The X-linked lymphoproliferative disease gene product SAP is expressed in activated T and NK cells

The unique manifestation of the inherited immunodeficiency, X-linked lymphoproliferative disease (XLP), is the impaired control of EBV infection. The gene, which carries mutations or is deleted in the patients, has been identified (Xq25). The encoded protein (SAP, 128 aa) contains a single SH2 domain and binds to signaling lymphocytic activation molecule (SLAM) and to other related surface molecules that are expressed on activated T, B and NK cells. SAP modifies signal transduction through its association with these molecules. Initially it was assumed that SAP acts passively by interfering and blocking active interactions involving other SH2 carrying molecules. We demonstrated that SAP protein is expressed in activated T and NK, but not in activated B cells. This finding is in line with the fact that in vitro performance of effector cells derived from XLP patients is impaired. However, it is still not known why the severe symptoms (fatal mononucleosis or malignant lymphoproliferation in the survivors of the primary infection) are elicited by EBV. We studied SAP expression in several Burkitt lymphoma (BL) derived lines. In contrast to normal B cells, certain lines expressed SAP. These were all type I cells in the Burkitt line nomenclature: they expressed only one of the EBV encoded proteins (EBNA-1) and their phenotype corresponded to resting B cells. Lymphoblastoid cell lines and type III BLs, whose phenotype resembled activated B cells and expressed all nine EBV encoded proteins, were devoid of SAP. The relationship between cell activation and SAP expression is reciprocal in T and B cells i.e. BL lines, activated T and NK cells express SAP, while BL blasts do not express SAP. This opposite relationship may be exploited for studies about the function of SAP.     

Molecular and cellular pathogenesis of X-linked lymphoproliferative disease

Summary: X‐linked lymphoproliferative disease (XLP) is an inherited immune defect caused by mutations in the Src homology 2 domain‐containing gene 1A, which encodes the adapter protein, signaling lymphocytic activation molecule (SLAM)‐associated protein (SAP). SAP is expressed in T cells, natural killer (NK) cells, and NKT cells, where it binds to the cytoplasmic domain of the surface receptor SLAM (CD150) and the related receptors, 2B4 (CD244), CD84, Ly9 (CD229), NK‐T‐B‐antigen, and CD2‐like receptor‐activating cytotoxic T cells. SAP also binds to the Src family tyrosine kinase Fyn and recruits it to SLAM, which leads to the generation of downstream phosphotyrosine signals. While the roles of the SLAM family receptors are only beginning to be understood, experiments suggest that these molecules regulate important aspects of lymphocyte function, such as proliferation, cytokine secretion, cytotoxicity, and antibody production. Thus, in XLP patients who lack functional SAP, the SLAM family receptors may not signal properly. This property likely contributes to the phenotypes of XLP, including fulminant infectious mononucleosis, lymphoma, and hypogammaglobulinemia. Further studies of SAP and the SLAM family receptors will provide insights into XLP and elucidate the signaling events regulating lymphocyte ontogeny and function.     

2B4: an NK cell activating receptor with unique specificity and signal transduction mechanism

2B4 is a cell surface glycoprotein of the Ig-superfamily structurally related to CD2-like molecules such as CD2, CD48, CD58, CD84, Ly-9, and SLAM. Engagement of 2B4 on NK cells with specific antibodies or with its ligand CD48 enhances NK cell-mediated cytotoxicity. 2B4 is also expressed on both CD8+ T cells and myeloid cells, but its function in these cells remains unknown. Signal transduction through 2B4 involves recruitment of the SH2-containing adapter molecule SAP to cytoplasmic tyrosines. SAP is deficient in patients affected by X-linked lymphoproliferative disorder (XLP), which is triggered following EBV infection. Thus, an interruption of signaling through 2B4 and related molecules may impair NK cell recognition of virally infected cells and contribute to XLP.     

