EBV Negative Lymphoma and Autoimmune Lymphoproliferative Syndrome Like Phenotype Extend the Clinical Spectrum of Primary Immunodeficiency Caused by STK4 Deficiency

Serine/threonine kinase 4 (STK4) deficiency is an autosomal recessive genetic condition that leads to primary immunodeficiency (PID) typically characterized by lymphopenia, recurrent infections and Epstein Barr Virus (EBV) induced lymphoproliferation and -lymphoma. State-of-the-art treatment regimens consist of prevention or treatment of infections, immunoglobulin substitution (IVIG) and restoration of the immune system by hematopoietic stem cell transplantation. Here, we report on two patients from two consanguineous families of Turkish (patient P1) and Moroccan (patient P2) decent, with PID due to homozygous STK4 mutations. P1 harbored a previously reported frameshift (c.1103 delT, p.M368RfsX2) and P2 a novel splice donor site mutation (P2; c.525+2 T>G). Both patients presented in childhood with recurrent infections, CD4 lymphopenia and dysregulated immunoglobulin levels. Patient P1 developed a highly malignant B cell lymphoma at the age of 10 years and a second, independent Hodgkin lymphoma 5 years later. To our knowledge she is the first STK4 deficient case reported who developed lymphoma in the absence of detectable EBV or other common viruses. Lymphoma development may be due to the lacking tumor suppressive function of STK4 or the perturbed immune surveillance due to the lack of CD4+ T cells. Our data should raise physicians' awareness of [1] lymphoma proneness of STK4 deficient patients even in the absence of EBV infection and [2] possibly underlying STK4 deficiency in pediatric patients with a history of recurrent infections, CD4 lymphopenia and lymphoma and unknown genetic make-up. Patient P2 experienced recurrent otitis in childhood, but when she presented at the age of 14, she showed clinical and immunological characteristics similar to patients suffering from Autoimmune Lymphoproliferative Syndrome (ALPS): elevated DNT cell number, non-malignant lymphadenopathy and hepatosplenomegaly, hematolytic anemia, hypergammaglobulinemia. Also patient P1 presented with ALPS-like features (lymphadenopathy, elevated DNT cell number and increased Vitamin B12 levels) and both were initially clinically diagnosed as ALPS-like. Closer examination of P2, however, revealed active EBV infection and genetic testing identified a novel STK4 mutation. None of the patients harbored typically ALPS-associated mutations of the Fas receptor mediated apoptotic pathway and Fas-mediated apoptosis was not affected. The presented case reports extend the clinical spectrum of STK4 deficiency

Die Rolle von Zink in der Signaltransduktion von T-Zellen

Zinc is an essential trace element with a variety of cellular functions in all organ systems. Disturbance of the cellular zinc availability by zinc deficiency leads to multiple disorders. Predominantly the cell-mediated immune response is influenced by zinc deprivation, and here in particular T-cell mediated functions. For example, the Th1/Th2 balance is disturbed leading to a reduced production of Th1 cytokines such as IFN-gamma and IL-2. Zinc supplementation in vivo causes reconstitution of Th1-mediated responses. To achieve a broad picture about the role of zinc in T-cell signal transduction, phosphorylation of several effector kinases in primary human T-cells and in the human T-cell line Jurkat was investigated. CD3-mediated activation and stimulation by zinc and the ionophore pyrithione led to differential activation of kinase Lck, MAPK p38, JNK1/2 and ERK1/2. Experiments showed that CD3-stimulated T-cells release intracellular zinc which has no effect on signal transduction itself. However, if cells were stimulated with zinc and pyrithione, the kinases Lck and p38 were activated, while CD3-induced ERK1/2 phosphorylation was reduced. To elucidate this differential zinc effect, several known zinc-mediated signalling mechanisms were investigated for their validity in T-cells. Lck is influenced by zinc in its activity at different levels. Experiments with Lck-negative cells showed that activation after stimulation with zinc and pyrithione additionally depends on protein tyrosine phosphatase CD45. Furthermore the observed zinc effect is independent from CD3 signalling and represents an intracellular event. MAPK phosphatase inhibition by zinc could be excluded as one possible explanation for p38 activation. Zinc induced phosphorylation of p38 by alternative activation mediated by Lck and ZAP70 could be also eliminated by inhibitor studies and the use of Lck and ZAP70 deficient cell lines. Further on, the involvement of the PKA signalling pathway was no explanation for the observed differential zinc effect, confirmed by the use of different inhibitors and activators. After verification of several signalling pathways, the molecular pathway of the observed zinc effect in T-cells seems to differ from established pathways in other cells. Although the mechanism could not be elucidated, it could be demonstrated that there is a connection between the induction of Th1 answers and the activation of p38/CREB cascade. For the first time the impact of zinc on Th1 cells could be linked to p38 activation. Further work on the molecular mechanism could have an influence on the treatment of T-cell mediated diseases and could open up new therapy perspectives

