Janet K. Dale

Dominant interfering fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome

Five unrelated children are described with a rare autoimmune lymphoproliferative syndrome (ALPS) characterized by massive nonmalignant lymphadenopathy, autoimmune phenomena, and expanded populations of TCR-CD3+CD4-CD8- lymphocytes. These findings, suggesting a genetic defect in the ability of T lymphocytes to respond to normal immunoregulatory mechanisms, prompted an evaluation of lymphocyte apoptosis. Each child had defective Fas-mediated T lymphocyte apoptosis associated with a unique, deleterious Fas gene mutation. One mutation appeared to cause a simple loss of function; however, four others had a dominant negative phenotype when coexpressed with normal Fas. Family studies demonstrated the inheritance of the mutant Fas alleles. The occurrence of Fas mutations together with abnormal T cell apoptosis in ALPS patients suggests an involvement of Fas in this recently recognized disorder of lymphocyte homeostasis and peripheral self-tolerance.

Inherited Human Caspase 10 Mutations Underlie Defective Lymphocyte and Dendritic Cell Apoptosis in Autoimmune Lymphoproliferative Syndrome Type II

Caspases are cysteine proteases that mediate programmed cell death in phylogenetically diverse multicellular organisms. We report here two kindreds with autoimmune lymphoproliferative syndrome (ALPS) type II, characterized by abnormal lymphocyte and dendritic cell homeostasis and immune regulatory defects, that harbor independent missense mutations in Caspase 10. These encode amino acid substitutions that decrease caspase activity and interfere with death receptor-induced apoptosis, particularly that stimulated by Fas ligand and TRAIL. These results provide evidence that inherited nonlethal caspase abnormalities cause pleiotropic apoptosis defects underlying autoimmunity in ALPS type II.

Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency

Apoptosis is a form of programmed cell death that is controlled by aspartate-specific cysteine proteases called caspases. In the immune system, apoptosis counters the proliferation of lymphocytes to achieve a homeostatic balance, which allows potent responses to pathogens but avoids autoimmunity. The CD95 (Fas, Apo-1) receptor triggers lymphocyte apoptosis by recruiting Fas-associated death domain (FADD), caspase-8 and caspase-10 proteins into a death-inducing signalling complex. Heterozygous mutations in CD95, CD95 ligand or caspase-10 underlie most cases of autoimmune lymphoproliferative syndrome (ALPS), a human disorder that is characterized by defective lymphocyte apoptosis, lymphadenopathy, splenomegaly and autoimmunity. Mutations in caspase-8 have not been described in ALPS, and homozygous caspase-8 deficiency causes embryonic lethality in mice. Here we describe a human kindred with an inherited genetic deficiency of caspase-8. Homozygous individuals manifest defective lymphocyte apoptosis and homeostasis but, unlike individuals affected with ALPS, also have defects in their activation of T lymphocytes, B lymphocytes and natural killer cells, which leads to immunodeficiency. Thus, caspase-8 deficiency in humans is compatible with normal development and shows that caspase-8 has a postnatal role in immune activation of naive lymphocytes.

Autoimmune Lymphoproliferative Syndrome with Defective Fas: Genotype Influences Penetrance

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance. Most patients have a heterozygous mutation in the APT1 gene, which encodes Fas (CD95, APO-1), mediator of an apoptotic pathway crucial to lymphocyte homeostasis. Of 17 unique APT1 mutations in unrelated ALPS probands, 12 (71%) occurred in exons 7-9, which encode the intracellular portion of Fas. In vitro, activated lymphocytes from all 17 patients showed apoptotic defects when exposed to an anti-Fas agonist monoclonal antibody. Similar defects were found in a Fas-negative cell line transfected with cDNAs bearing each of the mutations. In cotransfection experiments, Fas constructs with either intra- or extracellular mutations caused dominant inhibition of apoptosis mediated by wild-type Fas. Two missense Fas variants, not restricted to patients with ALPS, were identified. Variant A(-1)T at the Fas signal-sequence cleavage site, which mediates apoptosis less well than wild-type Fas and is partially inhibitory, was present in 13% of African American alleles. Among the ALPS-associated Fas mutants, dominant inhibition of apoptosis was much more pronounced in mutants affecting the intracellular, versus extracellular, portion of the Fas receptor. Mutations causing disruption of the intracellular Fas death domain also showed a higher penetrance of ALPS phenotype features in mutation-bearing relatives. Significant ALPS-related morbidity occurred in 44% of relatives with intracellular mutations, versus 0% of relatives with extracellular mutations. Thus, the location of mutations within APT1 strongly influences the development and the severity of ALPS.

