and ion trap mass spectroscopy of neutral cellular membranes extracted from aortic endothelial was used for the detection of sugar structures. Finally, the presence of iGb3 synthase mRNA was tested by RT-PCR in pig thymus, spleen, lymph node, kidney, lung, and liver tissue samples. RESULTS: Aortic endothelial cells derived from GalT knockout pigs expressed neither Galα1,3Gal nor iGb3 on their surface, and GalT enzymatic activity was also absent. Lectin staining showed an increase in the blood group H-type sugar structures present in GalT knockout cells as compared to wild-type pig aortic endothelial cells (PAEC). Mass spectroscopic analysis did not reveal Galα1,3Gal in membranes of GalT knockout PAEC; iGb3 was also totally absent, whereas a fucosylated form of iGb3 was detected at low levels in both pig aortic endothelial cell extracts. Isoglobotrihexosylceramide 3 synthase mRNA was expressed in all pig tissues tested whether derived from wild-type or GalT knockout animals. CONCLUSIONS: These results confirm unequivocally the absence of terminal Galα1,3Gal disaccharides in GalT knockout endothelial cells. Future work will have to focus on other mechanisms responsible for xenograft rejection, in particular non-Galα1,3Gal antibodies and cellular responses.

Erythrocyte and porcine intestinal glycosphingolipids recognized by F4 fimbriae of enterotoxigenic Escherichia coli.

Enterotoxigenic F4-fimbriated Escherichia coli is associated with diarrheal disease in neonatal and postweaning pigs. The F4 fimbriae mediate attachment of the bacteria to the pig intestinal epithelium, enabling an efficient delivery of diarrhea-inducing enterotoxins to the target epithelial cells. There are three variants of F4 fimbriae designated F4ab, F4ac and F4ad, respectively, having different antigenic and adhesive properties. In the present study, the binding of isolated F4ab, F4ac and F4ad fimbriae, and F4ab/ac/ad-fimbriated E. coli, to glycosphingolipids from erythrocytes and from porcine small intestinal epithelium was examined, in order to get a comprehensive view of the F4-binding glycosphingolipids involved in F4-mediated hemagglutination and adhesion to the epithelial cells of porcine intestine. Specific interactions between the F4ab, F4ac and F4ad fimbriae and both acid and non-acid glycosphingolipids were obtained, and after isolation of binding-active glycosphingolipids and characterization by mass spectrometry and proton NMR, distinct carbohydrate binding patterns were defined for each fimbrial subtype. Two novel glycosphingolipids were isolated from chicken erythrocytes, and characterized as GalNAcα3GalNAcß3Galß4Glcß1Cer and GalNAcα3GalNAcß3Galß4GlcNAcß3Galß4Glcß1Cer. These two compounds, and lactosylceramide (Galß4Glcß1Cer) with phytosphingosine and hydroxy fatty acid, were recognized by all three variants of F4 fimbriae. No binding of the F4ad fimbriae or F4ad-fimbriated E. coli to the porcine intestinal glycosphingolipids occurred. However, for F4ab and F4ac two distinct binding patterns were observed. The F4ac fimbriae and the F4ac-expressing E. coli selectively bound to galactosylceramide (Galß1Cer) with sphingosine and hydroxy 24:0 fatty acid, while the porcine intestinal glycosphingolipids recognized by F4ab fimbriae and the F4ab-fimbriated bacteria were characterized as galactosylceramide, sulfatide (SO(3)-3Galß1Cer), sulf-lactosylceramide (SO(3)-3Galß4Glcß1Cer), and globotriaosylceramide (Galα4Galß4Glcß1Cer) with phytosphingosine and hydroxy 24:0 fatty acid. Finally, the F4ad fimbriae and the F4ad-fimbriated E. coli, but not the F4ab or F4ac subtypes, bound to reference gangliotriaosylceramide (GalNAcß4Galß4Glcß1Cer), gangliotetraosylceramide (Galß3GalNAcß4Galß4Glcß1Cer), isoglobotriaosylceramide (Galα3Galß4Glcß1Cer), and neolactotetraosylceramide (Galß4GlcNAcß3Galß4Glcß1Cer).

Recognition of ß-linked self glycolipids mediated by natural killer T cell antigen receptors.

The most potent foreign antigens for natural killer T cells (NKT cells) are α-linked glycolipids, whereas NKT cell self-reactivity involves weaker recognition of structurally distinct ß-linked glycolipid antigens. Here we provide the mechanism for the autoreactivity of T cell antigen receptors (TCRs) on NKT cells to the mono- and tri-glycosylated ß-linked agonists ß-galactosylceramide (ß-GalCer) and isoglobotrihexosylceramide (iGb3), respectively. In binding these disparate antigens, the NKT cell TCRs docked onto CD1d similarly, achieving this by flattening the conformation of the ß-linked ligands regardless of the size of the glycosyl head group. Unexpectedly, the antigenicity of iGb3 was attributable to its terminal sugar group making compensatory interactions with CD1d. Thus, the NKT cell TCR molds the ß-linked self ligands to resemble the conformation of foreign α-linked ligands, which shows that induced-fit molecular mimicry can underpin the self-reactivity of NKT cell TCRs to ß-linked antigens.