X-linked lymphoproliferative syndrome presenting with systemic lymphocytic vasculitis

X-linked lymphoproliferative syndrome (XLP) is a rare, often fatal, primary immunodeficiency disease characterized by an abnormal response to Epstein-Barr virus (EBV) infection. The gene responsible for XLP has been identified as SH2D1A/DSHP/SLAM-associated protein (SAP). The major clinical manifestations include fulminant infectious mononucleosis, lymphoproliferative disorder, and dysgammaglobulinemia. Affected males uncommonly present with lymphocytic vasculitis in addition to aplastic anemia. In this study, we describe a Japanese XLP patient who presented with hypogammaglobulinemia following acute EBV-induced infectious mononucleosis in the infancy and later had systemic lymphocytic vasculitis and hemophagocytic lymphohistiocytosis in the adulthood, which resolved by steroid pulse therapy. The patient's SAP gene was found to harbor a missense mutation (His8Asp), presumably resulting in defective expression of SAP in T cells. Biopsy specimens of lung and skin disclosed that CD8+ T cells predominantly infiltrated vascular vessels. However, immunohistochemical examination showed that EBV-infected cells were not identifiable in the vessels. We propose that T-cell-mediated immune dysregulation in XLP can cause vasculitis by EBV infection-unrelated mechanism.     

Altered lymphocyte responses and cytokine production in mice deficient in the X-linked lymphoproliferative disease gene SH2D1A/DSHP/SAP

We have introduced a targeted mutation in SH2D1A/DSHP/SAP, the gene responsible for the human genetic disorder X-linked lymphoproliferative disease (XLP). SLAM-associated protein (SAP)-deficient mice had normal lymphocyte development, but on challenge with infectious agents, recapitulated features of XLP. Infection of SAP- mice with lymphocyte choriomeningitis virus (LCMV) or Toxoplasma gondii was associated with increased T cell activation and IFN-gamma production, as well as a reduction of Ig-secreting cells. Anti-CD3-stimulated splenocytes from uninfected SAP- mice produced increased IFN-gamma and decreased IL-4, findings supported by decreased serum IgE levels in vivo. The Th1 skewing of these animals suggests that cytokine misregulation may contribute to phenotypes associated with mutation of SH2D1A/SAP.     

Viral targeting of fibroblastic reticular cells contributes to immunosuppression and persistence during chronic infection

Many chronic viral infections are marked by pathogen persistence and a generalized immunosuppression. The exact mechanisms by which this occurs are still unknown. Using a mouse model of persistent lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate viral targeting of fibroblastic reticular cells (FRC) in the lymphoid organs. The FRC stromal networks are critical for proper lymphoid architecture and function. High numbers of FRC were infected by LCMV clone 13, which causes a chronic infection, whereas few were infected by the acute strain, LCMV Armstrong. The function of the FRC conduit network was altered after clone 13 infection by the action of CD8(+) T cells. Importantly, expression of the inhibitory programmed death ligand 1, which was up-regulated on FRC after infection, reduced early CD8(+) T cell-mediated immunopathology and prevented destruction of the FRC architecture in the spleen. Together, this reveals an important tropism during a persistent viral infection. These data also suggest that the inhibitory PD-1 pathway, which likely evolved to prevent excessive immunopathology, may contribute to viral persistence in FRC during chronic infection.     

Loss of circulating CD27+ memory B cells and CCR4+ T cells occurring in association with elevated EBV loads in XLP patients surviving primary EBV infection

Detailed longitudinal studies of patients with X-linked lymphoproliferative disease (XLP) may increase our understanding of the immunologic defects that contribute to the development of lymphoma and hypogammaglobulinemia in XLP. We describe progressive changes observed in immunoglobulin concentrations, lymphocyte subsets, and Epstein-Barr virus (EBV) loads occurring in a 2-year period in a newly infected, but otherwise healthy, carrier (patient 9). We compare these findings with those observed in the patient's brother, who had hypogammaglobulinemia and XLP (patient 4). Immunoglobulin G (IgG), IgM, and IgA concentrations increased in patient 9 during acute EBV infection, but thereafter they decreased steadily to concentrations consistent with hypogammaglobulinemia, reaching a plateau 5 months after infection. In both patients, CD19+ B-lymphocyte rates remained lower than 3%, with a contraction of the B-cell memory compartment (CD27+ CD19+/CD19+) to 20% (normal range, 32%-56%). T-lymphocyte subpopulations showed a reduction in CD4+ T-cell counts and a permanent CD8+ T-cell expansion. Interestingly, CXCR3 memory TH1 cells were expanded and CCR4+ TH2 lymphocytes were reduced, suggesting that abnormal skewing of memory T-cell subsets might contribute to reduced antibody synthesis. Despite an expanded number of CD3+CD8+ lymphocytes, increased EBV loads occurred in both patients without overt clinical symptoms of mononucleosis, lymphoproliferative disease, or lymphoma.     

