Figure 6. CD2 receptor-ligand interactions promote NT forma- tion in eNK cells. (A) NTs were visualized in PKH26 labeled eNK cells isolated from whole blood. (B) eNK cells were labeled with PKH26, treated with isotype or anti-CD2 blocking antibody for 15 minutes, then incubated with 721 cells on anti-CD48 coated plates for 30 minutes. Single images were obtained to determine NT frequency (n.850 cells). (C) eNK cells were sorted into CD2+ and CD22 populations using a non- blocking antibody to CD2. (D) CD2+ and CD22 eNK populations were labeled with PKH26, and single images were obtained to determine the frequency of NK-NK (black bar) and NK-TC (grey bar) NT formation (n.325 cells). (Scale bar: 5 mm.). doi:10.1371/journal.pone.0047664.g006 strate that the presence of CD2 and the CD2 receptor-ligand interaction play an essential role in enabling NT formation in NK cells directly ex vivo in their interaction with a susceptible target cell. Discussion These are the first data to demonstrate NT formation in YTS NK cells, but more importantly have thereby identified a receptor that plays a crucial role in NT formation. Previous research has documented NT formation in immortalized NKL cells, however those NTs differ from the ones formed by primary NK cells. NKL cell NTs have a much longer average length (21.4 mm) than do primary NK cell NTs (11.4 mm) [16]. The average length reported here in YTS cells (,10 mm) is more consistent with that of primary NK cell NTs, suggesting that YTS cells may form NTs more structurally similar to those of ex vivo NK cells. Because the YTS cell line inherently lacks CD2, it provided a fortuitous way to observe the effects of the CD2 receptor-ligand interaction on NT formation. YTS-CD2-GFP cells formed more NK to target cell NTs than did either YTS-MyoIIA-GFP or YTS- KIR-GFP cells, and blocking CD2’s interaction with its ligand through the use of a blocking antibody abrogated NK to target cell NT formation. Interestingly, the use of a non-blocking antibody had no impact on NT formation, suggesting that the CD2-CD58/ 48 receptor-ligand interaction plays a crucial role in NT formation. CD2 expression also increased the cytotoxic activity of YTS cells at levels commensurate with what has been previously attributed to the cytotoxicity promoting capacity of NTs [16]. Taken together, these data suggest that CD2 plays an essential role in the formation of NTs, which are known to enhance NK cell cytotoxicity [16]. We also show that CD2 plays a role in NT formation ex vivo, as blocking CD2 on eNK cells dramatically reduces the frequency of NT formation between NK cells and susceptible target cells. In addition, CD22 eNK cells form NTs with targets at a negligible frequency whereas CD2+ NK cells have robust numbers of NTs. Therefore, we show for the first time that CD2 plays a crucial role in the formation of NTs both in a cell line and ex vivo human NK cells. Using YTS-CD2-GFP cells, we demonstrated that, like NKG2D [16], CD2 is enriched at the NT tip. Through analysis of time-lapse video sequences, we also provided evidence that this enrichment occurs over time. The accumulation of CD2 at the NT tip suggests that it may play a role in maintaining NT stability. Alternatively, CD2 signaling may drive NT function, facilitating NT-mediated reformation of the conventional NK cell immuno- logical synapse or activating long-range cytotoxic activity, both of which have been previously proposed as possible NT functions [16]. Interestingly, we found that only interactions between NK cells and target cells result in the formation of multiple NTs and that CD2 Promotes NK Cell Nanotube Formation the number inversely correlates with their length. In other words, more NTs result in a shorter distance between the NK cell and its target. When NK cells possessing 4 NTs with a single target cell were identified, the intercellular distance was uniformly small. These findings further support the proposed function of NK cell NTs in facilitating contact with target cells. Additionally, the presence of CD2 was needed for multiple NT formation with a target cell as the few MyoIIA-GFP and KIR2DL1-expressing cells that formed NTs with target cells failed to demonstrate multiple intercellular NTs. Thus the presence of multiple NTs between an NK cell and target is suggested as a correlate of the previously proposed NT enhanced cytotoxicity [16] since the YTS-CD2-GFP cells had increased cytotoxic activity. At a mini- mum, multiple NTs between an NK cell and target cell can be viewed as a hallmark of a potentially cytotoxic interaction. We also demonstrate that not all receptor-ligand interactions promote NT formation, with KIR2DL1-HLA-Cw4 providing an example. Since this receptor-ligand pair provides an inhibitory signal, it will be important to explore if there might indeed be a decrease in NT frequency measured in the presence of the KIR2DL1-HLA-Cw4 ligation. Although the difference in NT formation in KIR2DL1-expressing YTS cells between targets expressing the cognate or non-cognate ligand was not significant, it raises the possibility that inhibitory signaling could interfere with NT formation. The converse logic suggests that, aside from receptor-ligand interaction, an activation signal is actually needed for NT generation, and certainly for the generation of multiple NTs between NK and target cells. Taken together, these results suggest that CD2 plays a crucial role in NK cell NT formation. Indeed, we also demonstrate that the activating and adhesion receptors LFA-1 and 2B4 fail to play as critical a role, since blocking these receptor-ligand interactions does not decrease NT formation. In fact, blockade of both receptors resulted in a potentially small, although statistically insignificant, increase in NT formation. Most likely, this is the result of decreased adhesion interactions increasing NK and target cell separation events, and thus increasing the formation of NTs. Surprisingly, our results of blockading LFA-1 interactions contrast previous reports that enhancing LFA-1 interactions through the use of Mn2+ promotes NK cell NT formation [16]. Our results, however, are limited to otherwise unactivated LFA-1 without an increase in LFA-1-ICAM interactions owing to the use of Mn2+. It is possible that in highly activated cells the role of LFA-1 is distinct. Thus, our data suggests that only specific receptor-ligand interactions, including but not limited to NKG2D [16] and CD2, promote NK to target cell NT formation. Further research is needed to fully appreciate why these specific receptors play such an essential role, their relative context-specific contributions, and if any other receptors provide similar promotion of NT formation. Our results are inconclusive on whether CD2 influences NK to NK cell NT formation. While YTS-CD2-GFP cells treated with a CD2 blocking antibody formed slightly fewer NK to NK cell NTs than did isotype control treated cells, eNK cells treated with CD2 blocking antibody formed the same number of NK to NK cell NTs as isotype control treated eNKs. Similarly, there was no significant difference in NK to NK cell NT formation between YTS-CD2-GFP and YTS-KIR-GFP cells. This difference between NK to NK cell and NK to target cell NT formation is intriguing, particularly since NK cells express both CD48 and CD58 [21]. Future research is needed to better understand this difference and the molecular interactions governing NK to NK cell NT formation. T cells [15,26], B cells [33,34], dendritic cells [14], macro- phages [35], neutrophils [36], and NK cells all form NTs. Some or PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e47664