CD2 Promotes NK Cell Nanotube Formation Figure 5. CD2, but not KIR2DL1, LFA-1, or 2B4, receptor-ligand interactions promote NT formation. (A) YTS-CD2-GFP cells were incubated with isotype or anti-CD2 blocking antibody for 15 minutes, followed by a 30 minute incubation with 721 target cells on anti-CD48 coated plates. Single time-point images were obtained to determine frequency of NT formation (n.350 cells). (B) YTS-KIR-GFP cells were incubated with 721- Cw4 cognate ligand-expressing cells or 721-Cw3 non-cognate ligand-expressing cells on anti-CD48 coated plates for 30 minutes. The frequency of YTS-CD2-GFP cells incubated with 721 cells (from Fig. 4A) is included for comparison (n$300 cells). (C) YTS-CD2-GFP cells were incubated with isotype or anti-LFA-1 blocking antibody for 15 minutes, followed by a 30 minute incubation with 721 target cells on anti-CD48 coated plates. Single time-point images were obtained to determine frequency of NT formation (n.500 cells). (D) YTS-CD2-GFP cells were incubated with isotype, anti- CD2 blocking antibody, or anti-2B4 blocking antibody for 15 minutes, followed by a 30 minute incubation with 721 target cells on anti-CD20 and FN coated plates. Single time-point images were obtained to determine frequency of NT formation (n.500 cells). Bars show the percentage of NK cells exhibiting NK to target cell NTs (black) and NK to NK cell NTs (grey). doi:10.1371/journal.pone.0047664.g005 mAb treated YTS-CD2-GFP cells formed NK to NK cell NTs (Figs. 5A). Although this difference is statistically significant, it represents a much smaller effect, suggesting that CD2-ligand binding may play a minor role in NK to NK cell NT formation. NKG2D-MICA and LFA-1-ICAM receptor-ligand interactions have been described to participate in NT formation, suggesting that perhaps receptor-ligand interactions in general enable NT formation [16]. To investigate whether any form of receptor- ligand interaction can promote NT formation, we chose to examine the inhibitory KIR2DL1-HLA-Cw4 interation. YTS- KIR-GFP cells were added to 721 cells expressing either HLA- Cw4, or a homologous but non-cognate ligand, HLA-Cw3. Only 1.861.4% of YTS-KIR-GFP cells formed NK to target cell NTs with 721-Cw4 cells (Fig. 5B). This is remarkably lower than both the 15.1% frequency for YTS-CD2-GFP cells (Fig. 4A) and the 4.763.2% formed by YTS-KIR-GFP cells when mixed with 721- Cw3 target cells (Fig. 5B). Clearly not all receptor-ligand pairs promote the formation of NTs, with the inhibitory KIR2DL1- HLA-Cw4 interaction providing an example. The comparison of NT formation in YTS-KIR-GFP cells between 721-Cw4 and 721- Cw3 cells was not significant. Because of the inhibitory nature of the KIR2DL1-HLA-Cw4 receptor-ligand interaction, it is not entirely surprising that it fails to promote NT formation. Thus we asked whether other activating and adhesion receptors also promote NT formation similarly to CD2. We initially targeted LFA-1 since, like CD2, it is well known to promote both adhesion and NK cell activation signaling [3,4]. Previous work has demonstrated that enhancing LFA-1-ICAM interactions to supraphysiologic levels through the use of Mn2+ promotes NK cell NT formation [16,29]. To determine if endogenous LFA-1 interactions play a role in NT formation, we abrogated LFA-1 receptor-ligand interactions using a blocking anti-LFA-1 mAb [22,23]. Surprisingly, only 3.462.3% of cells treated with isotype control Ab formed NTs with target cells as compared to 5.161.2% of cells treated with the LFA- PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e47664