title: The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells.
abstract: Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such "tonic" activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Barcelona, Spain;
authors.forename: Juan A
authors.initials: JA
authors.lastname: Torreno-Pina
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Barcelona, Spain;
authors.forename: Carlo
authors.initials: C
authors.lastname: Manzo
authors.affiliation: Medical Research Council (MRC) Human Immunology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
authors.forename: Mariolina
authors.initials: M
authors.lastname: Salio
authors.affiliation: Medical Research Council (MRC) Human Immunology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
authors.forename: Michael C
authors.initials: MC
authors.lastname: Aichinger
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Barcelona, Spain;
authors.forename: Anna
authors.initials: A
authors.lastname: Oddone
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Barcelona, Spain;
authors.forename: Melike
authors.initials: M
authors.lastname: Lakadamyali
authors.affiliation: Medical Research Council (MRC) Human Immunology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom;
authors.forename: Dawn
authors.initials: D
authors.lastname: Shepherd
authors.affiliation: School of Biosciences, University of Birmingham, Birmingham B11 2TT, United Kingdom;
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
authors.affiliation: Medical Research Council (MRC) Human Immunology Unit, Weatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, United Kingdom; vincenzo.cerundolo@imm.ox.ac.uk maria.garcia-parajo@icfo.es.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cerundolo
authors.affiliation: ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain vincenzo.cerundolo@imm.ox.ac.uk maria.garcia-parajo@icfo.es.
authors.forename: Maria F
authors.identifier: http://orcid.org/0000-0001-6618-3944
authors.initials: MF
authors.lastname: Garcia-Parajo
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000938:Antigen-Presenting Cells / Q000378:metabolism*; D055610:Antigens, CD1d / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004058:Diffusion; D005699:Galactosylceramides / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000473:pathology; D008213:Lymphocyte Activation; D008954:Models, Biological; D009000:Monocytes / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry*; D055611:Natural Killer T-Cells / Q000378:metabolism*; D021381:Protein Transport; D062211:Spatio-Temporal Analysis
chemical_list: D055610:Antigens, CD1d; D005699:Galactosylceramides
keywords: cell membrane compartmentalization; iNKT cell autoreactivity; protein nanoclustering; single-particle tracking; stimulated emission depletion nanoscopy