Inhibitory 2B4 contributes to NK cell education and immunological derangements in XLP1 patients

X‐linked lymphoproliferative disease 1 (XLP1) is an inherited immunodeficiency, caused by mutations in SH2D1A encoding Signaling Lymphocyte Activation Molecule (SLAM)‐associated protein (SAP). In XLP1, 2B4, upon engagement with CD48, has inhibitory instead of activating function. This causes a selective inability of cytotoxic effectors to kill EBV‐infected cells, with dramatic clinical sequelae. Here, we investigated the NK cell education in XLP1, upon characterization of killer Ig‐like receptor (KIR)/KIR‐L genotype and phenotypic repertoire of self‐HLA class I specific inhibitory NK receptors (self‐iNKRs). We also analyzed NK‐cell cytotoxicity against CD48⁺ or CD48^? KIR‐ligand matched or autologous hematopoietic cells in XLP1 patients and healthy controls. XLP1 NK cells may show a defective phenotypic repertoire with substantial proportion of cells lacking self‐iNKR. These NK cells are cytotoxic and the inhibitory 2B4/CD48 pathway plays a major role to prevent killing of CD48⁺ EBV‐transformed B cells and M1 macrophages. Importantly, self‐iNKR defective NK cells kill CD48^? targets, such as mature DCs. Self‐iNKR^? NK cells in XLP1 patients are functional even in resting conditions, suggesting a role of the inhibitory 2B4/CD48 pathway in the education process during NK‐cell maturation. Killing of autologous mature DC by self‐iNKR defective XLP1 NK cells may impair adaptive responses, further exacerbating the patients’ immune defect.     

XLP1 inhibitory effect by 2B4 does not affect DNAM‐1 and NKG2D activating pathways in NK cells

X‐linked lymphoproliferative disease 1 (XLP1) is a rare congenital immunodeficiency caused by SH2D1A (Xq25) mutations resulting in lack or dysfunction of SLAM‐associated protein adaptor molecule. In XLP1 patients, upon ligand (CD48) engagement, 2B4 delivers inhibitory signals that impair the cytolytic activity of NK (and T) cells. This causes the selective inability to control EBV infections and the occurrence of B‐cell lymphomas. Here, we show that in the absence of SLAM‐associated protein, co‐engagement of 2B4 with different activating receptors, either by antibodies or specific ligands on target cells, inhibits different ITAM‐dependent signaling pathways including activating killer Ig‐like receptors. In XLP1 NK cells, 2B4 affected both the cytolytic and IFN‐γ production capabilities, functions that were restored upon disruption of the 2B4/CD48 interactions. Notably, we provide evidence that 2B4 dysfunction does not affect the activity of DNAM‐1 and NKG2D triggering receptors. Thus, while CD48⁺ B‐EBV and lymphoma B cells devoid of NKG2D and DNAM‐1 ligands were resistant to lysis, the preferential usage of these receptors allowed XLP1 NK cells to kill lymphomas that expressed sufficient amounts of the specific ligands. The study sheds new light on the XLP1 immunological defect and on the cross‐talk of inhibitory 2B4 with triggering NK (and T) receptors.     

Missense mutations in SH2D1A identified in patients with X-linked lymphoproliferative disease differentially affect the expression and function of SAP

X-linked lymphoproliferative disease (XLP) is an immunodeficiency resulting from mutations in SH2D1A, which encodes signalling lymphocytic activation molecule (SLAM)-associated protein (SAP). In addition to SLAM, SAP associates with several other cell-surface receptors including 2B4 (CD244), Ly9 (CD229), CD84 and NTB-A. SAP contains a single src-homology-2 domain and acts as an intracellular adaptor protein by recruiting the protein tyrosine kinase FynT to the cytoplasmic domains of some of these receptors, which results in the initiation of specific downstream signal transduction pathways. XLP is likely to result from perturbed signalling through one or more of these SAP-associating receptors. In this study, we identified missense (Y54C, I84T and F87S) and insertion (fs82 --> X103) mutations in four different kindreds affected by XLP. Each mutation dramatically reduced the half-life of SAP, thus diminishing its expression in primary lymphocytes as well as in transfected cell lines. Interestingly, although the Y54C and F87S mutations compromised the ability of SAP to associate with different receptors, the I84T mutation had no effect on the ability of SAP to bind SLAM, CD84 or 2B4. However, signalling downstream of SLAM was reduced in the presence of SAP bearing the I84T mutation. These findings indicate that, irrespective of the type of mutation, signalling through SAP-associating receptors in XLP can be impaired by reducing the expression of SAP, the ability of SAP to bind surface receptors and/or its ability to activate signal transduction downstream of the SLAM-SAP complex.     

