CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation Colin J. Comerci1, Emily M. Mace2, Pinaki P. Banerjee2, Jordan S. Orange2* 1 Department of Pediatrics, Children’s Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, United States of America, 2 Department of Pediatrics, Center for Human Immunobiology, Baylor College of Medicine, Houston, Texas, United States of America Abstract Membrane nanotubes are thin membranous projections that physically connect two cells. While nanotubes have been studied in human natural killer (NK) cells and are implicated in aiding NK cell cytotoxic function, requirements for their formation to susceptible target cells remain incompletely understood. Here we demonstrate that the CD2-CD58/48 receptor-ligand interaction promotes and is required for nanotube formation in human NK cells. In the CD22 NK cell line YTS, a stable CD2 expression variant enabled effective nanotube formation, and was associated with better cytotoxic function. Importantly, only interactions between an NK cell and a susceptible target cell were associated with multiple nanotubes and the number of nanotubes was inversely correlated with their length. Quantitative live cell fluorescence microscopy of CD2 nanotubes revealed time-dependent enrichment and localization of CD2 to the nanotube tip, and blocking CD2 receptor-ligand interactions prevented nanotube formation. Increased nanotube formation was not simply a feature of receptor-ligand pairing, as a KIR-MHC interaction in the same cell line system failed to promote nanotube formation. Additionally, blocking LFA-1-ICAM and 2B4-CD48 receptor-ligand interactions failed to inhibit nanotube formation. Thus only specific receptor-ligand pairs promote nanotubes. CD2 also promoted nanotube formation in ex vivo NK cells suggesting that CD2 plays a crucial role in the generation of nanotubes between an NK cell and its target. Citation: Comerci CJ, Mace EM, Banerjee PP, Orange JS (2012) CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation. PLoS ONE 7(10): e47664. doi:10.1371/journal.pone.0047664 Editor: Roland Jacobs, Hannover Medical University (MHH), Germany Received June 20, 2012; Accepted September 14, 2012; Published October 24, 2012 Copyright: 2012 Comerci et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health grant AIR01AI067946 (to JSO). CJC was supported by the Vagelos Scholars Program in the Molecular Life Sciences at the University of Pennsylvania (http://www.sas.upenn.edu/biochem/vspmls.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: orange@bcm.edu Introduction Natural Killer (NK) cells are lymphocytes of the innate immune system that recognize and kill virally infected and tumorigenic cells. While NK cells can indirectly promote immune function via cytokine release and costimulation of other immune cells, direct NK cell cytotoxicity requires stable contact with target cells; in other words, transient interaction between an NK cell and its target must be stabilized by cell-cell adhesion forces prior to cytolysis [1]. One such adhesion force is provided via receptor- ligand interactions, some of which can also promote activation signaling like that of the NK cell receptors CD2 and its cognate ligands CD58 and CD48 [2], or LFA-1 and its cognate ligands ICAM-1 through 5 [3,4]. Upon appropriate signals of stress and other activating signals from the target cell, a favorable balance of receptor-ligand interactions promotes formation of the NK cell immunological synapse (NKIS) which can further support activation signaling and cellular reorganization [5]. Downstream signaling directs the polarization of the microtubule organizing center and associated lytic granules to the immunological synapse providing a means for directed secretion and lysis of the target cell of interest [6]. Given NK cells’ lethal potential, it is paramount that they can effectively target infected and transformed cells, while sparing healthy cells. This specificity is provided via particular receptor- ligand interactions. Some receptor-ligand interactions, such as CD2-CD58/48 [7], activate NK cells and promote signaling that drives directed secretion and cytolysis [8]. Other receptors, such as killer-cell immunoglobulin-like receptor 2DL1 (KIR2DL1) [9], recognize determinants of self (e.g. HLA-Cw4) and inhibit NK cytotoxicity. Both activating and inhibiting receptor-ligand inter- actions require contact between NK and target cells, and both, in turn, provide some degree of adhesive force. These adhesion forces, in combination with appropriate activating or inhibitory signals, help to regulate NK cell cytotoxic function [6]. Regulation of NK cell cytotoxicity, however, involves more than just simple receptor-ligand interactions. As an added complexity, numerous thin membranous tethers, known as membrane nanotubes, form between NK cells and their targets. Originally described to enable intercellular traffic [10], nanotubes (NT) range in size from 20–200 nm in diameter and extend up to several cell diameters in length [10,11]. They can connect a variety of immune cells, including T cells, T cells and their target cells, dendritic cells, NK cells, and NK cells and their target cells [11]. NTs are believed to perform a variety of functions, including assisting in intercellular communication by transporting vesicles and membrane proteins [11,12,13], transmitting calcium fluxes [14], and even transporting viruses between cells [12,15]. Primary human NK cells and the human NK cell tumor line NKL readily form NTs with themselves and various target cells [16]. Functionally, NK cell NTs facilitate cytotoxic function both by aiding in the retrieval of motile target cells to form an NK cell PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e47664