Monica G. Lawrence

Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation–polyendocrinopathy–enteropathy–X-linked–like syndrome

BACKGROUND Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

Immune deficiencies, infection, and systemic immune disordersDominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation–polyendocrinopathy–enteropathy–X-linked–like syndrome

BACKGROUND Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

Loss-of-function mutations in the IL-21 receptor gene cause a primary immunodeficiency syndrome

A primary immunodeficiency syndrome caused by loss-of-function mutations in the IL-21 receptor exhibits impaired B, T, and NK cell function.

Gain-of-function STAT1 mutations are associated with PD-L1 overexpression and a defect in B-cell survival

To the Editor: Heterozygous gain-of-function mutations in the coiled-coil domain of STAT1 were recently identified as a cause of chronic mucocutaneous candidiasis (CMC) (1, 2). As with STAT3 mutations in hyper-IgE syndrome, the candidal susceptibility associated with gain-of-function STAT1 mutations appears secondary to TH17 cell deficiency (3). The mechanistic link between constitutive STAT1 activity and diminished TH17 cells has yet to be clearly defined. Here we present a kindred with a novel gain-of-function STAT1 mutation associated with a complex clinical phenotype including candidiasis, humoral immunodeficiency, overexpression of programmed cell death protein ligand 1 (PD-L1) and increased B-cell apoptosis. We have identified four related individuals each heterozygous for a novel E235A missense mutation in a highly conserved segment of the coiled-coil domain of STAT1 (Fig 1, A). To our knowledge this is the first mutation described in exon 9 of the STAT1α gene locus to be associated with CMC. The mutation is not present in unaffected adult family members. The index patient (II.6) is a 60-year-old woman with CMC and progressive antibody deficiency. Beginning in infancy, the patient experienced candidal infections at sites including the oral cavity, esophagus, vagina, skin and nails. Later, as a young woman, the patient was diagnosed with IgG2 subclass deficiency that progressed to frank hypogammaglobulinemia (IgG 514, IgA 7, IgM<10 mg/dl) and B-cell lymphopenia (2 CD20+ cells/µl, nml range 97–440) requiring IVIG. Over her lifetime, the patient suffered recurrent pulmonary infections from P. aeruginosa, S. pneumonia, Serratia species, M. avium and respiratory syncytial virus that resulted in severe bronchiectasis requiring lobectomy. Consistent with published reports of patients with gain-of-function STAT1 mutations, our index patient (II.6) has experienced HPV+ squamous cell carcinoma of palate, basal cell carcinoma, shingles and fibromuscular dysplasia with carotid and celiac/splenic artery dissection. FIG 1 Gain-of-function STAT1 mutations increase PD-L1 expression. A, Pedigree with patients (black) and carriers (gray) heterozygous at STAT1 nucleotide 705. B, Increased STAT1 phosphorylation in IL-21 treated T-cells from patients (filled), compared to controls ... The index patient’s daughter (III.2) is a 30-year-old female with CMC, B-cell lymphopenia (6 CD20+ cells/µl) and IgG2 subclass deficiency manifesting in adolescence. Despite antifungal therapy, the patient experienced candidal infections at sites including the vagina, skin and nails. She has also experienced non-candidal infections including pneumonia, otitis media, sinusitis and chronic bronchitis. The third and forth persons carrying the E235A allele are children of patient III.2, two males ages 6 weeks and 24 months (IV.1 and IV.2). They have not yet manifested symptoms of immunodeficiency. Stimulation of T cells from patients II.6 and III.2 with IL-21 significantly increased phosphorylation of STAT1 compared to a healthy control (Fig 1, B). Furthermore, stimulation of patient PBMCs with PMA/ionomycin demonstrated diminished IL-17 secreting CD4+ cells compared to a related healthy control (Fig 1, C). Hence the E235A mutation confers to STAT1 a gain-of-function and is associated with TH17-cell deficiency. A remarkable feature of our kindred is overexpression of PD-L1 on the surface of naive CD4+ T cells. All four family members carrying the E235A allele had higher PD-L1 staining compared to members without it (Fig 1, D). To investigate if overexpression of PD-L1 was a common feature to gain-of-function STAT1 mutations, we obtained PBMCs from two additional patients carrying either the I156T or the E353K missense STAT1 mutation. Both patients, unrelated to our kindred, revealed a similar increase in PD-L1 expression on their naive T cells (Fig 1, D). Recent data from mice demonstrate that the expression of PD-L1 on undifferentiated naive T cells prevents commitment to the TH17 lineage through a PD-1/PD-L1 interaction. In this context, PD-L1 expression is dependent on IL27/IL27R binding and STAT1 (4). Accordingly, constitutively active STAT1 molecules in subjects carrying gain-of-function STAT1 mutations may be responsible for increased PD-L1 expression on naive T cells thereby discouraging differentiation into TH17 cells. As previously described, the clinical phenotype of patients with gain-of-function STAT1 mutations is quite broad and can include candidiasis, anti-thyroid autoimmunity, squamous cell carcinoma and vascular anomalies. In this issue two reports show broader phenotypes to gain-of-function STAT1 mutations with an IPEX-like autoimmune syndrome in one report, and disseminated coccidioidomycosis and histoplasmosis in the other. Here, we report a not yet appreciated feature associated with gain-of-function STAT1 mutations: humoral immunodeficiency. STAT1’s function as a key modulator of cell death is thoroughly described. Perhaps the best illustration of this is growth arrest of the STAT1-negative U3A fibroblast line upon transfection with wild type STAT1α and treatment with interferon-γ (5). The interferon-γ receptor requires STAT1 for intracellular signaling. Moreover, transfection of the same cell line with constitutively activated STAT1 initiates caspase mediated apoptosis (6). Related experiments implicate STAT1 activation during apoptosis in B-cell lymphoma cells (7). Interestingly, progressive B-cell lymphopenia is a remarkable feature of patients (II.6 and III.2) in our kindred suggesting a defect in cell survival. Indeed, CD19+ B cells from subjects carrying the E235A allele appear apoptotic with increased Annexin V staining (Fig 2, A top row) and elevated caspase activity (Fig 2, B). In culture for 24 hours, B cells from patient III.2 demonstrated even greater Annexin V and considerable 7-AAD staining, evidence of accelerated cell death (Fig 2, A middle row). The patient’s B cells were only partially rescued by stimulation of their B-cell receptors (Fig 2,A bottom row). We also found enhanced caspase activity in B cells from the two additional patients that were heterozygous for either the E353K or the I156T missense STAT1 mutation (Fig 2, B). B cell lymphopenia was a significant finding in the former patient (39 cells/µl) but not the latter (335 cells/ul). Altogether, our data reveal that gain-of-function STAT1 mutations increase B-cell apoptosis. Over time this may result in B-cell lymphopenia and antibody deficiency. FIG 2 Gain-of-function STAT1 mutations promote B-cell death. A, Annexin V and 7-AAD staining of CD19+ cells immediately ex-vivo (top row) and after 24 hours in culture with (middle row) or without rescue with F(ab’)2-anti-IgM (bottom row). B, Active ... In summary, we identified individuals heterozygous for gain-of-function STAT1 mutation with two unappreciated features. The first is the overexpression of PD-L1 on naive T cells which provides a general mechanism for how constitutively active STAT1 blocks the development of the TH17 lineage. The second feature, accelerated B-cell apoptosis that may result in progressive B-cell lymphopenia and humoral immunodeficiency, further broadens the clinical phenotype associated with gain-of-function STAT1 mutations.

