SAP Deficiency Research Articles

Natural killer T cells and X-linked lymphoproliferative syndrome

Recent progress in elucidating the physiopathology of X-linked lymphoproliferative syndrome (XLP) has raised novel and important issues regarding the biology of natural killer T cells. Here I will review this information and discuss the issues involved. XLP is a rare inherited immunodeficiency characterized by a high susceptibility to severe infection by the Epstein-Barr virus. Mutations in the gene SH2D1A (or alternatively SAP) underlie 80% of familial XLP (XLP-1) cases. Recently the remaining 20% of familial XLP (XLP-2) cases were shown to harbor mutations in the gene XIAP (X-linked inhibitor of apoptosis protein). Both SAP and XIAP deficiencies are associated with a defect in the development and/or homeostasis of natural killer T cells. It can be hypothesized that the susceptibility to Epstein-Barr virus in XLP might result from the defect of natural killer T cells. The role of these cells in viral infection is unclear, but several herpes viruses have developed strategies to escape natural killer T cells. The discovery that SAP and XIAP deficiency leads to a defect in natural killer T cells has also shed light on novel signaling pathways required for natural killer T cell development and/or homeostasis.

SAP (SH2D1A), the Immunomodulator Deficient in X-Linked Lymphoproliferative Syndrome (XLP), Is Profoundly Decreased in Aplastic Anemia: An Immunologic Link between Constitutional and Acquired Bone Marrow Failure.

Ligation of the signaling lymphocyte activation molecule (SLAM), a member of the immunoglobulin superfamily expressed in T and B cells, results in T cell activation and Th1 cytokine production. SAP is a small cytoplasmic protein expressed in T- and NK cells that controls the activation signals mediated by SLAM. On T cell activation, SAP binds to Fyn kinase; Fyn is activated and phosphorylates tyrosine residues on SLAM; phosphorylation results in the formation of a complex that selectively down-regulates co-stimulatory signals in activated T cells, resulting in inhibition of IFN-γ production. Thus SAP acts as a natural suppressor of SLAM-mediated T cell activation, and, in the absence of SAP, T cells are constitutively activated and overproduce IFN-γ. Mutations in the SAP gene lead to abnormal T cell activation and enhanced Th1 cytokine production in mouse models and in humans: about half of patients with X-linked lympoproliferative disease (XLP) have functionally disabling SAP mutations. Acquired aplastic anemia (AA) is a bone marrow failure syndrome in which hematopoietic cell destruction is effected by cytotoxic T cells and type 1 cytokines. We have recently shown that T cells from patients with AA have increased protein levels of T-bet, resulting in IFN-γ overproduction (Solomou EE et al, Blood 2006; 107:3983). IFN-γ inhibits hematopoietic stem cell proliferation and induces Fas-mediated apoptosis; stem cell depletion results in marrow hypoplasia and peripheral blood pancytopenia. We examined SAP expression as an explanation for aberrant T cell activation and extreme Th1 polarization. SAP protein expression on immunoblot was very low to absent in unstimulated T cells from 16 of 20 AA patients examined, as compared to normal levels of expression in equivalent numbers of healthy donors (p<0.001). No significant differences were detected in Fyn and SLAM protein levels between AA and controls. SAP mRNA levels were also significantly decreased in T cells from those AA patients with low SAP protein levels, as determined by RT-PCR. Peripheral blood DNA samples from 18 patients with AA were analyzed for SAP mutations: three novel intronic mutations, not present in controls, were identified among 7 unrelated patients: one mutation was in the promoter region of SAP (position 106, C to T; 3 patients), and two mutations in the intron-exon junction between exons 1 and 2 (position 38975, C toT; 3 patients) and 3 and 4 (position 62771, C to A; 1 patient). IFN-γ, as measured by ELISA, in three patients with undetectable SAP protein levels was significantly increased compared to healthy controls (n=5, p<0.001). Increased IFN-γ levels and Th1 polarization in AA can in part be explained by functional SAP deficiency. SAP-deficient T cells in AA would be unable to block co-stimulatory signals, leading to an activated T cell phenotype and ultimately hematopoietic cell destruction and bone marrow failure. The SAP-deficient phenotype in T cells from patients with aplastic anemia may be secondary to subtle genetic alteration in the gene's regulation (abnormal promoter binding sites or epigenetic modulation due to mutations in introns) or as yet unidentified aberrant upstream pathways (Ets-1 and Ets-2, the transcription factors that regulate SAP expression).

