Aberrant T-cell antigen receptor-mediated responses in autoimmune lymphoproliferative syndrome

Autoimmune Lymphoproliferative Syndrome (ALPS) is a disorder of defective lymphocyte apoptosis due to mutations of the Fas receptor and other molecules in the Fas signaling pathway. In addition to accumulation of CD4(-) CD8(-) double-negative (DN) T cells, many patients display a dysregulated cytokine pattern with dysfunctional T cells, suggesting Fas defects may impact pathways of T-cell activation/differentiation. Here, we report two novel mutations in the Fas receptor resulting in an ALPS phenotype. Utilizing flow cytometry, we found anti-CD3 activated CD4(+) T cells from these patients were incapable of fully upregulating activation markers (CD25, CD69, and CD40L) or producing interferon-gamma and IL-2. Additionally, DN T cells were unable to transduce proximal T-cell antigen receptor signals or produce cytokines. Furthermore, DN T cells overexpressed CD57 and phenotypically resembled end-stage effector cells. As DN T cells were essentially anergic, the clinical manifestations of autoimmunity are more likely to be a consequence of aberrant cytokine secretion within the CD4(+) T-cell subpopulation.     

Expression in transgenic mice of dominant interfering Fas mutations: a model for human autoimmune lymphoproliferative syndrome.

Most humans with autoimmune lymphoproliferative syndrome (ALPS) carry heterozygous dominant mutations in one allele of the gene encoding Fas/APO-1/CD95. ALPS patients, like Fas-deficient MRL lpr/lpr mice, have lymphoproliferation, autoimmunity, increased CD4(-)/CD8(-) T lymphocytes, and apoptosis defects. Consistent with the phenotypic variability of lpr/lpr mice of different background strains, human genetic studies indicate that a Fas mutation is insufficient to induce ALPS in all mutation carriers. To investigate the dominant function of human Fas mutations and the additional genetic factor(s) involved in the development of ALPS, we generated transgenic mice expressing, in addition to endogenous Fas, mouse Fas molecules bearing mutations in the intracellular death domain corresponding to mutations identified in ALPS patients. Transgenic mice developed mild features of ALPS, including hepatosplenomegaly, elevated proportions of lymphocytes in spleen and lymph nodes, apoptotic defects, and hepatic lymphocytic infiltrates. Therefore defective murine Fas proteins act in a dominant manner to impair apoptosis of activated lymphocytes and disrupt lymphocyte homeostasis. The influence of genetic background on phenotype was studied by comparing transgenic mice on FVB/N and (FVB/N x MRL) backgrounds with syngenetic control mice and with MRL and MRL lpr/lpr mice. While expression of transgenic mutant Fas contributed mainly to hepatosplenomegaly and accumulation of lymphocytes, MRL background genes played a major role in the production of autoantibodies and elevated serum immunoglobulin levels. Moreover, compared to FVB/N (+/+) mice, a substantial Fas-specific apoptotic defect was found in MRL (+/+) mice, suggesting a mechanism for the known tendency of this strain to develop autoimmunity.     

Cytomegalovirus infection in infants with autoimmune lymphoproliferative syndrome (ALPS)

Fas-mediated apoptosis may be one of the effector pathways leading to the elimination of virus-infected cells. Cytomegalovirus (CMV) infection in two brothers with Fas deficiency associated with autoimmunity and benign lymphoproliferation (ALPS) provided a unique opportunity to study the clinical course of CMV infection in children with defective apoptosis. The clinical courses of two brothers with autosomal dominant ALPS who were infected with CMV in the neonatal period are described. CMV was detected from throat and urine culture from the brothers. ALPS was confirmed by in vitro anti-CD95 MoAb-induced T lymphocyte apoptosis assay and subsequent sequencing and identification of mutations in the Fas gene. A de novo mutation in the Fas gene, leading to a truncated cytoplasmic Fas product, was associated with autosomal dominant ALPS in a mother and her two sons. Both boys had evidence of CMV infection acquired early in infancy which cleared by the age of 2-3 years. There were no neurodevelopmental sequelae. The natural history of CMV infection in two infants with ALPS was similar to that described in normal children.     

