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Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapses
Bebhinn Treanor1,
Peter M.P. Lanigan2,
Sunil Kumar1,2,
Chris Dunsby2,
Ian Munro2,
Egidijus Auksorius2,
Fiona J. Culley1,
Marco A. Purbhoo1,
David Phillips3,
Mark A.A. Neil2,
Deborah N. Burshtyn4,
Paul M.W. French2, , and
Daniel M. Davis1
1 Division of Cell and Molecular Biology, 2 Department of Physics, and 3 Department of Chemistry, Imperial College London, London SW7 2AZ, England, UK 4 Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta T6G 2E1, Canada
Correspondence to Daniel M. Davis: d.davis{at}imperial.ac.uk
Abstract:
We report the supramolecular organization of killer Ig–likereceptor (KIR) phosphorylation using a technique applicableto imaging phosphorylation of any green fluorescent protein–taggedreceptor at an intercellular contact or immune synapse. Specifically,we use fluorescence lifetime imaging (FLIM) to report Försterresonance energy transfer (FRET) between GFP-tagged KIR2DL1and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody.Visualization of KIR phosphorylation in natural killer (NK)cells contacting target cells expressing cognate major histocompatibilitycomplex class I proteins revealed that inhibitory signalingis spatially restricted to the immune synapse. This explainshow NK cells respond appropriately when simultaneously surveyingsusceptible and resistant target cells. More surprising, phosphorylatedKIR was confined to microclusters within the aggregate of KIR,contrary to an expected homogeneous distribution of KIR signalingacross the immune synapse. Also, yellow fluorescent protein–taggedLck, a kinase important for KIR phosphorylation, accumulatedin a multifocal distribution at inhibitory synapses. Spatialconfinement of receptor phosphorylation within the immune synapsemay be critical to how activating and inhibitory signals areintegrated in NK cells.
Abbreviations used in this paper: FLIM, fluorescence lifetimeimaging; FRET, Förster resonance energy transfer; HLA,human leukocyte antigen; IS, immunological synapse; ITIM, immunoreceptortyrosine-based inhibition motif; KIR, killer Ig–like receptor;MHC, major histocompatibility complex; mYFP, monomeric YFP;NK, natural killer; SHP, Src homology protein tyrosine phosphatase.
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