2009

ABSTRACT © F e r r a t a S t o r t i F o u n d a t i o n For instance, forced expression of the Fas regulating miR146a caused an immune disorder similar to ALPS in transgenic mice. Methods Study cohorts and DNA isolation Twenty-six ALPS patients, relatives and healthy controls were enrolled in the study. Written informed consent was obtained from all participants. Experiments were approved by the Ethical Review Boards of Hadassah, the Israeli Ministry of Health and the local Ethics committee of the University of Düsseldorf. Mononuclear cells were derived from peripheral blood by Ficoll (Biochrom, Berlin, Germany) density centrifugation. DNT cells were magnetically selected employing the double-negative T-cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). Genomic DNA was isolated from whole blood or DNT cells using the DNA blood kit (Qiagen, Hilden, Germany). Whole-exome sequencing and data analysis After exclusion of mutations in known ALPS-associated genes by targeted Sanger sequencing (Online Supplementary Methods, Online Supplementary Table S1) whole-exome sequencing was carried out as described elsewhere. Sequencing data were aligned against the human reference genome hg19 (GRCh37, statistics provided in Online 39 Single nucleotide variations, small insertions and deletions were annotated using Variant Effect Predictor 40 (based on Ensemble database v70). Variations were imported into a proprietary MySQL database driven workbench (termed Single Nucleotide Polymorphism Database, SNuPy). STRING 9.1 41 was used to identify high confidence (≥0.900) Fas pathway interaction partners (Online Primary T-cell culture Primary T cells were cultured in RPMI1640 (Life Technologies, Darmstadt, Germany) and Panserin 401 (PAN-Biotech, Aidenbach, Germany) mixed 1:1, supplemented with 10% fetal calf serum, 100 mg gentamycin (Life Technologies) and 30 U/mL IL2 (Miltenyi). They were activated with 7 mg/mL phytohemagglutinin (Life Technologies) for 4 days. Immunophenotyping and enzyme-linked immunosorbent assays DNT cells in peripheral blood were measured using a FACSCalibur equipped with CellQuestPro software (Becton Dickinson, BD, Heidelberg, Germany) employing anti-CD3, anti-TCRαβ (both from BD), anti-CD4 and anti-CD8 (both from Miltenyi) antibodies. Immunophenotyping was performed using: anti-B220, anti-HLA-DR, anti-CD27, anti-CD19, anti-CD25 (all from BD) and anti-CD45R (Beckman Coulter, Krefeld, Germany). Expression of Fas, FasL and IL12RB1 was measured using anti-CD95 (BD), anti-CD178/FasL (Miltenyi) and anti-CD212 antibodies (BD). FasL, IL10 and IFNγ levels in plasma and cell culture supernatants of activated T cells were measured by enzymelinked immunosorbent assays (R&D-Systems, Wiesbaden, Germany) employing an Infinite M200 microplate reader equipped with Magellan software (Tecan, Maennedorf, Switzerland). T cells were stimulated with IL12, IL23 and IL2/IL27 (Miltenyi) as indicated. Measurement of apoptosis Activated primary T cells were stimulated with recombinant SuperFasL (100 ng/mL, Enzo Life Sciences, Loerrach, Germany), 1 mM staurosporine (LC Laboratories, Woburn, MA, USA), IL12 Results Interleukin-12 induces upregulation of FasL and FasL-dependent apoptosis in healthy T cells, whereas FasL-deficient T cells from patients with autoimmune lymphoproliferative syndrome lack this response Of 26 analyzed ALPS cases, 20 had no known ALPSassociated mutation. Four patients harbored heterozygous germline mutations in the Fas receptor gene. Two siblings had a homozygous truncating FASLG mutation (g.172628545insT, p.P69Afs*75) that led to loss of FasL surface expression ( 1190 haematologica | 2015; 100(9) © F e r r a t a S t o r t i F o u n d a t i o n IL12 signaling may be protected against this physiological apoptosis trigger. Identification of a homozygous c.698G>A, p.R212* mutation in the IL12RB1 gene To analyze whether mutations in the IL12 pathways or related genes may cause a phenotype similar to ALPS, we sequenced the exomes of the remaining 20 ALPS-U patients who had classical ALPS symptoms without a known genetic cause, and their relatives. Sanger sequencing of all exons and exon/intron boundaries of FAS, FASLG and CASP10 was used to exclude classical disease-causing germline or somatic mutations. By whole-exome sequencing we identified an IL12RB1 mutation in one of the ALPS families. A KEGG-based protein interaction analysis interface of our in-house developed proprietary MySQL database driven workbench (termed Single Nucleotide Polymorphism Database, SNuPy) gave this candidate disease-causing mutation highest priority because of its predicted interaction with FasL ( As the mutated sequence seemed to encode a truncated protein that lacks the transmembrane domain necessary for membrane anchorage, we carried out FACS analyses of the patient's lymphocytes to test for IL12RB1 expression on the cell surface. To this end, primary lymphocytes from the patient and healthy individuals were stimulated for 4 days with phytohemagglutinin/IL2. Whereas lympho- + cells specifically induced in IL12-treated compared to untreated cells is shown. (C) Cultivated patient's T cells are resistant to apoptosis induced by stimulation with IL12. T cells were activated as in (A). Apoptosis was induced by incubation with 100 ng/mL IL12 for 2 days and measured employing flow cytometric detection of annexin V-FITC and propidium iodide. The difference in the apoptosis rate compared to that of an untreated control is depicted. Specific apoptosis ranged from 4-12% in the healthy controls between comparable experiments and was absent in the patient's cells. (D) T cells were activated by phytohemagglutinin /IL2 treatment as described in (A). Fas receptor-mediated apoptosis was triggered by application of 100 ng/mL optimized and preoligomerized recombinant FasL for 16 h and apoptosis was