Ligand Size Is Important for NK Signal Integration Figure 5. Size differences drive segregation of different length variants of HLA-Cw6 within immune synapses. 221 cells double- transfected with normal-length or CD4-elongated forms of Cw6 fused to either GFP (green) or mCherry (red) were incubated for 30 min with KIR2DL1-expressing YTS transfectants and conjugates were imaged by live-cell confocal microscopy. En-face views of the synapse were reconstructed and the Pearson correlation coefficient (r) for GFP and mCherry fluorescence were determined. Mean 6 SD of the total number of conjugates is shown. The scale bar represents 10 mm. doi:10.1371/journal.pone.0015374.g005 KIR2DL1-Cw6 complex, and the fact that signaling through KIR2DL1 is initiated by tyrosine phosphorylation of its cytoplas- mic domain ITIM motif by Src kinases, it is plausible that KIR2DL1 signals through the kinetic-segregation mechanism. An alternative possibility is that KIR2DL1 is phosphorylated by Src kinases associated with other nearby small receptors, and that elongation of Cw6 inhibits signalling by preventing colocalization of KIR2DL1 with these receptors. Further studies are required to distinguish between these mechanisms. Our finding that normal-length and elongated Cw6 spontane- ously segregated at the IS shows that size differences alone are sufficient to drive segregation of receptor/ligand complexes in the IS. It has previously been shown that HLA-Cw6 segregated from regions enriched in ICAM-1 in a manner largely independent of cytoskeletal or ATP-dependent processes [17,37,50]. In addition, proteins were seen to segregate at synapses when expressed in insect cells thought not to express other ligands for human NK cells [51]. Our finding that differences in the molecular size alone are sufficient to drive segregation at the NK IS provides an explanation for these observations. Inhibitory NK receptors such as KIR2DL1 abrogate NK cell activation by recruiting to the plasma membrane tyrosine phosphatases such as SHP-1 and SHP-2 and the inositol lipid phosphatases SHIP, where they directly oppose or reverse phosphorylation triggered by engagement of activating receptors [2]. This suggests that for inhibitory receptors to be effective they would need to co-localise with engaged activating receptors in the IS. This is supported by a recent study of NK activation on surfaces presenting micropatterned NK receptor ligands [52]. Our observa- tion that differences in size alone result in segregation suggests that inhibitory receptor-ligand complexes would need to be similar in size to activating receptor-ligand complexes to colocalise with them and inhibit the activating signals. To test this hypothesis, we used cells expressing both normal-length or elongated Cw6 and normal-length or elongated MICA, in all 4 possible combinations. Our results confirmed that matched sizes of MICA and Cw6 proteins were important for co-localization within the IS and for signal integration; Cw6 and MICA colocalised best when they were similar in size. Similarly, normal sized Cw6 could only inhibit activation by normal- length MICA, while elongated Cw6 could only inhibit stimulation by elongated MICA. Thus, optimal signal integration between NKG2D and KIR2DL1 requires that NKG2D/MICA and KIR2DL1/Cw6 complexes are matched in size, probably because this is required for their colocalisation within the IS. PLoS ONE | www.plosone.org 7 November 2010 | Volume 5 | Issue 11 | e15374