Defect In Fas-mediated Apoptosis Research Articles

Detection of Signature Double-Negative T Cells Is a Reliable Marker for ALPS Associated with FAS Mutation

Defects in FAS mediated apoptosis lead to a rare autoimmune disease called autoimmune lymphoproliferative syndrome (ALPS), which is manifested by a variety of symptoms including chronic lymphoproliferation, splenomegaly and autoimmune cytopenia. ALPS is mainly caused by germline or somatic FAS mutations, but gene analysis is time and cost intensive. Therefore, a tool is needed to screen for patients that are likely to harbor such a mutation to reduce costs and allow a faster diagnosis and treatment. One hallmark of the disease is the pathognomonic accumulation of TCRαβ+ CD4-/CD8- double-negative T (DNT) cells, which show high mitotic activity and unique expression profile. In this study, we compared the immune phenotype of ALPS patients with germline or somatic FAS mutation (ALPS-FAS/sFAS, n=11), patients who fulfill ALPS diagnostic criteria without FAS mutation (ALPS-U, n=11), and patients with ALPS-like disorders (two major diagnostic criteria without further criteria or at least one major criterion and autoimmune cytopenia, n=22). In addition, healthy donors, patients with chronic autoimmune cytopenia (AC, n=9), and patients with chronic non-malignant lymphoproliferation (LP, n=9) with no other major or minor criteria for ALPS diagnosis were used as controls. ALPS-FAS/sFAS patients showed accumulation of DNT cells with increased mitotic activity, terminal differentiation and expression of CD45RA/CD38 compared to healthy donors, AC and LP patients. Using receiver operating characteristic analyses, we could demonstrate that Ki67 and CD45RA/CD38 expression within DNT cells clearly distinguish ALPS-FAS/sFAS patients and ALPS-U and ALPS-like patients. To further strengthen specificity and sensitivity of the test, we assessed whether combination of DNT cell frequency with one additional marker is useful in supporting ALPS diagnosis. We found that a combination of DNT cell frequency with the expression of CD38 and CD45RA or Ki67 is most efficient to predict FAS mutation in ALPS patients. Taken together, flow cytometric analysis of these signature markers is a highly effective tool to discriminate patients with ALPS-FAS/sFAS and ALPS-related diseases and may also be useful for monitoring of treatment efficacy.

The contribution of rare copy number variants in FAS toward pathogenesis of autoimmune lymphoproliferative syndrome

AbstractAutoimmune lymphoproliferative syndrome (ALPS) is characterized by chronic nonmalignant lymphadenopathy, splenomegaly, cytopenias, and other autoimmune manifestations. ALPS is caused by lymphocyte accumulation from defects in FAS-mediated apoptosis. Heterozygous germline or somatic pathogenic single nucleotide variants in FAS are the most common molecular etiology of ALPS. Through the Centralized Sequencing Program at the National Institute of Allergy and Infectious Diseases, we performed exome sequencing on subjects with a clinical diagnosis of ALPS, with a subset receiving copy number variant (CNV) analysis. In this cohort, we identified 3 subjects from unrelated families with CNVs at the FAS locus. One subject had a de novo ∼0.828 Mb copy number loss encompassing all of FAS. The second subject had a maternally inherited ∼1.004 Mb copy number loss encompassing all of FAS. The third subject had a paternally inherited ∼0.044 Mb copy number loss encompassing exons 7 through 9 of FAS. Subjects with deletions in FAS had clinical presentations and biomarker profiles similar to those with ALPS and with germline and somatic FAS variants. We demonstrate that CNV analysis should be pursued if there is clinical and biomarker evidence of ALPS because it can lead to a molecular diagnosis and appropriate treatment when FAS sequencing is inconclusive.

ITK deficiency presenting as autoimmune lymphoproliferative syndrome

Activated T cells use apoptosis as a regulatory mechanism to prevent an exaggerated immune response. Repetitive or sustained stimulation of the T-cell receptor (TCR) induces surface expression of Fas and its ligand (FasL). Binding of FasL to its receptor initiates a signaling pathway resulting in the death of Fas-expressing T cells. Defects in Fas-mediated apoptosis cause autoimmune lymphoproliferative syndrome (ALPS), which is characterized by lymphoproliferation, autoimmune cytopenia, susceptibility to lymphoma, and accumulation of TCRαβ+CD4–CD8– (double-negative) T cells.

Autoimmune Lymphoproliferative Syndrome: An Overview.