Signaling lymphocytic activation molecule (SLAM) regulates T cellular cytotoxicity

Signaling lymphocytic activation molecule (SLAM) is a CD2-related surface receptor expressed by activated T cells and B cells. SLAM is a self ligand and enhances T cellular proliferation and IFN-gamma production. A defective SLAM associated protein (SAP) causes X-linked lymphoproliferative syndrome (XLP), a frequently lethal mononucleosis based on the inability to control EBV. We report that SLAM augments TCR-mediated cytotoxicity. In normal CD4(+) and CD8(+) T cells, SLAM enhanced TCR-mediated cytotoxicity. In CD4(+) and CD8(+) Herpesvirus saimiri (H.saimiri) infected T cells, SLAM engagement alone triggered cytotoxicity. Using H.saimiri-transformed T cells as a model system we found that SLAM-engagement promotes the release of lytic granules and a CD95-independent killing that requires extracellular Ca(2+), cytoskeletal rearrangements, and signaling mediated by mitogen-activated protein kinase kinases MEK1/2. SLAM-enhanced cytotoxicity implies an immunoregulatory function by facilitating the elimination of APC and a role in overcoming infections with pathogens requiring a cytotoxic immune response.     

The impact of telomere erosion on memory CD8+ T cells in patients with X-linked lymphoproliferative syndrome

Patients with X-linked lymphoproliferative syndrome (XLP) experience excessive T cell proliferation after primary Epstein-Barr virus (EBV) infection, due to mutations in the signalling lymphocyte activation molecule (SLAM) associated protein (SAP) molecule. We examined the impact of dysfunctional proliferative control on the extent of CD8+ T cell differentiation in XLP patients who recovered from primary EBV infection. Although these young patients have normal numbers of lytic and latent EBV-epitope-specific CD8+ T cells, they were extremely differentiated as defined by loss of CCR7 and CD27, low telomerase activity and very short telomeres. This was not a direct effect arising from the loss of SAP, but was due to excessive T cell stimulation due to this defect. Thus, transduction of XLP CD8+ T cells with the catalytic component of telomerase (hTERT), but not SAP, prevented telomere loss and considerably extended proliferative lifespan in vitro. These results indicate that excessive proliferation in CD8+ T cells in XLP patients may lead to end-stage differentiation and loss of functional EBV-specific CD8+ T cells through replicative senescence. This may contribute to the defective immunity found in XLP patients who survive acute EBV infection who develop EBV-related B cell lymphomas before the fourth decade of life.     

WhatSAP — 2B4 sends mixed messages in the absence of SAP

2B4 (CD244), a member of the SLAM‐related receptor family, has important immuno‐regulatory functions including coactivating the cytotoxicity and cytokine secretion of NK cells. Immune modulation by 2B4 is dependent on the small intracellular signaling molecule SAP. In patients suffering from X‐linked lymphoproliferative disease (XLP1), SAP is nonfunctional, not only abolishing the activating function of 2B4, but rendering this receptor inhibitory. In this issue of European Journal of Immunology, Meazza et al. [Eur. J. Immunol. 2014. 44: 1526–1534.] demonstrate that 2B4‐mediated inhibition in NK cells from XLP1 patients is selective. While the activation of NK cells via ITAM‐based receptors is blocked by inhibitory 2B4, DNAM‐1, and NKG2D‐dependent NK‐cell activation is not affected by SAP deficiency. These findings provide an important insight into the different defective NK‐cell functions in XLP1 patients and demonstrate the differential integration of redundant receptor signaling pathways in NK cells.     

