Signal Transducer And Activator Of Transcription 1 Gain-of-function Mutation Research Articles

Deep Immune Profiling of Patients with STAT1 Gain-of-Function: Revealing New Mechanisms of Pathology

Background: Patients with heterozygous signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) pathogenic variants exhibit an array of clinical phenotypes including susceptibility to multiple infections, autoimmunity, and cancer predisposition. Previous studies to characterize the pathways involved and explore therapeutic interventions have been constrained by the technology to perform in-depth immunophenotyping. Mass cytometry has allowed us to perform extensive immune profiling of patients with inborn errors of immunity (IEI) to help gain a better understanding of disease pathology. STAT1 gain-of-function (GOF) mutations have demonstrated higher levels of phosphorylated STAT1 in response to type I and II interferons, but the response to other cytokines is less understood. Using advanced cytometry, we demonstrate a unique pattern of STAT1 phosphorylation in response to IL-6 stimulation in T-cell subsets and this differential pattern may play a role in T-cell differentiation and memory in STAT1 GOF patients.Cases: We report two patients with heterozygous STAT1 GOF mutations in the coiled-coil domain. For both patients, the clinical phenotype was largely consistent with other STAT1 GOF patients, one (P1, c.800C>T; p.ala267Val) presented with secondary HLH due to histoplasmosis, and the second (P2, c.866A>G; p.Tyr289Cys) presented with presumed vaccine strain varicella zoster virus (VZV) meningitis and subsequent history of recurrent herpes simplex virus (HSV) skin lesions. Patient peripheral blood mononuclear cells (PBMCs) were evaluated by fluorescence flow cytometry and cytometry by time of flight (CyTOF) as previously described (Roussel et al. J Leukoc Biol. 2017) to evaluate the impact of STAT1 GOF mutations on T-cell immunophenotype and cytokine signaling.Results: Utilizing cytometric data, we were able to identify similar patterns of T cell distribution on t-distributed stochastic neighbor embedding (t-SNE) plots for both patients with STAT1 GOF that were distinct compared to healthy controls (Fig 1a). In the T-cell compartment, both patients had decreased Th17 and Treg populations and an increased Th1/Th2 ratio compared to healthy donor (Fig 1b). In response to stimulation with IFNg or IL-6, there were also clear patterns with the two patients compared to healthy controls. Levels of p-STAT1 and p-STAT3 were assessed in STAT1 GOF and health donor PBMCs at several times points between 15 and 120 minutes, after stimulation with either IFNg or IL-6. Using fluorescence flow, we found that IL-6 stimulation led to greater than anticipated p-STAT1 response at all timepoints compared to a much more muted response to IFNg. The cell subsets highlighted in the t-SNE after IL-6 stimulation differ from the cell subsets that respond to IFNg stimulation in patients and healthy control (Fig 2a), suggesting that distinct cell populations are driving the response to IL-6. By evaluating these IL-6 responsive subsets in comparison with healthy control by CyTOF, we identified an exaggerated p-STAT1 response to IL-6 in the memory T-cell populations in P2 (Fig 2b).Conclusions: These two unique clinical presentations demonstrate that with similar mutations in the coiled-coil domain of STAT1, yet largely differing clinical presentations, the immune profiling patterns of the patients compared to healthy controls can drive further work on disease characterization and therapeutic interventions. This is relevant for this patient cohort, as treatment recommendations for STAT1 GOF are not well established. Long-term outcomes with JAK inhibition are lacking and transplant survival rates to date have been very poor compared to other immune diseases including familial HLH. With identification of IL-6 signaling playing a potential role in T-cell maturation, further studies will need to be performed to determine if IL-6 modulation could be used as a treatment modality. We conclude that performing deep immunophenotyping of patients with inborn errors of immunity (IEIs) such as STAT1 GOF can point to new disease mechanisms of human immunity and inflammation and lead to improved understanding of the role of cytokine and cellular signaling in normal hematopoiesis and cellular maturation. [Display omitted] DisclosuresRathmell: Sitryx: Consultancy, Current equity holder in publicly-traded company, Research Funding; Caribou: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Nirogy: Consultancy, Current holder of stock options in a privately-held company; Merck: Speakers Bureau; Pfizer: Speakers Bureau; Mitobridge: Consultancy; Incyte: Research Funding; Calithera: Research Funding; Tempest: Research Funding.