Autoimmune lymphoproliferative syndrome (ALPS) in a patient with a new germline Fas gene mutation

Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder characterized by chronic lymphoproliferation, autoimmune manifestations and expansion of TCRalphabeta+CD4-CD8- lymphocytes. The main pathogenic factor is a defective Fas-mediated apoptosis generally caused by mutations in the Fas gene. This report describes a new heterozygous Fas gene mutation in a boy with clinical and immunological features of ALPS. In vitro, T-cell blasts from the patient are completely resistant to the effects on the anti-Fas cytotoxic mAb CH-11, they also have a higher proliferation rate than T cells from healthy donors, while PHA-induced AICD is normal. The location of the mutation (I246S) found in the intracytoplasmic death domain, and the conservation of that residue in four different species from human suggest that I246 is an essential amino acid for Fas function. The patient has inherited the mutation from his father who also shows defective Fas-mediated apoptosis but the clinical and immunological manifestations are much less severe. These results provide evidence that the penetrance of genetic defects in Fas is variable and that other factors may influence the phenotype of the disease.     

TcR-alpha/beta(+) CD4(-)CD8(-) T cells in humans with the autoimmune lymphoproliferative syndrome express a novel CD45 isoform that is analogous to murine B220 and represents a marker of altered O-glycan biosynthesis

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor alpha/beta(+) CD4(-)CD8(-) T cells (alpha/beta(+) double-negative T cells [alpha/beta(+)-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The alpha/beta(+)-DNT cells are immunophenotypically and functionally similar to alpha/beta(+)-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, alpha/beta(+)-DNT cells express the B-cell-specific CD45R isoform B220. We show that alpha/beta(+)-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4(+) T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell-cell interactions, and access to alternative apoptosis pathways.     

Defective function of Fas in T cells from paediatric patients with autoimmune thyroid diseases

Triggering of the Fas receptor induces T cell apoptosis and is involved in shutting-off the immune response. Inherited defects impairing Fas function cause the autoimmune lymphoproliferative syndrome, and may play a role in other autoimmune diseases. The aim of this work was to analyse the Fas function in paediatric patients with thyroid autoimmunities. We found that T cells from 24/28 patients with Graves' disease (GD) and 12/35 patients with Hashimoto's thyroiditis (HT) displayed defective Fas function. In HT, the defect was more frequent in patients requiring replacement therapy (11/20) than in those not requiring (1/15); moreover, in untreated HT the highest defect was displayed by patients with the highest levels of autoantibodies. Fas was always expressed at normal levels and no Fas mutations were detected. Analysis of the healthy parents of seven Fas-resistant patients showed that several of them were Fas-resistant, which suggests a genetic component. Fusion of Fas-resistant T cells with the Fas-sensitive HUT78 T cell line generated Fas-resistant hybrid cells, which suggests the presence of molecules exerting a dominant negative effect on Fas function. Analysis of Fas-induced activation of caspase-8 and -9 showed decreased activity of both caspases in HT, whereas activity of caspase-9 was increased and that of caspase-8 was decreased in GD. These data suggest that heterogeneous inherited defects impairing Fas function favour the development of thyroid autoimmunities.     