2B4 utilizes ITAM-containing receptor complexes to initiate intracellular signaling and cytolysis

2B4 is a member of the SLAM receptor family capable of activating NK cell cytotoxicity in the context of EBV infection. SAP (SLAM Associated Protein) deficiency causes defective signaling downstream of SLAM family receptors and high susceptibility to EBV. 2B4 costimulates natural cytotoxicity receptor (NCR) and TCR initiated signals to induce cellular cytotoxicity and cytokine release. The 2B4-SAP signal transduction pathway is not predicted to overlap with the TCR-ITAM pathway, although SAP is required for some TCR-induced signals. We therefore examined the functional relationship between SLAM family receptor 2B4 and ITAM-containing adaptor complexes. Removal of Fc?RIγ or CD3ζ-containing complexes, using genetically manipulated cell lines or siRNA specific suppression, significantly reduces 2B4-initiated functions in NK and T cells, respectively. Consistent with this relationship, Syk and ZAP-70 are capable of transducing 2B4 signals for calcium mobilization and cytolysis. Furthermore, ITAM-containing molecules constitutively associate with SAP. These results suggest a potential physical association between 2B4 and the ITAM receptor complexes that is required for 2B4-initiated signaling and cell-mediated killing.     

Distinct interactions of the X-linked lymphoproliferative syndrome gene product SAP with cytoplasmic domains of members of the CD2 receptor family.

X-linked lymphoproliferative syndrome (XLP; Duncan's disease) is a primary immunodeficiency disease that manifests as an inability to regulate the immune response to Epstein-Barr virus (EBV) infection. Here we examine the ability of the product of the gene defective in XLP, SAP (DSHP/SH2D1A), to associate with the cytoplasmic domains of several members of the CD2 subfamily of cell surface receptors, including SLAM, 2B4, and CD84. While recruitment of SAP to SLAM occurred in a phosphorylation-independent manner, SAP was found to bind preferentially to tyrosine-phosphorylated cytoplasmic domains within 2B4 and CD84. Missense or nonsense mutations in the SAP open reading frame were identified in five of seven clinically diagnosed XLP patients from different kindreds. Four of these variants retained the ability to bind to the cytoplasmic tails of SLAM and CD84. While ectopic expression of wild-type SAP was observed to block the binding of SHP-2 to SLAM, mutant SAP derivatives that retained the ability to bind SLAM did not inhibit recruitment of SHP-2 to SLAM. In contrast, SAP binding to CD84 had no effect on the ability of CD84 to recruit SHP-2, but instead displaced SHP-1 from the cytoplasmic tail of CD84. These results suggest that mutations in the gene encoding the XLP protein SAP lead to functional defects in the protein that include receptor binding and SHP-1 and SHP-2 displacement and that SAP utilizes different mechanisms to regulate signaling through the CD2 family of receptors.     

XLP: Clinical Features and Molecular Etiology due to Mutations in SH2D1A Encoding SAP

X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency affecting approximately 1–2 per 1 million males. A key feature of XLP is the exquisite sensitivity of affected individuals to disease induced following EBV infection. However, patients can also develop hypogammaglobulinemia and B-cell lymphoma independently of exposure to EBV. XLP is caused by loss-of function mutations in SH2D1A, which encodes the intracellular adaptor molecule SAP. SAP is predominantly expressed in T cells and NK cells, and functions to regulate signal transduction pathways downstream of the SLAM family of surface receptors to control CD4+ T cell (and by extension B cells), CD8+ T cell and NK cell function, as well as the development of NKT cells. The study of XLP had shed substantial light on the requirements for lymphocyte differentiation and immune regulation, which in turn have the potential to be translated into novel treatments for not only XLP patients but individuals affected by EBV-induced disease, impaired humoral immunity and malignancy.     

