immunological synapse (approximately 12% of NK cell cytotoxic events) and by transporting lytic granules along their length to mediate long-distance killing (approximately 5.4% of NK cell cytotoxic events) [16]. The specifics of NT formation, however, remain incompletely understood. Here we identify CD2 as a crucial promoter of NK cell NT formation. Materials and Methods Ethics Statement All human samples were obtained using written informed donor consent and were used with the approval of the Children’s Hospital of Philadelphia and/or Baylor College of Medicine Institutional Review Board for the Protection of Human Subjects. Cell lines and ex vivo NK Cells Immortalized NK cell lines derivative from the parental YTS cell line stably expressing a myosin IIA-GFP fusion protein (YTS- MyoIIA-GFP) [17], a CD2-GFP fusion protein (YTS-CD2-GFP) [18], or a KIR2DL1-GFP fusion protein (YTS-KIR-GFP) [9] were used as model NK cell systems. 721.221 B-lymphoblastoid cells (721), and 721.221 cells stably expressing HLA-Cw3 (721- Cw3) or HLA-Cw4 (721-Cw4) [19] were used as target cells (TC). Ex vivo NK (eNK) cells were prepared either from whole blood obtained from volunteer donors by negative depletion using the RosetteSep human NK cell isolation reagent (StemCell Technol- ogies), or from anonymized leukophoresis-derived PBMC via negative depletion using magnetic particles and the NK Cell Isolation Kit (Miltenyi Biotech). Alternatively, PBMCs were prepared from whole blood using centrifugation through Ficoll- Paque Plus lymphocyte isolation medium (Amersham Bioscience). eNK cells in PBMCs were identified as CD56+CD32 and then sorted into CD2+ and CD22 populations using a non-blocking CD2 antibody (BD Bioscience, clone L303.1) [20] and a Coulter MoFlo (Beckman Coulter). In all cases, cells were used immedi- ately following preparation. Plate Coating Anti-CD48 (BD Biosciences) or anti-CD20 (Rituximab, Gen- entech) were diluted to 8 mg/mL in PBS or in 0.2 mm filter- sterilized 10 mg/mL human fibronectin (Collaborative Biomedical Products or BD) in PBS prior to addition to DT dishes (Bioptechs). Only certain experiments were performed with added human fibronectin (FN) as specified. Dishes were incubated at 37uC for one hour in anti-CD48/PBS solution, or left overnight at 4uC in anti-CD48 or anti-CD20/fibronectin solution and then washed 3 times with PBS. Prior to use in experiments, fibronectin coated dishes were blocked with 0.2 mm filter-sterilized 3% BSA in PBS for one hour at room temperature. IL-2 Activation YTS-MyoIIA-GFP cells were incubated for 24 hours in RPMI 1640 with 10% FCS supplemented with 125 U/mL human recombinant IL-2 (NIH AIDS reagents program). Live Cell Microscopy NK cells (YTS-MyoIIA-GFP, YTS-CD2-GFP, YTS-KIR-GFP) and target cells (721, 721-Cw3, 721-Cw4) were washed and resuspended in RPMI 1640 with 10% FCS. eNK cells were labeled using the PKH26 red fluorescent dye (Sigma-Aldrich), and resuspended in RPMI 1640 with 10% FCS. For time course imaging, target cells were adhered to a coated DT dish for 20 minutes at 37uC. NK cells were added to the DT dish at a NK:TC ratio of 1.5:1. Cells were imaged in a single z-plane using an CD2 Promotes NK Cell Nanotube Formation Olympus IX-81 spinning disk confocal microscope, with Hama- matsu EM-CCD camera and Volocity Software (Perkin Elmer). Images were captured at maximum speed for the required exposure times (,3 images/min) with both differential interfer- ence contrast microscopy and 488 nm or 568 nm excitation while the dishes were maintained at 37uC using a DT dish heater unit (Bioptechs). For single time point analyses, NK cells and target cells were incubated in a coated DT dish for 30 minutes at 37uC. After scanning planes for NT presence, cells were imaged in a single z-plane as above, or using a Zeiss Axio Observer Z1 with Yokogawa CSU10 spinning disk, Hamamatsu Orca-AG camera and Volocity Software for LFA-1 and 2B4 blocking antibody experiments. Images obtained using the Zeiss microscope were obtained using transmitted light and 488 nm excitation with cells maintained in a DT dish heater unit. 3D images were obtained using the Zeiss microscope at a z-axis spacing of 0.2 mm. For blocking antibody experiments, YTS-CD2-GFP cells were in- cubated with 10 mg/mL of biotinylated mouse IgG1 anti-human CD2 (BioLegend, clone RPA-2.10) [20,21], biotinylated mouse IgG1 isotype control (BD), purified mouse IgG1 anti-human 2B4 (BioLegend, clone C1.7) [21], mouse IgG1 anti-human LFA-1 (clone TS1/22) [22,23], or mouse IgG1 isotype control (BioLe- gend) for 15 minutes at 37uC prior to incubation on DT dishes. 51Cr Release Cytotoxicity Assay Parental YTS or YTS-CD2-GFP cells were synchronized for 3 weeks by culturing in RPMI 1640 with 10% FCS. NK cell cytotoxic function was measured using a 4 hour 51Cr release assay as described previously [24]. Data Analysis Image analysis was conducted using Volocity software (Perkin Elmer). For Fig. 1A, frequency of NK to target cell NTs was calculated as a percentage of NK cells (NK cells with NTs divided by the total number of NK cells observed), or as a percentage of cell interactions (where an interaction is defined as a NK to target cell contact lasting longer than a minute). For other figures, NT frequency was calculated by dividing the number of NK cells containing NTs, either NK to target cell NTs (NK-TC NT) or NK to NK cell NTs (NK-NK NT), by the total number of NK cells observed in single time point images. For the kinetic assessment of NTs, photobleaching was corrected using an exponential curve fit to the total cell fluorescent intensity (cell body and NT). Videos were then normalized to a relative time scale, ranging from 0 to 1. Time was split into 21 time bins (0.05 units), and the average value for the fluorescence or area of each NT tip was calculated for each time bin. Graphs represent the average of these values for all NTs. Trend lines were created using a linear regression in Prism (GraphPad). Time weighted averages were determined by calculating the fraction of time that a NT lasts compared to all of the NTs (the sum of all NT times), multiplying this fraction by the fluorescent value (or other measurement) of interest, and then summing across all NTs. Statistical Evaluations The minimum number of cells to be evaluated in each microscopy experiment was determined using a sample size calculation based on preliminary data. Sample size calculations were performed using the DSS sample size calculator (DSS Research) and a and b errors of 1%. Statistical analysis was performed using Prism (GraphPad). All error bars represent the standard deviation. In Fig. 1D, individual ratios were analyzed using a Student’s two tailed t test while overall the data was analyzed using a paired two tailed t test. Fig. 2D was analyzed PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e47664