Pelleted cells were sonicated for 5 s, and the samples then analysed using SDS-PAGE gel and Western Blot. Membranes were stained with Ab against SLP-76 phospho-tyrosine 113 (Abcam, Cam- bridge, UK) (1:1000), followed by peroxidase-conjugated second- ary antibody (1:4000), and visualized by X-ray film. Microscopy and Image Analysis Protein localization at NK/target cell synapses was determined by imaging live cells at 37uC/5% CO2 in 8-well glass bottom chamberslides (Labtek) using an inverted laser-scanning confocal fluorescence microscope (SP5, Leica) equipped with a 63x 1.2 water immersion objective, and a resonance scanner. Laser wavelengths of 488 and 591 nm were used for the excitation of GFP and mCherry, respectively. En-face views of the intercellular contact were rendered from multiple optical slices (Volocity, Improvision, Waltham, MA, USA). Quantification of fluorescence at the intercellular contact, and the determination of Pearson correlation coefficient r for the GFP and mCherry channel at the IS were performed using ImageJ (National Institute of Health). Supporting Information Figure S1 MICA elongation affects NK cell activation. (A) MICA elongation affects intracellular IFN-c. NKL cells were incubated for 1 h with equal numbers of P815 cells expressing the indicated length variant (or normal-length) MICA before adding Brefeldin A (5 mg/ml) and incubating for a further 4 h. Cells were then fixed and permeabilized, stained with PE-conjugated IFN-c mAb, and analysed by flow cytometry. The percentage of IFN-c+ NK cells is shown. (B) MICA elongation affects degranulation. NKL cells were incubated for 5 h in the presence of monensin with equal numbers of P815 cells expressing the indicated MICA. Degranulation was measured by CD107a antibody staining. The percentage of CD107a-positive NK cells is shown. These data represent the mean and SD of two independent experiments. Found at: doi:10.1371/journal.pone.0015374.s001 (TIF) Figure S2 NKG2D-bead binding to MICA-expressing P815 cells in the presence of blocking antibody. Fluorescent streptavidin beads (Spherotech) were functionalized with NKG2D as described for Figure 2A. The indicated P815 cells were pre- treated with a MICA blocking Ab (R&D) at the indicated dilution (stock solution 50 mg/ml) in PBS +5% mouse serum for 30 min. NKG2D-coated beads were then incubated with these cells for 1 h at room temperature the number of bead-conjugated cells determined by flow cytometry. The data are a representative of 2 independent experiments. Found at: doi:10.1371/journal.pone.0015374.s002 (TIF) Figure S3 Cw6 elongation abrogates inhibition of NK cell activation. YTS-KIR2DL1 cells were co-incubated with 221 cells expressing the indicated variant of Cw6 and intracellular IFN-c was measured as described in Figure S1A. Data represent the mean and range of two independent experiments. Found at: doi:10.1371/journal.pone.0015374.s003 (TIF) References 1. Vivier E, Nunes JA, Vely F (2004) Natural killer cell signaling pathways. Science 306: 1517–1519. 2. Lanier LL (2008) Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol 9: 495–502. 3. Bauer S, Groh V, Wu J, Steinle A, Phillips JH, et al. (1999) Activation of NK cells and T cells by NKG2D, a receptor for stress- inducible MICA. Science 285: 727–729. 4. Wu J, Song Y, Bakker AB, Bauer S, Spies T, et al. (1999) An activating immunoreceptor complex formed by NKG2D and DAP10. Science 285: 730–732. 5. Raulet DH, Guerra N (2009) Oncogenic stress sensed by the immune system: role of natural killer cell receptors. Nat Rev Immunol 9: 568–580. Ligand Size Is Important for NK Signal Integration Figure S4 KIR2DL1-bead binding to Cw6-expressing 221 cells in the presence of blocking antibody. Fluorescent streptavidin beads were functionalized with KIR2DL1-Fc (a kind gift from Peter Parham, Stanford), as described for Figure 2A, and then pre-incubated with the indicated dilution of HP3E4 (stock solution 1:10 hybridoma supernatant). These KIR2DL1-coated beads were then incubated in PBS +5% mouse serum for 1 h at room temperature with the indicated 221 cells, and the number of bead-conjugated cells determined by flow cytometry. The data is representative of 2 independent experiments. Found at: doi:10.1371/journal.pone.0015374.s004 (TIF) Figure S5 Elongation of Cw6 increases clustering at the synapse periphery. 221 cells expressing GFP-tagged versions of HLA-Cw6 heavy chain (i.e. wild type Cw6) or normal-length (Cw6) or elongated (Cw6-CD4) single chain trimer versions of HLA-Cw6 were incubated with YTS-KIR2DL1 cells for 30 min and conjugates imaged by confocal microscopy. The relative location of the GFP clusters was determined by dividing the distance between the cluster centre and synapse centre by the diameter of the synapse, plotted at the y-axis. Each point represents an individual cell/cell contact with the mean and SD shown as lines, * denotes p,10-5. Found at: doi:10.1371/journal.pone.0015374.s005 (TIF) Figure S6 The size of Cw6 and MICA influences the integration of their receptor signals. (A) NKL cells expressing the inhibitory receptor KIR2DL1 were incubated with the indicated P815 cells for 16 h before supernatants were harvested and IFN-c measured. To augment IFN-c secretion triggered by elongated MICA, IL-2 (100 U/ml) and IL-12 (10 ng/ ml) were added exogenously. Data shown is representative of three independent experiments (mean 6 SD of triplicates). (B) P815 cells expressing the indicated molecules were incubated with equal numbers of KIR2DL1-expressing NKL cells for 30 min. The number of conjugates was determined as in Figure 2B. Found at: doi:10.1371/journal.pone.0015374.s006 (TIF) Acknowledgments We thank Katsumi Maenaka for making the plasmid encoding KIR2DL1- CDd34 and Peter Parham for the KIR2DL1-Fc construct, Marion H. Brown for help with using it for bead binding experiments. We thank Martin Spitaler and Christian Liebig (FILM facility, Imperial College) for expert technical assistance in microscopy, Niga Nawroly, Fredrik Wallberg, Nigel Rust and Jane Srivastava for help with flow cytometry and cell sorting, Marcus Bridge for help with plasmid purification and ELISA and all members of our laboratories for discussions. Author Contributions Conceived and designed the experiments: KK SX MM PE AH SC SO KG PV DD. Performed the experiments: KK SX MM AH SC. Analyzed the data: KK SX MM PE AH SC SO VM KG PV DD. Contributed reagents/materials/analysis tools: PE SO VM KG NB. Wrote the paper: KK SX PV DD. 6. Gleimer M, Parham P (2003) Stress management: MHC class I and class I-like molecules as reporters of cellular stress. Immunity 19: 469–477. 7. Billadeau DD, Upshaw JL, Schoon RA, Dick CJ, Leibson PJ (2003) NKG2D- DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathway. Nat Immunol 4: 557–564. 8. Colonna M, Samaridis J (1995) Cloning of immunoglobulin-superfamily members associated with HLA-C and HLA-B recognition by human natural killer cells. Science 268: 405–408. 9. Hoglund P, Glas R, Ohlen C, Ljunggren HG, Karre K (1991) Alteration of the natural killer repertoire in H-2 transgenic mice: specificity of rapid lymphoma PLoS ONE | www.plosone.org 10 November 2010 | Volume 5 | Issue 11 | e15374