Activation-dependent T cell expression of the X-linked lymphoproliferative disease gene product SLAM-associated protein and its assessment for patient detection

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterized by extreme vulnerability to Epstein-Barr virus (EBV) infection, resulting in fatal infectious mononucleosis, dysgammaglobulinemia and malignant lymphoma. Recently, mutations in the SH2D1A gene, which encodes SLAM-associated protein (SAP), have been found to cause XLP. Although the molecular events behind XLP are largely unknown, there is evidence that affected males exhibited some immunohematological abnormalities, such as hypogammaglobulinemia or lymphoma, even prior to EBV infection. Because of the poor prognosis in XLP, an early diagnosis to patients and families is clinically of great importance. A glutathione-S-transferase-SAP fusion protein was used to immunize rats and generate mAb against human SAP to investigate its pathogenic role in XLP and develop a flow cytometric assay for detection of XLP. By flow cytometric and Western immunoblot analyses using an established anti-SAP mAb, termed KST-3, we determined that SAP was expressed intensely in thymocytes, but at lower levels in peripheral T cells and NK cells. In contrast, expression of SAP was negligible in B cells, monocytes or granulocytes. We found that SAP expression in T cells increased upon in vivo as well as in vitro activation. In two XLP survivors with SH2D1A mutations, a flow cytometric evaluation of activated T cells using KST-3 could demonstrate SAP deficiency as a diagnostic indicator of XLP. Through this approach, we identified three novel XLP families with SH2D1A mutations in Japan. A flow cytometric assessment of SAP expressed in activated T cells would lead to easy detection of XLP patients.     

X-linked lymphoproliferative disease: genetics and biochemistry

Primary immunodeficiencies comprise a broad group of disorders due to germline mutations in genes regulating lymphocyte development and function. One of these genes, DSHP (also known as SH2D1A, SAP), is mutated in X-linked lymphoproliferative syndrome (XLP), an inherited immunodeficiency characterized by increased susceptibility to primary Epstein-Barr virus (EBV) infection, hypogammaglobulinenia, and lymphoma. Expressed primarily in T and NK cells, DSHP consists of a single SH2 domain and short carboxyl-terminal tail. The presence of a single SH2 domain, without other functional motifs, suggests that DSHP may be a physiologic competitor of other SH2 domain-containing proteins whose binding to phosphotyrosine controls lymphocyte activation and/or function. DSHP binds to the cytoplasmic domains of CDw150 (Signaling Lymphocyte Activation Molecule, SLAM) and 2B4, and may regulate signals transmitted by these receptors in T and NK cells, respectively. Unlike other SH2 domain-containing proteins, DSHP associates with both phosphorylated and non-phosphorylated tyrosine residues, and crystallography studies have revealed novel properties of the DSHP SH2 domain. Future studies exploring the function of DSHP during lymphocyte proliferation and activation should improve our ability to diagnose and treat XLP and possibly other human diseases associated with EBV.     

Molecular and immunological basis of X-linked lymphoproliferative disease

Summary: X‐linked lymphoproliferative (XLP) disease is a human immune dysfunction characterized primarily by an inappropriate response to Epstein–Barr virus infection. In 1998, it was discovered that XLP is caused by inactivating mutations in the SAP/SH2D1A/DSHP gene. This gene codes for an immune cell‐specific polypeptide termed SAP (SLAM‐associated protein) that is composed almost exclusively of an Src homology 2 (SH2) domain. By way of its SH2 domain, SAP interacts with tyrosine‐based motifs located in the cytoplasmic region of members of the SLAM (signaling lymphocyte activation molecule) family of receptors. Recent findings indicate that SAP is required for the function of SLAM‐related receptors, as a consequence of its capacity to promote the recruitment and activation of the Src‐related protein tyrosine kinase FynT, thereby allowing SLAM receptor‐mediated protein tyrosine phosphorylation signals in immune cells. Functional and genetic analyses suggest that the phenotype associated with XLP is caused in large part by defects in the functions of SLAM‐related receptors due to SAP deficiency.     