Pathological Findings in Human Autoimmune Lymphoproliferative Syndrome

The defects in lymphocyte apoptosis that underlie the autoimmune lymphoproliferative syndrome (ALPS) are usually attributable to inherited mutations of the CD95 (Fas) gene. In this report, we present the histopathological and immunophenotypic features seen in the lymph nodes (n = 16), peripheral blood (n = 10), bone marrow (n = 2), spleen (n = 3), and liver (n = 2) from 10 patients with ALPS. Lymph nodes showed marked paracortical hyperplasia. Interfollicular areas were expanded and populated by T cell receptor-alphabeta CD3+ CD4-CD8- (double-negative, DN) T cells that were negative for CD45RO. CD45RA+ T cells were increased in all cases studied. The paracortical infiltrate was a result of both reduced apoptosis and increased proliferation, as measured by in situ detection of DNA fragmentation and staining with MIB-1, respectively. The paracortical proliferation may be extensive enough to suggest a diagnosis of malignant lymphoma. Many of the paracortical lymphocytes expressed markers associated with cytotoxicity, such as perforin, TIA-1, and CD57. CD25 was negative. In addition, most lymph nodes exhibited florid follicular hyperplasia, often with focal progressive transformation of germinal centers; in some cases, follicular involution was seen. A polyclonal plasmacytosis also was present. The spleens were markedly enlarged, more than 10 times normal size. There was expansion of both white pulp and red pulp, with increased DN T cells. DN T cells also were observed in liver biopsies exhibiting portal triaditis. In the peripheral blood, the T cells showed increased expression of HLA-DR and CD57 but not CD25. CD45RA+ T cells were increased in the four cases studied. Polyclonal B cell lymphocytosis with expansion of CD5+ B cells was a characteristic finding. Taken together, the histopathological and immunophenotypic findings, particularly in lymph nodes and peripheral blood, are sufficiently distinctive to suggest a diagnosis of ALPS. Of note, two affected family members of one proband developed lymphoma (T-cell-rich B-cell lymphoma and nodular lymphocyte predominance Hodgkins disease, respectively).

NRAS mutation causes a human autoimmune lymphoproliferative syndrome

The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization, and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous, and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4−, CD8− αβ T cells. Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving TNF receptor superfamily proteins, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis. The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling, which markedly decreases the proapoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects. Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyltransferase inhibitors should be examined in human autoimmune and lymphocyte homeostasis disorders.

Autoimmune lymphoproliferative syndrome.

Autoimmune lymphoproliferative syndrome arises early in childhood in people who inherit mutations in genes that mediate lymphocyte apoptosis, or programed cell death. In the immune system, antigen-induced lymphocyte apoptosis maintains immune homeostasis by limiting lymphocyte accumulation and minimizing reactions against self-antigens. In autoimmune lymphoproliferative syndrome, defective lymphocyte apoptosis manifests as chronic, nonmalignant adenopathy and splenomegaly; the expansion of an unusual population of CD4−CD8− T cells; and the development of autoimmune disease. Most cases of autoimmune lymphoproliferative syndrome involve heterozygous mutations in the lymphocyte surface protein Fas (CD95, Apo1) that impair a major apoptotic pathway. Prospective evaluations of patients and their families have revealed an ever-expanding spectrum of autoimmune lymphoproliferative syndrome and its major complications.

Characterization and treatment of chronic active Epstein-Barr virus disease: a 28-year experience in the United States

Chronic active EBV disease (CAEBV) is a lymphoproliferative disorder characterized by markedly elevated levels of antibody to EBV or EBV DNA in the blood and EBV RNA or protein in lymphocytes in tissues. We present our experience with CAEBV during the last 28 years, including the first 8 cases treated with hematopoietic stem cell transplantation in the United States. Most cases of CAEBV have been reported from Japan. Unlike CAEBV in Japan, where EBV is nearly always found in T or natural killer (NK) cells in tissues, EBV was usually detected in B cells in tissues from our patients. Most patients presented with lymphadenopathy and splenomegaly; fever, hepatitis, and pancytopenia were common. Most patients died of infection or progressive lymphoproliferation. Unlike cases reported from Japan, our patients often showed a progressive loss of B cells and hypogammaglobulinemia. Although patients with CAEBV from Japan have normal or increased numbers of NK cells, many of our patients had reduced NK-cell numbers. Although immunosuppressive agents, rituximab, autologous cytotoxic T cells, or cytotoxic chemotherapy often resulted in short-term remissions, they were not curative. Hematopoietic stem cell transplantation was often curative for CAEBV, even in patients with active lymphoproliferative disease that was unresponsive to chemotherapy. These studies are registered at http://www.clinicaltrials.gov as NCT00032513 for CAEBV, NCT00062868 and NCT00058812 for EBV-specific T-cell studies, and NCT00578539 for the hematopoietic stem cell transplantation protocol.

Allergy and the chronic fatigue syndrome

The chronic fatigue syndrome is a heterogeneous disorder characterized by easy fatigability, feverishness, diffuse pains, and depression. Many patients also report inhalant, food, or drug allergies. This article reviews the clinical features of the syndrome and hypotheses of its pathogenesis, especially those regarding the Epstein-Barr virus and cellular immune mechanisms. Also summarized are recent studies of the validity of atopic complaints in the syndrome. The results of epicutaneous skin testing demonstrated a high correlation with history in 24 patients. Atopy coexists with the chronic fatigue syndrome in greater than 50% of patients.

Lymphocyte phenotype and function in the chronic fatigue syndrome

Lymphocytes of 18 patients meeting the Centers for Disease Control (CDC) case definition for the chronic fatigue syndrome (CFS), 10 similar, chronically fatigued patients not fully conforming to the CDC case definition, and 17 matched, healthy individuals were studied to determine the presence of abnormalities of peripheral cell phenotype and function. Extensive phenotypic analyses of B- and T-cell subsets, natural killer (NK) cells, and macrophages were performed using single-, dual-, and three-color flow cytometry. Compared to controls, in CFS patients the percentage of CD4 T cells and CD4, CD45RA, or naive T cells, was reduced. The CD4, CD45RO, or memory T-cell, subset was numerically normal but expressed increased levels of adhesion markers (CD29, CD54, and CD58). CFS patient lymphocytes showed reduced proliferative responses to phytohemagglutinin, concanavalin A, and staphylococcal enterotoxin B. Lymphocytes from fatigue patients not meeting the CDC definition showed similar abnormalities. These data indicate that peripheral T cells manifest an increased state of differentiation in CFS and related conditions. This may arise as a consequence of an underlying neuropsychiatric and/or neuroendocrine disorder or because of exposure to antigens or superantigens of an infectious agent.