Loss of B Cells in Patients with Heterozygous Mutations in IKAROS.

BACKGROUND Common variable immunodeficiency (CVID) is characterized by late-onset hypogammaglobulinemia in the absence of predisposing factors. The genetic cause is unknown in the majority of cases, and less than 10% of patients have a family history of the disease. Most patients have normal numbers of B cells but lack plasma cells. METHODS We used whole-exome sequencing and array-based comparative genomic hybridization to evaluate a subset of patients with CVID and low B-cell numbers. Mutant proteins were analyzed for DNA binding with the use of an electrophoretic mobility-shift assay (EMSA) and confocal microscopy. Flow cytometry was used to analyze peripheral-blood lymphocytes and bone marrow aspirates. RESULTS Six different heterozygous mutations in IKZF1, the gene encoding the transcription factor IKAROS, were identified in 29 persons from six families. In two families, the mutation was a de novo event in the proband. All the mutations, four amino acid substitutions, an intragenic deletion, and a 4.7-Mb multigene deletion involved the DNA-binding domain of IKAROS. The proteins bearing missense mutations failed to bind target DNA sequences on EMSA and confocal microscopy; however, they did not inhibit the binding of wild-type IKAROS. Studies in family members showed progressive loss of B cells and serum immunoglobulins. Bone marrow aspirates in two patients had markedly decreased early B-cell precursors, but plasma cells were present. Acute lymphoblastic leukemia developed in 2 of the 29 patients. CONCLUSIONS Heterozygous mutations in the transcription factor IKAROS caused an autosomal dominant form of CVID that is associated with a striking decrease in B-cell numbers. (Funded by the National Institutes of Health and others.).

