Molecular Pathogenesis of EBV Susceptibility in XLP as Revealed by Analysis of Female Carriers with Heterozygous Expression of SAP

Abstract

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency caused by mutations in SH2D1A which encodes SAP. SAP functions in signalling pathways elicited by the SLAM family of leukocyte receptors. A defining feature of XLP is exquisite sensitivity to infection with EBV, a B-lymphotropic virus, but not other viruses. Although previous studies have identified defects in lymphocytes from XLP patients, the unique role of SAP in controlling EBV infection remains unresolved. We describe a novel approach to this question using female XLP carriers who, due to random X-inactivation, contain both SAP⁺ and SAP⁻ cells. This represents the human equivalent of a mixed bone marrow chimera in mice. While memory CD8⁺ T cells specific for CMV and influenza were distributed across SAP⁺ and SAP⁻ populations, EBV-specific cells were exclusively SAP⁺. The preferential recruitment of SAP⁺ cells by EBV reflected the tropism of EBV for B cells, and the requirement for SAP expression in CD8⁺ T cells for them to respond to Ag-presentation by B cells, but not other cell types. The inability of SAP⁻ clones to respond to Ag-presenting B cells was overcome by blocking the SLAM receptors NTB-A and 2B4, while ectopic expression of NTB-A on fibroblasts inhibited cytotoxicity of SAP⁻ CD8⁺ T cells, thereby demonstrating that SLAM receptors acquire inhibitory function in the absence of SAP. The innovative XLP carrier model allowed us to unravel the mechanisms underlying the unique susceptibility of XLP patients to EBV infection in the absence of a relevant animal model. We found that this reflected the nature of the Ag-presenting cell, rather than EBV itself. Our data also identified a pathological signalling pathway that could be targeted to treat patients with severe EBV infection. This system may allow the study of other human diseases where heterozygous gene expression from random X-chromosome inactivation can be exploited. X-linked lymphoproliferative disease (XLP) is an immunodeficiency caused by mutations in the SH2D1A gene, which encodes a cytoplasmic component, SAP involved in a signalling pathway in certain populations of immune cells. The Achilles' heel in XLP is extreme sensitivity to Epstein-Barr virus (EBV) infection. Although EBV infection in normal individuals is generally innocuous, in XLP it can be fatal. Strikingly, individuals with XLP do not display this same vulnerability to other viruses, and here we investigate what immune defects underlie this specific susceptibility. We developed a system to examine the behaviour of immune cells that are identical with the exception of whether or not they have a functional SH2D1A gene. This approach uses human female carriers of XLP (one of their X chromosomes carries the mutation). Following the process of X-chromosome inactivation in female cells, which is random, individuals harbour T cells that express the normal SH2D1A gene as well as cells that express the mutated version. We found that SAP-deficient CD8⁺ T cells fail to be activated by antigen-presenting B cells, but are activated by other antigen-presenting cell types. Since EBV selectively infects B cells, the exquisite sensitivity in XLP to EBV infection results from the ability of the virus to sequester itself in B cells, which can only induce a cytotoxic T cell response in SAP-sufficient cells. Thus, the functional defect in SAP-deficient CD8⁺ T cells does not relate to a specific virus but rather to the nature of the target cell presenting viral epitopes.