CD2 Promotes NK Cell Nanotube Formation 1 mAb (Fig. 5C). Thus, endogenous LFA-1 fails to play a role in NT formation in this context. Next, we asked whether another CD2 family member, such as 2B4, may play a role in NT formation. 2B4, like CD2 and LFA-1, plays a role in NK cell adhesion [3,4] and activation [2,30]. Importantly, 2B4 shares CD48 as a cognate ligand with CD2, however in the case of 2B4, CD48 is its high-affinity ligand. Because of this, CD48 could no longer be utilized to immobilize the 721 cells to the imaging chambers. Instead, chambers were coated with anti-CD20 and FN. Because NK cells are more motile under these imaging conditions, they were more likely to form clumps of cells, and thus NTs were formed less frequently than on anti-CD48 coated imaging chambers. However, treatment with anti-CD2 blocking antibody still inhibited NT formation, with 3.561.0% of isotype control Ab treated cells and 0.861.0% of anti-CD2 mAb treated cells forming NK to target cell NTs (Fig. 5D). Additionally, treating YTS-CD2-GFP cells with a blocking anti-2B4 mAb [21] had no effect on NK to target cell NT formation, with 2.161.0% of isotype control Ab treated cells and 2.560.9% of anti-2B4 mAb treated cells forming NK to target cell NTs (Fig. 5D). This demonstrates that like LFA-1, endogenous levels of 2B4 fail to play a role in NK to target cell NT formation. CD2 Receptor-ligand Interactions Promote NT Formation in eNK Cells Although the YTS cell line has faithfully reproduced the biology of human NK cells in a number of contexts (eg. [27,31,32]), we wanted to examine the effect of CD2 receptor-ligand interactions on NT formation in ex vivo NK (eNK) cells. eNK cells were labeled with the fluorescent membrane dye PKH26 and pre- treated with either CD2 blocking mAb or isotype control for 15 minutes prior to incubation with 721 target cells. NTs were readily identified (Fig. 6A). 1.660.9% of eNK cells treated with isotype Ab formed NTs with target cells compared to only 0.360.4% of eNK cells treated with the CD2 blocking antibody (Fig. 6B). This decrease in NT frequency further supports the hypothesis that the CD2 receptor-ligand interaction promotes NT formation. 0.460.4% of eNK cells treated with isotype and 0.660.6% of those treated with CD2 blocking mAb exhibited NK to NK cell NTs. These latter results are not statistically different, supporting our previous hypothesis that CD2’s role, if any, in NK-NK NT formation is minor thus further substantiating a function for CD2 in promoting target cell-specific NTs in NK cells. To further confirm the importance of CD2 in NK cell to target cell NT formation, we isolated highly purified CD2+ and CD22 eNK cell populations by FACS using a non-blocking CD2 antibody (Fig. 6C) [20]. To confirm that this antibody did not interfere with NT formation, it was first evaluated for any effect on the formation of NTs when added to YTS-CD2-GFP cells combined with susceptible 721 target cells. While YTS-CD2- GFP cells treated with the blocking antibody demonstrated an 88.064.7% decrease in NT frequency to target cells compared to cells treated with an isotype control, treatment with the non- blocking anti-CD2 antibody resulted in only a 5.0613.0% de- crease (p = 0.001, n.325 cells). Therefore, eNK cells sorted using this non-blocking anti-CD2 antibody were used to determine if the presence of CD2 might enable NT formation in fresh human NK cells. Directly following sorting using the non-blocking antibody, the CD2+ and CD22 eNK cells were labeled with PKH26 and incubated with 721 target cells for 30 minutes. 2.560.8% of CD2+ eNK cells formed NK to target cell NTs, while 0.260.6% of CD22 eNK cells formed NTs (Fig. 6D). No NK to NK cell NTs were observed. Taken together, the above experiments demon- PLOS ONE | www.plosone.org 9 October 2012 | Volume 7 | Issue 10 | e47664