A composite picture of TcR alpha/beta(+) CD4(-)CD8(-) T Cells (alpha/beta-DNTCs) in humans with autoimmune lymphoproliferative syndrome

The discovery of an unusual T-cell subset characterized by the expression of the alpha/beta T-cell receptor without expression of either CD4 or CD8 [alpha/beta-double-negative T cells (alpha/beta-DNTCs)] provided critical insights in the evaluation of a "new" lymphoproliferative disorder known as autoimmune lymphoproliferative syndrome (ALPS). ALPS is a disorder of defective Fas-mediated lymphocyte apoptosis, manifested by accumulation of alpha/beta-DNTCs and other lymphocyte subsets, leading to lymphadenopathy and splenomegaly, autoimmunity, and an increased risk of lymphoma. The expanded population of alpha/beta-DNTCs from ALPS patients has a remarkable uniform phenotype that is for the most part similar to alpha/beta-DNTCs from mice with defective Fas (lpr) or Fas ligand (gld). This is in contrast to the minor alpha/beta-DNTC compartment in healthy individuals that contains multiple, immunophenotypically distinct subpopulations. Current data indicate that alpha/beta-DNTCs from ALPS patients are derived from cytotoxic CD8(+) T cells, chronically activated in vivo but anergic in vitro. Their anergic state may be related to persistent modifications of O-linked carbohydrates on cell surface molecules, such as CD43 and CD45, as well as to the increased presence of interleukin-10. Although largely consistent with a model of (linear) CD8(+) cytotoxic T-cell differentiation, the expression patterns of certain surface molecules, such as CD27 and CD28, are not consistent with this model. This may be the result of the perturbed homeostasis of lymphocytes in ALPS, thereby revealing pathways of differentiation and immunophenotypes, including phenotypes pertaining to cell surface glycosylation that are hidden from view in healthy individuals.     

The role of T cell apoptosis in nervous system autoimmunity

Fas is a transmembrane receptor involved in the death program of several cell lines, including T lymphocytes. Deleterious mutations hitting genes involved in the Fas pathway cause the autoimmune lymphoprolipherative syndrome (ALPS). Moreover, defective Fas function is involved in the development of common autoimmune diseases, including autoimmune syndromes hitting the nervous system, such as multiple sclerosis (MS) and chronic inflammatory demyelinating polyneuropathy (CIDP). In this review, we first explore some peculiar aspects of Fas mediated apoptosis in the central versus peripheral nervous system (CNS, PNS); thereafter, we analyze what is currently known on the role of T cell apoptosis in both MS and CIDP, which, in this regard, may be seen as two faces of the same coin. In fact, we show that, in both diseases, defective Fas mediated apoptosis plays a crucial role favoring disease development and its chronic evolution.     

Autoimmune lymphoproliferative syndrome in a patient with common variable immunodeficiency: dichotomy of apoptosis.

BACKGROUND: Autoimmune lymphoproliferative syndrome (ALPS) is a disorder usually associated with hypergammaglobulinemia and defective apoptosis mostly due to Fas or Fas ligand mutation. Common variable immunodeficiency (CVID) is a disorder with hypogammaglobulinemia commonly associated with increased Fas expression and spontaneous apoptosis. OBJECTIVE: To describe a patient with a unique combination of hypogammaglobulinemia and ALPS with Fas deficiency but high spontaneous apoptosis. METHODS: Fas expression on freshly isolated lymphocytes was evaluated by means of immunofluorescence using polyclonal rabbit anti-Fas IgG antibody. Apoptosis of cultured lymphocytes was quantitated using acridine orange and ethidium bromide staining. RESULTS: We describe a male patient diagnosed as having CVID at the age of 10 years receiving monthly intravenous immunoglobulin. The patient developed ALPS at the age of 21 years manifested by persistent lymphadenopathy and hepatosplenomegaly. The percentage of double-negative T lymphocytes was estimated to be 9%. Freshly isolated lymphocytes showed low Fas expression (3.6% for the patient and 11.2% for the control). The spontaneous apoptosis rate was high (15% for the patient and 5% for the control). CONCLUSIONS: Autoimmune lymphoproliferative syndrome can be associated with hypogammaglobulinemia and should be looked for in chronic nonmalignant lymphoproliferation in CVID. Common variable immunodeficiency might involve Fas-independent pathways or recruitment of Fas downstream molecules for apoptosis. There is a subset of patients with both CVID and ALPS in whom Fas deficiency could be associated with enhanced spontaneous apoptosis.     

