complex [22]. A number of studies have provided support for this model [23]. Firstly, molecules at the T cell IS do segregate according to size [15,16,24], although active, cytoskeletally-driven transport processes are also involved [25]. Secondly, CD45 and CD148 are excluded from the site of TCR engagement and triggering [26,27]. Thirdly, truncation of CD45 and CD148 ectodomains [28,29] and elongation of TCR ligands [30,31] abrogate TCR triggering. However, whereas T cell activation is controlled primarily by a single receptor, i.e. the TCR, NK cells are regulated by the integration of signals from a number of activating and inhibitory receptors [32]. Thus, it is not immediately obvious how the dimensions of protein complexes would influence signal integration by multiple receptors expressed by NK cells. Intriguingly, both the inhibitory HLA/KIR and activating NKG2D/MICA complexes are small, spanning 10–15 nm [33,34,35], and have been shown to segregate within the IS from the larger [,40 nm [21,36]] integrin/ ligand complex LFA1/ICAM-1, even in the absence of any active or cytoskeletally-driven process [17,19,37,38]. Here we explore the role of ectodomain size in NK cell activation by manipulating the length of activating and/or inhibitory receptor ligands. Specifically, we analysed the effects of elongating MICA and/or HLA-Cw6 on NK cell activation or inhibition, and examine how size influences segregation within the NK cell IS. We show that ligand elongation abrogates both activating and inhibitory receptor function. We also demonstrate that size compatibility is important for the colocalization of, and signal integration between, engaged activating and inhibitory receptors, and that differences in ectodomain size are sufficient to drive segregation within the IS. This suggests a general principle for the importance of proteins size in how signals integrate from multiple receptor/ligand interactions across intercellular contacts. Results The size of MICA is important in triggering NKG2D- dependent lysis The size of MICA was varied by inserting 2, 3 or 4 independently-folding Ig domains from CD2, CD22, or CD4 respectively [30,31], in the stalk region linking the transmembrane and extracellular domains (Figure 1A). These inserts were chosen because they are structurally well-characterised and have previ- ously been shown to elongate cell surface proteins without disrupting their ligand binding properties [30,31]. These differ- ently-sized variants of MICA were then expressed in the mouse mastocytoma cell line P815, which does not express any ligand for human NKG2D endogenously. Transfected P815 cells were selected for comparable expression levels by fluorescence-activated cell sorting (Figure 1A) and then tested for their susceptibility to lysis by NK cells (Figure 1B). Expression of normal-length MICA was sufficient to induce lysis of P815 cells by the immortal human NK cell tumor line NKL. However, elongation of the MICA- ectodomain significantly reduced its ability to trigger lysis. Indeed, the ability of MICA to trigger NK cell cytotoxicity varied inversely with its length. To test whether elongation affected other functional responses, we also compared the ability of normal-length and elongated MICA to trigger IFN-c production by NKL cells. While normal- length MICA was sufficient to trigger IFN-c, MICA elongated by 2 or 3 Ig domains was less stimulatory and MICA extended by 4 Ig domains did not elicit any IFN-c response (Figure 1C). Similar results were obtained when we assayed intracellular IFN-c production by flow cytometry, confirming that this effect was the result of decreased IFN-c production rather than increased Ligand Size Is Important for NK Signal Integration consumption (Figure S1A). Elongation of MICA also abrogated NK cell degranulation as measured by induction of CD107a expression on the cell surface (Figure S1B). Finally, elongation of MICA also inhibited target cell lysis and IFN-c production by primary NK cells (Figure 1D, E). In order to examine the effect of elongation of MICA on earlier activation events we also examined the phosphorylation of SLP- 76, an adapter protein involved in the early stages of NKG2D signaling [39]. Incubation of NKL cells with MICA-expressing P815 cells induced SLP-76 phosphorylation within 2 min of co- incubation. The extent of phosphorylation was dramatically decreased by elongation of MICA (Figure 1F). Thus, elongation of MICA abrogates the early stages of NKG2D signaling as well as later cellular responses. Elongated MICA proteins bind to NKG2D To test the ability of elongated MICA to be bound by NKG2D, we compared the binding of NKG2D-coated beads to P815 cells expressing normal-length versus elongated forms of MICA (Figure 2A). NKG2D-coated beads bound at similar levels to P815 cells expressing normal-length or elongated MICA both in the absence (Figure 2A) and presence (Figure S2) of a titrated blocking antibody, indicating that the different length variants of MICA had a comparable ability to bind NKG2D. To examine whether elongated variants of MICA could bind NKG2D in the context of an intercellular contact, we used flow cytometry to measure conjugate formation between NKL cells and P815 transfectants (Figure 2B). Expression of all MICA variants on P815 cells resulted in a comparable increase in conjugate formation, indicating that the elongated forms of MICA are able to bind NKG2D in a cell-cell interface (Figure 2B). As a final test of the ability of elongated MICA to bind NKG2D we examined their clustering in IS between NKL and P815 cells expressing variants of MICA tagged with GFP, using confocal fluorescent microscopy. Normal-length MICA-GFP accumulated at the IS, leading to a 6–8 fold increase in fluorescence intensity compared to elsewhere at the cell surface (Figure 2C). MICA elongated with 2 (MICA-CD2) or 4 (MICA-CD4) Ig domains clustered at the IS to a similar extent (Figure 2C), providing further confirmation that elongation of MICA does not abrogate binding to NKG2D at the cell-cell interface. Elongation of HLA-Cw6 reduces KIR2DL1-mediated inhibition Since the length of MICA was shown to be important for NKG2D-dependent activation, we next examined whether recognition by inhibitory receptors was also influenced by the size of their ligands. For this we used the well-characterised cell line YTS, which is activated principally via engagement of the 2B4 and CD28 receptors [40,41] and does not endogenously express inhibitory receptors for HLA-C. YTS cells transfected to express the inhibitory receptor KIR2DL1 (YTS-KIR2DL1) can be inhibited by expression on target cells of the KIR2DL1 ligand HLA-Cw6 [42]. To allow us to vary the size of HLA-Cw6 without confounding effects on peptide loading and HLA-Cw6 assembly we expressed it as a single-chain trimer fusion protein (hereafter termed Cw6) comprising a peptide, b2-microglobulin, and the HLA-Cw6 heavy chain joined by flexible glycine/serine linkers [43]. The size of Cw6 was varied, as with MICA, by inserting Ig domains from CD2, CD22, or CD4 into the stalk region linking the transmembrane and extracellular domains (Figure 3A). Transfectants were selected to express these constructs at comparable (high and low) levels on 721.221 cells (hereafter PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e15374