Matched Sizes of Activating and Inhibitory Receptor/ Ligand Pairs Are Required for Optimal Signal Integration by Human Natural Killer Cells Karsten Ko¨ hler1.¤, Shiqiu Xiong2.¤, Joanna Brzostek3, Maryam Mehrabi1, Philipp Eissmann 1 , Alice Harrison2, Shaun-Paul Cordoba2, Stephane Oddos1, Vladimir Miloserdov4, Keith Gould3, Nigel J. Burroughs4, Philip Anton van der Merwe 2 * " , Daniel M. Davis1*" 1 Division of Cell and Molecular Biology, Imperial College London, London, United Kingdom, 2 Sir William Dunn School of Pathology, Oxford University, Oxford, United Kingdom, 3 Wright-Fleming Institute, Imperial College London, London, United Kingdom, 4 Warwick Systems Biology Centre, University of Warwick, Coventry, United Kingdom Abstract It has been suggested that receptor-ligand complexes segregate or co-localise within immune synapses according to their size, and this is important for receptor signaling. Here, we set out to test the importance of receptor-ligand complex dimensions for immune surveillance of target cells by human Natural Killer (NK) cells. NK cell activation is regulated by integrating signals from activating receptors, such as NKG2D, and inhibitory receptors, such as KIR2DL1. Elongating the NKG2D ligand MICA reduced its ability to trigger NK cell activation. Conversely, elongation of KIR2DL1 ligand HLA-C reduced its ability to inhibit NK cells. Whereas normal-sized HLA-C was most effective at inhibiting activation by normal-length MICA, only elongated HLA-C could inhibit activation by elongated MICA. Moreover, HLA-C and MICA that were matched in size co- localised, whereas HLA-C and MICA that were different in size were segregated. These results demonstrate that receptor- ligand dimensions are important in NK cell recognition, and suggest that optimal integration of activating and inhibitory receptor signals requires the receptor-ligand complexes to have similar dimensions. Citation: Ko¨ hler K, Xiong S, Brzostek J, Mehrabi M, Eissmann P, et al. (2010) Matched Sizes of Activating and Inhibitory Receptor/Ligand Pairs Are Required for Optimal Signal Integration by Human Natural Killer Cells. PLoS ONE 5(11): e15374. doi:10.1371/journal.pone.0015374 Editor: Jacques Zimmer, Centre de Recherche Public de la Sante´ (CRP-Sante´), Luxembourg Received August 9, 2010; Accepted August 19, 2010; Published November 5, 2010 Copyright: 2010 Ko¨ hler et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the Biotechnology and Biological Sciences Research Council (www.bbsrc.ac.uk; BB/D011663/1) and the MRC (www.mrc.ac.uk; G0500563). DMD is supported by a Wolfson Royal Society Research Merit Award (royalsociety.org). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: anton.vandermerwe@path.ox.ac.uk (PAvdM); d.davis@imperial.ac.uk (DMD) . These authors contributed equally to this work. " These authors also contributed equally to this work. ¤ Current address: Department of Pathology, Cambridge University, Cambridge, United Kingdom Introduction NK cell activation is regulated by a balance of signals from various germ-line encoded activating receptors and inhibitory receptors [1,2]. One of the best studied activating receptors is the lectin-like receptor NKG2D, which recognises proteins that are usually not expressed on cells but are upregulated in response to cellular stress [3,4,5,6]. Signaling downstream from activating receptors commonly involves the phosphorylation of the immu- noreceptor-tyrosine based activation motifs (ITAMs). NKG2D signals via an adapter DAP10 that contains an ITAM-like motif (YXNM), which can be phosphorylated by Src-family kinases [7]. Human inhibitory NK cell receptors include members of the Killer-cell Immunoglobulin-like Receptor (KIR) and the Leuko- cyte Ig-like receptor (LILR) families, and the CD94:NKG2A heterodimer, all of which recognise class I MHC molecules. Ligation of KIR2DL1 by a subset of class I MHC proteins that includes HLA-Cw4 and -Cw6 inhibits NK cell-mediated lysis [8]. In this manner, loss of class I MHC protein from the surface, i.e. ‘‘missing self’’, can render cells susceptible to NK lysis [9,10,11,12]. KIR2DL1 signal transduction is mediated via immunoreceptor-tyrosine based inhibition motifs (ITIMs) which are phosphorylated by tyrosine kinases and then recruit SH2- domain containing phosphatases [13,14]. Lymphocyte activation involves the engagement of multiple cell surface receptors with ligands presented on the surface of other cells in a contact area termed the immunological or immune synapse (IS) [15,16,17,18,19,20]. Because lymphocyte cell surface molecules vary considerably in size it has been suggested that they may segregate according to size within the IS [21]. This hypothesis specifically suggests that receptor-ligand complexes with similar dimensions would be expected to co-localise, whereas those differing in size would be expected to segregate. Lymphocyte signaling is initiated by tyrosine phosphorylation, which is regulated by the balance between tyrosine kinases and phospha- tases at the cell membrane. The kinetic-segregation model of T cell antigen recognition proposed that signaling is initiated by size- based segregation of the engaged T cell receptor (TCR) from receptor tyrosine phosphatases with large ectodomains such as CD45 and CD148, leading to phosphorylation of the TCR/CD3 PLoS ONE | www.plosone.org 1 November 2010 | Volume 5 | Issue 11 | e15374