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited nonmalignant lymphoproliferative disorder characterized by heterozygous mutations within the first apoptosis signal receptor (FAS) signaling pathway. Defects in FAS-mediated apoptosis cause an expansion and accumulation of autoreactive CD4- and CD8- (double-negative) T cells, leading to cytopenias, splenomegaly, lymphadenopathy, autoimmune disorders, and a greatly increased lifetime risk of lymphoma. The differential diagnosis of ALPS includes infection, other inherited immunodeficiency disorders, primary and secondary autoimmune syndromes, and lymphoma. The most consistent pathologic feature is a florid paracortical expansion of double-negative T cells in lymph nodes. A presumptive clinical diagnosis can be made from symptoms and a constellation of laboratory test results. However, a definitive diagnosis requires ancillary testing and enables disease subclassification. Recognition of ALPS is critical, as treatment with immunosuppressive therapies can effectively reduce or ameliorate symptoms for most patients.

Role of Fas and RANKL Signaling in Peripheral Immune Tolerance

The death receptor, Fas, has been well-characterized and is a critical factor in apoptosis in immune cells. Fas also has an important role in maintaining immune tolerance as demonstrated in the autoimmune-prone MRL/lpr mouse strain which carries a defect in Fas-mediated apoptosis of T cells. However, the role of Fas-independent apoptosis remains to be characterized in autoimmune diseases. In dendritic cells (DCs), binding of receptor activator of nuclear factor-κB ligand (RANKL) to RANK perpetuates the survival of mature DCs. However, cross-talk between the RANK/RANKL pathway and Fas-mediated signaling during the function or activation of DCs has not been well-studied. This short communication review describes a mechanism involving interactions between activated DCs and T cells in the autoimmune response of MRL/lpr mice and a novel Fas-independent apoptosis pathway in T cells that maintains peripheral tolerance, and controls autoimmunity in MRL/lpr mice.

A FAS-ligand variant associated with autoimmune lymphoproliferative syndrome in cats.

British shorthair (BSH) kittens in multiple litters died as a result of a severe non-neoplastic lymphoproliferative disease that showed many similarities with human autoimmune lymphoproliferative syndrome (ALPS). Human ALPS is caused by inherited defects in FAS-mediated lymphocyte apoptosis and the possibility of similar defects was investigated in BSH cats. The whole genomes of two affected kittens were sequenced and compared to 82 existing cat genomes. Both BSH kittens had homozygous insertions of an adenine within exon 3 of the FAS-ligand gene. The resultant frameshift and premature stop codon were predicted to result in a severely truncated protein that is unlikely to be able to activate FAS. Three additional affected BSH kittens were homozygous for the variant, while 11 of 16 unaffected, but closely related, BSH cats were heterozygous for the variant. All BSH cats in the study were from a population with significant inbreeding. The variant was not identified in a further survey of 510 non-BSH cats. Identification of a genetic defect in the FAS-mediated apoptosis pathway confirms that the lymphoproliferative disease in BSH cats fulfills the diagnostic criteria for ALPS in humans. These results will enable the development of a genetic test to detect BSH carrier animals.

Impact of loss of NF-κB1, NF-κB2 or c-REL on SLE-like autoimmune disease and lymphadenopathy in Fas(lpr/lpr) mutant mice.

Defects in apoptosis can cause autoimmune disease. Loss-of-function mutations in the 'death receptor' FAS impair the deletion of autoreactive lymphocytes in the periphery, leading to progressive lymphadenopathy and systemic lupus erythematosus-like autoimmune disease in mice (Fas(lpr/lpr) (mice homozygous for the lymphoproliferation inducing spontaneous mutation)) and humans. The REL/nuclear factor-κB (NF-κB) transcription factors regulate a broad range of immune effector functions and are also implicated in various autoimmune diseases. We generated compound mutant mice to investigate the individual functions of the NF-κB family members NF-κB1, NF-κB2 and c-REL in the various autoimmune pathologies of Fas(lpr/lpr) mutant mice. We show that loss of each of these transcription factors resulted in amelioration of many classical features of autoimmune disease, including hypergammaglobulinaemia, anti-nuclear autoantibodies and autoantibodies against tissue-specific antigens. Remarkably, only c-REL deficiency substantially reduced immune complex-mediated glomerulonephritis and extended the lifespan of Fas(lpr/lpr) mice. Interestingly, compared with the Fas(lpr/lpr) animals, Fas(lpr/lpr)nfkb2(-/-) mice presented with a dramatic acceleration and augmentation of lymphadenopathy that was accompanied by severe lung pathology due to extensive lymphocytic infiltration. The Fas(lpr/lpr)nfkb1(-/-) mice exhibited the combined pathologies caused by defects in FAS-mediated apoptosis and premature ageing due to loss of NF-κB1. These findings demonstrate that different NF-κB family members exert distinct roles in the development of the diverse autoimmune and lymphoproliferative pathologies that arise in Fas(lpr/lpr) mice, and suggest that pharmacological targeting of c-REL should be considered as a strategy for therapeutic intervention in autoimmune diseases.