X-linked lymphoproliferative syndrome: a genetic condition typified by the triad of infection, immunodeficiency and lymphoma

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterized by the clinical triad of increased susceptibility to primary Epstein-Barr virus (EBV) infection, dysgammaglobulinaemia and lymphoma. Most cases are caused by germline mutations in the SH2D1A gene, which encodes the adaptor molecule Signalling Lymphocytic Activation Molecule (SLAM)-associated protein (SAP). Recently, a subset of patients with an XLP-like phenotype was found to carry mutations in XIAP, the gene encoding the X-linked inhibitor of apoptosis protein (XIAP). Studies of XLP patients and Sap-/- mice reveal that loss of SAP expression impairs immune cell activities, such as natural killer and CD8+ T cell cytotoxicity, T cell cytokine production, activation-induced cell death, germinal centre formation and natural killer T cell development. Efforts to dissect the diverse roles of SAP and XIAP are enhancing our understanding of immune cell biology and defining how genetic defects in these molecules predispose to EBV-specific as well as more general cellular and humoral immune dysfunction. These studies are also highlighting critical signalling pathways that might be amenable to pharmacological targeting to improve the treatment of XLP and other disorders associated with impaired antiviral and antitumour immunity.     

Lymphocytic vasculitis in X-linked lymphoproliferative disease

Systemic vasculitis is an uncommon manifestation of X-linkedlymphoproliferative disease (XLP), a disorder in which there is aselective immune deficiency to Epstein-Barr virus (EBV). The molecularbasis for XLP has recently been ascribed to mutations withinSLAM-associated protein (SAP), an SH2 domain-containing proteinexpressed primarily in T cells. The authors describe a patient who diedas a result of chronic systemic vasculitis and fulfilled clinicalcriteria for the diagnosis of XLP. Sequencing of this patient'sSAP gene uncovered a novel point mutation affecting the SH2domain. The patient presented with virus-associated hemophagocytic syndrome (VAHS) and later had chorioretinitis, bronchiectasis, andhypogammaglobulinemia develop. He further developed mononeuritis andfatal respiratory failure. Evidence of widespread small and mediumvessel vasculitis was noted at autopsy with involvement of retinal,cerebral, and coronary arteries as well as the segmental vessels of thekidneys, testes, and pancreas. Immunohistochemical analysis usingantibodies to CD20, CD45RO, and CD8 revealed that the vessel wallinfiltrates consisted primarily of CD8⁺ T cells,implying a cytotoxic T-lymphocyte response to antigen. EBV DNA wasdetected by polymerase chain reaction (PCR) in arterial wall tissuemicrodissected from infiltrated vessels further suggesting that theCD8⁺ T cells were targeting EBV antigens within theendothelium. The authors propose that functional inactivation of theSAP protein can impair the immunologic response to EBV, resulting insystemic vasculitis.     

Large deletion of the X-linked lymphoproliferative disease gene detected by fluorescence in situ hybridization

The X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterized by an abnormal responses to infection with Epstein-Barr virus (EBV), resulting in fatal infectious mononucleosis, hypogammaglobulinemia, virus-associated hemophagocytic syndrome, and malignant lymphoma. Mutations in the gene coding for a T cell-specific SLAM-associated protein (SAP) have been recently identified in XLP patients. We report on a 1-year-old boy representing fulminant hemophagocytic syndrome. He developed high fever, lymphadenopathy, hepatosplenomegaly with liver dysfunction, and pancytopenia with marrow hemophagocytosis. EBV DNA was abnormally increased in the blood. Polymerase chain reaction failed to amplify SAP mRNA and genomic DNA products from the patient' As peripheral blood. A large deletion of the SAP gene was confirmed by fluorescence in situ hybridization (FISH). FISH analysis also disclosed that the patient's mother was a carrier. We conclude that FISH can be useful in the diagnosis of XLP with large deletions of the SAP gene and its carrier state.     

