Association of the X-linked lymphoproliferative disease gene product SAP/SH2D1A with 2B4, a natural killer cell-activating molecule, is dependent on phosphoinositide 3-kinase

Natural killer (NK) cells express an activating receptor, 2B4, that enhances cellular cytotoxicity. Upon NK cell activation by ligation of 2B4, the intracellular domain of 2B4 associates with the X-linked lymphoproliferative disease (XLP) gene product, signaling lymphocytic activation molecule-associated protein/SH2D1A (SAP/SH2D1A). Defective intracellular association of 2B4 with mutated SAP/SH2D1A is likely to underlie the defects in cytotoxicity observed in NK cells from patients with XLP. We report here a role for phosphoinositide 3-kinase (PI3K) in the recruitment and association of SAP/SH2D1A to 2B4 in human NK cells. The activation of normal NK cells by ligation of 2B4 leads to the phosphorylation of 2B4, recruitment of SAP/SH2D1A, and association of the p85 regulatory subunit of PI3K. The inhibition of PI3K enzymatic activity with either wortmannin or LY294002 prior to 2B4 ligation does not alter the association of 2B4 with the p85 subunit but prevents the recruitment of SAP/SH2D1A to 2B4. In addition, PI3K inhibitors significantly diminish the cytotoxic function of primary NK cells. This observed inhibition of cytotoxicity, present in normal NK cells, was less apparent or absent in NK cells derived from a patient with XLP. These data indicate that the cytotoxicity of activated NK cells is mediated by the association of 2B4 and SAP/SH2D1A, and that this association is dependent upon the activity of PI3K.     

Functional requirement for SAP in 2B4-mediated activation of human natural killer cells as revealed by the X-linked lymphoproliferative syndrome

X-linked lymphoproliferative syndrome (XLP) is an immunodeficiency characterized by life-threatening infectious mononucleosis and EBV-induced B cell lymphoma. The gene mutated in XLP encodes SLAM (signaling lymphocytic activation molecule-associated protein)-associated protein (SAP), a small SH2 domain-containing protein. SAP associates with 2B4 and SLAM, activating receptors expressed by NK and T cells, and prevents recruitment of SH2 domain-containing protein tyrosine phosphatase-2 SHP-2) to the cytoplasmic domains of these receptors. The phenotype of XLP may therefore result from perturbed signaling through SAP-associating receptors. We have addressed the functional consequence of SAP deficiency on 2B4-mediated NK cell activation. Ligating 2B4 on normal human NK cells with anti-2B4 mAb or interaction with transfectants bearing the 2B4 ligand CD48 induced NK cell cytotoxicity. In contrast, ligation of 2B4 on NK cells from a SAP-deficient XLP patient failed to initiate cytotoxicity. Despite this, CD2 or CD16-induced cytotoxicity of SAP-deficient NK cells was similar to that of normal NK cells. Thus, selective impairment of 2B4-mediated NK cell activation may contribute to the immunopathology of XLP.     

SLAM-associated protein deficiency causes imbalanced early signal transduction and blocks downstream activation in T cells from X-linked lymphoproliferative disease patients

Deficiency of SAP (SLAM (signaling lymphocyte activation molecule)-associated protein) protein is associated with a severe immunodeficiency, the X-linked lymphoproliferative disease (XLP) characterized by an inappropriate immune reaction against Epstein-Barr virus infection often resulting in a fatal clinical course. Several studies demonstrated altered NK and T cell function in XLP patients; however, the mechanisms underlying XLP disease are still largely unknown. Here, we show that non-transformed T cell lines obtained from XLP patients were defective in several activation events such as IL-2 production, CD25 expression, and homotypic cell aggregation when cells were stimulated via T cell antigen receptor (TCR).CD3 but not when early TCR-dependent events were bypassed by stimulation with phorbol 12-myristate 13-acetate/ionomycin. Analysis of proximal T cell signaling revealed imbalanced TCR.CD3-induced signaling in SAP-deficient T cells. Although phospholipase C gamma 1 phosphorylation and calcium response were both enhanced in T cells from XLP patients, phosphorylation of VAV and downstream signal transduction events such as mitogen-activated protein kinase phosphorylation and IL-2 production were diminished. Importantly, reconstitution of SAP expression by retroviral-mediated gene transfer completely restored abnormal signaling events in T cell lines derived from XLP patients. In conclusion, SAP mutation or deletion in XLP patients causes profound defects in T cell activation, resulting in immune deficiency. Moreover, these data provide evidence that SAP functions as an essential integrator in early TCR signal transduction.     

