of exogenously expressed GFP-cell surface receptors. Interestingly, there was no statistical difference in the frequency of NK cell to NK cell NT formation between YTS-CD2-GFP cells and YTS- KIR-GFP cells (3.562.9% and 3.762.8%, respectively, Fig. 4A). This suggests that NK to NK cell NT formation may rely on different factors than does NK to susceptible target cell NT formation. NT characteristics of YTS-CD2-GFP and YTS-KIR-GFP cells were also compared. While the number of NTs formed differed greatly between YTS-CD2-GFP and YTS-KIR-GFP cells, the mean NT length did not. YTS-CD2-GFP NTs had a mean length of 10.468.0 mm, while YTS-KIR-GFP NTs had a mean length of 9.366.5 mm (Fig. 4B). These values lacked statistical difference and agreed closely with the reported mean NT length of 11.4 mm for primary human NK cells [16]. In contrast, very few YTS-KIR- GFP NTs exhibited KIR enrichment at the distal NT tip (Fig. 4C and 4C inset), particularly compared to the strong enrichment seen with YTS-CD2-GFP cells (Fig. 4D). No YTS-KIR-GFP cells exhibited KIR tip enrichment compared to the cell body, and only 10% (1 NT) exhibited KIR enrichment at the tip compared to the overall length of the NT. These observations support the hypothesis that the CD2 receptor plays a specific role in NT CD2 Promotes NK Cell Nanotube Formation formation. It also provides specificity regarding the observation that membranous CD2 preferentially relocates to the NT tip. Thus, it is likely that the interaction of CD2 with its ligand on target cells promotes NT formation. CD2, but not KIR2DL1, LFA-1, or 2B4, Receptor-ligand Interactions Promote NT Formation To more directly address the role of CD2 in NT formation, we blocked CD2 receptor-ligand interactions using a blocking anti- CD2 mAb [20,21]. YTS-CD2-GFP cells were incubated with mAb or isotype control for 15 minutes prior to incubation with 721 target cells on anti-CD48 coated plates. Again, thorough scanning through focal planes was performed to ensure NT detection. 11.564.4% of cells treated with isotype control Ab formed NTs with target cells as compared to only 1.561.2% of cells treated with the CD2 mAb (Fig. 5A). This decrease in frequency of NT formation to the level of YTS-MyoIIA-GFP and YTS-KIR-GFP cell NT formation supports the hypothesis that the specific interaction of CD2 with its ligand (which is highly expressed on 721 cells, not shown) plays a crucial role in NT formation. In contrast, 4.361.2% of isotype and 2.360.9% of Figure 4. YTS-CD2-GFP cells form more NTs than YTS-KIR-GFP cells. (A) YTS-CD2-GFP and YTS-KIR-GFP cells were incubated with 721 cells for 30 minutes on anti-CD48 coated plates, then single images were obtained using live-cell confocal microscopy and scanning of z-planes to ensure NT detection. Bars show the percentage of NK cells exhibiting NK to target cell NTs (black) and NK to NK cell NTs (grey). See Materials and Methods section for a detailed description of frequency calculations. (B) Average NT length did not vary. Representative images of (C) YTS-KIR2DL1-GFP, with inset showing a lack of KIR enrichment at the tip and (D) YTS-CD2-GFP NTs. (n.320 cells) (Scale bars: 5 mm, Inset: 1 mm.) doi:10.1371/journal.pone.0047664.g004 PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e47664