title: Species Specific Differences of CD1d Oligomer Loading In Vitro.
abstract: CD1d molecules are MHC class I-like molecules that present glycolipids to iNKT cells. The highly conserved interaction between CD1d:α-Galactosylceramide (αGC) complexes and the iNKT TCR not only defines this population of αβ T cells but can also be used for its direct identification. Therefore, CD1d oligomers are a widely used tool for iNKT cell related investigations. To this end, the lipid chains of the antigen have to be inserted into the hydrophobic pockets of the CD1d binding cleft, often with help of surfactants. In this study, we investigated the influence of different surfactants (Triton X-100, Tween 20, Tyloxapol) on in vitro loading of CD1d molecules derived from four different species (human, mouse, rat and cotton rat) with αGC and derivatives carrying modifications of the acyl-chain (DB01-1, PBS44) and a 6-acetamido-6-deoxy-addition at the galactosyl head group (PBS57). We also compared rat CD1d dimers with tetramers and staining of an iNKT TCR transductant was used as readout for loading efficacy. The results underlined the importance of CD1d loading efficacy for proper analysis of iNKT TCR binding and demonstrated the necessity to adjust loading conditions for each oligomer/glycolipid combination. The efficient usage of surfactants as a tool for CD1d loading was revealed to be species-specific and depending on the origin of the CD1d producing cells. Additional variation of surfactant-dependent loading efficacy between tested glycolipids was influenced by the acyl-chain length and the modification of the galactosyl head group with PBS57 showing the least dependence on surfactants and the lowest degree of species-dependent differences.
authors.affiliation: Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Paletta
authors.affiliation: Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany.
authors.forename: Alina Suzann
authors.initials: AS
authors.lastname: Fichtner
authors.affiliation: Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Starick
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, United States of America.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Porcelli
authors.affiliation: Department of Chemistry, Brigham Young University, Provo, UT 84602, United States of America.
authors.forename: Paul B
authors.initials: PB
authors.lastname: Savage
authors.affiliation: Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Herrmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D055610:Antigens, CD1d / Q000737:chemistry* / Q000378:metabolism*; D002460:Cell Line; D006017:Glycolipids / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D055611:Natural Killer T-Cells / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D051381:Rats; D016415:Sequence Alignment; D013045:Species Specificity; D013501:Surface-Active Agents / Q000494:pharmacology
chemical_list: D055610:Antigens, CD1d; D006017:Glycolipids; D013501:Surface-Active Agents