CS1, a novel member of the CD2 family,is homophilic and regulates NK cell function

CS1 is a novel member of the CD2 subset of immunoglobulin superfamily (IgSF) expressed on NK, T and stimulated B cells. The cytoplasmic domain of CS1 contains immunoreceptor tyrosine-based switch motif (ITSM) which is present in 2B4, SLAM and CD84. The signaling adaptor molecule SAP/SH2D1A, the defective gene in X-linked lymphoproliferative disease (XLPD), binds to ITSM and regulates immune cell function. However, recent studies indicate that CS1 may be regulated by a SAP-independent mechanism. In this study, we have examined the ligand specificity of CS1 and the effect of CS1 interaction with its ligand on the cytolytic activity of YT, a human NK cell line. Recombinant fusion protein, CS1-Ig, containing the CS1 extracellular domain and Fc portion of the human IgG bound cells transfected with CS1. CS1-Ig did not show any binding to cells expressing other members of the CD2 family. The cytolytic activity of YT was enhanced in presence of soluble CS1-Ig fusion protein. These results demonstrate that CS1 is a self-ligand and homophilic interaction of CS1 regulates NK cell cytolytic activity.     

Regulation of SLAM-mediated signal transduction by SAP, the X-linked lymphoproliferative gene product

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative (XLP) disease. Although the exact role and mechanism of action of SAP are not known, it has the capacity to interact with the cytoplasmic region of SLAM and other related immune cell receptors. As SAP is composed almost exclusively of a Src homology 2 (SH2) domain, it has been proposed that it functions as a natural blocker of SH2 domain--mediated interactions. We report here that the SLAM receptor is capable of triggering a protein tyrosine phosphorylation signal in T cells via a mechanism that is strictly dependent on SAP expression. This signal involves the SH2 domain--containing inositol phosphatase (SHIP); the adaptor molecules Dok2, Dok1 and Shc; and Ras GTPase--activating protein RasGAP. SAP is essential for this pathway because it facilitates the selective recruitment and activation of the Src-related protein tyrosine kinase FynT. We also show that signaling via the SLAM-SAP pathway in an established T cell line can alter the profile of cytokine production during T cell activation. These findings identify a mechanism by which a putative adaptor molecule is required for receptor-mediated signaling events in the immune system. They also provide insights into the pathophysiology of a severe human lymphoproliferative disease.     

SAP regulates T(H)2 differentiation and PKC-theta-mediated activation of NF-kappaB1

XLP is caused by mutations affecting SAP, an adaptor that recruits Fyn to SLAM family receptors. SAP-deficient mice recapitulate features of XLP, including increased T cell activation and decreased humoral responses post-infection. SAP-deficient T cells also show increased TCR-induced IFN-gamma and decreased T(H)2 cytokine production. We demonstrate that the defect in IL-4 secretion in SAP-deficient T cells is independent of increased IFN-gamma production. SAP-deficient cells respond normally to polarizing cytokines, yet show impaired TCR-mediated induction of GATA-3 and IL-4. Examination of TCR signaling revealed normal Ca(2+) mobilization and ERK activation in SAP-deficient cells, but decreased PKC-theta recruitment, Bcl-10 phosphorylation, IkappaB-alpha degradation, and nuclear NF-kappaB1/p50 levels. Similar defects were observed in Fyn-deficient cells. SLAM engagement amplified PKC-theta recruitment in wt but not SAP- or Fyn-deficient cells, arguing that a SAP/Fyn-mediated pathway enhances PKC-theta/NF-kappaB1 activation and suggesting a role for this pathway in T(H)2 regulation.     

Signaling pathways involved in the T‐cell‐mediated immunity against Epstein‐Barr virus: Lessons from genetic diseases

Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein‐Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non‐malignant and malignant B‐cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV – notably the central role of T‐cell expansion and its regulation in the pathophysiology of EBV‐associated diseases. On one hand, the defective expansion of EBV‐specific CD8 T cells results from mutations in genes involved in T‐cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co‐stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4‐1BB)) leads to impaired elimination of proliferating EBV‐infected B cells and the occurrence of lymphoma. On the other hand, protracted T‐cell expansion and activation after the defective killing of EBV‐infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T‐ and NK cell‐cytotoxicity. In this setting, the persistence of EBV‐infected cells results in HLH, a condition characterized by unleashed T‐cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co‐receptor molecules (CD27, CD137, and SLAM‐R) selectively involved in immune responses against infected B cells via specific T‐B cell interactions.