Lack of basophil CD203c-upregulating activity as an immunological marker to predict response to treatment with omalizumab in patients with symptomatic chronic urticaria

Chronic urticaria (CU) is defined as recurrent, transitory, pruritic-raised wheals present on most days of the week for more than 6 weeks. The pathogenesis of CU is unknown, but it is thought to be the result of autoimmunity, at least in some patients. Evidence supporting this comes from assays suggesting the presence of functional autoantibodies. IgG autoantibodies to IgE or to the high-affinity IgE receptor (FceRI) have been directly detected in approximately 40%-50% of patients with CU. Although of interest, identifying these antibodies is laborious and not fully validated; therefore, numerous surrogate assays have been developed. A positive autologous serum skin test (ASST) can be identified in 30%-67% of patients with CU and is an indirect reflection of autoantibodies. However, the ASST is positive in 37% of patients with non-CU, making it of less certain clinical relevance. Alternatively, the sera of 40%-50% of patients with CU can induce the release of histamine from the basophils of healthy subjects (basophil histamine-releasing assay, [HRA]). Limitations to this test are based on interlaboratory reproducibility and variation in healthy basophil donor characteristics; and as with the ASST, the HRA lacks diagnostic specificity for CU. More recently, flow cytometry has been used to evaluate the ability of serum of patients with CU to activate donor basophils as determined by the upregulation of CD203c (ectonucleotide pyrophosphatase and/or phosphodiesterase). In the validation studies for this assay, the upregulation of CD203c surface expression was evaluated on basophils obtained from a healthy atopic donor after exposure for 10 minutes to serum obtained from 32 patients with CU and 11 healthy controls. (See Yasnowsky et al for a more detailed description.) The data are expressed as the percentage of basophils expressing more CD203c than 99% of basophils incubated with buffer only. A result 5% or more is highly specific for CU as this result was never seen using serum obtained from healthy controls. Serum factors driving increased CD203c expression in this assay are unknown and as with the HRA are assumed to reflect autoantibodies; however, this has never been determined. Furthermore, factors driving histamine release from basophils may differ from those responsible for CD203c upregulation. Treating CU is challenging, as many patients are refractory to the standard pharmacological therapy. Omalizumab, a humanized anti-IgE monoclonal antibody, has been approved for the treatment of CU. During phase 3 trials, omalizumab was effective in 52% of patients with CU based on Urticaria Activity Score (UAS7) of less than 6, but only 34% had complete clinical response (UAS7 of 0). Given this modest response rate, the requirement to administer this agent for up to 3 months before recognizing treatment failure, and the expense associated with its use, the identification of biomarkers predicting responsiveness would prove valuable. In the omalizumab CU clinical trials, the HRA was positive in only 25%-30% of subjects and had no value in predicting therapeutic response. The greater specificity of the CD203c assay for CU suggests that it may have superior predictive value in determining omalizumab responsiveness. Therefore, we performed a retrospective chart review of 41 consecutive adult patients with antihistamine-refractory CU seen at the University of Virginia outpatient allergy clinic between 2011 and 2013 to investigate the use of the basophil CD203c assay as a biomarker of responsiveness to omalizumab in CU. This study was approved by the University of Virginia Health System Institutional Review Board (IRB-HSR #18100). Basophil CD203c-upregulating activity was obtained in all subjects before omalizumab administration (National Jewish Health Advance Diagnostic Laboratories, Denver, Colo). Clinical response was evaluated after a minimum of 3 months of treatment based on the patient’s and physician’s subjective determination. Fisher’s exact test was used to compare basophil CD203c-upregulating activity in responders and nonresponders (GraphPad Prism 6.0) and P < .05 was considered significant. Patients’ characteristics are summarized in Table I. CD203cupregulating activity was present in 18 of the 41 subjects (43.9%). Subjects were treated with omalizumab 300 mg SC q4weeks for at least 3 months. Omalizumab was effective in 29 of the 41 (71%) patients with CU overall, slightly higher than what has been reported in published studies. Of the 18 subjects demonstrating CD203c-upregulating activity, only 9 (50%) had clinical improvement with omalizumab (Figure 1). In contrast, of the 23 without CD203c-upregulating activity, 20 (87%) had a clinical response to omalizumab (P 1⁄4 .02, Fisher’s exact test). Thus, having a negative result predicts a much greater likelihood of responding to omalizumab with the odds ratio of a negative test being 6.7 (95% CI, 1.4-31). No correlation of efficacy was found with age, sex, or the presence of thyroid autoantibodies (not shown). Although not proven, basophil CD203c-upregulating activity is thought to reflect the presence of autoantibodies to IgE and/or FceRIa, suggesting that the presence of these autoantibodies unexpectedly predicted a lower likelihood of clinical response. Clearly, this observation needs to be repeated in a prospective fashion, but if confirmed in those studies, this holds promise as a clinically useful biomarker predicting response to treatment. The mechanism of action of omalizumab in CU is not known, and this understanding will ultimately be central to predicting

