CD2 Promotes NK Cell Nanotube Formation Figure 2. YTS-CD2-GFP cells can simultaneously form multiple NTs. YTS-CD2-GFP cells were incubated with 721 cells for 30 minutes on anti- CD48 coated plates then imaged live using a spinning disc confocal microscope at a single time point. (A) Multiple NTs form between a single NK and target cell, or (B) connect an NK cell to multiple target cells. (C) The number of NK cells forming multiple NTs is inversely proportional to the number of NTs per cell (n.100 NTs). (D) As the number of NTs per cell increases, the average NT length decreases (1–3 NTs/Cell: n.25 NTs; 4 NTs/Cell: n.5 NTs). (Scale bars: 5 mm.) doi:10.1371/journal.pone.0047664.g002 In an attempt to better reproduce culture systems known to promote NT formation, imaging chambers were coated with fibronectin (FN) [16], which is implicated in NK cell motility [25], in addition to using the anti-CD48 to immobilize the 721 cells. We hypothesized that FN would facilitate NK cell motility and thereby increase cell-cell contacts. Both cell-cell contact [16] and a sub- stratum supporting optimal cell motility [15] are considered central to NT formation. On FN coated imaging chambers, however, only 1.1% of all observed YTS-MyoIIA-GFP cells formed NTs. Furthermore, only 2.0% of all YTS-MyoIIA-GFP and 721 cell interactions (defined as physical contact between the two cells lasting longer than one minute) resulted in NT formation (Fig. 1A). NTs that did form lacked the stability and characteristics of NTs described elsewhere [10,15,16,26]. The rare NTs observed typically broke free of the target cell within seconds of their detection, leaving a long membranous projection that was eventually reintegrated back into the NK cell (Fig. 1B). These rare NTs also exhibited unusually thick diameters (,400 nm) as compared to those reported previously [10,15,16,26]. Cytokine stimulation of NK cells has been shown to approx- imately double the frequency of NK cell NTs [16]. We hypothesized that by using an NK cell line that does not depend on exogenous cytokine for growth, we removed a signal necessary for NT formation. Since YTS cells respond to IL-2 [27], however, we tested this hypothesis, by stimulating YTS-MyoIIA-GFP cells with IL-2 for 24 hours prior to addition to target cells in anti- CD48 and FN coated imaging chambers. Surprisingly, this failed to increase the frequency of NT formation; in fact, no NTs were identified following IL-2 pre-activation (Fig. 1A). Another distinguishing feature of YTS cells is that they lack the cell surface receptor CD2. Given that receptor-ligand interactions are important mediators of cell-cell adhesion and several interactions have been demonstrated relevant to NT formation [16], we hypothesized that CD2 is important in promoting NT formation. To test this, we utilized YTS cells stably expressing a CD2-GFP fusion protein. Interestingly, 9.8% of all YTS-CD2- GFP cells formed NTs and 14.3% of all YTS-CD2-GFP and 721 cell interactions resulted in NT formation. Unexpectedly, this percentage was reduced on FN coated image chambers, with only 3.9% of all YTS-CD2-GFP cells forming NTs and 5.0% of all YTS-CD2-GFP and 721 cell interactions resulting in NT formation (Fig 1A). The NTs observed in these experiments exhibited usual characteristics. They remained connected to target cells for long periods of time (sometimes over 2 hours) and had more typical diameters of ,100 nm (Fig. 1C). This increase in NT formation suggested that the presence of CD2 in YTS cells promotes their ability to form NTs. PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e47664