[57]. Primary NK cells were stimulated in 200 U/ml IL-2 for 4–5 days before the functional assays unless stated otherwise. Ethical approval was obtained from the Riverside Health Centre Ethics Committee (London, UK) and written informed consent was obtained from donors before obtaining and using their cells. Fusion protein constructs Constructs encoding MICA and a single chain trimer version of HLA-Cw6 (hereafter termed Cw6) were generated in pcDNA3.1+ using a similar approach to that previously described [30,31]. The junctions between MICA or Cw6 and C-terminal GFP/mCherry were (single letter amino acid code) EGAADPPVATMVSKG and ACKAGGRSMVSKG, respectively (junctional sequences under- lined). The sequences preceding the additional junctional sequences are the C-termini of MICA and Cw6, and the sequence following them are the identical N-termini of GFP or mCherry. To facilitate the insertion of protein spacers, in-frame BamHI sites were introduced immediately after Thr292 and Trp288 in the stalk regions of MICA and Cw6, respectively. This required the mutation to Asp of His293 and Glu289 in MICA and Cw6, respectively. Fragments encoding the ectodomains of human CD2 and CD4, and the three N-terminal Ig domains of the mouse CD22 ectodomain were generated and inserted into these BamHI sites, as previously described [30,31]. To facilitate the transfer of these constructs into the lentiviral vector pHR-SIN-BX-IRES-Em, an MluI site was introduced 6 base pairs upstream of the Not I site. The fragments encoding normal-length and elongated MICA and Cw6 with C-terminal tags of GFP or mCherry were amplified using forward and reverse primers that added Xho I and Mlu I sites, respectively, and then inserted into the XhoI/MluI sites of pHR-SIN-BX-IRES-Em. NKL-KIR2DL1-CFP transfectants was generated as follows: First, EGFP in the pEGFPN1 vector was exchanged with mCFP as a BamHI/BsrGI fragment, resulting in pMCFPN1. Then, KIR2DL1 was subcloned into pMCFPN1 from pcDNA3.1- KIR2DL1-CGFP (a kind gift from M. Purbhoo) using the HindIII/BamHI restriction sites. KIR2DL1-CFP was then subcloned from pMCFPN1-KIR2DL1 into the pIB2 vector transferring a blasticidin resistance (a kind gift from M. Purbhoo). Expression of cell surface proteins Normal-length and elongated versions of Cw6 and MICA fused to GFP or mCherry [58] were expressed in target cells using lentiviral transduction. For this, 70–80% confluent 293T cells in a 175 cm2 flask were transfected with 14 mg expression vector pHR- SIN-BX-IRES-Em; 21 mg Vpack 11 mg VSVG (Stratagene, La Jolla, CA, USA), in 10 mM polyethylenimine. Supernatants cultured from 293T transfectants were taken 2 days later and concentrated 20 times. 26106 221 or P815 cells were transduced with 1 ml lentivirus-containing supernatant for 4 hours, then cultured in complete medium for another 2 days. NKL were transduced by retrovirus produced by transfection of the Phoenix packaging cell line and selected with blasticidin. Transduced cells were sorted by flow cytometry (FACS DiVa/Aria II, BD/DAKO) based on fusion protein fluorescence and/or surface antigen staining. Fusion proteins were detected using anti-MICA staining (R&D, Abingdon, UK), followed by anti-mouse Alexa633, or Cy5- labeled anti-HLA (clone W6/32, ATCC). Fc receptors of P815 cells were pre-blocked by adding 2 mg/ml anti-CD16/CD32 (Fc block, BD, Oxford, UK) prior to antibody staining. Functional and binding assays For NK cell activation assays, sorted transfectants of matching expression levels of the different length MICA/HLA were Ligand Size Is Important for NK Signal Integration selected. NK cell cytotoxicity was determined using standard [35S]-Met radioactive release assays for 5 h. In order to validate the ability of the MICA-fusion proteins to engage NKG2D, transfected cells were analyzed using conjugation assays with NKG2D beads. For this, streptavidin beads (Bangs Laboratories, Fishers, IN, USA) were coated with biotinylated anti-human Fc (Sigma, 5 mg/ml in PBS +0.1% BSA) at 4uC for 1 h under agitation. After washing three times with PBS, beads were coated with 10 mg/ml NKG2D-Fc fusion protein, or 10 mg/ ml MICA-Fc fusion protein as a negative control (both R&D), followed by labelling with additionally anti-mouse-Alexa 633 (Molecular Probes). Following three washing steps with PBS, the beads were mixed with the target cells in a 96-well plate to a final density of 106 cells each, briefly spun down and allowed to form conjugates for 1 h before fixing with ice-cold paraformaldehyde (total concentration 1%), followed by flow cytometry. A similar bead binding assay was used to check the ability of the normal-length and elongated Cw6-fusion proteins to bind KIR2DL1. KIR2DL1 was expressed as a fusion protein KIR2DL1-CD4d34 comprising the entire KIR2DL1 ectodomain followed by rat CD4 domains 3 and 4 and the BirA biotinylation tag added at the C-terminus [59]. The sequence at the junction between KIR2DL1 and the CD4 was GNPRHLHGSTSITAYK (KIR2DL1 underlined). KIR2DL1-CD4d34 and a control protein CD4d34 lacking the KIR2DL1 portion were expressed, biotiny- lated, and coated onto avidin fluorescent beads (Spherotech) as previously described [59]. KIR2DL1-CD4d34 or CD4d34-coated fluorescent beads (15 mL) were incubated with 0.5 * 106 221 cells expressing various Cw6 proteins at 4uC for 2 hours and bead binding to 221 cells was measured by flow cytometry. For antibody-mediated blocking of bead binding, antibodies against MICA (R&D, stock concentration 0.5 mg/ml), or KIR2DL1 (HP3E4 hybridoma supernatant) were added to the cell/bead mix diluted as indicated. Cell conjugation assays were performed based on previous protocols [60]. For the analysis of NKL conjugation with P815 transfectants, NK cells were stained with the red fluorescent dye PKH26 (2 mM), target cells with the green fluorescent dye PKH67 (2 mM) (both Sigma-Aldrich, Dorset, UK). After washing, 50 mL of 2 * 106 cells/ml each of NK cell and target cell were mixed in a 96 well V-bottom plate, briefly spun down, and incubated for the times indicated. Conjugation was stopped by adding ice-cold parafor- maldehyde to a final concentration of 1%, and conjugates were analysed by flow cytometry. The percentage of PKH26-labeled NK cells forming conjugates with PKH67-labeled target cells was determined as the number of double-positive events [100*(NK cells in conjugate)/(total number of NK cells)] (FloJo software). IFN-c expression and secretion were quantified by intracellar cytokine staining and ELISA, respectively, using standard protocols (antibodies from BD bioscience). The assay for degranulation was performed as described previously [61]: 106 target cells/ml were cocultured with a different ratio of effector cells in a 48-well plate. Control wells contained either NK cells alone or NK cells stimulated with PMA (2.5 mg/ml) and ionomycin (0.5 mg/ml). PE-conjugated anti-CD107a mAb (Santa Cruz Biotechnology) was added and incubated for 1 h at 37uC. The exocytosis inhibitor monensin was added to a final concentration of 6 mg/ml and the incubation continued for a further 4 hours. Cells were washed 2 times with PBS, and in some experiments NK cells were stained for CD2, before analysis by flow cytometry (FACSCalibur). For the analysis of SLP-76 phosphorylation by Western Blotting, 106 NKL cells were stimulated by 106 control or MICA-transduced P815 cells for 2 minutes in a 37uC water bath. PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e15374