X-Linked Lymphoproliferative Disease in an Adult

X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterized by an extreme susceptibility to Epstein-Barr virus (EBV) infection. Patients with XLP mainly present with the 3 clinical manifestations of fulminant infectious mononucleosis, lymphoproliferative disorder, and dysgammaglobulinemia and in rare cases have aplastic anemia and lymphocytic vasculitis.The causative gene for XLP was identified as SH2D1A/DSHP/SLAM-associated protein (SAP) in 1998, and genetic analysis has been used for the definite diagnosis of XLP. Diagnosis for most patients occurs at ages younger than 10 years, and there are few adult patients. Here we describe a 23-year-old man with hypogammaglobulinemia and EBV-associated hemophagocytic lymphohistiocytosis and a diagnosis of XLP. In addition, the patient showed type 1 helper T-cell (Th1) skewing, as has been described in Sap knock-out mice. Th1/Th2 imbalance in humans, as well as in mice, may play an important role in the pathogenesis of XLP.     

SAP mediates specific cytotoxic T-cell functions in X-linked lymphoproliferative disease

Cytotoxic T cells (CTLs) and natural killer cells play a major role in the immune response to Epstein-Barr virus (EBV) infection. In X-linked lymphoproliferative (XLP) disease, a severe immunodeficiency, immunodysregulatory phenomena are observed following EBV infection, suggesting that defects exist in these effector populations. The gene defective in XLP is SAP (signaling lymphocytic activation molecule [SLAM]-associated protein), an adaptor protein that mediates signals through SLAM and other immunoglobulin superfamily receptors including 2B4. We generated EBV-specific T-cell lines from controls and XLP patients and examined CTL function in response to different stimuli. We show that XLP patients can generate EBV-T-cell lines that are phenotypically similar to those from controls. XLP patient EBV-T-cell lines showed a significant decrease in interferon-gamma (IFN-gamma) production in response to 2B4 and autologous EBV-transformed lymphoblastoid cell line (LCL) stimulation but not in response to SLAM. Furthermore, XLP EBV-T-cell lines demonstrated markedly decreased cytotoxic activity against autologous LCLs. By retroviral gene transfer of the SAP gene into XLP EBV-T-cell lines, we show reconstitution of IFN-gamma production and of cytotoxic activity confirming SAP-dependent defects. These studies demonstrate that in XLP the lack of SAP affects specific signaling pathways resulting in severe disruption of CTL function.     

SH2D1A mutations in Japanese males with severe Epstein-Barr virus--associated illnesses

X-linked lymphoproliferative disease (XLP), a genetic disorder characterized by immunodeficiency to Epstein-Barr virus (EBV) infection, has been linked to mutations in the SH2D1A gene. To search for the occurrence of SH2D1A mutations in Japan, we performed genetic analysis of the SH2D1A gene in 40 males presenting with severe EBV-associated illnesses, including fulminant infectious mononucleosis, EBV-positive lymphoma, and severe chronic active EBV infection. SH2D1A mutations were detected in 10 of these 40 patients. Five of these 10 cases were sporadic. Patients with SH2D1A mutations displayed severe acute infectious mononucleosis with hyperimmunoglobulin M, hypogammaglobulinemia, and B-cell malignant lymphoma. By contrast, chronic active EBV infection was not associated with SH2D1A mutations. XLP survivors exhibited normal levels of circulating EBV-DNA during convalescence, suggesting that SH2D1A protein is not directly responsible for control of EBV replication. Thus, genetic analysis of the SH2D1A gene is particularly useful in the diagnosis of sporadic cases and carriers of XLP. (Blood. 2001;98:1268-1270)     

Cross-Reactive T Cell Response Exists in Chronic Lymphocytic Choriomeningitis Virus Infection upon Pichinde Virus Challenge

Immunological memory to a previously encountered pathogen can influence the outcome of a sequential infection, which is called heterologous immunity. Lymphocytic choriomeningitis virus (LCMV) immune mice develop a NP205-specific T cell response that is cross-reactive to Pichinde virus infection (PICV). So far, limited data are available if cross-reactive T cell responses appear also during chronic infections with exhausted T cell responses. Exhaustion in chronic viral infections can be treated with checkpoint inhibitors, which might affect heterologous outcomes unexpectedly. The aim of this study was to investigate the cross-reactive immune response in chronic LCMV clone 13 (LCMVcl13) infection during primary PICV infection at phenotypic, functional, and T cell receptor (TCR) level. Moreover, the influence of checkpoint inhibitor therapy with αPD-L1 was investigated. Cross-reactive NP205-specific responses were present and functional in the chronic environment. Additionally, chronically infected mice were also protected from PICV mediated weight loss compared to naive PICV mice. An altered phenotype of NP205-specific T cells was detectable, but no major differences in the clonality and diversity of their TCR repertoire were observed. Checkpoint inhibitor treatment with αPD-L1 did alter chronic LCMV infection but had no major effect on heterologous immunity to PICV. Our study demonstrated that cross-reactive CD8⁺ T cells also exist in the setting of chronic infection, indicating a clinically relevant role of cross-reactive T cells in chronic infections.     

