CD2 Promotes NK Cell Nanotube Formation Figure 1. YTS-CD2-GFP but not YTS-MyoIIA-GFP NK cells form stable NTs. (A) The frequency of NT formation between NK cells and 721 cells was calculated as a percentage of NK cells forming NTs (black bars) and as a percentage of NK and 721 cell interactions leading to NT formation (white bars). See Materials and Methods for a detailed description of frequency calculations. YTS-MyoIIA-GFP cells were imaged with 721 cells on anti- CD48 coated plates, anti-CD48 and fibronectin (FN) coated plates, and on anti-CD48/FN coated plates with IL-2 pre-activation (n.100); YTS-CD2-GFP cells were imaged with 721 cells on anti-CD48 coated plates and on anti-CD48/FN coated plates (n.50). (B) YTS-MyoIIA-GFP NTs broke quickly, leaving thick projections ,400 nm in diameter. (C) YTS-CD2-GFP NTs had a diameter of ,100 nm and stably connected NK and target cells, sometimes for longer than 2 hours. (D) Cytotoxicity against 721 target cells is enhanced in YTS-CD2-GFP (black) cells in comparison to YTS (gray) cells (n = 3). (Scale bars: 5 mm.) doi:10.1371/journal.pone.0047664.g001 using one-way ANOVA as well as a series of individual t tests. Data in Figs. 2D, 3D, 3E, and 3F were fit using a linear regression model. The coefficient of determination was calculated for Fig. 2D, and Figs. 3D, 3E, and 3F were analyzed to determine whether the slope differed significantly from 0. Figs. 4A, 5A, 5B, 5C, 5D, 6B, and 6D were analyzed using two-way ANOVA followed by a series of individual t tests. Fig. 4B was analyzed using a Student’s two tailed t test. Results YTS-CD2-GFP but not YTS-MyoIIA-GFP NK Cells form Stable NTs Primary human NK cells and the immortalized human NK cell line NKL readily form NTs between themselves as well as various target cells [16]. Our first attempts to observe NT formation employed YTS cells stably expressing a myosin IIA-GFP fusion protein (YTS-MyoIIA-GFP). The immortalized NK cell line YTS has a limited receptor-ligand repertoire and does not require the presence of cytokines for growth in vitro. We utilized myosin IIA- GFP expressing cells since myosin is an abundant cytoplasmic and cortical protein, which we hypothesized would provide an opportunity to observe the formation of NK to target cell NTs. While these cells adhere to and mediate cytotoxicity against 721 B- lymphoblastoid target cells [17], they did not form NTs observable by either DIC or fluorescence microscopy for myosinIIA-GFP (Fig. 1A). In contrast, others had shown that 6.3% of primary NK cells and 7.5% of NKL cells were connected to 721 cells by NTs [16]. PLOS ONE | www.plosone.org 3 October 2012 | Volume 7 | Issue 10 | e47664