SAP binds to CD22 and regulates B cell inhibitory signaling and calcium flux

The signaling lymphocyte activation molecule (SLAM)-associated protein (SAP or SH2D1A) is an important regulator of immune function which, when mutated or deleted, causes the X-linked lymphoproliferative syndrome (XLP). Because B cell lymphoma is a major phenotype of XLP, it is important to understand the function of SAP in B cells. Here we report that SAP is expressed endogenously in mouse splenic B cells, is inducibly expressed in the human BJAB cells, and co-localizes and interacts with CD22. We also show that SAP binding to the inhibitory immunoreceptor CD22 regulates calcium mobilization in B cells. Moreover, forced expression of SAP leads to constitutive CD22 tyrosine phosphorylation and decreased Ca²⁺ response in B cells. Biochemical analysis reveals that, in response to IgM cross-linking, the phosphorylation of Syk, Blnk, or PLCγ2 and their interactions with one another were either diminished or completely abolished in SAP-expressing cells compared to cells that lack SAP. Collectively our work identifies a novel role for SAP in B cells and extends its function to inhibitory immunoreceptor signaling and calcium mobilization.     

Cutting edge: human 2B4, an activating NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor signaling protein SAP.

The genetic defect in X-linked lymphoproliferative syndrome (XLP) is the Src homology 2 domain-containing protein SAP. SAP constitutively associates with the cell surface molecule, signaling lymphocytic activation molecule (SLAM), and competes with SH2-domain containing protein tyrosine phosphatase-2 (SHP-2) for recruitment to SLAM. SLAM exhibits homology with the mouse cell surface receptor 2B4. The human homologue of 2B4 has now been identified. It is recognized by the c1.7 mAb, a mAb capable of activating human NK cells. Human 2B4 became tyrosine phosphorylated following pervanadate-treatment of transfected cells and recruited SHP-2. SAP was also recruited to 2B4 in activated cells. Importantly, the 2B4-SAP interaction prevented the association between 2B4 and SHP-2. These results suggest that the phenotype of XLP may result from perturbed signaling not only through SLAM, but also other cell surface molecules that utilize SAP as a signaling adaptor protein.     

Disease-causing SAP mutants are defective in ligand binding and protein folding

The X-linked lymphoproliferative (XLP) syndrome is caused by mutations or deletions in the SH2D1A gene that encodes an SH2 domain protein named SH2D1A or SAP. The identification of a number of missense mutations within the protein's SH2 domain, each of which can directly cause disease, provides a unique opportunity to investigate the function of an interaction protein module, SH2, in the pathogenesis of XLP. We show here that SAP mutants found in XLP patients are defective in binding its physiological ligands signaling lymphocyte activating molecule (SLAM), a co-receptor in T cell activation, and Fyn, a Src family protein tyrosine kinase. Consequently, these mutants are deficient in signaling through the SLAM receptor. This is reflected by compromised abilities for the mutants to recruit Fyn to SLAM and to activate Fyn, by reduced phosphorylation of the receptor, and by deficiencies for the mutants in blocking binding of SHP-2 to SLAM. Furthermore, all mutants examined are defective in protein folding as manifested by their significantly reduced melting temperatures upon thermal denaturation, compared to that of SAP. Taken together, these results suggest that defects in ligand binding, receptor signaling, and protein folding collectively contribute to the loss of function for disease-causing SAP mutants.     

Src homology 2 domain-containing inositol 5-phosphatase 1 mediates cell cycle arrest by FcgammaRIIB

We previously found that low affinity receptors for the Fc portion of IgG, FcgammaRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcgammaRIIB, FcgammaRIIB-deficient mast cells reconstituted with wild-type or mutated FcgammaRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcgammaRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcgammaRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcgammaRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcgammaRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcgammaRIIB can use other effectors that specifically inhibit cell proliferation.     