measured as in (C). In (B-D) mean values and standard deviations of representative experiments repeated at least three times and carried out in duplicate are shown. Similar results were obtained using samples from two individuals with homozygous FASLG (g.172628545insT, p.P69Afs*75) mutation and five wild-type controls. A B C D © F e r r a t a S t o r t i F o u n d a t i o n cytes from healthy individuals upregulated IL12RB1 surface expression upon activation, IL12RB1 remained absent in the patient's lymphocytes ( The homozygous c.698G>A, p.R212* mutation in the IL12RB1 gene was associated with an autoimmune lymphoploliferative syndrome-like phenotype The male patient harboring the homozygous IL12RB1 c.698G>A, p.R212* mutation originated from a consanguineous family of Palestinian descent. He was referred in 1996 at the age of 4 years because of the suspicion of lymphoma. In the follow up of this patient for more than 16 years he presented with classical clinical features for the diagnosis of ALPS (Table 1, The heterozygous parents and siblings appeared clinically normal, although immune phenotyping revealed increased DNT cell counts in two of them In vitro the apoptotic response of the patient's lymphocytes to treatment with recombinant FasL and a classical apoptosis-inducing agent (staurosporine) was similar to that of age-and gender-matched healthy blood donors [FasL-induced apoptosis: 58% ± 5% (patient), 57% ± 4% (healthy control); staurosporine-induced apoptosis: 86% ± 6% (patient), 86% ± 7% (healthy control)]. This demon- S. Nabhani et al. 1192 haematologica | 2015; 100(9) To analyze whether the defect in IL12RB1 affects FasL signaling, we first tested protein expression of FasL. Activation and expansion of T cells usually leads to upregulation of Fas signaling pathway components. However, in the absence of IL12RB1 expression the patient's T lymphocytes showed a significantly lower expression of both membrane-bound and soluble FasL protein compared to that of healthy controls ( The IL12RB1 c.698G>A, p.R212* mutation abrogates responsiveness of T cells to interleukin-12 To test whether lack of IL12RB1 expression affects IL12 signaling we analyzed phosphorylation of STAT4, a crucial downstream component of the pathway STAT4 activation eventually leads to transcription and production of IFNg. To test whether this is deficient in the patient we measured the induction of IFNG transcription by quantitative real-time polymerase chain reaction after 24 and 48 h of treatment with IL12 ( Finally, we tested the responsiveness of the patient's primary lymphocytes to apoptosis mediated by prolonged IL12 treatment ( Discussion Although ALPS is frequently caused by mutations in known genes, such as FAS, FASLG or CASP10, in 20-30% of cases the defect is still unknown. It is highly likely that defects in or overexpression of regulators of these genes such as miR-146a It might be reasoned that the observed autoimmunity and lymphoproliferation are likely a side-effect of recurrent infections, because IL12RB1 mutations have previously been associated with a predisposition to mycobacterial infections. However, the patient experienced only one non-recurrent episode of infection with Salmonella in 16 years of follow-up arguing against a secondary effect. Consistently, it was recently demonstrated, employing an IL12RB2 knockout mice model, that lack of IL12 signaling predisposes to spontaneous lymphoproliferation, autoimmunity and B-cell lymphoma. 45 A similar phenotype is described for targeted IL12RB1 knockout mice. Heterozygous human carriers appear clinically normal with normal IL12/IL23 signaling and IFNg production. Our study demonstrates for the first time that loss of IL12RB1 expression in a patient leads to reduced upregu- S. Nabhani et al. 1194 haematologica | 2015; 100(9) © F e r r a t a S t o r t i F o u n d a t i o n Deregulation of FasL as a cause of ALPS-like disease haematologica | 2015; 100(9) 1195 Densitometric measurement of signals derived by the western blot in the left panel carried out on a LAS-3000 equipped with LAS-3000 Image Reader software (Fujifilm, Düsseldorf, Germany). The difference of relative arbitrary units of pSTAT4 expression related to STAT4 expression is shown. The β-actin control was used to compensate differences due to loading. A representative result of two independent experiments is shown. A B C D E © F e r r a t a S t o r t i F o u n d a t i o n lation of FasL and loss of the apoptotic response of T cells to prolonged treatment with IL12. In addition, the general level of FasL expression in activated T cells and the level of secreted FasL in the plasma or cell culture supernatant were much lower in the patient than in controls. Lower levels of FasL expression in the patient are probably attributable to a lack of IFNg, because transcription of the FASLG promoter is positively regulated by the interferonregulatory factors IRF-1 and IRF-2. 48 IL12 is known as a factor that can stimulate growth and function of T cells and the differentiation of naive T cells into Th1 cells. However, prolonged stimulation of T cells with IL12 leads to apoptosis and stimulation of T cells with IL12 during activation enhances the tendency to undergo Fas-mediated activation induced cell death due to upregulation of FasL and downregulation of the inhibitor FLIPs. 27 Therefore, similar to defective Fas/FasL signaling, absence of IL12 signaling could lead to decreased death of T cells and accumulation of autoreactive T cells. In addition, it has been shown that low levels of IL12 drive the differentiation of activated T cells to long-lived selfrenewing memory CD8 + T cells rather than to short-lived effector cells when acute infections resolve. 49 This is dependent on IL12-controlled expression of T-bet and reflected in the phenotype of T-bet knockout mice. Acknowledgment