A new disorder of lymphocyte apoptosis: combination of autoimmunity,infectious lymphadenopathy,double negative T cells,and impaired activation-induced cell death

A new symptom-complex is described characterized by manifestations of autoimmune disease, infectious lymphadenopathy, double negative T cells, and impaired activation-induced cell death that developed in late adolescence. Similarities, but also significant differences, to autoimmune lymphoproliferative syndromes (ALPS, Canale-Smith syndrome) and autoimmune lymphoproliferative disease (ALD, Dianzani syndrome), were observed. The main clinical features were recurrent bacterial infections with subsequent lymphadenopathy due to autoimmune neutropenia. Laboratory results revealed a large proportion of alphabetaTCR positive, CD4 negative, CD8 negative, peripheral T cells, and a decreased apoptosis upon activation with phytohemagglutinin and interleukin 2, but normal Fas-mediated apoptosis. Genetic investigations excluded mutations in Fas gene death domain and in the 4 exons of Fas ligand gene. Despite unknown pathogenesis, this new syndrome might belong to the growing group of diseases with defects in apoptosis.     

The autoimmune lymphoproliferative syndrome: an experiment of nature involving lymphocyte apoptosis

Autoimmune lymphproliferative syndrome (ALPS) is a human disorder that has been characterized in the past two decades at both a functional and a genetic level. The underlying basis for this disorder is a defect in lymphocyte apoptosis that alters immune homeostasis resulting in an expansion of a normally rare circulating lymphocyte, the alpha beta double negative T cell. The abnormality in Fas mediated apoptosis underlying ALPS serves as a risk factor for autoimmunity involving blood cells and the development of lymphoma. There remain patients with a diagnosis of ALPS but without a defined genetic defect and current investigations are focusing on fully characterizing this patient subgroup.     

HLA B44 is associated with decreased severity of autoimmune lymphoproliferative syndrome in patients with CD95 defects (ALPS type Ia)

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte apoptosis characterized by non-malignant lymphadenopathy and splenomegaly, expansion of T cells without either CD4 or CD8 surface markers, and increased incidence of autoimmune diseases and lymphoma. Most patients with ALPS have dominant, heterozygous mutations in tumor necrosis factor receptor superfamily member 6 (TNFRSF6), which encodes CD95, also known as Fas, a mediator of apoptosis. Penetrance and range of disease manifestations in ALPS are highly variable, even among family members who share the same dominant TNFRSF6 mutation. To evaluate HLA as a candidate modifier locus, we typed HLA A, B (including subtypes), and DQB alleles in 356 individuals from 63 unrelated families with defined TNFRSF6 mutations associated with ALPS. We also developed a quantitative severity score and performed statistical analysis. Among the healthier, mutation-bearing individuals, transmission of HLA B44 was significantly overrepresented (nominal P<0.0074) as compared to transmission in patients with severe clinical features of ALPS. The B44 allele may exert a protective role in ALPS.     

Cell-death signaling and human disease

The autoimmune lymphoproliferative syndrome (ALPS) in humans and the lpr mouse strain are the first examples of primary apoptosis defects caused by inherited death-receptor mutations. They illustrate the role of Fas and Fas ligand in the control of autoimmune T-cell and B-cell proliferation. Subsequent analyses of ALPS in humans have highlighted the role of caspase 10 in the induction of apoptosis. The recent identification of a human caspase 8 defect has revealed a potential connection between apoptosis pathways and immune-receptor signaling. The genetic basis of ALPS is extremely complex, as multiple factors are potentially involved in the pathogenesis of this disease, thus offering an interesting model with which to unravel the mechanisms involved in T-cell homeostasis and self-tolerance.     