The XLP syndrome protein SAP interacts with SH3 proteins to regulate T cell signaling and proliferation

The gene sap/shd1a, which encodes a 128-residue SH2 domain protein, is frequently deleted or mutated in the X-linked lymphoproliferative syndrome (XLP). The SAP SH2 domain differs from others in the same class in that it is not only capable of binding to a phosphotyrosine-containing peptide, it can also associate with an SH3 domain using a distinct surface. This novel mode of ligand-binding is initially discovered in the SLAM-SAP-Fyn complex that plays a critical role in T cell and natural killer cell activation. To identify additional binding partners for SAP, we screened a panel of 12 SH3 domains derived from regulatory proteins and identified NCK1 as a novel target of SAP in T cells. NMR analysis demonstrated that the NCK1 and Fyn SH3 domains possessed comparable affinities for SAP and engaged the same set of residues on the surface of the SAP SH2 domain. Depletion of SAP by siRNA caused a significant decrease in NCK1 tyrosine phosphorylation as well as the phosphorylation of other T cell receptor (TCR) downstream proteins such as LAT and SLP-76. Moreover, SAP was shown to regulate T cell proliferation through the MAP-kinase Erk. Taken together, our work identifies NCK1 as a novel physiological partner for SAP and a direct regulator of TCR signaling and T cell proliferation.     

Signaling lymphocytic activation molecule-associated protein controls NKT cell functions

X-linked lymphoproliferative disease (XLP) is a fatal immunological disorder that typically manifests following EBV infection. XLP patients exhibit a number of immune defects including abnormal T, B, and NK lymphocyte function. These defects have been attributed to mutations of Src homology 2 domain-containing gene 1A (SH2D1A), the gene encoding signaling lymphocytic activation molecule-associated protein (SAP), an intracellular adaptor molecule expressed in lymphocytes. We have observed that SAP knockout (SAPKO) mice and humans with XLP have a complete lack of CD1d-restricted NKT cells. As expected, SAPKO mice injected with the NKT cell agonist, alpha-galactosylceramide failed to generate NKT cell IFN-gamma or IL-4. Furthermore, in contrast to wild-type littermates, SAPKO mice coinjected with OVA and alpha-galactosylceramide failed to mount OVA-specific CTL responses. These data suggest that an absence of NKT cells may underlie part of the immune dysregulation seen in SAPKO mice and in XLP patients.     

Genomic organization and characterization of mouse SAP, the gene that is altered in X-linked lymphoproliferative disease

X-linked lymphoproliferative (XLP) disease is a fatal immunological disorder that renders the immune system unable to respond effectively to Epstein-Barr virus (EBV) infection. The gene that encodes a protein termed SAP or SH2D1A is either deleted or mutated in XLP patients, resulting in uncontrolled B- and T-cell proliferation upon EBV infection. Here, we report the cloning and characterization of the mouse SAP gene. It is localized on the mouse X chromosome and comprises four exons spanning approximately 25 kb. Its expression appears to be restricted to T lymphocytes. Whereas a high level of SAP expression is observed in Thl cells, only small amounts are detectable in Th2 cells. Moreover, SAP expression is down-regulated upon in vitro activation of T cells, including CD4+, CD8+ single-positive T cells, and Thl and Th2 cells. This study provides valuable information for in-depth genetic and biochemical analysis of the function of SAP in the immune system.     

The activation and subsequent regulatory roles of Lyn and CD19 after B cell receptor ligation are independent

The cell surface glycoprotein CD19 and the Src-related protein tyrosine kinase Lyn are key mediators of, respectively, positive and negative signaling in B cells. Despite the apparent opposition of their regulatory functions, a recent model of the biochemical events after B cell receptor (BCR) ligation intimately links the activation of Lyn and CD19. We examined the biochemical consequences of BCR ligation in mouse B cells lacking either Lyn or CD19 for evidence of interaction or codependence. In contrast to published results, we found CD19 phosphorylation after BCR ligation to be unaffected by the absence of Lyn, yet dependent on Src family protein tyrosine kinases as it was inhibited fully by PP2, an Src family-specific inhibitor. Consistent with normal CD19 phosphorylation in lyn(-/-) B cells, the recruitment of phosphoinositide-3 kinase to CD19 and the ability of CD19 to enhance both intracellular calcium flux and extracellular signal-regulated kinase 1/2 activation after coligation with the BCRs were intact in the absence of Lyn. Similarly, unique functions of Lyn were found to be independent of CD19. CD19(-/-) B cells were normal for increased Lyn kinase activity after BCR ligation, inhibition of BCR-mediated calcium flux after CD22 coligation, and inhibition of extracellular signal-regulated kinase phosporylation after FcgammaRIIB coligation. Collectively, these data show that the unique functions of Lyn do not require CD19 and that the signal amplification mediated by CD19 is independent of Lyn. We conclude that the roles of Lyn and CD19 after BCR ligation are independent and opposing, one being primarily inhibitory and the other stimulatory.     

NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244)

2B4 is an NK cell activation receptor that can provide a co-stimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-gamma release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor.     

Defective NK cell activation in X-linked lymphoproliferative disease

X-linked lymphoproliferative disease (XLP) is characterized by a selective immune deficiency to EBV. The molecular basis of XLP has been attributed to mutations of signaling lymphocytic activation molecule-associated protein, an intracellular molecule known to associate with the lymphocyte-activating surface receptors SLAM and 2B4. We have identified a single nucleotide mutation in SLAM-associated protein that affects the NK cell function of males carrying the mutated gene. In contrast to normal controls, both NK and lymphokine-activated killer cell cytotoxicity was significantly reduced in two XLP patients. In addition to decreased baseline cytotoxicity, ligation of 2B4 significantly augmented NK lytic function in normal controls but failed to enhance the cytotoxicity of NK cells from XLP patients. These findings suggest that association of SAP with 2B4 is necessary for optimal NK/lymphokine-activated killer cytotoxicity and imply that alterations in SAP/2B4 signaling contribute to the immune dysfunction observed in XLP.     

Functional requirements for interactions between CD84 and Src homology 2 domain-containing proteins and their contribution to human T cell activation

Cell surface receptors belonging to the CD2 subset of the Ig superfamily of molecules include CD2, CD48, CD58, 2B4, signaling lymphocytic activation molecule (SLAM), Ly9, CD84, and the recently identified molecules NTB-A/Ly108/SLAM family (SF) 2000, CD84H-1/SF2001, B lymphocyte activator macrophage expressed (BLAME), and CRACC (CD2-like receptor-activating cytotoxic cells)/CS-1. Some of these receptors, such as CD2, SLAM, 2B4, CRACC, and NTB-A, contribute to the activation and effector function of T cells and NK cells. Signaling pathways elicited via some of these receptors are believed to involve the Src homology 2 (SH2) domain-containing cytoplasmic adaptor protein SLAM-associated protein (SAP), as it is recruited to SLAM, 2B4, CD84, NTB-A, and Ly-9. Importantly, mutations in SAP cause the inherited human immunodeficiency X-linked lymphoproliferative syndrome (XLP), suggesting that XLP may result from perturbed signaling via one or more of these SAP-associating receptors. We have now studied the requirements for SAP recruitment to CD84 and lymphocyte activation elicited following ligation of CD84 on primary and transformed human T cells. CD84 was found to be rapidly tyrosine phosphorylated following receptor ligation on activated T cells, an event that involved the Src kinase Lck. Phosphorylation of CD84 was indispensable for the recruitment of SAP, which was mediated by Y(262) within the cytoplasmic domain of CD84 and by R(32) within the SH2 domain of SAP. Furthermore, ligating CD84 enhanced the proliferation of anti-CD3 mAb-stimulated human T cells. Strikingly, this effect was also apparent in SAP-deficient T cells obtained from patients with XLP. These results reveal a novel function of CD84 on human lymphocytes and suggest that CD84 can activate human T cells via a SAP-independent mechanism.     

The SH2 domain containing tyrosine phosphatase-1 down-regulates activation of Lyn and Lyn-induced tyrosine phosphorylation of the CD19 receptor in B cells

SHP-1 is a cytosolic tyrosine phosphatase implicated in down-regulation of B cell antigen receptor signaling. SHP-1 effects on the antigen receptor reflect its capacity to dephosphorylate this receptor as well as several inhibitory comodulators. In view of our observation that antigen receptor-induced CD19 tyrosine phosphorylation is constitutively increased in B cells from SHP-l-deficient motheaten mice, we investigated the possibility that CD19, a positive modulator of antigen receptor signaling, represents another substrate for SHP-1. However, analysis of CD19 coimmunoprecipitable tyrosine phosphatase activity in CD19 immunoprecipitates from SHP-1-deficient and wild-type B cells revealed that SHP-1 accounts for only a minor portion of CD19-associated tyrosine phosphatase activity. As CD19 tyrosine phosphorylation is modulated by the Lyn protein-tyrosine kinase, Lyn activity was evaluated in wild-type and motheaten B cells. The results revealed both Lyn as well as CD19-associated Lyn kinase activity to be constitutively and inducibly increased in SHP-1-deficient compared with wild-type B cells. The data also demonstrated SHP-1 to be associated with Lyn in stimulated but not in resting B cells and indicated this interaction to be mediated via Lyn binding to the SHP-1 N-terminal SH2 domain. These findings, together with cyanogen bromide cleavage data revealing that SHP-1 dephosphorylates the Lyn autophosphorylation site, identify Lyn deactivation/dephosphorylation as a likely mechanism whereby SHP-1 exerts its influence on CD19 tyrosine phosphorylation and, by extension, its inhibitory effect on B cell antigen receptor signaling.     