Primary Immunodeficiencies Inducing EBV-Associated Severe Illnesses

Epstein-Barr virus (EBV) is a ubiquitous human gamma-herpesvirus that infects about 95% of the adult population. The majority of primary infections occurs in early childhood and is generally subclinical; it can cause infectious mononucleosis (IM), which is usually a self-limiting lymphoproliferative disorder. However, infection of EBV occasionally results in severe, often lethal diseases, which include fatal IM, hemophagocytic syndrome, polyclonal lymphoproliferative disorders, and malignant lymphoma. These severe EBV-related illnesses occur secondary to some primary immunodeficiency diseases showing inefficient immune reaction to EBV. One example is X-linked lymphoproliferative disease (XLP), which is caused by mutations in the SLAM-associated protein (SAP) gene. The major clinical manifestations of XLP are fulminant IM, malignant lymphoma and dysgammaglobulinemia. Aplastic anemia, virus-associated hemophagocytic syndrome, and vasculitis have also been reported in XLP. We have developed a flow cytometric method using the anti-SAP monoclonal antibody to search for XLP. This clinically useful assay has successfully been used to identify XLP patients in Japan. In this review, clinical and mutational characteristics of XLP in Japan are mainly described. In addition, it is shown that the similar situations to XLP can occur in other primary immunodeficiencies involving T-cell killing function, such as autoimmune lymphoproliferative syndrome caused by Fas gene mutations or familial hemophagocytic lymphohistiocytosis caused by perforin gene mutations. Finally, the EBV-related terrible disease condition, namely chronic active EBV infection, which is common in Asian areas but its genetic background remains to be elucidated, will be touched on.     

Antigen-specific CD4 T-cell help rescues exhausted CD8 T cells during chronic viral infection

CD4 T cells play a critical role in regulating CD8 T-cell responses during chronic viral infection. Several studies in animal models and humans have shown that the absence of CD4 T-cell help results in severe dysfunction of virus-specific CD8 T cells. However, whether function can be restored in already exhausted CD8 T cells by providing CD4 T-cell help at a later time remains unexplored. In this study, we used a mouse model of chronic lymphocytic choriomeningitis virus (LCMV) infection to address this question. Adoptive transfer of LCMV-specific CD4 T cells into chronically infected mice restored proliferation and cytokine production by exhausted virus-specific CD8 T cells and reduced viral burden. Although the transferred CD4 T cells were able to enhance function in exhausted CD8 T cells, these CD4 T cells expressed high levels of the programmed cell death (PD)-1 inhibitory receptor. Blockade of the PD-1 pathway increased the ability of transferred LCMV-specific CD4 T cells to produce effector cytokines, improved rescue of exhausted CD8 T cells, and resulted in a striking reduction in viral load. These results suggest that CD4 T-cell immunotherapy alone or in conjunction with blockade of inhibitory receptors may be a promising approach for treating CD8 T-cell dysfunction in chronic infections and cancer.     

High antigen levels are the cause of T cell exhaustion during chronic viral infection

Many persistent viral infections induce dysfunctional T cell responses. Although a negative correlation exists between viral load and T cell responses during chronic infection, it is not known whether high antigen levels are the cause or just the consequence of T cell exhaustion. Furthermore, it is unclear what role antigen presentation by bone-marrow (BM) derived versus infected parenchymal cells has on T cell exhaustion. To address these issues, we examined the influence of antigen presentation by different cell types on CD8⁺ T cell responses during persistent infection of mice with lymphocytic choriomeningitis virus (LCMV) clone 13. We generated BM chimeric mice, in which non-BM derived cells were MHC class I deficient. Virus-specific CD8⁺ T cells in lymphoid and nonlymphoid tissues were increased in both number and ability to produce cytokines in these mice soon after infection. However, viral clearance from infected MHC I^−/− parenchyma was significantly impaired, despite increased populations of cytokine producing CTL. The CD8⁺ T cell response was overwhelmed by sustained antigen persistence, becoming increasingly exhausted within 4–6 weeks. Thus, we find that (i) sustained antigen presentation directly drives T cell exhaustion during a chronic viral infection, (ii) CTL require direct antigen-MHC interactions to clear virus-infected cells, and (iii) persistent interactions with antigen presented on both hematopoietic and nonhematopoietic cells negatively impacts virus-specific T cell responses during chronic infection.     

Recombinant Listeria monocytogenes as a live vaccine vehicle for the induction of protective anti-viral cell-mediated immunity.