A selective defect in Fas-mediated apoptosis in HAM/TSP: An ex vivo, in vitro and in silico study

Fas/FasL-mediated apoptosis is crucial for a functional immune response, with deficient Fas/FasL expression being linked to several autoimmune diseases. In addition, a FAS-670 polymorphism in an interferon (IFN)-regulated STAT1-binding site has been associated to both ATL (Farre et al., 2008) and HAM/TSP (Vallinotto et al., 2012) susceptibility. Recently, Fas has also been identified as part of an IFN-regulated gene signature in HAM/TSP (Tattermusch et al., 2012). Therefore, we examined Fas expression and function in lymphocyte activation, apoptosis, lymphoproliferation and gene expression profiling, using polychromatic flow cytometry, thymidine incorporation and microarray analysis (Affymetrix) respectively, in PBMCs from 20 HAM/TSP patients, 30 asymptomatic HTLV-1-infected individuals and 34 healthy controls (Salvador-Bahia, Brazil). Fas expression was increased in both asymptomatic HTLV-1-infected individuals and HAM/TSP patients, as compared to uninfected controls. In HAM/TSP, Fas expression correlated positively to lymphocyte activation markers (HLA-DR, CD86), but negatively to disease duration, suggesting that Fas-mediated pro-apoptotic capacity decreases over time in patients. Likewise, increased Fas expression in HAM/TSP did not lead to increased apoptosis upon in vitro culture. However, in HAM/TSP patients, IFN-alpha-induced Fas expression was paralleled by decreased lymphoproliferation, suggesting a link between defective Fas-mediated apoptosis and excessive lymphoproliferation, a hallmark of HTLV-1 infection, which can be restored by IFN-alpha treatment. Taken together, our results suggest defective Fas-mediated apoptosis might be an additional factor of HAM/TSP pathogenesis, besides its recently identified IFN gene signature.

Autoimmune Lymphoproliferative Syndrome and Epstein-Barr Virus-Associated Lymphoma: An Adjunctive Diagnostic Role for Monitoring EBV Viremia?

Background. Autoimmune lymphoproliferative syndrome (ALPS) is a genetic disorder of lymphocyte homeostasis due to defects in FAS-mediated apoptosis. ALPS is characterized by childhood onset of chronic lymphadenopathy and splenomegaly, autoimmunity, an expanded population of double-negative T cells (DNTCs), and an increased risk of lymphoma. This propensity for lymphoma in ALPS is not well understood. It is possible that lymphomagenesis in some of these patients may result from Epstein-Barr virus (EBV) infection exploiting the defective T-cell surveillance resulting from impaired FAS-mediated apoptosis. Case Presentation. We report the first case, to our knowledge, of lymphoma in a patient with ALPS that was clinically heralded by progressively increasing EBV viremia. We discuss its practical implications and the possible immune pathways involved in the increased risk for EBV-associated lymphoproliferative disorders in ALPS patients. Conclusion. In patients with ALPS, distinguishing chronic lymphadenopathy from emerging lymphoma is difficult, with few practical recommendations available. This case illustrates that, at least for some patients, monitoring for progressively increasing EBV viremia may be useful.

Clinico-Pathological Characteristics of RAS Associated Autoimmune Lymphoproliferative Syndrome Like Disease (RALD), and RAS Mutated Juvenile Myelomonocytic Leukemia (JMML)

