title: Presentation of alpha-galactosylceramide by murine CD1d to natural killer T cells is facilitated by plasma membrane glycolipid rafts.
abstract: CD1 molecules are non-polymorphic major histocompatibility complex class I-related proteins that bind and present glycolipid antigens to T-cell antigen receptors (TCR) expressed by alphabeta T cells or natural killer-like T cells (NKT). Anti-metastatic properties of NKT cells reactive to the CD1d-binding antigen alpha-galactosylceramide (alpha-GalCer) are now being explored as a contributor to tumour cell killing. In this study, we tested the hypothesis that presentation of alpha-GalCer by murine CD1d (mCD1d) to mCD1d-restricted NKT cells was facilitated by plasma membrane glycolipid rafts. Confocal microscopy of mCD1d-transfected A20 B cells (A20mCD1d) demonstrated that mCD1d was raft-localized. This observation was confirmed by immunoblotting of raft fractions isolated on sucrose density gradients. Raft disruption by the cholesterol-binding agent nystatin, or short-chain ceramides, inhibited presentation of low concentrations of alpha-GalCer to NKT cells. Inhibition of antigen presentation was reversed by treatment of A20mCD1d cells with higher alpha-GalCer concentrations, or removal of raft-disrupting agents. These data indicate that partitioning of mCD1d into membrane rafts increases the capacity of antigen-presenting cells to present limiting quantities of glycolipid antigens, perhaps by stabilizing mCD1d/antigen structures on the plasma membrane and optimizing TCR engagement on NKT cells.
authors.affiliation: Department of Microbiology and Immunology, Dartmouth Medical School, Lebanon, NH 03756, USA.
authors.forename: Gillian A
authors.initials: GA
authors.lastname: Lang
authors.forename: Sergei D
authors.initials: SD
authors.lastname: Maltsev
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.forename: Mark L
authors.initials: ML
authors.lastname: Lang
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D018949:Antigens, CD1 / Q000276:immunology*; D055610:Antigens, CD1d; D001402:B-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005699:Galactosylceramides / Q000276:immunology*; D006017:Glycolipids / Q000032:analysis* / Q000378:metabolism; D015151:Immunoblotting / Q000379:methods; D007376:Interleukin-2 / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology*; D051379:Mice; D018613:Microscopy, Confocal
chemical_list: D018949:Antigens, CD1; D055610:Antigens, CD1d; D005699:Galactosylceramides; D006017:Glycolipids; D007376:Interleukin-2