Human STAT1 gain of function with chronic mucocutaneous candidiasis: A comprehensive review for strengthening the connection between bedside observations and laboratory research.

Germline human heterozygous STAT1 gain-of-function (GOF) variants were first discovered a common cause of chronic mucocutaneous candidiasis (CMC) in 2011. Since then, numerous STAT1 GOF variants have been identified. A variety of clinical phenotypes, including fungal, viral, and bacterial infections, endocrine disorders, autoimmunity, malignancy, and aneurysms, have recently been revealed for STAT1 GOF variants, which has led to the expansion of the clinical spectrum associated with STAT1 GOF. Among this broad range of complications, it has been determined that invasive infections, aneurysms, and malignancies are poor prognostic factors for STAT1 GOF. The effectiveness of JAK inhibitors as a therapeutic option has been established, although further investigation of their long-term utility and side effects is needed. In contrast to the advancements in treatment options, the precise molecular mechanism underlying STAT1 GOF remains undetermined. Two primary hypotheses for this mechanism involve impaired STAT1 dephosphorylation and increased STAT1 protein levels, both of which are still controversial. A precise understanding of the molecular mechanism is essential for not only advancing diagnostics but also developing therapeutic interventions. Here, we provide a comprehensive review of STAT1 GOF with the aim of establishing a stronger connection between bedside observations and laboratory research.

Immunophenotyping and Therapeutic Insights from Chronic Mucocutaneous Candidiasis Cases with STAT1 Gain-of-Function Mutations.

PURPOSE: Heterozygous STAT1 Gain-of-Function (GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC) among Inborn Errors of Immunity. Clinically, these mutations manifest as a broad spectrum of immune dysregulation, including autoimmune diseases, vascular disorders, and malignancies. The pathogenic mechanisms of immune dysregulation and its impact on immune cells are not yet fully understood. In treatment, JAK inhibitors have shown therapeutic effectiveness in some patients. METHODS: We analyzed clinical presentations, cellular phenotypes, and functional impacts in five Taiwanese patients with STAT1 GOF. RESULTS: We identified two novel GOF mutations in 5 patients from 2 Taiwanese families, presenting with symptoms of CMC, late-onset rosacea, and autoimmunity. The enhanced phosphorylation and delayed dephosphorylation were displayed by the patients' cells. There are alterations in both innate and adaptive immune cells, including expansion of CD38+HLADR +CD8+ T cells, a skewed activated Tfh cells toward Th1, reduction of memory, marginal zone and anergic B cells, all main functional dendritic cell lineages, and a reduction in classical monocyte. Baricitinib showed therapeutic effectiveness without side effects. CONCLUSION: Our study provides the first comprehensive clinical and molecular characteristics in STAT1 GOF patient in Taiwan and highlights the dysregulated T and B cells subsets which may hinge the autoimmunity in STAT1 GOF patients. It also demonstrated the therapeutic safety and efficacy of baricitinib in pediatric patient. Further research is needed to delineate how the aberrant STAT1 signaling lead to the changes in cellular populations as well as to better link to the clinical manifestations of the disease.

Stepwise Reversal of Immune Dysregulation Due to STAT1 Gain-of-Function Mutation Following Ruxolitinib Bridge Therapy and Transplantation.

PURPOSE: Patients with heterozygous gain-of-function (GOF) mutations in STAT1 frequently exhibit chronic mucocutaneous candidiasis (CMC), immunodeficiency and autoimmune manifestations. Several treatment options including targeted therapies and hematopoietic stem cell transplantation (HSCT) are available for STAT1 GOF patients but modalities and outcomes are not well established. Herein, we aimed to unravel the effect of ruxolitinib as a bridge therapy in a patient with sporadic STAT1 T385M mutation to manage infections and other disease manifestations. METHODS: Peripheral blood mononuclear cells were isolated from the patient prior to, during ruxolitinib treatment and 6 months after HSCT. IFN-ß-induced STAT1 phosphorylation/dephosphorylation levels and PMA/ionomycin-stimulated intracellular IL-17A/IFN-γ production in CD4+ T cells were evaluated. Differentially expressed genes between healthy controls and the patient prior to, during ruxolitinib treatment and post-transplantation were investigated using Nanostring nCounter Profiling Panel. RESULTS: Ruxolitinib provided favorable responses by controlling candidiasis and autoimmune hemolytic anemia in the patient. Dysregulation in STAT1 phosphorylation kinetics improved with ruxolitinib treatment and was completely normalized after transplantation. TH17 deficiency persisted after ruxolitinib treatment, but normalized following HSCT. Consistent with the impairment in JAK/STAT signaling, multiple immune related pathways were found to be dysregulated in the patient. At baseline, genes related to type I IFN-related pathways, antigen processing, T-cell and B-cell functions were upregulated, while NK-cell function and cytotoxicity related genes were downregulated. Dysregulated gene expression was partially improved with ruxolitinib treatment and normalized after transplantation. CONCLUSION: Our findings suggest that improved disease management and immune dysregulatory profile can be achieved with ruxolitinib treatment before transplantation and this would be beneficial to reduce the risk of adverse outcome of HSCT.

