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Published online 26 June 2006. doi:10.1083/jcb.200601108
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 174, Number 1, 153-161
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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

We report the supramolecular organization of killer Ig–like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein–tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein–tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.

Abbreviations used in this paper: FLIM, fluorescence lifetime imaging; FRET, Förster resonance energy transfer; HLA, human leukocyte antigen; IS, immunological synapse; ITIM, immunoreceptor tyrosine-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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