Prevalence and Pathology of Amyloid in Atherosclerotic Arteries

To the Editor: Apolipoprotein AI (AApoAI)–associated amyloidosis is characterized by the deposition of apolipoprotein AI (apoAI) and occurs as a hereditary and a nonhereditary form. Hereditary AApoAI amyloidosis is a systemic disease leading to the deposition of amyloid in various organs and tissues and is caused by germline mutations in the APOA1 gene. Nonhereditary AApoAI amyloid is far more prevalent and characterized by deposits of nonvariant protein in atherosclerotic arteries.1–3 Despite being linked to the most common cause of morbidity and mortality in Western societies, nonhereditary AApoAI amyloid has achieved only little attention.1–4 It shares several striking similarities with secondary or reactive AA amyloidosis. Nonhereditary AApoAI amyloid occurs in the background of a local chronic inflammatory reaction, it originates from an apolipoprotein that largely associates with high-density lipoproteins (HDLs), and apoAI-containing HDL is endocytosed and retroendocytosed by macrophages, which, in themselves, are able to form amyloid in vitro. Finally, AApoAI amyloidosis is characterized by the deposition of proteolytic fragments of the precursor protein, leading us to speculate that proteolysis is involved in the pathogenesis of AApoAI amyloid.1,3 The aim of this study was to gain further insights into the pathology of nonhereditary AApoAI amyloid. See cover The prevalence and spatial distribution of amyloid, macrophages, cathepsin B (CathB), cathepsin K (CathK), cathepsin L (CathL), and carboxy methyl lysine (CML) was studied using carotid artery specimens obtained from a consecutive series of all 225 patients undergoing carotid endarterectomy with polyester patch angioplasty (Table I, available online at http://atvb.ahajournals.org) during the period from 1997 to 2003. All patients were scheduled for elective therapeutic endarterectomy and gave written informed consent in the surgical procedure. Patient characteristics were retrieved from hospital records. This study was in accordance with the guidelines of the ethics committee of the University of Magdeburg. Tissue samples were …

Regulation of NKT cell development by SAP, the protein defective in XLP

The adaptor molecule SAP is expressed in T lymphocytes and natural killer (NK) cells, where it regulates cytokine production and cytotoxicity. Here, we show that SAP, encoded by the SH2D1A gene locus, also has a crucial role during the development of NKT cells, a lymphocyte subset with immunoregulatory functions in response to infection, cancer and autoimmune disease. Following stimulation with the NKT cell-specific agonist alpha-galactosyl ceramide (alphaGC), Sh2d1a-/- splenocytes did not produce cytokines or activate other lymphoid lineages in an NKT cell-dependent manner. While evaluating the abnormalities in alphaGC-induced immune responses, we observed that Sh2d1a-/- animals lacked NKT cells in the thymus and peripheral organs. The defect in NKT cell ontogeny was hematopoietic cell autonomous and could be rescued by reconstitution of SAP expression within Sh2d1a-/- bone marrow cells. Seventeen individuals with X-linked lymphoproliferative disease (XLP), who harbored germline mutations in SH2D1A, also lacked NKT cells. Furthermore, a female XLP carrier showed completely skewed X chromosome inactivation within NKT cells, but not T or B cells. Thus, SAP is a crucial regulator of NKT cell ontogeny in humans and in mice. The absence of NKT cells may contribute to the phenotypes of SAP deficiency, including abnormal antiviral and antitumor immunity and hypogammaglobulinemia.

Reply to: Avoiding the effect of linked genes is crucial to elucidate the role of Apcs in autoimmunity

Our original conclusion that serum amyloid P-component (Sap) deficiency in mice, created by targeted deletion of Apcs, causes antinuclear autoantibody production and associated immune complex glomerulonephritis1, was based on our earlier discovery that human SAP binds avidly and specifically to DNA, nuclei, apoptotic cells, long chromatin and nucleosomes core particles, both in vitro and in vivo2, 3, 4, 5. But we were aware of the possibility of confounding by unexpected genetic interactions incurred during gene deletion and subsequent interstrain mouse breeding1. Following our original publication of the presumed Sap knockout phenotype1, we backcrossed the gene deletion into pure-line C57BL/6 mice and extensively investigated their phenotype in very large cohorts of animals up to the age of 12 months6. Although they also developed autoantibodies and glomerulonephritis, this phenotype was not abrogated by introduction of transgenic human SAP6. We also showed that, in contrast to our earlier observations of whole-body clearance of labeled long chromatin in 129/Sv C57BL/6 mice, the plasma clearance of neither long chromatin nor nucleosome core particles was substantially affected by Sap deficiency in C57BL/6 animals6. Furthermore, we found that mouse Sap binds much more weakly than human SAP to DNA and chromatin, and scarcely at all to apoptotic cells6. Notably, we also found that Sap deficiency in pure 129/Sv mice was not associated with any autoimmunity6. Our recent publication of these results concluded that the autoimmune phenotype caused by Apcs deletion was likely to be a consequence of unintended genetic modification and/or interstrain interactions, rather than Sap deficiency6. This was subsequently independently confirmed by Bygrave et al., who produced a congenic strain of C57BL/6 mice carrying just the relevant Apcs-containing segment of 129/Sv chromosome 1 (ref. 7). These animals developed spontaneous antinuclear autoimmunity, although notably not glomerulonephritis. The study of Maeda et al. again confirms these results. An important general conclusion is that attribution of phenotype to loss of function of the deficient product of a targeted deleted gene must include consideration of both mouse strain genetic background and unexpected effects of interstrain genetic interactions.