The pathogenic T387A missense mutation in the gene encoding signal transducer and activator of transcription 1 exhibits a differential gene expression profile

Heterozygous gain-of-function (GOF) mutations in the interferon-driven transcription factor STAT1 (signal transducer and activator of transcription 1) cause chronic mucocutaneous candidiasis (CMC). In this study, we characterized the molecular basis of a CMC-associated missense mutation by introducing a threonine-to-alanine exchange in the STAT1 DNA-binding domain at position 387. This substitution had previously been described in a CMC patient with suppurative eyelid infection and cutaneous abscesses, which are both unusual symptoms in this immunodeficiency. The STAT1-T387A mutant generated was compared to the wild-type protein and, in addition, to the missense mutant in the neighbouring position 386. Our results showed that the T387A mutant displayed distinct properties different from the wild-type molecule, namely elevated levels of tyrosine phosphorylation in conjunction with increased DNA-binding activity, hyperactive transcriptional regulation, and prolonged nuclear accumulation. The elevated tyrosine phosphorylation of the T387A mutant did not result in an increased mRNA production above the level of the wild-type molecule for all transcripts tested, indicating that the transcriptional activity of this mutant is largely gene-dependent. Nonetheless, these data demonstrate that the pathogenic T387A mutation associated with an atypical CMC symptomatology is biochemically similar to other well-characterized GOF mutants, while the H386A mutant was indistinguishable from the wild-type molecule. Our findings are in line with the assumption that the phenotype of this dominant STAT1 GOF mutation probably results from a disturbed shift in the equilibrium between the parallel and antiparallel dimer conformation, which is required for physiological gene activation.

Live Cell Imaging Demonstrates Multiple Routes Toward a STAT1 Gain-of-Function Phenotype.

Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in a primary immunodeficiency (PID) characterized typically by chronic mucocutaneous candidiasis (CMC), but a wider phenotypic range is reported and remains unexplained from a pathophysiological point-of-view. We hypothesized that different STAT1 GOF mutations may result in distinct molecular mechanisms, possibly explaining the variable phenotypes observed in patients. We selected STAT1 GOF mutants (R274W, R321S, T419R, and N574I) that are spread over the protein and studied their dynamic behavior in vitro in U3A and HeLa cell lines. All GOF mutants showed increased STAT1 phosphorylation compared to STAT1 WT. Real-time imaging demonstrated three underlying mechanisms for STAT1 GOF: (i) R274W showed a faster nuclear accumulation, (ii) both R321S and N574I showed a reduced nuclear mobility and slower dephosphorylation, whereas (iii) T419R was near-immobile in the nucleus, potentially due to enhanced binding to chromatin.

Jakinibs for the Treatment of Immune Dysregulation in Patients With Gain–of–Function Signal Transducer and Activator of Transcription 1 (STAT1) or STAT3 Mutations

To investigate the efficacy of jakinib therapy in patients with gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) or signal transducer and activator of transcription 3 (STAT3) mutations.The study included 17 total patients with either a STAT1 GOF (n = 11) or STAT3 GOF (n = 6) mutation who were treated with jakinibs (ruxolitinib or tofacitinib). Patients came from 11 international centers.This was a retrospective study to describe disease-related clinical manifestations and determine indication for treatment, dosage, length of treatment, treatment response, and treatment-associated complications. All subjects underwent thorough pre- and posttreatment clinical and laboratory evaluations and follow-up (follow-up period: 1–34 months).Fourteen patients (82%) responded favorably with clinical improvement to jakinib treatment. Observed clinical improvements included resolution of enteropathy leading to total parenteral nutrition independence, better lung function, improved autoimmune cytopenias, decreased arthritis symptoms, and remission of hemophagocytic lymphohistiocytosis. All patients with STAT1 GOF had resolved chronic mucocutaneous candidiasis with treatment. Most patients tolerated the jakinibs well with minimal or transient adverse effects. An increase in herpes zoster infections (none systemic and all cleared with antiviral agents) was observed in some patients. Four patients died despite treatment with a jakinib. The majority of these patients were critically ill with severe invasive infection or progressive lung disease. One patient died due to posttransplant complications.Jakinibs are both safe and effective in the treatment of patients with STAT1 or STAT3 GOF mutations with clinical manifestations of autoimmunity and/or immune dysregulation.Patients with STAT1 and STAT3 GOF mutations often present in early childhood with severe and/or refractory infections, lymphoproliferation, autoinflammation, and autoimmunity. Jakinibs allow for a targeted therapeutic approach to block the cytokine-induced JAK activation in STAT1 and STAT3 GOF diseases. This is the first study to evaluate the use and clinical impact of jakinibs in STAT1 and STAT3 GOF. The results are overwhelmingly encouraging, with the majority of patients having clinical improvement and reduction or resolution of autoimmune manifestations after treatment with a jakinib. Use of jakinibs late in disease progression or when critically ill was associated with mortality, suggesting early initiation may improve efficacy. This study has significant clinical impact in the treatment of STAT1 and STAT3 GOF and reinforces the notion that early genetic diagnosis of primary immunodeficiency diseases may lead to targeted therapeutic options and improved clinical outcomes.

