The Size of Activating and Inhibitory Killer Ig-like Receptor Nanoclusters Is Controlled by the Transmembrane Sequence and Affects Signaling

Kevin Stacey,Philippa R. Kennedy,David J. Williamson,Anna Oszmiana,David J. Morgan,Shaun-Paul Cordoba,Daniel M. Davis

doi:10.1016/j.celrep.2016.04.075

Kevin Stacey, Philippa R. Kennedy + Show 5 more

Open Access

https://doi.org/10.1016/j.celrep.2016.04.075

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Abstract

SummarySuper-resolution microscopy has revealed that immune cell receptors are organized in nanoscale clusters at cell surfaces and immune synapses. However, mechanisms and functions for this nanoscale organization remain unclear. Here, we used super-resolution microscopy to compare the surface organization of paired killer Ig-like receptors (KIR), KIR2DL1 and KIR2DS1, on human primary natural killer cells and cell lines. Activating KIR2DS1 assembled in clusters two-fold larger than its inhibitory counterpart KIR2DL1. Site-directed mutagenesis established that the size of nanoclusters is controlled by transmembrane amino acid 233, a lysine in KIR2DS1. Super-resolution microscopy also revealed two ways in which the nanoscale clustering of KIR affects signaling. First, KIR2DS1 and DAP12 nanoclusters are juxtaposed in the resting cell state but coalesce upon receptor ligation. Second, quantitative super-resolution microscopy revealed that phosphorylation of the kinase ZAP-70 or phosphatase SHP-1 is favored in larger KIR nanoclusters. Thus, the size of KIR nanoclusters depends on the transmembrane sequence and affects downstream signaling.

Highlights

Natural killer (NK) cells are part of our defense against cancer and viral infections and are of medical importance in cancer immunotherapy and bone marrow transplantation (Vivier et al, 2012; Davis, 2014; Della Chiesa et al, 2014; Foley et al, 2014)
As ligation of KIR2DL1-HA inhibited the formation of a dense ring of peripheral F-actin at the contact interface, and the secretion of interferon (IFN)-g, in cells activated via NKG2D (Figures S1D and S1G)
The nanoscale organization of KIR2DL1 and KIR2DS1 at the cell surface was compared using ground state depletion microscopy followed by individual molecule return (GSDIM)

Summary

Introduction

Natural killer (NK) cells are part of our defense against cancer and viral infections and are of medical importance in cancer immunotherapy and bone marrow transplantation (Vivier et al, 2012; Davis, 2014; Della Chiesa et al, 2014; Foley et al, 2014) Their activity depends on the balance of signals from germ-line encoded activating and inhibitory receptors. The KIR family includes activating receptors, which share ligand specificity with their inhibitory counterparts due to structural homology of extracellular domains (Ivarsson et al, 2014; Biassoni et al, 1997) One example of such a pairing are receptors KIR2DL1 and KIR2DS1, which bind to human leukocyte antigen (HLA) proteins from the C2 group (Stewart et al, 2005; Sivori et al, 2011). KIR2DS1 in the telomeric region of the KIR B haplotype was shown to have a protective effect against complications in pregnancy (Xiong et al, 2013; Hiby et al, 2010)

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