Triggering of 2B4 (CD244) can induce natural killer (NK)-cell activation, costimulation, or even inhibition of NK-cell activity. Here, we investigate the molecular basis for the different signals generated by 2B4. We show that the first immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of 2B4 is sufficient for 2B4-mediated NK-cell activation, whereas the third ITSM can negatively influence 2B4 signaling. We further identify signaling molecules that associate with 2B4. Signaling lymphocyte activation molecule-associated protein (SAP) can bind to all 4 ITSMs of 2B4 in a phosphorylation-dependent manner. The phosphorylated third ITSM can additionally recruit the phosphatases SHP-1, SHP-2, SHIP, and the inhibitory kinase Csk. SAP acts as an inhibitor of interactions between 2B4 and these negative regulatory molecules, explaining how 2B4 inhibits NK-cell activation in the absence of functional SAP, as occurs in cells from patients with X-linked lymphoproliferative syndrome (XLP IntroductionThe activity of natural killer (NK) cells is regulated by a multitude of different surface receptors. 1 Depending on their function they can be divided roughly into activating and inhibiting receptors. In human NK cells inhibiting receptors are represented by members of the KIR (killer cell immunoglobulin-like receptor) family and by the CD94/NKG2 heterodimer. 2 Inhibiting receptors recognize self-major histocompatibility complex (MHC) class I and thereby protect normal cells from NK-cell attack. 3 Many activating NK-cell receptors have been discovered only recently and include NKp30, NKp44, NKp46, NKp80, NKG2D, DNAM-1, 2B4, NTB-A, and CS1 (CRACC). 4 2B4 binds to CD48, which is widely expressed in the hematopoietic system. 5,6 NTB-A and CS1 (CRACC) are homophilic, 7-9 and NKG2D recognizes several different ligands, some of which are inducible by cellular stress. 10 In most cases, however, the ligands or structures recognized by activating NK-cell receptors remain unknown.Signals by activating and inhibiting receptors are finely balanced to ensure the tolerance of NK cells toward normal cells. 11 This balance can be disturbed by a reduction of the negative signals or by an enhancement of positive signals, both leading to the activation of NK cells. A loss of negative signal is usually observed when target cells lose MHC class I expression, for example, during viral infection or transformation, and thereby become sensitive to NK-cell attack. 12 Recently, it became clear that the induced expression of ligands for activating NK-cell receptors on target cells with normal MHC class I expression is also sufficient to lead to This underscores the fine balance of positive and negative signals that regulate NK-cell activity.2B4 was first identified as an activating NK-cell receptor. 14 In humans 2B4 is expressed on all NK cells, ␥␦ T cells, monocytes, basophils, and on a subset of CD8 ϩ T cells. [15][16][17] On engagement 2B4 can stimulate NK-cell cytotoxicity, production of interferon ␥, and granule exocytosis....