Functional analysis of two STAT1 gain-of-function mutations in two Iranian families with autosomal dominant chronic mucocutaneous candidiasis.

Candidiasis is characterized by susceptibility to recurrent or persistent infections caused by Candida spp., typically Candida albicans, of cutaneous and mucosal surfaces. In this report, function and frequency of Th17 cells as well as genetics of patients susceptible to mucocutaneous candidiasis were studied. For patients, T-cell proliferation tests in response to Candida antigen, Th17 cell proportions, and STAT1 phosphorylation were evaluated through flow cytometry. Expression of IL17A, IL17F and IL22 genes were measured by real-time quantitative PCR. At the same time, whole exome sequencing was performed for all patients. We identified two heterozygous substitutions, one: c.821G > A (p. R274Q) was found in a multiplex family with three individuals affected, the second one: c.812A > C (p. Q271P) was found in a sporadic case. Both mutations are located in the coiled-coil domain (CCD) of STAT1. The frequency of Th17 cells, IL17A, IL17F, and IL22 gene expression in patients' peripheral blood mononuclear cells (PBMCs), and T-cell proliferation to Candida antigens were significantly reduced in the patients as compared to healthy controls. An increased STAT1 phosphorylation was observed in patients' PBMCs upon interferon (IFN)-γ stimulation as compared to healthy controls. We report two different but neighboring heterozygous mutations, located in exon 10 of the STAT1 gene, in four Iranian patients with CMC, one of whom also had hypothyroidism. These mutations were associated with impaired T cell proliferation to Candida antigen, low Th17 cell proportions, and increased STAT1 phosphorylation upon IFN-γ. We suggest that interfering with STAT1 phosphorylation might be a promising way for potential therapeutic measurements for such patients.

Interferon signature in patients with STAT1 gain-of-function mutation is epigenetically determined.

STAT1 gain-of-function (GOF) variants lead to defective Th17 cell development and chronic mucocutaneous candidiasis (CMC), but frequently also to autoimmunity. Stimulation of cells with STAT1 inducing cytokines like interferons (IFN) result in hyperphosphorylation and delayed dephosphorylation of GOF STAT1. However, the mechanism how the delayed dephosphorylation exactly causes the increased expression of STAT1-dependent genes, and how the intracellular signal transduction from cytokine receptors is affected, remains unknown. In this study we show that the circulating levels of IFN-α were not persistently elevated in STAT1 GOF patients. Nevertheless, the expression of interferon signature genes was evident even in the patient with low or undetectable serum IFN-α levels. Chromatin immunoprecipitation (ChIP) experiments revealed that the active chromatin mark trimethylation of lysine 4 of histone 3 (H3K4me3), was significantly enriched in areas associated with interferon-stimulated genes in STAT1 GOF cells in comparison to cells from healthy donors. This suggests that the chromatin binding of GOF STAT1 variant promotes epigenetic changes compatible with higher gene expression and elevated reactivity to type I interferons, and possibly predisposes for interferon-related autoimmunity. The results also suggest that epigenetic rewiring may be responsible for treatment failure of Janus kinase 1/2 (JAK1/2) inhibitors in certain patients.

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.

BACKGROUND: Gain-of-function (GOF) mutations in the human signal transducer and activator of transcription 1 (STAT1) manifest in immunodeficiency and autoimmunity with impaired TH17 cell 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. OBJECTIVE: 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. METHODS: We used in vitro polarization assays, as well as phenotypic and functional analysis of STAT1-mutated patient cells. RESULTS: 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 TH17 cell 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. CONCLUSIONS: 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.

