2B4 localizes toward NK/NK interaction sites even in the presence of CD48-expressing targets. 2B4-GFP fusion protein is expressed in WT LAK cells, and the accumulation at the NK/NK interface was analyzed as described in “Materials and methods.” Briefly, for detection of NK-cell couples, cells in PBS with 10% FCS, 1 mM CaCl, and 0.5 mM MgCl were placed on an inverted epifluorescence microscope equipped with a Nikon Plan Apo 20 ×/0.75 air objective lens (Nikon, Melville, NY) and analyzed using videomicroscopy (Princeton Scientific MicroMax, Trenton, NJ). The Metamorph 6.6/6.0 imaging system was also used for analysis (Universal Imaging, Downingtown, PA). (A) The percentage of NK-cell/NK-cell couples showing 2B4-GFP interface accumulation is given. Briefly, 2B4-GFP accumulation, designated as strong, partial, minimal, or no accumulation in the figure, was classified as follows. Average 2B4-GFP fluorescence intensity of the area of 2B4 accumulation at a cellular interface was measured with Metamorph Software (Universal Imaging, Downingtown, PA). Strong accumulation at interface is greater than 40% of anywhere else in the cell. Partial accumulation is greater than 40% of the background but greater than anywhere else in the cell. Minimal is an interface accumulation of greater than 40% above background but less than another area of accumulation in the cell. Addition of all 4 patterns of accumulation equals 100%. NK/NK + targets indicates 2B4 accumulation at the NK-cell/NK-cell interface in a cell cluster where a particular NK cell also had a NK-cell/target-cell interface; Jasp, the presence of Jasplakinolide, which disrupts actin cytoskeleton. Twenty (on average 25) cell couples from at least 5 independent experiments were analyzed per condition. Statistical significance as determined with a Mann-Whitney U test was P < .005 between NK/NK or NK/NK⁺ targets and NK/NK + targets + Jasp. (B) A time-lapse video image of the 2B4-GFP distribution in a cell cluster of 3 NK cells (NK) and 3 target cells (T) as labeled in the first panel; productive NK-cell/NK-cell and NK-cell/target-cell interfaces are labeled in the top middle panel with arrows pointing at the center of the interfaces. The leftmost target cell only binds to the bottom of the cluster at 10:00 minutes. 2B4-GFP fluorescence intensity is shown as a differential interference contrast (DIC) image on top as well as the top-down projection of three-dimensional data in a false color scale (increasing from purple to red). As the punctuate 2B4-GFP distribution makes alignment of fluorescence and DIC images difficult, the 2B4-GFP fluorescence is also directly laid over the DIC image in a green intensity scale. At the bottom right of the bottom NK cell, 2B4-GFP is enclosed in a bright vesicle that is not continuous with the rest of the cell membrane. In the first 4 panels, the translocation of a 2B4-GFP cluster on the same NK cell (blue-green to yellow in the GFP intensity image) toward an NK-cell/NK-cell interface can be seen on the top of the cell. A minimal 2B4-GFP accumulation at the interface of the same NK cell with the bottom target cell can also be seen in the last 2 panels.