Abstract 5169Autoimmune lympho proliferative syndrome (ALPS) is a disease characterized by dysfunction of the FAS-mediated apoptotic pathway, currently categorized as Type Ia, germline TNFRSF6/FAS mutation; Type Ib, germline FAS ligand mutation; Type Is, somatic TNFRSF6/FAS mutation; and Type II, germline Caspase 10 mutation. Patients exhibit lymphadenopathy, hepatosplenomegaly, and autoimmune diseases such as immune cytopenia and hyper-γ-globulinemia. An additional subclassification has been proposed that includes Types III, which has been defined as that with no known mutation but with a defect in FAS-mediated apoptosis. Juvenile myelomonocytic leukemia (JMML) is a chronic leukemia in children. Patients show lymphadenopathy, hepatosplenomegaly, leukocytosis associated with monocytosis, anemia, thrombocytopenia. About 80% of patients with JMML have been shown to have a genetic abnormality in their leukemia cells including mutations of NF1, RAS family, CBL, or PTPN11. HSCT is currently considered the only curative treatment. However, some patients have been shown to achieve remission without HSCT. Interestingly, there are several cases of JMML reported simultaneously having clinical and laboratory findings compatible with autoimmune disease. Germline RAS pathway mutations cause Costello (HRAS), Noonan (PTPN11, KRAS, and SOS1), and cardio-facio-cutaneous (CFC) syndromes (KRAS, BRAF, MEK1, and MEK2). Patients with Costello and Noonan syndromes have an increased propensity to develop solid and hematopoietic tumors, respectively.We have recently reported a unique disease which lies between ALPS and JMML. The patients were characterized by prominent autoimmune cytopenia, lymphoadenopathy/splenomegaly and moderate monocytosis resembling to ALPS and JMML, but they did not satisfy the diagnostic criteria for ALPS or JMML. KRAS mutation in hemopoietic precursors including T and myeloid linage is the common molecular feature, and we have recently proposed these cases to be defined under a new disease entity of RAS associated ALPS like disease (RALD). These patients were characterized by activated T cells resistant to IL-2 depletion but not to FAS-dependent apoptosis associated with the decreased expression of pro-apoptotic protein BIM.Since somatic RAS mutation has been identified in classical JMML and RALD, it is tempting to speculate that the developmental stage dependent RAS mutation of hemopoietic stem/precursors may determine the clinical features of JMML and RALD. Clinico-pathological characteristics of three RALD cases and three JMML cases cured without SCT will be presented and discussed. Disclosures:No relevant conflicts of interest to declare.

Epigenetic Causes of Apoptosis Resistance in Cutaneous T-Cell Lymphomas

In cutaneous T-cell lymphomas (CTCLs) defects in Fas-mediated apoptosis have been suggested to be involved in disease pathogenesis. Decreased or absent Fas expression has been reported in a significant proportion of CTCL patients, but the molecular mechanisms of such impaired Fas expression have hardly been investigated to date. In this issue, Jones et al. show that defective Fas expression is attributable to positional methylation of the Fas gene.

Transcriptional and post-translational regulation of Flip, an inhibitor of Fas-mediated apoptosis, in human gut inflammation

Objective:Defects in Fas-mediated apoptosis are supposed to contribute to the accumulation of T lymphocytes in the gut of patients with Crohn’s disease (CD). This phenomenon has been functionally linked with...

Histologic Features of Sinus Histiocytosis With Massive Lymphadenopathy in Patients With Autoimmune Lymphoproliferative Syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder associated with defects in Fas-mediated apoptosis, characterized most often by childhood onset of lymphadenopathy, splenomegaly, hypergammaglobulinemia, and autoimmune phenomena. Children with sinus histiocytosis with massive lymphadenopathy (SHML) have a somewhat similar clinical phenotype in which prominent adenopathy also is associated with hypergammaglobulinemia, and autoimmune phenomena are reported in 10-15% of cases. We observed histopathological features of SHML in the lymph nodes of some of our ALPS patients, further suggesting an association between these two disorders. We, thus, reviewed the lymph nodes from 44 patients ALPS type Ia, all of whom were confirmed to have germline mutations in the TNFRSF6 gene encoding Fas (CD95/Apo-1). Eighteen of 44 (41%) patients had a histiocytic proliferation resembling SHML. The affected patients included 15 males and 3 females ranging in age from 11 months to 30 years at the time of the LN biopsy. The lymph nodes contained S-100+ histiocytes with characteristic nuclear features of SHML, and showed evidence of emperipolesis in both hematoxylin and eosin (H and E) and immunostained sections. The extent of the histiocytic proliferation was variable, being confluent in 2 cases, multifocal in 13, and only evident as isolated SHML-type histiocytes in 3. In lymph nodes without confluent SHML changes, increased numbers of CD3+CD4-CD8+ (double negative) alphabeta T-cells, also negative for CD45RO, a feature of ALPS, could be identified in the paracortex. Furthermore, because SHML shares many clinical features with ALPS, we sought evidence of ALPS in sporadic SHML. We attempted to sequence TNFRSF6 DNA from archived tissue of 14 cases of Rosai-Dorfman disease. Full sequencing of the gene was successful in 4 of the cases; no mutations were identified. Nevertheless, our observations suggest that histologic features of SHML are part of the pathologic spectrum of ALPS type Ia. It remains to be determined if some cases of apparently sporadic SHML may be associated with heritable defects in Fas-mediated apoptosis.