Progressive Multifocal Leukoencephalopathy in Primary Immune Deficiencies: Stat1 Gain of Function and Review of the Literature.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is a rare, severe, otherwise fatal viral infection of the white matter of the brain caused by the polyomavirus JC virus, which typically occurs only in immunocompromised patients. One patient with dominant gain-of-function (GOF) mutation in signal transducer and activator of transcription 1 (STAT1) with chronic mucocutaneous candidiasis and PML was reported previously. We aim to identify the molecular defect in 3 patients with PML and to review the literature on PML in primary immune defects (PIDs). METHODS: STAT1 was sequenced in 3 patients with PML. U3C cell lines were transfected with STAT1 and assays to search for STAT1 phosphorylation, transcriptional response, and target gene expression were performed. RESULTS: We identified 3 new unrelated cases of PML in patients with GOF STAT1 mutations, including the novel STAT1 mutation, L400Q. These STAT1 mutations caused delayed STAT1 dephosphorylation and enhanced interferon-gamma-driven responses. In our review of the literature regarding PML in primary immune deficiencies we found 26 cases, only 54% of which were molecularly characterized, the remainder being syndromically diagnosed only. CONCLUSIONS: The occurrence of PML in 4 cases of STAT1 GOF suggests that STAT1 plays a critical role in the control of JC virus in the central nervous system.

Gain-of-function STAT1 mutations impair STAT3 activity in patients with chronic mucocutaneous candidiasis (CMC).

Signal transducer and activator of transcription 3 (STAT3) triggered production of Th-17 cytokines mediates protective immunity against fungi. Mutations affecting the STAT3/interleukin 17 (IL-17) pathway cause selective susceptibility to fungal (Candida) infections, a hallmark of chronic mucocutaneous candidiasis (CMC). In patients with autosomal dominant CMC, we and others previously reported defective Th17 responses and underlying gain-of-function (GOF) STAT1 mutations, but how this affects STAT3 function leading to decreased IL-17 is unclear. We also assessed how GOF-STAT1 mutations affect STAT3 activation, DNA binding, gene expression, cytokine production, and epigenetic modifications. We excluded impaired STAT3 phosphorylation, nuclear translocation, and sequestration of STAT3 into STAT1/STAT3 heterodimers and confirm significantly reduced transcription of STAT3-inducible genes (RORC/IL-17/IL-22/IL-10/c-Fos/SOCS3/c-Myc) as likely underlying mechanism. STAT binding to the high affinity sis-inducible element was intact but binding to an endogenous STAT3 DNA target was impaired. Reduced STAT3-dependent gene transcription was reversed by inhibiting STAT1 activation with fludarabine or enhancing histone, but not STAT1 or STAT3 acetylation with histone deacetylase (HDAC) inhibitors trichostatin A or ITF2357. Silencing HDAC1, HDAC2, and HDAC3 indicated a role for HDAC1 and 2. Reduced STAT3-dependent gene transcription underlies low Th-17 responses in GOF-STAT1 CMC, which can be reversed by inhibiting acetylation, offering novel targets for future therapies.

Orf Infection in a Patient with Stat1 Gain-of-Function.

PURPOSE: Chronic Mucocutaneous Candidiasis (CMC) refers to a group of immunodeficiencies, characterized by persistent or recurrent infections of the skin, nails, and mucosae caused by Candida. It is typically caused by inborn errors of IL-17 immunity. Orf, also known as contagious ecthyma, is a zoonotic infection caused by a dermatotropic parapoxvirus that commonly infects sheep and goats; it is transmitted to humans through contact with an infected animal or fomites. While orf is usually a benign self-limiting illness, it can be progressive and even life-threatening in immune-compromised hosts. METHODS AND RESULTS: A 34-year-old man with autosomal dominant CMC due to a heterozygous STAT1 gain-of-function (GOF) mutation cut his hand with a knife during slaughter. Giant orf infection developed in 2 weeks. He was successfully treated by cidofovir injections every other week for 4 months. CONCLUSIONS: This is the first patient with severe orf in the context of a well-defined genetically identified PID: CMC and inborn error of IL-17 immunity due to a GOF STAT1 mutation.

Case report: A STAT1 gain-of-function mutation causes a syndrome of combined immunodeficiency, autoimmunity and pure red cell aplasia.

