Massimo Morra

The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAM

In addition to triggering the activation of B- or T-cell antigen receptors, the binding of a ligand to its receptor at the cell surface can sometimes determine the physiological outcome of interactions between antigen-presenting cells, T and B lymphocytes. The protein SLAM (also known as CDw150), which is present on the surface of B and T cells, forms such a receptor–ligand pair as it is a self-ligand. We now show that a T-cell-specific, SLAM-associated protein (SAP), which contains an SH2 domain and a short tail, acts as an inhibitor by blocking recruitment of the SH2-domain-containing signal-transduction molecule SHP-2 to a docking site in the SLAM cytoplasmic region. The gene encoding SAP maps to the same area of the X chromosome as the locus for X-linked lymphoproliferative disease (XLP) and we found mutations in the SAP gene in three XLP patients. Absence of the inhibitor SAP in XLP patients affects T/B-cell interactions induced by SLAM, leading to an inability to control B-cell proliferation caused by Epstein–Barr virus infections.

Crystal Structures of the XLP Protein SAP Reveal a Class of SH2 Domains with Extended, Phosphotyrosine-Independent Sequence Recognition

SAP, the product of the gene mutated in X-linked lymphoproliferative syndrome (XLP), consists of a single SH2 domain that has been shown to bind the cytoplasmic tail of the lymphocyte coreceptor SLAM. Here we describe structures that show that SAP binds phosphorylated and nonphosphorylated SLAM peptides in a similar mode, with the tyrosine or phosphotyrosine residue inserted into the phosphotyrosine-binding pocket. We find that specific interactions with residues N-terminal to the tyrosine, in addition to more characteristic C-terminal interactions, stabilize the complexes. A phosphopeptide library screen and analysis of mutations identified in XLP patients confirm that these extended interactions are required for SAP function. Further, we show that SAP and the similar protein EAT-2 recognize the sequence motif TIpYXX(V/I).

Structural basis for the interaction of the free SH2 domain EAT-2 with SLAM receptors in hematopoietic cells

The T and natural killer (NK) cell‐specific gene SAP (SH2D1A) encodes a ‘free SH2 domain’ that binds a specific tyrosine motif in the cytoplasmic tail of SLAM (CD150) and related cell surface proteins. Mutations in SH2D1A cause the X‐linked lymphoproliferative disease, a primary immunodeficiency. Here we report that a second gene encoding a free SH2 domain, EAT‐2, is expressed in macrophages and B lympho cytes. The EAT‐2 structure in complex with a phosphotyrosine peptide containing a sequence motif with Tyr281 of the cytoplasmic tail of CD150 is very similar to the structure of SH2D1A complexed with the same peptide. This explains the high affinity of EAT‐2 for the pTyr motif in the cytoplasmic tail of CD150 but, unlike SH2D1A, EAT‐2 does not bind to non‐phosphorylated CD150. EAT‐2 binds to the phosphorylated receptors CD84, CD150, CD229 and CD244, and acts as a natural inhibitor, which interferes with the recruitment of the tyrosine phosphatase SHP‐2. We conclude that EAT‐2 plays a role in controlling signal transduction through at least four receptors expressed on the surface of professional antigen‐presenting cells.

CD38 is functionally dependent on the TCR/CD3 complex in human T cells

One of the functions of surface CD38 is the induction of phosphorylation of discrete cytoplasmic substrates and mobilization of cytoplasmic calcium (Ca2+). The present work addresses the issue of whether the signaling mediated via CD38 operates through an independent pathway or, alternatively, is linked to the TCR/CD3 signaling machinery. We studied the signals elicited through CD38 by the specific agonistic IB4 monoclonal antibody (mAb) by monitoring the levels of cytoplasmic Ca2+ and the induced phenotypic and functional variations in T cell growth. IB4 mAb presented the unique ability to increase cytoplasmic Ca2+ levels, which correlated with the phosphorylation of the PLC‐γ1. These effects were blocked by phorbol 12‐myristate 13‐acetate (PMA) and were dependent on the presence of a functional TCR/CD3 surface complex, no effects being recorded on mutant Jurkat cells lacking part of the CD3 structures. CD38 signaling appeared to share with TCR/CD3 the ability to induce apoptotic cell death in Jurkat T cells, an event paralleled by specific up‐regulation of the Fas molecule and inhibited by cyclosporin A. CD28, a costimulatory molecule, is synergized by increasing CD38‐induced apoptotic cell death. The results indicate the existence of a strong functional interdependence between CD38 and TCR/CD3.

