Ligand Size Is Important for NK Signal Integration Figure 2. Elongated MICA binds NKG2D. (A) Binding of NKG2D beads. P815 expressing normal-length or elongated MICA were incubated with uncoated fluorescent beads, or beads coated with NKG2D-Fc or (control) MICA-Fc. Bead binding was analysed by flow cytometry and expressed as percentage of cells forming conjugates to bead. The mean (normalized to the number of conjugates for NKG2D beads to P815 MICA) and SD of three independent experiments are shown. (B) Conjugation of P815 and NKL cells. PKH67-labeled P815 cells expressing the indicated form of MICA were incubated for 30 min with the equal number of PKH26-labeled NKL cells and analysed by flow cytometry. The percentage of the NKL cells forming conjugates with P815 cells is shown. The data represents the mean and SD of three independent experiments. p = 0.04; 0.06; 0.02; 0.007 for P815 vs. MICA; MICA-CD2; MICA-CD22; MICA-CD4 respectively (one-tailed t-test). (C) Clustering of MICA in NKL immune synapses. P815 cells expressing the indicated length variant (or normal-length) of MICA-GFP were allowed to form contacts with NKG2D-expressing NKL cells for 10 min and imaged by live-cell confocal microscopy. Clustering was quantified as fold-enrichment of GFP fluorescence intensity within the contact area versus outside the contact area. Each point represents an individual cell/cell contact with the mean and SD shown. The scale bars represent 10 mm. doi:10.1371/journal.pone.0015374.g002 KIR2DL1-coated fluorescent beads to 221 cells expressing the various forms of Cw6. KIR2DL1-coated beads bound at comparable levels to cells expressing wild type and elongated Cw6 both in the absence (Figure 4A) and presence (Figure S4) of a titrated blocking antibody, indicating that elongation of Cw6 does not disrupt its interaction with KIR2DL1. We next examined whether elongated Cw6 could bind KIR2DL1 in the context of an intercellular contact. To do this we examined conjugate formation between KIR2DL1- expressing NKL cells and P815 cells expressing normal- length or elongated Cw6. Expression of normal-length Cw6 on P815 cells resulted in a substantial increase in conjugate formation, with over 50% of NKL cells forming conjugates (Figure 4B). A similar level of conjugate formation was seen with Cw6-CD4, indicating effective binding of KIR2DL1 at the IS (Figure 4B). As a final test of whether elongation of Cw6 affected KIR2DL1 binding we used live-cell confocal microscopy to visualize the accumulation of KIR2DL1-GFP and Cw6-mCherry at the interface between YTS and 221 transfectants (Figure 4C). This revealed that both normal-length and CD4-elongated Cw6 accumulated to similar extents and induced a similar level of accumulation of KIR2DL1; KIR2DL1 was enriched 6.262.0 fold (n = 14) at the IS with target cells expressing Cw6 and 5.061.5 fold (n = 13) for target cells expressing Cw6-CD4 (Figure 4C). The fact that KIR enrichment is not significantly different for elongated or normal-length Cw6 (t-test p.0.1) provides further evidence that both normal-length and elongated Cw6 are similarly able to bind KIR2DL1 at immune synapses. Segregation of Cw6 by size at the immune synapse We next tested whether Cw6 and Cw6-CD4 segregate or co- localise within the IS. We expressed normal-length and/or elongated Cw6 fused to different fluorescent proteins in 221 cells and visualized by confocal microscopy their location at inhibitory synapses with YTS-KIR2DL1 (Figure 5). Strikingly, elongated Cw6-CD4-GFP, segregated from normal-length Cw6-mCherry (Pearson coefficient r = 20.2860.17 (SD), n = 10; Figure 5). Interestingly, the longer Cw6 tended to accumulate at the periphery of the IS (Figure S5). Importantly, normal-length Cw6-GFP and Cw6-mCherry co-localised at the IS, confirming that the type of the fluorescent tag itself did not affect segregation (Pearson coefficient r = 0.7760.14, n = 6; Figure 5). Finally, elongated Cw6-CD4-GFP co-localised with Cw6-CD4-mCherry (r = 0.7160.22, n = 7). These results demonstrate that differences in size are sufficient to drive segregation of proteins within the IS. Matching activating and inhibitory receptor/ligand size is important for signal integration Our finding that size-compatibility may be required for colocalisation raised the possibility that the elongation of HLA- Cw6 abrogates its inhibitory effect on NK cell activation because it prevents colocalisation of engaged KIR2DL1 with the engaged activating receptor(s) within the IS. We tested this possibility by varying the dimensions of both activating and inhibitory ligands simultaneously. Specifically, we compared lysis by KIR2DL1- expressing NKL cells of P815 cells expressing different combina- tions of normal-length or elongated MICA and Cw6 at matching PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e15374