Ligand Size Is Important for NK Signal Integration Figure 3. Elongation of HLA-Cw6 abrogates its inhibitory effect. (A) Normal-length and elongated forms of HLA-Cw6 were expressed on 221 cells at matched (high or low) levels. (B) 221 target cells expressing normal-length or elongated Cw6 were incubated with KIR2DL1-expressing YTS effector cells for 5 h at the indicated E:T ratios. Lysis of 221 cells was assayed as in Figure 1B. (C) YTS-KIR2DL1 cells were stimulated with 221 cells expressing no, low or high levels of the indicated form of Cw6 for 16 h. Supernatants were harvested and IFN-c secretion quantified by ELISA. Data in B and C are representative results from four independent experiments. Error bars are SD of triplicate measurements. doi:10.1371/journal.pone.0015374.g003 levels of expression (Figure 6A). As shown previously, expression of normal-length MICA on P815 cells provoked killing by NKL cells (Figure 6B). Simultaneous expression on these cells of normal- length Cw6 was able to inhibit killing more effectively than expression of elongated Cw6 (Figure 6B). We next examined the effect of varying Cw6 length when MICA is elongated. Since elongation of MICA abrogates NKL activation (see Figure 1C, E) we augmented NKL activation by adding IL-2 and IL-12 to these assays. Under these conditions elongated MICA elicited killing, albeit more weakly that normal length MICA (Figure 6B). In contrast to the results obtained with normal length MICA, in P815 cells expressing elongated MICA simultaneous expression on normal length Cw6 did not inhibit killing, whereas expression of elongated Cw6 was able to inhibit killing (Figure 6B). Thus optimal inhibition by Cw6 of MICA-mediated killing was observed when Cw6 and MICA where matched in size. Similar results were obtained using IFN-c secretion as a readout of NKL cell activation (Figure S6A). Simultaneous expression of normal-length Cw6, but not elongated Cw6, was able to inhibit IFN-c secretion induced by normal-length MICA (Figure S6A). Indeed, elongated Cw6 actually augmented IFN-c secretion induced by MICA (Figure S6A). Conversely, normal length Cw6 but not elongated Cw6 augmented IFN-c secretion induced by elongated MICA (Figure S6A). These positive effects probably arises from the greatly improved adhesion of NKL cells to P815 cells mediated by the KIR2DL1 interaction with normal-length or elongated Cw6 (Figure S6B), and may be particularly evident here as NKL and P815 cells are from different species and so lack endogenous adhesion receptor/ligand pairs. Furthermore, the secretion of cytokines is more dependent on the stability of the intercellular contact than target cell killing [45]. Taken together, these results indicate that the ligands MICA and Cw6 need to be matched in size for appropriate integration of positive and negative signals from their respective receptors. A plausible explanation for this is that the inhibitory KIRL2DL1- Cw6 complex needs to span the same distance as the activating NKG2D-MICA complex in order to colocalise with it. To examine this directly we used confocal microscopy to determine the distribution of normal-length and elongated forms of MICA- GFP and Cw6-mCherry within IS between KIR2DL1-expressing NKL and 221 cells (Figure 6C). Normal-length HLA-Cw6 co- localised with normal-length MICA (Pearson’s correlation coefficient, r = 0.6560.14 (SD), n = 12) whilst elongated MICA- CD4 segregated from normal-length Cw6 (r = 20.1660.33, n = 11). Conversely elongated Cw6-CD4 co-localised with elongated MICA-CD4 (r = 0.5160.19, n = 12) but not with normal-length MICA (r = 20.0760.33, n = 13). This indicates that co-localization of activating and inhibitory receptors within the IS is dependent on matching ligand/receptor complex dimensions. PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e15374