Molecular and Phenotypic Characterization of Nine Patients with STAT1 GOF Mutations in China.

Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that mediates cellular responses to interferons (IFNs) and other cytokines and growth factors in diverse cell types. STAT1 gain-of-function (GOF) mutations result in an unexpectedly wide range of clinical features. It remains unclear why STAT1 GOF mutations result in such a broad spectrum of phenotypes. We analyzed the clinical, molecular, and phenotypic characteristics of nine Chinese patients with STAT1 GOF mutations. This study enrolled nine patients with STAT1 GOF mutations including five novel mutations. We discuss the molecular and phenotypic characterization such as unique Penicillium marneffei lymphadenitis. Patients with STAT1 GOF mutations had defects in both innate and adaptive immunity, including impaired T cell receptor (TCR) diversity; reduced numbers of naïve and effector memory CD4+ T cells, memory B cells, and NK cells; and defects in the production of IL-17A and IFN-γ. In addition, experiments with primary immune cells revealed that enhanced STAT1 phosphorylation resulted from not only lower rates of STAT1 dephosphorylation but also increased total STAT1 expression. Our report provides the first comprehensive overview of the molecular genetics, clinical heterogeneity, and underlying immunological abnormalities of patients with STAT1 GOF mutations in China. In further study, to find the relationship between different STAT1 GOF mutations and clinical phenotype as well as the mechanism of increased total STAT1 expression will be needed.

The activating p.Ser466Arg change in STAT1 causes a peculiar phenotype with features of interferonopathies

Signal Transducer and Activator of Transcription 1 (STAT1) is a DNA-binding signal transducer that regulates transcription of specific genes in response to IFNγ and IFNα/β stimulation. Loss-of-function mutations impairing STAT1 activity are known to confer susceptibility to intracellular bacterial and viral diseases. Conversely, the few known activating mutations of STAT1 allow predisposition to chronic mucocutaneous candidiasis disease, and occur in patients with combined immunodeficiency and defective Th1 and Th17 responses. Here, we report on a de novo gain-of-function (GoF) STAT1 mutation (c.1398C>G, p.Ser466Arg) identified by exome sequencing in an individual with brain calcification, arthritis, recurrent pericarditis, leukopenia, thrombocytopenia and low C3 levels, a phenotype resembling an interferonopathy. The Ser466Arg change affects a highly conserved residue located in the DNA binding domain of the protein and the amino acid substitution was documented to have an activating role both in vitro and in vivo. Altogether, clinical features and functional studies are compatible with hyperactivation of the Interferon pathways, highlighting a role of STAT1 GoF mutation in clinical phenotypes fitting interferonopathies.

Ruxolitinib partially reverses functional natural killer cell deficiency in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations

Natural killer (NK) cells are critical innate effector cells whose development is dependent on the Janus kinase-signal transducer and activator of transcription (STAT) pathway. NK cell deficiency can result in severe or refractory viral infections. Patients with STAT1 gain-of-function (GOF) mutations have increased viral susceptibility. We sought to investigate NK cell function in patients with STAT1 GOF mutations. NK cell phenotype and function were determined in 16 patients with STAT1 GOF mutations. NK cell lines expressing patients' mutations were generated with clustered regularly interspaced short palindromic repeats (CRISPR-Cas9)-mediated gene editing. NK cells from patients with STAT1 GOF mutations were treated invitro with ruxolitinib. Peripheral blood NK cells from patients with STAT1 GOF mutations had impaired terminal maturation. Specifically, patients with STAT1 GOF mutations have immature CD56dim NK cells with decreased expression of CD16, perforin, CD57, and impaired cytolytic function. STAT1 phosphorylation was increased, but STAT5 was aberrantly phosphorylated in response to IL-2 stimulation. Upstream inhibition of STAT1 signaling with the small-molecule Janus kinase 1/2 inhibitor ruxolitinib invitro and invivo restored perforin expression in CD56dim NK cells and partially restored NK cell cytotoxic function. Properly regulated STAT1 signaling is critical for NK cell maturation and function. Modulation of increased STAT1 phosphorylation with ruxolitinib is an important option for therapeutic intervention in patients with STAT1 GOF mutations.

