Consistent with the reported role for NTs in enhancing NK cell cytotoxicity by contributing to ,17.4% of target cell deaths [16], NT-forming cells should exhibit similar enhanced killing com- pared with those unable to form stable NTs. We tested this hypothesis in aggregate using a 51Cr release assay to evaluate the cytotoxic activity of YTS-CD2-GFP and YTS cells. YTS cells exhibited less target cell lysis than YTS-CD2-GFP cells, with ,16% less lysis at the largest effector to target cell ratio (Fig. 1D). While activation- and adhesion-promoting properties of CD2 are likely to contribute to this decrease in cytotoxic function, these results are also consistent with the previously reported role that NTs are described to serve in NK cell cytotoxicity [16]. In addition to being consistent with these previously published predictions, the present results support the observation that CD2-expressing NK cells stably form NTs. YTS-CD2-GFP Cells can Simultaneously Form Multiple NTs Microscopy carried out after cells had been incubated together for 30 minutes revealed that a high percentage of YTS-CD2-GFP cells form more than one NT when interacting with target cells. Multiple NTs formed between an NK cell and a single target cell (Fig. 2A) or between an NK cell and multiple target cells (Fig. 2B). Approximately 40% of all NK cells with at least one NT formed more than one, and the number of cells exhibiting multiple NTs was inversely related to the number of NTs per cell (Fig. 2C). Importantly, multiple NTs were not identified between just two NK cells (n.320 cells), although a single NK cell could form NTs to multiple NK cells (data not shown). Thus the formation of multiple NTs between two cells was specific to interactions between NK and target cells. The mean length of NTs emanating from a single NK cell was inversely related to NT number (Fig. 2D). For cells containing 1, 2, 3, or 4 NTs, the mean NT lengths were 12.567.3, 10.9610.2, 8.465.8, and 3.261.3 mm, respectively, and could be described in aggregate by a linear trend line with a correlation coefficient of r2 = 0.93. This linear trend suggests there is a finite limit to how many NTs a single cell can maintain, supporting the hypothesis that NTs are created from a single membrane reservoir [28]. As the cell creates more NTs, it is presumably able to devote less membrane to each NT, ultimately resulting in shorter NTs. Furthermore, the shorter length of multiple NTs substantiates their previously proposed role in target cell traction or anchoring [16]. The observation that NK cells with 4 NTs were always close to the target cell also suggests that they can synergize in this capacity. Thus NK cells can likely fix to a susceptible target by taking advantage of multiple NTs. While two-dimensional imaging allows for the detection of multiple NTs, they are likely to exist in orientations that are underestimated when viewed in a single plane. Thus we utilized three-dimensional imaging of living YTS-CD2-GFP cells to provide further detail. This approach demonstrated more NTs than can be identified in the plane of perceived maximal NT fluorscence in a two-dimensional image (Video S1). However, due to the limits of spatial resolution within a given plane, the number of NTs that escape identification using two-dimensional images is likely proportional to the number of NTs identified. In other words, more NTs will likely be missed in cells that contain a larger number of identifiable NTs. The method employed here of scanning through z-planes to identify NTs ensures that if an NK cell contains a NT it will be recognized, even if defining the exact number present is not feasible. This further supports our observations regarding NT frequency, as unexpected dispropor- tional concentrations of NTs outside of the maximal two- CD2 Promotes NK Cell Nanotube Formation dimensional fluorescence plane were not observed (n = 8 cell-to- cell interactions with NTs). Moreover the estimation of frequency only relies upon identification of NTs and not their total number. CD2 Localized to the Distal Tip of the NK Cell NT Previous studies have shown that receptors associated with NT formation are found in increased numbers at the distal tip of the NT [16]. Similarly, we observed bright patches of fluorescence at the tips of YTS-CD2-GFP NTs, consistent with an enrichment of CD2 (Figs. 1C, 2A, 2B, 3A). To quantify this enrichment, we compared the mean fluorescence intensity of the NT tip to that of the cell body (Fig. 3B) and the NT as a whole (Fig. 3C). Approximately 65% of NTs exhibited an enrichment of CD2 at the tip compared to the cell body and 85% exhibited an enrichment compared to the overall NT. Enrichment persisted over the life of a NT and appeared to increase over time (Fig. 3A). While two-dimensional images provided enough detail to quantify this enrichment, a three-dimensional image demonstrates more clearly the magnitude of the CD2 enrichment at the NT tip (Video S1). In order to accurately quantify time-dependent enrichment of CD2, we generated linear trend lines for CD2 fluorescence intensity. This was performed using time-lapse video sequences, which were corrected for photobleaching. Videos in which NTs exhibited substantive tip enrichment of CD2 were considered separately from those that did not exhibit enrichment (Fig. S1). We first examined sum fluorescent intensity at the NT tip in order to gain insight into the relative number of GFP molecules present over time (Figs. 3D and S1A). A majority (85%) of NTs exhibiting CD2 enrichment showed an increase in CD2 at the tip over time, as demonstrated by a positively sloped trend line for the sum fluorescent intensity. In contrast, all NTs that failed to exhibit CD2 enrichment showed a decrease in CD2 sum fluorescence intensity over time (data not shown). The decrease in this case was likely to have been a feature of time-dependent photobleaching. A time-weighted average of the slopes in which CD2 accumulated at the NT tip shows the average increase in sum fluorescent intensity to be 55847 a.u./min. Thus CD2 accumulated at and relocalized to the NT tip over time. Since it appeared that increasing fluorescence of CD2 at the NT tip might not be limited to the original precise point of formation with the target cell, the video sequences used to define accumulation of CD2 were more closely examined. Quantitative evaluation of the area of CD2 fluorescence revealed that the tips of many NTs increased slightly in size over time (Fig. S1B). Most (77%) of NTs exhibiting CD2 enrichment showed a time-de- pendent increase in tip area, as demonstrated by the mild trend line slope (Fig. 3E). In contrast, all of the NTs that failed to exhibit CD2 enrichment showed a decrease in tip area over time (which, again, was likely to have been a feature of photobleaching, not shown). A time-weighted average of the slopes in which CD2 was 2 enriched shows the average increase in tip area to be 0.0066 mm / min. Finally, we examined the mean fluorescence intensity as a marker for CD2 density within a given area. Only 46% of NTs exhibiting CD2 enrichment showed an increase in CD2 density at the tip over time (Fig. S1C). This enrichment, however, was substantive enough to lead to a positively sloped trend line for the mean fluorescent intensity with the time-weighted average of these slopes showing the average mean fluorescent intensity to increase with a slope of 20630 a.u./(mm2)/min (Fig. 3F). 100% of the NTs that failed to exhibit CD2 enrichment showed a decrease in CD2 density (data not shown). Taken together, these data demonstrated that CD2 quantity and area at the NT tip is dynamic. PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e47664