A ‘three-pronged’ binding mechanism for the SAP/SH2D1A SH2 domain: structural basis and relevance to the XLP syndrome

The SH2 domain protein SAP/SH2D1A, encoded by the X‐linked lymphoproliferative (XLP) syndrome gene, associates with the hematopoietic cell surface receptor SLAM in a phosphorylation‐independent manner. By screening a repertoire of synthetic peptides, the specificity of SAP/SH2D1A has been mapped and a consensus sequence motif for binding identified, T/S‐x‐x‐x‐x‐V/I, where x represents any amino acid. Remarkably, this motif contains neither a Tyr nor a pTyr residue, a hallmark of conventional SH2 domain–ligand interactions. The structures of the protein, determined by NMR, in complex with two distinct peptides provide direct evidence in support of a ‘three‐pronged’ binding mechanism for the SAP/SH2D1A SH2 domain in contrast to the ‘two‐pronged’ binding for conventional SH2 domains. Differences in the structures of the two complexes suggest considerable flexibility in the SH2 domain, as further confirmed and characterized by hydrogen exchange studies. The structures also explain binding defects observed in disease‐causing SAP/SH2D1A mutants and suggest that phosphorylation‐independent interactions mediated by SAP/SH2D1A likely play an important role in the pathogenesis of XLP.

Cutting Edge: The SLAM Family Receptor Ly108 Controls T Cell and Neutrophil Functions

Ly108, a glycoprotein of the signaling lymphocytic activation molecule family of cell surface receptors expressed by T, B, NK, and APCs has been shown to have a role in NK cell cytotoxicity and T cell cytokine responses. In this study, we describe that CD4+ T cells from mice with a targeted disruption of exons 2 and 3 of Ly108 (Ly108ΔE2+3) produce significantly less IL-4 than wild-type CD4+ cells, as judged by in vitro assays and by in vivo responses to cutaneous infection with Leishmania mexicana. Surprisingly, neutrophil functions are controlled by Ly108. Ly108ΔE2+3 mice are highly susceptible to infection with Salmonella typhimurium, bactericidal activity of Ly108ΔE2+3 neutrophils is defective, and their production of IL-6, IL-12, and TNF-α is increased. The aberrant bactericidal activity by Ly108ΔE2+3 neutrophils is a consequence of severely reduced production of reactive oxygen species following phagocytosis of bacteria. Thus, Ly108 serves as a regulator of both innate and adaptive immune responses.

Identification and characterization of SF2000 and SF2001, two new members of the immune receptor SLAM/CD2 family.

Abstract. The SLAM family of human genes currently consists of seven related members of the immunoglobulin superfamily, membrane-associated proteins, including CD150 (SLAM), CD244 (2B4), CD84, CD229 (Ly-9), BLAME, CD48, and 19A. These genes are expressed to varying degrees in subsets of immune cells (T, B, natural killer, and myeloid cells) and may function as ligands or receptors. This set of genes, related to CD2 and CD58 on Chromosome (Chr) 1p98, are found clustered close together in the human genome on Chr 1q22. Four of these family members (CD150, CD244, CD84, CD229) contain conserved tyrosine motifs in their cytoplasmic tails that enable them to bind intracellular signaling molecules SAP and EAT-2. SAP is mutated in human X-linked lymphoproliferative disease (XLP), and studies in XLP patients have shown that improper signaling via molecules that bind SAP contributes to the disease. We have identified two new members of the SLAM family (SF), which we term SF2000 and SF2001, which are expressed in immune cells and map in the SLAM gene cluster. SF2001 does not contain SAP-binding motifs in its short cytoplasmic tail. SF2000, which is co-expressed with SAP in T cells, binds both SAP and EAT-2. The data suggest that signaling through SF2000, together with CD150, CD244, CD84, and CD229, is controlled by SAP and therefore contributes to the pathogenesis of XLP.

The gene defective in X-linked lymphoproliferative disease controls T cell dependent immune surveillance against Epstein–Barr virus

Our understanding of the X-linked lymphoproliferative syndrome (XLP) has advanced significantly in the past two years. The gene that is aberrant in the condition - SH2D1A/SAP, which encodes SAP (signaling lymphocytic activation molecule [SLAM]-associated protein) - was cloned, the crystal structure of its product was solved and insights into the signaling mechanisms of this small SH2-domain-containing protein via the cell surface receptors SLAM and 2B4 have been provided. SAP mutation, and not Epstein-Barr virus infection per se, may be critical for XLP.

IgA and IgG hypogammaglobulinemia in Waldenström’s macroglobulinemia

Background Hypogammaglobulinemia is common in Waldenström’s macroglobulinemia. The etiology of this finding remains unclear, but it has been speculated to be based on tumor-induced suppression of the ‘uninvolved’ immunoglobulin production Design and Methods We evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 untreated patients with Waldenström’s macroglobulinemia and investigated the associated clinicopathological findings and impact of therapy. We also sequenced eight genes (AICDA, BTK, CD40, CD154, NEMO, TACI, SH2D1A, UNG) implicated in immunoglobulin deficiency in 19 Waldenström’s macroglobulinemia patients with IgA and/or IgG hypogammaglobulinemia. Results At baseline 63.3%, 58.0% and 49.3% of the 207 patients had abnormally low serum levels of IgA, IgG, or both. No association between IgA and IgG hypogammaglobulinemia and disease burden, serum IgM levels, β2-microglobulin, International Prognostic Scoring System score, or incidence of recurrent infections was observed, although the presence of adenopathy and/or splenomegaly was associated with a lower incidence of hypogammaglobulinemia. Lower IgA and IgG levels were associated with disease progression in patients managed with a ‘watch and wait’ strategy. IgA and/or IgG levels remained abnormally low despite response to treatment, including complete remissions. A missense mutation in the highly conserved catalytic site of UNG was observed in a patient with hypogammaglobulinemia, warranting further study of this pathway in Waldenström’s macroglobulinemia. Conclusions IgA and IgG hypogammaglobulinemia is common in Waldenström’s macroglobulinemia and persists despite therapeutic intervention and response. IgA and IgG hypogammaglobulinemia does not predict the risk of recurrent infections in patients with Waldenström’s macroglobulinemia, although lower levels of serum IgA and IgG are associated with disease progression in Waldenström’s macroglobulinemia patients being managed with a ‘watch and wait’ strategy.

Auto-antibody production and glomerulonephritis in congenic Slamf1−/− and Slamf2−/− [B6.129] but not in Slamf1−/− and Slamf2−/− [BALB/c.129] mice

Several genes in an interval of human and mouse chromosome 1 are associated with a predisposition for systemic lupus erythematosus. Congenic mouse strains that contain a 129-derived genomic segment, which is embedded in the B6 genome, develop lupus because of epistatic interactions between the 129-derived and B6 genes, e.g. in B6.129chr1b mice. If a gene that is located on chromosome 1 is altered through homologous recombination in 129-derived embryonic stem cells (ES cells) and if the resultant knockout mouse is backcrossed with B6, interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. Here, we report that knockout mice of two adjacent chromosome 1 genes, Slamf1(-/-) and Slamf2(-/-), which were generated with the same 129-derived ES cell line, develop features of lupus, if backcrossed on to the B6 genetic background. By contrast, Slamf1(-/-) [BALB/c.129] and Slamf2(-/-) [BALB/c.129] do not develop disease. Surprisingly, Slamf1(-/-) [B6.129] mice develop both auto-antibodies and glomerulonephritis between 3 and 6 months of age, while disease fully develops in Slamf1(-/-) [B6.129] mice after 9-14 months. Functional analyses of CD4(+) T cells reveals that Slamf2(-/-) T cells are resistant to tolerance induction in vivo. We conclude that the Slamf2(-/-) mutation may have a unique influence on T-cell tolerance and lupus.