Novel Fas (CD95/APO-1) mutations in infants with a lymphoproliferative disorder

Fas is an apoptosis-signaling receptor important for homeostasis of the immune system. In this study, Fas-mediated apoptosis and Fas mutations were analyzed in three Japanese children from two families with a lymphoproliferative disorder characterized by lymphadenopathy, hepatosplenomegaly, pancytopenia, hypergammaglobulinemia and an increase in TCR alphabeta+ CD4- CD8- T cells. Apoptosis induced by anti- Fas mAb was defective in both activated T cells and B cells, and granulocytes from these patients. Truncated Fas receptor lacking the cytoplasmic death domain caused by a point mutation in the splice region of intron 7 were demonstrated in two siblings. A homozygous point mutation in the splice acceptor of intron 3 was found in the Fas gene of the third patient, which resulted in the skipping of exon 4 and complete loss of Fas expression. Corresponding to these mutations, soluble Fas concentrations were decreased and reciprocally soluble Fas ligands were increased in patients' sera. Interestingly, co-stimulation by immobilized anti-Fas mAb in T cells from the two siblings was comparable to that seen in normal T cells. These results suggest that Fas-mediated apoptosis plays a pivotal role in immunological homeostasis in vivo, especially regarding clonal deletion of immune cells in humans.      

TcR-α/β CD4CD8 T Cells in Humans with the Autoimmune Lymphoproliferative Syndrome Express a Novel CD45 Isoform That Is Analogous to Murine B220 and Represents a Marker of Altered O-Glycan Biosynthesis

Autoimmune lymphoproliferative syndrome (ALPS), caused by inherited defects in apoptosis secondary to mutations in genes encoding Fas/CD95/APO-1 and Fas ligand (Fasl)/CD95L, is characterized by nonmalignant lymphadenopathy and splenomegaly, increased T cell receptor α/β⁺ CD4⁻CD8⁻ T cells (α/β⁺ double-negative T cells [α/β⁺-DNT cells]), autoimmunity, hypergammaglobulinemia, and cytokine abnormalities. The α/β⁺-DNT cells are immunophenotypically and functionally similar to α/β⁺-DNT cells that accumulate in lpr and gld mice, which bear genetic mutations in Fas and FasL. In these mice, α/β⁺-DNT cells express the B-cell-specific CD45R isoform B220. We show that α/β⁺-DNT cells of ALPS patients, with either Fas or FasL mutations, also express B220. In addition, also similar to LPR/gLD mice, they have an unusual population of B220-positive CD4⁺ T cells. B220 expression, together with our finding of characteristic lectin binding profiles, demonstrates that cell surface O-linked glycoproteins have undergone specific modifications, which may have consequences for lymphocyte trafficking, cell–cell interactions, and access to alternative apoptosis pathways.     

Genome-wide association of echocardiographic dimensions,brachial artery endothelial function and treadmill exercise responses in the Framingham Heart Study

Background:

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance due primarily to genetic defects in Fas (CD95/APO-1; TNFRSF6), a cell surface receptor that regulates apoptosis and its signaling apparatus.

Methods:

Fas ligand gene mutations from ALPS patients were identified through cDNA and genomic DNA sequencing. Molecular and biochemical assessment of these mutant Fas ligand proteins were carried out by expressing the mutant FasL cDNA in mammalian cells and analysis its effects on Fas-mediated programmed cell death.

Results:

We found an ALPS patient that harbored a heterozygous A530G mutation in the FasL gene that replaced Arg with Gly at position 156 in the protein's extracellular Fas-binding region. This produced a dominant-interfering FasL protein that bound to the wild-type FasL protein and prevented it from effectively inducing apoptosis.

Conclusion:

Our data explain how a naturally occurring heterozygous human FasL mutation can dominantly interfere with normal FasL apoptotic function and lead to an ALPS phenotype, designated Type Ib.     