Autoimmune haemolytic anaemia is associated with a defect in Fas-mediated apoptosis: a case report

Autoimmune haemolytic anaemia is associated with a defect in Fas-mediated apoptosis: a case report

Expression of active protein kinase B in T cells perturbs both T and B cell homeostasis and promotes inflammation.

The molecular mechanisms that contribute to autoimmunity remain poorly defined. While inflammation is considered to be one of the major checkpoints in autoimmune disease progression, very little is known about the initiating events that trigger inflammation. We have studied transgenic mice expressing the prosurvival molecule protein kinase B/Akt under control of a T cell-specific CD2 promoter. In this study, we demonstrate that aged mice develop lymphadenopathy and splenomegaly that result from an accumulation of CD4, CD8, and unexpectedly B cells. An increased proportion of T cells express activation markers, while T cell proliferative responses remain normal. B cells are hyperproliferative in response to anti-IgM F(ab')(2) and anti-CD40, and increased IgA and IgG2a were found in the sera. In addition, a profound multiorgan lymphocytic infiltration is observed, and T cells from these mice display a defect in Fas-mediated apoptosis, which may be the mechanism underlying this phenotype. Therefore, T cell expression of active protein kinase B can alter T cell homeostasis, indirectly influence B cell homeostasis, and promote inflammation in vivo.

Investigation of suspected deficient Fas‐mediated apoptosis in a father and son

Background A 2-year-old boy presented with symptoms consistent with a diagnosis of autoimmune lymphoproliferative syndrome (ALPS). His father had been splenectomized at age 12 with similar symptoms. ALPS is a rare hereditary syndrome that may result from a functional defect in Fas-mediated apoptosis. Methods Peripheral blood lymphocytes (PBL) and splenic lymphocytes from the patient and PBL from his father and a normal control were analyzed for surface Fas expression. They were then stimulated with an anti-Fas monoclonal antibody (DX2). Apoptosis was assayed by flow cytometry at 0, 20, 28, and 34 h. Results There was no significant difference in expression of Fas (CD95) in the PBL of the patient, his father, or the normal control, or the splenic lymphocytes. Compared with the normal control, the PBL of the patient and his father failed to progress to apoptosis. They also contained a markedly elevated proportion of CD3+CD4−CD8− “double-negative” cells. Conclusions PBL from both the patient and his father expressed CD95, but failed to proceed to apoptosis after stimulation, suggesting a functional defect. These results and the clinical presentation are consistent with published descriptions of ALPS. Cytometry 43:195–198, 2001. © 2001 Wiley-Liss, Inc.

A novel murine anti-human Fas mAb which mitigates lymphadenopathy without hepatotoxicity

Defects in Fas-mediated apoptosis are implicated in autoimmune diseases including rheumatoid arthritis (RA). Although induction of Fas-mediated apoptosis could have therapeutic effects on these diseases, it might cause deleterious effects in liver as Fas ligand or an agonistic anti-murine Fas antibody Jo2 causes severe hepatic injury in mice. We report here on the interesting characteristics of the newly obtained anti-Fas mAb, HFE7A, which cross-reacts with the Fas molecules of various species ranging from human to mouse and mitigates autoimmune symptoms without hepatotoxicity in mice. The administration of HFE7A to mice induced apoptosis in the thymocytes, although administration of HFE7A to mice or to marmosets did not induce any sign of hepatitis. The effect of HFE7A on liver is different from that of anti-murine Fas antibody Jo2, which causes acute and lethal hepatic injury to mice. Administration of HFE7A reduced lymphadenopathy and abnormal T cells in MRL-gld/gld mice. HFE7A induced apoptosis in synovial cells prepared from RA patients. Surprisingly, HFE7A protected mice from fulminant hepatitis induced by Jo2. Therefore, HFE7A is a potential therapeutic antibody not only for autoimmune diseases including RA but also for fulminant hepatitis.