Abnormal T cell receptor signal transduction of CD4 Th cells in X-linked lymphoproliferative syndrome

The molecular basis of X-linked lymphoproliferative (XLP) disease has been attributed to mutations in the signaling lymphocytic activation molecule-associated protein (SAP), an src homology 2 domain-containing intracellular signaling molecule known to interact with the lymphocyte-activating surface receptors signaling lymphocytic activation molecule and 2B4. To investigate the effect of SAP defects on TCR signal transduction, herpesvirus saimiri-immortalized CD4 Th cells from XLP patients and normal healthy individuals were examined for their response to TCR stimulation. CD4 T cells of XLP patients displayed elevated levels of tyrosine phosphorylation compared with CD4 T cells from healthy individuals. In addition, downstream serine/threonine kinases are constitutively active in CD4 T cells of XLP patients. In contrast, TCR-mediated activation of Akt, c-Jun-NH(2)-terminal kinases, and extracellular signal-regulated kinases in XLP CD4 T cells was transient and rapidly diminished when compared with that in control CD4 T cells. Consequently, XLP CD4 T cells exhibited severe defects in up-regulation of IL-2 and IFN-gamma cytokine production upon TCR stimulation and in MLRs. Finally, SAP specifically interacted with a 75-kDa tyrosine-phosphorylated protein upon TCR stimulation. These results demonstrate that CD4 T cells from XLP patients exhibit aberrant TCR signal transduction and that the defect in SAP function is likely responsible for this phenotype.     

B cell induction of IL-13 expression in NK cells: role of CD244 and SLAM-associated protein

NK cells are an important component of the innate immune system that can also interact with B cells in a mutually productive manner. We have previously shown that activated B cells can induce NK cells to up-regulate their secretion of IFN-gamma. In this study, we show that B cells, and, particularly, marginal zone B cells, can, in addition, induce NK cells via direct cell-cell interactions to express mRNA encoding the Th2 cytokine IL-13. The induction of NK cell IL-13 mRNA expression requires the ligation of the CD244 receptor by the CD48 ligand on B cells via signaling pathways that depend upon expression of the X-linked lymphoproliferative disease gene product, SH2D1A/DSHP/SAP (SLAM-associated protein, or SAP) in NK cells. Thus, the positive signals attributed to the B cell activation of CD244 on murine NK cells appears to be more similar to the activity of CD244 on human cells. The induction of IL-13 mRNA by B cells may account for the effect of NK cells on the generation of Th2-type responses in the presence of some adjuvants.     

Mutational analysis reveals multiple distinct sites within Fc gamma receptor IIB that function in inhibitory signaling

The low-affinity receptor for IgG, FcgammaRIIB, functions broadly in the immune system, blocking mast cell degranulation, dampening the humoral immune response, and reducing the risk of autoimmunity. Previous studies concluded that inhibitory signal transduction by FcgammaRIIB is mediated solely by its immunoreceptor tyrosine-based inhibition motif (ITIM) that, when phosphorylated, recruits the SH2-containing inositol 5'- phosphatase SHIP and the SH2-containing tyrosine phosphatases SHP-1 and SHP-2. The mutational analysis reported here reveals that the receptor's C-terminal 16 residues are also required for detectable FcgammaRIIB association with SHIP in vivo and for FcgammaRIIB-mediated phosphatidylinositol 3-kinase hydrolysis by SHIP. Although the ITIM appears to contain all the structural information required for receptor-mediated tyrosine phosphorylation of SHIP, phosphorylation is enhanced when the C-terminal sequence is present. Additionally, FcgammaRIIB-mediated dephosphorylation of CD19 is independent of the cytoplasmic tail distal from residue 237, including the ITIM. Finally, the findings indicate that tyrosines 290, 309, and 326 are all sites of significant FcgammaRIIB1 phosphorylation following coaggregation with B cell Ag receptor. Thus, we conclude that multiple sites in FcgammaRIIB contribute uniquely to transduction of FcgammaRIIB-mediated inhibitory signals.     

The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling

Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex.