Molecular and functional analysis of the SAP/SLAM pathway in XLP and other EBV-associated disorders

Research project P 13973 SAP and SLAM in EBV associated disorders Ornella PAROLINI 11.10.1999 The Epstein-Barr virus is a human herpes virus that preferentially infects B lymphocytes. In healthy individuals EBV infection occurs either without any clinical symptoms or induces an overshooting lymphoproliferative response, the so called infectious mononucleosis with fever, lymphonode swelling and the appearance of atypical blast cells in blood. Such atypical cells are a selected population of activated T lymphocytes that protect the individual from potentially oncogenic effects of EBV. However there are situations where the immune system is not protective and either there is uncontrolled growth of T cells that infiltrate different organs: liver, brain, heart, causing organ failure and subsequent death, or there is unlimited growth of infected B cells with potential malignant transition as often observed in immunocompromised patients such as transplant recipients and in those with inherited or acquired immunodeficiencies. So far the general mechanisms that, control the normal immune response to EBV and the reasons underlying a defective immune response are still unresolved questions. Recently the molecular basis of one of the most outspoken EBV related disease, the. X-linked lymphoproliferative disease (XLP), a fatal. disorder that occurs in pediatric age and manifest both symptoms of overshooting immune response and development of lymphomas, has been identified. A mutation in a gene called SAP is the cause of this disease. This finding is going to open new possibilities of investigation and may contribute in the understanding of the mechanisms underlying EBV immune response in healthy individuals and of the one involved in the pathogenesis of other EBV associated diseases. We therefore propose to explore this issue in our project. These findings will have evident importance in diagnostic and therapeutic approaches in a broader spectrum of EBV related disease.

Regulation and coordination of cellular and humoral responses to infection are key features of the normal immune system. Inefficient and/or unbalanced immune response often results in incomplete eradication of pathogens and persistent activation of immune cells with harmful consequences to the host. A clear example of such dysregulated immune response is the X-linked lymphoproliferative syndrome (XLP), Purtilo Syndrome or Duncan`s Disease, a fatal disorder that occurs in pediatric age. XLP patients present with inappropriate immune reaction against Epstein Barr virus (EBV) infection, often resulting in a fatal clinical course. The gene defective in this disease has been identified and named SH2D1A/SAP gene. The main goal of our project was to clarify some questions still undefined in the XLP disease with the clear aim to open new possibilities of investigation of the mechanisms underlying EBV immune response in healthy individuals and of the one involved in the pathogenesis of other EBV related diseases. In particular, we aimed at defining: 1) SAP gene expression in cells and tissues and how is the tissues specific expression regulated 2) whether defects of SAP/SLAM pathway are also involved in other EBV-associated disorders 3) pathophysiology of the disease at the biochemical level We have accomplished with success the reported aims, indeed we have defined the expression pattern of the gene, identified potential regulatory regions in the 5` part of the SAP gene and demonstrate a clear correlation between differential methylation and specific tissue expression. We have largely investigated the biochemical role of the SAP-SH2D1A gene in T cell and demonstrated a fundamental function of the gene during T cell activation. XLP derived T cell lines clearly showed defects in proliferation, interleukins production and alterations in the main downstream signaling events after T cell stimulation. Strikingly we could demonstrate that in vitro reconstitution of SAP expression by retroviral-mediated gene transfer restored defective T cell function in T cell lines derived from XLP patients. These findings beside bringing important information in the understanding of the mechanisms underlying XLP disease, open new investigation possibilities for therapeutical strategies of this genetic disorder.