Inherited autosomal dominant gain-of-function (GOF) mutations of signal transducer and activator of transcription 1 (STAT1) cause a wide range of symptoms affecting multiple systems, including chronic mucocutaneous candidiasis (CMC), infections, and autoimmune disorders. We describe a rare case of STAT1 mutation with recurrent CMC, lung infections, and anemia. According to the whole-exome sequencing (WES), the patient was genetically mutated in STAT1 GOF (c.854A>G, p.Q285R), and bone marrow biopsy suggested pure red cell aplasia (PRCA). As a functional verification, STAT1 levels and phosphorylation (p-STAT1) of peripheral blood mononuclear cells (PBMCs) following IFN-γ stimulation in STAT1 GOF patient was higher than in the healthy control. Combination therapy of blood transfusion, antimicrobials, intravenous immunoglobulin, methylprednisolone, and the Janus Kinase (JAK) specific inhibitor ruxolitinib was used during treatment of patients. The patient also received a hematopoietic stem cell transplant (HSCT) to help with infections and anemia. This is the first reported case of STAT1 GOF disease complicated with PRCA. This complication might be attributed to immune disorders caused by STAT1 GOF. Furthermore, ruxolitinib may be a viable therapeutic option before HSCT to improve disease management.

JAK inhibition in a patient with a STAT1 gain-of-function variant reveals STAT1 dysregulation as a common feature of aplastic anemia.

BACKGROUND: Idiopathic aplastic anemia is a potentially lethal disease, characterized by T cell-mediated autoimmune attack of bone marrow hematopoietic stem cells. Standard of care therapies (stem cell transplantation or immunosuppression) are effective but associated with a risk of serious toxicities. METHODS: An 18-year-old man presented with aplastic anemia in the context of a germline gain-of-function variant in STAT1. Treatment with the JAK1 inhibitor itacitinib resulted in a rapid resolution of aplastic anemia and a sustained recovery of hematopoiesis. Peripheral blood and bone marrow samples were compared before and after JAK1 inhibitor therapy. FINDINGS: Following therapy, samples showed a decrease in the plasma concentration of interferon-γ, a decrease in PD1-positive exhausted CD8+ T cell population, and a decrease in an interferon responsive myeloid population. Single-cell analysis of chromatin accessibility showed decreased accessibility of STAT1 across CD4+ and CD8+ T cells, as well as CD14+ monocytes. To query whether other cases of aplastic anemia share a similar STAT1-mediated pathophysiology, we examined a cohort of 9 patients with idiopathic aplastic anemia. Bone marrow from six of nine patients also displayed abnormal STAT1 hyper-activation. CONCLUSIONS: These findings raise the possibility that STAT1 hyperactivition defines a subset of idiopathic aplastic anemia patients for whom JAK inhibition may be an efficacious therapy. FUNDING: Funding was provided by the Massachusetts General Hospital Department of Medicine Pathways Program and NIH T32 AI007387. A trial registration is at https://clinicaltrials.gov/ct2/show/NCT03906318.

Precision Medicine in the Treatment of Primary Immune Deficiency Patients With Disorders of Immune Dysregulation.

There are now more than 450 described monogenic germline mutations for inborn errors of immunity that result in the loss of expression, loss of function (LOF), or gain in function (GOF) of the encoded protein. Molecular characterization of these inborn errors of immunity has not only allowed us to characterize on a genetic basis these immune deficiency disorders but has provided a better understanding of the immunobiology of these inborn errors of immunity. More recently, these advances have allowed us to apply targeted therapy or precision medicine in their treatment. Of particular interest related to this review are those inborn errors of immunity that result in gain-of-function (GOF) genetic abnormalities. Many of these inborn errors of immunity fall into a new category referred to as diseases of immune dysregulation in which many of the patients not only exhibit an increased susceptibility to infection but also have a clinical phenotype associated with autoimmune processes and lymphoproliferative disease.

Impaired B-Cell Differentiation in a Patient With STAT1 Gain-of-Function Mutation.

Hypogammaglobulinemia is a rare complication of STAT1 gain-of-function (GOF) mutations. We report an adult patient diagnosed with hypogammaglobulinemia caused by B-cell depletion during the treatment of disseminated cryptococcosis. The patient carried the STAT1 GOF mutation (c.820C>T, p.R274W). The flow cytometric analysis of his bone marrow revealed that B-cell differentiation was blocked in the stages between pre-B1b and pre-B2 cells. On the other hand, his brother who carried the same mutation displayed normal B-cell counts, thereby indicating that the unrecognized variants in same or other gene might be associated with abnormal B-cell differentiation in the patients. In conclusion, impaired B-cell differentiation in the bone marrow can cause hypogammaglobulinemia in patients with STAT1 GOF mutations.