Dynamic Redistribution of the Activating 2B4/SAP Complex at the Cytotoxic NK Cell Immune Synapse

Abstract

The 2B4 molecule (CD244) has been described as a coreceptor in human NK cell activation. However, the behavior of 2B4 during the cytotoxic NK cell immune synapse (NK-IS) formation remains undetermined. In this study, we demonstrate the redistribution of 2B4 and the signaling adaptor molecule, signaling lymphocyte activation molecule-associated protein (SAP), to the cytotoxic NK-IS upon formation of conjugates between resting NK cells and EBV-infected 721.221 human cells. Confocal microscopy showed that 2B4 localized at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing talin within NK cell and ICAM-1 on target cells. Videomicroscopy studies with 2B4-GFP-transfected NK cells revealed that 2B4 redistributed to cytotoxic NK-IS as soon as the cell contact occurred. Simultaneously, a SAP-GFP also clustered at the contact site, where it remained during the interaction period. The 2B4 molecular clusters remained bound to the target cell even after NK cell detachment. These results underscore the function of 2B4 as an adhesion molecule and suggest a relevant role in the initial binding, scanning of target cells, and formation of cytotoxic NK-IS. Finally, these findings are indicative of an important role of the activating 2B4/signaling lymphocyte activation molecule-associated protein complex during the recognition of EBV-infected cells.

Assembly and function of the natural killer cell immune synapse

“…In other experiments, cell conjugates formed between NK cells and target cells expressing the 2B4 ligand CD48 showed the accumulation of the activating receptor complex 2B4/LAT at the cSMAC, surrounded by a pSMAC characterized by the accumulation of talin in the NK cell and ICAM-1 on the target cell. Moreover, CD48 also accumulated at this synapse (29). The topography of this synapse observed at 5 minutes of the conjugate formation is consistent with the recruitment of 2B4 and LAT to lipid rafts observed in different systems, and with the organization of signaling molecules (cSMAC) and LFA-1 (pSMAC) observed at the cNK-IS in activated NK cells (14,55).…”

“…This early accumulation of 2B4 underscores the role of this molecule in cell-cell adhesion and NK cell activation. In addition, the adaptor molecule SAP (SLAM-associated protein) also accumulates at the cNK-IS and co-localizes with 2B4 during this prolonged time, which demonstrates the relevance of this adaptor molecule for the cytotoxic process in NK cells (29). In this regard, SAP is encoded by the SH2D1A gene, which is mutated in patients with the inherited immunodeficiency X-linked lymphoproliferative disease (XLP), a human disorder characterized by a dysfunction of the T/B-cell interactions and NK cell function, leading to a reduced ability to control the EpsteinBarr virus (EBV) infection (30,31).…”

Inappropriate Recruitment and Activity by the Src Homology Region 2 Domain-Containing Phosphatase 1 (SHP1) Is Responsible for Receptor Dominance in the SHIP-Deficient NK Cell

“…This dramatic increase of SHP1 at 2B4 receptor complexes could alter the balance of signaling in the SHIP-deficient NK cell. Importantly, 2B4 has been shown to be recruited to the NK synapse (48,49). Therefore, the increased presence of SHP1 at the NK synapse in SHIP Ϫ/Ϫ NK cells is likely to terminate activating signals before they propagate to more distal effectors required for NK function.…”

SAP controls the cytolytic activity of CD8+ T cells against EBV-infected cells

“…35,36 Recent studies show that 2B4 and SAP are rapidly recruited to the central part of the NK immunologic synapse that forms at the contact with CD48 ϩ target cells. 37,38 In addition, it has been proposed that killer cell inhibitory receptors (KIRs) negatively control NK immunologic synapse assembly and lytic activity by blocking recruitment of 2B4 into lipid rafts and by blocking clustering of lipid rafts at the synapse in a SHP-1-and SHP-2-dependent manner. 34,39 Since SAP has the properties to compete with SHP-2 and to bind to 2B4, it is possible that SAP regulates the activity of both activatory (2B4) and inhibitory (KIR) receptors controlling the assembly of the lytic synapse.…”