Induction of Allograft Tolerance in the Absence of Fas-Mediated Apoptosis

Using certain immunosuppressive regimens, IL-2 knockout (KO) mice, in contrast to wild-type (wt) controls, are resistant to the induction of allograft tolerance. The mechanism by which IL-2 regulates allograft tolerance is uncertain. As IL-2 KO mice have a profound defect in Fas-mediated apoptosis, we hypothesized that Fas-mediated apoptosis of alloreactive T cells may be critical in the acquisition of allograft tolerance. To definitively study the role of Fas in the induction of transplantation tolerance, we used Fas mutant B6.MRL-lpr mice as allograft recipients of islet and vascularized cardiac transplants. Alloantigen-stimulated proliferation and apoptosis of Fas-deficient cells were also studied in vivo. Fas mutant B6.MRL-lpr (H-2b) mice rapidly rejected fully MHC-mismatched DBA/2 (H-2d) islet allografts and vascularized cardiac allografts with a tempo that is comparable to wt control mice. Both wt and B6.MRL-lpr mice transplanted with fully MHC-mismatched islet allografts or cardiac allografts can be readily tolerized by either rapamycin or combined costimulation blockade (CTLA-4Ig plus anti-CD40L mAb). Despite the profound defect of Fas-mediated apoptosis, Fas-deficient T cells can still undergo apoptotic cell death in vivo in response to alloantigen stimulation. Our study suggests that: 1) Fas is not necessarily essential for allograft tolerance, and 2) Fas-mediated apoptosis is not central to the IL-2-dependent mechanism governing the acquisition of allograft tolerance.

The Development of Autoimmune Inflammatory Arthropathy in Mice Transgenic for the Human T Cell Leukemia Virus Type-1env-pXRegion Is Not Dependent on H-2 Haplotypes and Modified by the Expression Levels of Fas Antigen

AbstractPreviously, we reported that human T cell leukemia virus type-1 env-pX region-introduced transgenic (pX-Tg) mice develop an inflammatory polyarthropathy. Although autoimmune pathogenesis was suggested, the detailed mechanisms remain to be elucidated. In this report, we examined effects of the MHC and fas genes on the development of the disease. When pX-Tg mice were backcrossed with different inbred strains, the incidence of arthritis differed among strains; 64% and 72% in BALB/cAn (H-2d), 25% and 46% in C3H/HeN (H-2k), and 0% and 2% in C57BL/6J (H-2b) background at 3 and 6 months of age, respectively. Rheumatoid factor levels in the serum correlated with the susceptibility to the disease, whereas IL-1β and MHC gene expression were similarly elevated in all of these strains, suggesting involvement of immune regulatory genes in this strain difference. However, introduction of the H-2d locus into C57BL/6J pX-Tg mice did not increase the incidence of arthritis, and substitution of the BALB/cAn H-2 locus with the H-2b did not decrease it. The results indicate that the H-2 locus is not the major determinant of the disease. Then, since previous study indicated a defect in Fas-mediated apoptosis of transgenic T cells, the effects of fas gene modification on the disease were examined. The incidence increased when these pX-Tg mice were crossed with lpr/lpr mice, while it decreased when crossed with fas-transgenic mice. These observations suggest that aberration of Fas-mediated apoptosis of peripheral lymphocytes, rather than negative selection in the thymus, is involved in the development of autoimmune arthropathy in pX-Tg mice.

Retarded thymic involution and massive germinal center formation in NF-ATp-deficient mice.

NF-ATp and NF-ATc are the most prominent nuclear NF-AT transcription factors in peripheral T lymphocytes. After T cell activation both factors bind to and control the promoters and enhancers of numerous lymphokine and receptor ligand genes. In order to define a specific role for NF-ATp in vivo we have inactivated the NF-ATp gene by gene targeting in mice. We show that NF-ATp deficiency leads to the accumulation of peripheral T cells with a "preactivated" phenotype, enhanced immune responses of T cells after secondary stimulation in vitro and severe defects in the proper termination of antigen responses, as shown by a reduced deletion of superantigen-reactive CD4+ T cells. These alterations in the function of the immune system are correlated with drastic changes in the morphology of lymphoid organs. Approximately 25 % of NF-ATp-deficient mice older than 6 months develop large germinal centers in the spleen and peripheral lymph nodes. In addition, they exhibit a pronounced retardation in the involution of the thymus. The thymus of these NF-ATp-deficient mice exhibits large cortical areas typical for newborn mice and a massive infiltration of IgM+/ IgD+ B lymphocytes. Contrary to the T lymphocytes from IL-2-deficient mice which develop a phenotype similar to the NF-ATp-/- mice, NF-ATp-/- T cells do not show obvious defects in Fas-mediated apoptosis. This might indicate defects in other types of programmed cell death which are controlled by the activity of NF-ATp.