Listeria monocytogenes (LM) is a Gram-positive bacterium that is able to enter host cells, escape from the endocytic vesicle, multiply within the cytoplasm, and spread directly from cell to cell without encountering the extracellular milieu. The ability of LM to gain access to the host cell cytosol allows proteins secreted by the bacterium to efficiently enter the pathway for major histocompatibility complex class I antigen processing and presentation. We have established a genetic system for expression and secretion of foreign antigens by recombinant strains, based on stable site-specific integration of expression cassettes into the LM genome. The ability of LM recombinants to induce protective immunity against a heterologous pathogen was demonstrated with lymphocytic choriomeningitis virus (LCMV). LM strains expressing the entire LCMV nucleoprotein or an H-2Ld-restricted nucleoprotein epitope (aa 118-126) were constructed. Immunization of mice with LM vaccine strains conferred protection against challenge with virulent strains of LCMV that otherwise establish chronic infection in naive adult mice. In vivo depletion of CD8+ T cells from vaccinated mice abrogated their ability to clear viral infection, showing that protective anti-viral immunity was due to CD8+ T cells.     

Epstein-Barr virus-induced diseases in boys with the X-linked lymphoproliferative syndrome (XLP): update on studies of the registry

Analyses of 100 subjects with the X-linked lymphoproliferative syndrome (XLP) in 25 kindreds revealed four major interrelated phenotypes: infectious mononucleosis, malignant B-cell lymphoma, aplastic anemia, and hypogammaglobulinemia. Eighty-one of the patients died. Two male subjects were asymptomatic but showed immunodeficiency to Epstein-Barr virus (EBV). Seventy-five subjects had the infectious mononucleosis phenotype and concurrently, 17 subjects of this group had aplastic anemia. All subjects with aplastic anemia died within a week. Aplastic anemia did not accompany hypogammaglobulinemia or malignant lymphoma phenotypes. Hypogammaglobulinemia had been detected before infectious mononucleosis in three subjects, after infectious mononucleosis in five subjects, and was not associated with infectious mononucleosis in 11 boys with hypogammaglobulinemia. In nine subjects infectious mononucleosis appeared to have evolved into malignant lymphoma; however, the majority of patients with malignant lymphoma showed no obvious antecedent infectious mononucleosis. One subject had infectious mononucleosis following recurrent malignant lymphoma. Twenty-six of 35 lymphomas were in the terminal ileum. Results of immunologic and virologic studies of 15 survivors revealed combined variable immunodeficiency and deficient antibody responses to EBV-specific antigens. Mothers of boys with XLP exhibited abnormally elevated titers of antibodies of EBV. Subjects of both sexes with phenotypes of XLP should be investigated for immunodeficiency to EBV. Persons with inherited or acquired immunodeficiency may be vulnerable to life-threatening EBV-induced diseases.     

Intrinsic defects in CD8 T cells with aging contribute to impaired primary antiviral responses

Aging is associated with altered immune responses, particularly with a diminished CD8 T cell response. Although both intrinsic and extrinsic factors are hypothesized to impact this decreased T cell response, the direct evidence of an intrinsic deficiency in virus-specific CD8 T cells is limited. In this study, a TCR transgenic (Tg) P14 mouse model was utilized to compare the activation and proliferation of the Tg CD8 T cells of young and aged P14 mice upon stimulation with antigen or infection with virus. The proliferation of purified Tg CD8 T cells of aged mice was significantly lower than that of young mice when cultured in vitro with both the LCMV specific peptide and antigen presenting cells from young wild type mice. In addition, expression of the activation markers, CD69, CD25, and CD44, was delayed on Tg T cells of aged mice after stimulation. Importantly, while adoptive transfer of purified Tg CD8 T cells of young or aged mice into young wild type mice resulted in expansion of the Tg CD8 T cells of both ages after LCMV infection, the expansion of the Tg T cells from aged mice was significantly decreased compared with that of the Tg T cells from young mice. However, while the number of IFN-γ secreting Tg CD8 T cells from aged mice was significantly decreased compared to that of young mice, the percentages of Tg CD8 T cells producing IFN-γ were similar in young and aged mice, demonstrating that proliferation, but not function, of the Tg CD8 T cells of aged mice was impaired. Importantly, chronological age alone was not sufficient to predict an altered proliferative response; rather, expression of high levels of CD44 on CD8 T cells of aged mice reflected a decreased proliferative response. These results reveal that alterations intrinsic to CD8 T cells can contribute to the age-associated defects in the primary CD8 T cell response during viral infection.