A Novel Heterozygous Mutation in the STAT1 SH2 Domain Causes Chronic Mucocutaneous Candidiasis, Atypically Diverse Infections, Autoimmunity, and Impaired Cytokine Regulation.

Chronic mucocutaneous candidiasis (CMC) is a primary immunodeficiency characterized by persistent or recurrent skin and mucosal surface infections with Candida species. Different gene mutations leading to CMC have been identified. These include various heterozygous gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) that are not only associated with infections but also with autoimmune manifestations. Recently, two STAT1 GOF mutations involving the Src homology 2 (SH2) domain have been reported, while so far, over 50 mutations have been described mainly in the coiled coil and the DNA-binding domains. Here, we present two members of a Dutch family with a novel STAT1 mutation located in the SH2 domain. T lymphocytes of these patients revealed STAT1 hyperphosphorylation and higher expression of STAT1 target genes. The clinical picture of CMC in our patients could be explained by diminished production of interleukin (IL)-17 and IL-22, cytokines important in the protection against fungal infections.

Gain-of-function STAT1 mutations are associated with intracranial aneurysms

Chronic mucocutaneous candidiasis, characterized by persistent or recurrent fungal infections, represents the clinical hallmark in gain-of-function (GOF) signal transducer and activator of transcription 1 (STAT1) mutation carriers. Several cases of intracranial aneurysms have been reported in patients with GOF STAT1 mutation but the paucity of reported cases likely suggested this association still as serendipity. In order to endorse this association, we link the development of intracranial aneurysms with STAT1 GOF mutation by presenting the two different cases of a patient and her mother, and demonstrate upregulated phosphorylated STAT4 and IL-12 receptor β1 upon stimulation in patient's blood cells. We also detected increased transforming growth factor (TGF)-β type 2 receptor expression, particularly in CD14+ cells, and a slightly higher phosphorylation rate of SMAD3. In addition, the mother of the patient developed disseminated bacille Calmette-Guérin disease after vaccination, speculating that GOF STAT1 mutations may confer a predisposition to weakly virulent mycobacteria.

Ruxolitinib reverses dysregulated T helper cell responses and controls autoimmunity caused by a novel signal transducer and activator of transcription 1 (STAT1) gain-of-function mutation

Gain-of-function (GOF) mutations in the human signal transducer and activator of transcription 1 (STAT1) manifest in immunodeficiency and autoimmunity with impaired TH17cell differentiation and exaggerated responsiveness to type I and II interferons. Allogeneic bone marrow transplantation has been attempted in severely affected patients, but outcomes have been poor. We sought to define the effect of increased STAT1 activity on T helper cell polarization and to investigate the therapeutic potential of ruxolitinib in treating autoimmunity secondary to STAT1 GOF mutations. We used invitro polarization assays, as well as phenotypic and functional analysis of STAT1-mutated patient cells. We report a child with a novel mutation in the linker domain of STAT1 who had life-threatening autoimmune cytopenias and chronic mucocutaneous candidiasis. Naive lymphocytes from the affected patient displayed increased TH1 and follicular T helper cell and suppressed TH17cell responses. The mutation augmented cytokine-induced STAT1 phosphorylation without affecting dephosphorylation kinetics. Treatment with the Janus kinase 1/2 inhibitor ruxolitinib reduced hyperresponsiveness to type I and II interferons, normalized TH1 and follicular T helper cell responses, improved TH17 differentiation, cured mucocutaneous candidiasis, and maintained remission of immune-mediated cytopenias. Autoimmunity and infection caused by STAT1 GOF mutations are the result of dysregulated T helper cell responses. Janus kinase inhibitor therapy could represent an effective targeted treatment for long-term disease control in severely affected patients for whom hematopoietic stem cell transplantation is not available.