Deregulation of Fas ligand expression as a novel cause of autoimmune lymphoproliferative syndrome-like disease

Autoimmune lymphoproliferative syndrome is frequently caused by mutations in genes involved in the Fas death receptor pathway, but for 20–30% of patients the genetic defect is unknown. We observed that treatment of healthy T cells with interleukin-12 induces upregulation of Fas ligand and Fas ligand-dependent apoptosis. Consistently, interleukin-12 could not induce apoptosis in Fas ligand-deficient T cells from patients with autoimmune lymphoproliferative syndrome. We hypothesized that defects in the interleukin-12 signaling pathway may cause a similar phenotype as that caused by mutations of the Fas ligand gene. To test this, we analyzed 20 patients with autoimmune lymphoproliferative syndrome of unknown cause by whole-exome sequencing. We identified a homozygous nonsense mutation (c.698G>A, p.R212*) in the interleukin-12/interleukin-23 receptor-component IL12RB1 in one of these patients. The mutation led to IL12RB1 protein truncation and loss of cell surface expression. Interleukin-12 and -23 signaling was completely abrogated as demonstrated by deficient STAT4 phosphorylation and interferon γ production. Interleukin-12-mediated expression of membrane-bound and soluble Fas ligand was lacking and basal expression was much lower than in healthy controls. The patient presented with the classical symptoms of autoimmune lymphoproliferative syndrome: chronic non-malignant, non-infectious lymphadenopathy, splenomegaly, hepatomegaly, elevated numbers of double-negative T cells, autoimmune cytopenias, and increased levels of vitamin B12 and interleukin-10. Sanger sequencing and whole-exome sequencing excluded the presence of germline or somatic mutations in genes known to be associated with the autoimmune lymphoproliferative syndrome. Our data suggest that deficient regulation of Fas ligand expression by regulators such as the interleukin-12 signaling pathway may be an alternative cause of autoimmune lymphoproliferative syndrome-like disease

Human RAD52: a novel player in DNA repair in cancer and immunodeficiency.

No abstract available