CD2 Promotes NK Cell Nanotube Formation Figure 3. CD2 localizes to the distal tip of the NK cell NT. (A) A bright patch of CD2 fluorescence is seen at the distal tip of a YTS-CD2-GFP cell NT. Magnification of the boxed region depicts the NT tip becoming brighter over time as CD2 accumulates. (B) 65% of NK cell NTs exhibited an enrichment of CD2 at the NT tip as compared to the cell body. Enrichment persisted for the life of the NT (n.100 NTs). (C) Similarly, 85% of NK cell NTs exhibit enrichment of CD2 at the tip as compared to the overall length of the NT (n.100 NTs). (D), (E) and (F) Videos of NTs that show CD2 enrichment of the tip compared to the length of the NT were photo bleach-corrected, normalized to a unit time scale, time-binned, and averaged. Both sum (D) and mean fluorescent intensity (F) increased significantly over time, indicating that CD2 accumulates at the junction over time. (E) Tip area increased, but only marginally over time. Trend lines are shown as red dashed lines (n = 13 NTs). (Scale bars: 5 mm, Inset: 1 mm.) doi:10.1371/journal.pone.0047664.g003 YTS-CD2-GFP Cells Form More NTs than YTS-KIR-GFP Cells Our initial attempts to observe NT formation in YTS-MyoIIA- GFP cells suggested that unmodified YTS cells may not stably form NTs and that CD2 may play a crucial role in NT formation. An alternative hypothesis is that fluorescently tagging CD2 (a membrane bound receptor) merely aided visualization of NTs. Since MyoIIA is not an integral membrane protein, perhaps YTS- MyoIIA-GFP NTs escaped detection. To ensure that NT formation was not simply a feature of exogenously expressing a GFP-fused membrane bound receptor, we compared the frequency of NT formation of YTS-CD2-GFP cells to that of YTS-KIR-GFP cells. While the 721 target cells utilized in both experiments contain the CD2 cognate ligands CD58 and CD48, they lack HLA-Cw4, the cognate ligand of KIR2DL1. As such, the KIR2DL1 receptor-ligand pair should not influence NT forma- tion. Thus, both of these pairings represent an NK cell interacting with a susceptible target cell, and the YTS-KIR-GFP cells provide a control for an NK cell expressing a GFP-labeled surface receptor. YTS cells and 721 cells were incubated for 30 minutes on an anti-CD48 coated plate and then imaged using live-cell confocal microscopy, and z-planes were scanned to ensure that all NTs were noted. 15.163.3% of YTS-CD2-GFP cells exhibited NTs, compared to just 2.561.5% of YTS-KIR-GFP cells exhibiting NTs (Fig. 4A). Importantly, no YTS-KIR-GFP cells formed multiple NTs, either with a single or multiple target cells. The low frequency of NT formation in YTS-KIR-GFP cells mirrored that of YTS-MyoIIA-GFP cells, confirming that NT formation between NK and susceptible target cells is not merely a function PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e47664