Impaired natural killer cell functions in patients with signal transducer and activator of transcription 1 (STAT1) gain-of-function mutations

Gain-of-function (GOF) mutations affecting the coiled-coil domain or the DNA-binding domain of signal transducer and activator of transcription 1 (STAT1) cause chronic mucocutaneous candidiasis disease. This condition is characterized by fungal and bacterial infections caused by impaired generation of TH17cells; meanwhile, some patients with chronic mucocutaneous candidiasis disease might also have viral or intracellular pathogen infections. We sought to investigate the effect of STAT1 GOF mutations on the functioning of natural killer (NK) cells. Because STAT1 is involved in the signaling response to several cytokines, we studied NK cell functional activities and STAT1 signaling in 8 patients with STAT1 GOF mutations. Functional analysis of NK cells shows a significant impairment of cytolytic and degranulation activities in patients with STAT1 GOF mutations. Moreover, NK cells from these patients display lower production of IFN-γ in response to IL-15 and reduced proliferation after stimulation with IL-2 or IL-15, suggesting that STAT5 signaling is affected. In addition, signaling studies demonstrate that the increased phosphorylation of STAT1 in response to IFN-α is associated with detectable activation of STAT1 and increased STAT1 binding to the interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) promoter in response to IL-15, whereas STAT5 phosphorylation and DNA binding to IL-2 receptor α (IL2RA) are reduced or not affected in response to the same cytokine. These observations suggest that persistent activation of STAT1 might affect NK cell proliferation and functional activities.

Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation

Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3+-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms.

Successful Therapy of a Patient with a Novel STAT1 Gain of Function Mutation and Life-Threatening Cytopenias with Janus Kinase Inhibitor Ruxolitinib

Monoallelic gain of function (GOF) mutations in the human Signal Transducer and Activator of Transcription 1 (STAT1) manifest in immunodeficiency and autoimmunity. The immunodeficiency is caused by impaired IL-17 immunity and typically presents with chronic mucocutaneous candidiasis, variably associated with other opportunistic infections. The autoimmune manifestations due to exaggerated responsiveness to interferon are more diverse and include SLE, thyroiditis, hepatitis, and alopecia areata. The phenotypic spectrum of STAT1 GOF mutations is extremely wide, ranging from intermittent localized disease to chronic systemic illness and fatality. Allogeneic bone marrow transplantation has been attempted in severely affected patients but outcomes have been poor, leaving a void for alternate long-term strategies to control the disease and maintain remission. Recently, we diagnosed a patient suffering from chronic mucocutaneous candidiasis and life-threatening autoimmune cytopenias with a novel monoallelic mutation in the linker domain of STAT1 (c.1633G>A; p.E545K). The mutation caused a profound increase in STAT1 phosphorylation in response to type 1 and 2 interferon without affecting dephosphorylation kinetics, which is mechanistically distinct from all reported STAT1 GOF mutations to date. The potential of naïve patient CD4+ T cells to become IFN- γ or IL-17 producing cells was investigated under TH0 (anti-CD3/28), TH1 (anti-CD3/28, IL-12) and TH17 (anti-CD3/28, IL6, IL-23, TGF-β1) conditions and revealed that patient CD4+ T cells are biased to produce IFN-γ irrespective of polarizing conditions, and resistant to TH17 induction upon stimulation with IL6, IL-23 and TGF-β1. In addition, the patient's proportion of T follicular helper cells (TFH) relative to regulatory T cells (Treg) was increased. Treatment with the Janus Kinase (JAK) 1/2 inhibitor Ruxolitinib reduced the hyperresponsiveness to interferon, normalized the TH1 response, enabled naïve T cells to differentiate into TH17 cells and decreased the TFH to Treg ratio. Under therapeutic dose Ruxolitinib the patient maintained clinical remission with respect to both autoimmunity and mucocutaneous candidiasis.Conclusion: Clinical vigilance for an underlying immune dysregulation syndrome due to abnormal JAK-STAT signaling is critical when evaluating patients with autoimmunity combined with opportunistic infections as JAK-inhibitors represents an effective targeted therapy for long-term disease control even in severely affected patients for whom hematopoietic stem cell transplantation is not available. DisclosuresOff Label Use: Ruxolitinib use for STAT1 GOF mutation.