Activation of cAMP signaling enhances Fas-mediated apoptosis and activation-induced cell death through potentiation of caspase 8 activation

Defects in Fas receptor signaling lead to compromised maintenance of lymphocyte homeostasis and peripheral immune tolerance, leading in turn to autoimmune disorders. Therefore, agents that can enhance Fas-mediated apoptosis may be therapeutically useful in management of such disorders. In this study, we focused on the effect of cAMP on Fas-mediated apoptosis in human T cells. We show that elevation of intracellular cAMP levels by forskolin, an activator of adenylyl cyclase, 3-isobutyl-1-methylxanthine, an inhibitor of cyclic nucleotide phosphodiesterases, or prostaglandin E(2) potentiates Fas-induced apoptosis in Jurkat cells. Accordingly, cAMP was found to enhance the cleavage of caspase 8 at death-inducing signaling complex and lead to augmentation of the processing of Fas effector proteins. We also demonstrate that cAMP enahnaces Fas-induced apoptosis in normal human T cells and activation-induced cell death in Jurkat cells. These findings provide a rationale for investigating the feasibility of using cAMP-elevating agents to potentiate apoptosis in T cells with aberrant Fas signaling.     

Prominent dominant negative effect of a mutant Fas molecule lacking death domain on cell-mediated induction of apoptosis

Using a panel of transfectant B lymphoma cells expressing varying amounts of the mutant Fas together with the endogenous wild type Fas, semi-quantitative studies on the dominant negative effect of a murine mutant Fas molecule lacking death domain were carried out. In anti-Fas antibody-mediated induction of apoptosis, the mutant molecules exerted significant dominant-negative effect only when their expression level was comparable to or higher than that of wild type molecules, or when exposed to low amounts of the antibody. The inhibitory effect was accompanied by the failure in DISC formation in spite of Fas aggregation. When they were subjected to T cell-mediated Fas-based induction of apoptosis, however, the dominant negative effect was prominent such that the expression of even a small amount of the mutant molecules resulted in significant inhibition. Such a strong inhibitory effect explains the dominant phenotype of this type of mutant Fas molecules in ALPS heterozygous patients and also implies that the physiological effectors for Fas in vivo are cells, i.e., FasL-expressing activated T cells.     

Elevated Double Negative T Cells in Pediatric Autoimmunity

Purpose

Autoimmune diseases are thought to be caused by a loss of self-tolerance of the immune system. One candidate marker of immune dysregulation in autoimmune disease is the presence of increased double negative T cells (DNTs) in the periphery. DNTs are characteristically elevated in autoimmune lymphoproliferative syndrome, a systemic autoimmune disease caused by defective lymphocyte apoptosis due to Fas pathway defects. DNTs have also been found in the peripheral blood of adult patients with systemic lupus erythematosus (SLE), where they may be pathogenic. DNTs in children with autoimmune disease have not been investigated.

Methods

We evaluated DNTs in pediatric patients with SLE, mixed connective tissue disease (MCTD), juvenile idiopathic arthritis (JIA), or elevated antinuclear antibody (ANA) but no systemic disease. DNTs (CD3⁺CD56^?TCRαβ⁺CD4^?CD8^?) from peripheral blood mononuclear cells were analyzed by flow cytometry from 54 pediatric patients including: 23 SLE, 15 JIA, 11 ANA and 5 MCTD compared to 28 healthy controls.

Results

Sixteen cases (29.6 %) had elevated DNTs (≥2.5 % of CD3⁺CD56^?TCRαβ⁺ cells) compared to 1 (3.6 %) control. Medication usage including cytotoxic drugs and absolute lymphocyte count were not associated with DNT levels, and percentages of DNTs were stable over time. Analysis of multiple phenotypic and activation markers showed increased CD45RA expression on DNTs from patients with autoimmune disease compared to controls.

Conclusion

DNTs are elevated in a subset of pediatric patients with autoimmune disease and additional investigations are required to determine their precise role in autoimmunity.