Rapid Detection of SAP Deficiency in Cytotoxic Lymphocytes from Patients with X-Linked Lymphoproliferative Disease and Their Family Members.

Background: X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency, which is characterized by an extreme susceptibility to Epstein-Barr virus (EBV). In half of the XLP patients, primary EBV infection can be fatal with explosive activation and proliferation of lymphocytes in many organs, which leads to fluminant hepatitis and bone marrow failure with hemophagocytosis. Mutations in the SH2D1A gene, which encodes the SAP protein, have been described in a proportion of patients with the clinical syndrome of XLP. The diagnosis of XLP is still difficult given its clinical heterogeneity, and the lack of a readily available rapid diagnostic laboratory test, particularly in cases without a family history of XLP. XLP should always be a consideration in males with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH).Methods: Four-color flow cytometric analysis was used to establish normal patterns of SAP expression for control subjects, and patterns of SAP staining in cytotoxic lymphocytes (CD8+ T cells, CD56+ T cells and natural killer [NK] cells). This assay was used to study patients with clinical syndromes consistent with XLP, and in some cases; their family members.Results: 6 patients with XLP confirmed by detection of mutations in the coding regions of SH2D1A showed lack of intracellular SAP in all cytotoxic cell types; one was tested presymptomatically. Carriers of SH2D1A mutations tested (mother and sister) had decreased SAP staining patterns below normal levels. Eleven males with clinical syndromes consistent with XLP, predominantly EBV-HLH who were demonstrated to have normal or increased perforin expression were also studied. All patients showed normal SAP expression including one case with a convincing X-linked family history; all were subsequently shown to have no mutations in SH2D1A. Additionally, one asymptomatic adult male with an intronic mutation previously published as causing XLP was found to have normal SAP expression.Conclusions: Four-color flow cytometry provides diagnostic information that may speed the identification of this fatal disease, differentiating it from other EBV-HLH. We also suspect it will prove useful in substantiating or excluding disease-causing genetic variants in the SH2D1A gene.

The SAP family of adaptors in immune regulation

SAP and EAT-2 define a new class of adaptor proteins composed almost exclusively of a Src homology 2 (SH2) domain. By way of their SH2 domain, SAP-like adaptors interact with tyrosine-based motifs in the cytoplasmic region of SLAM-related receptors, a family of immune cell-specific molecules involved in immunoregulation. Recent findings indicate that SAP is required for the functions of SLAM family receptors, as a consequence of its ability to promote recruitment of Src-related protein tyrosine kinase FynT and allow SLAM-related receptors to transduce tyrosine phosphorylation signals. SAP is mutated in X-linked lymphoproliferative (XLP) syndrome, a rare inherited human disease characterized by an deregulated immune response to Epstein-Barr virus infection. Several lines of evidence indicate that defects in the activities of SLAM-related receptors caused by SAP deficiency account for the immune dysfunctions associated with XLP.

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.

Sphingolipid activator protein deficiency in a 16-week-old atypical Gaucher disease patient and his fetal sibling: Biochemical signs of combined sphingolipidoses

We describe a patient who presented shortly after birth with hyperkinetic behaviour, myoclonia, respiratory insufficiency and hepatosplenomegaly. Gaucher-like storage cells were found in bone marrow. A liver biopsy showed massive lysosomal storage morphologically different to that in known lipid storage disorders. Biochemically, the patient had partial deficiencies of beta-galactocerebrosidase, beta-glucocerebrosidase and ceramidase in skin fibroblast extracts, but the sphingomyelinase activity was normal. Glucosyl ceramide and ceramide were elevated in liver tissue. Loading of cultured fibroblasts with radioactive sphingolipid precursors indicated a profound defect in ceramide catabolism. Immunological studies in fibroblasts showed a total absence of cross-reacting material to sphingolipid activator protein 2 (SAP-2). The patient died at 16 weeks of age. The fetus from his mother's next pregnancy was similarly affected. The possibility that the disorder results from a primary defect at the level of SAP-2 is discussed. We have named this unique disorder SAP deficiency.