Pre-existing anti-PEG antibodies are associated with severe immediate allergic reactions to pegnivacogin, a PEGylated aptamer

To the Editor: PEGylation is commonly used to extend half-life and limit volume of distribution of an increasing number of nucleic acid, peptide, and small molecule therapeutics. Pegnivacogin is a modified 31-nucleotide RNA aptamer that binds to and inhibits factor IXa conjugated to an inert 40-kD branched methoxypolyethylene glycol polymer. Although early clinical testing did not identify any safety concerns, the phase IIb Randomized, Partially Blinded, Multicenter, Active-Controlled, Dose-Ranging Study Assessing the Safety, Efficacy, and Pharmacodynamics of the REG1 Anticoagulation System in Patients with Acute Coronary Syndromes (RADAR) trial was stopped after 3 allergic reactions. An extensive investigation demonstrated elevated levels of IgG anti-PEG antibodies in the 3 patients with allergic events, suggesting that the PEG moiety, and not the oligonucleotide, was the causative allergic agent. On the basis of previous safety record of PEGylated products, investigators and regulatory authorities agreed that pegnivacogin should undergo additional definitive testing incorporating a risk mitigation and action plan in a phase III trial (Randomized, Open-label, Multi-Center, Active-Controlled, Parallel Group Study to Determine the Efficacy and Safety of the REG1 Anticoagulation System Compared to Bivalirudin in Patients Undergoing Percutaneous Coronary Intervention [REGULATE-PCI]) in which subjects undergoing percutaneous coronary intervention were randomized to pegnivacogin or bivalirudin.Methodology of the trial, planned biochemical analyses, statistical analyses, and allergy definitions are available in the first and second sections in this article’s Online Repository at www.jacionline.org. REGULATE-PCI was ultimately terminated after enrollment of 3,232 of a planned 13,200 patients after an excess of allergic reactions in pegnivacogin-treated patients. The incidence and timing of allergic reactions are summarized in Table I. Descriptions of allergies meeting reporting criteria, as judged by the investigators, are provided in the third section in this article’s Online Repository at www.jacionline. org. Assignment to pegnivacogin was associated with a statistically significant increase in allergic reactions. Of the clinical variables assessed, female sex, allergic reactions in the past year, current smoking, and previous percutaneous coronary

FOXP3+ Tregs require WASP to restrain Th2-mediated food allergy

In addition to the infectious consequences of immunodeficiency, patients with Wiskott-Aldrich syndrome (WAS) often suffer from poorly understood exaggerated immune responses that result in autoimmunity and elevated levels of serum IgE. Here, we have shown that WAS patients and mice deficient in WAS protein (WASP) frequently develop IgE-mediated reactions to common food allergens. WASP-deficient animals displayed an adjuvant-free IgE-sensitization to chow antigens that was most pronounced for wheat and soy and occurred under specific pathogen-free as well as germ-free housing conditions. Conditional deletion of Was in FOXP3+ Tregs resulted in more severe Th2-type intestinal inflammation than that observed in mice with global WASP deficiency, indicating that allergic responses to food allergens are dependent upon loss of WASP expression in this immune compartment. While WASP-deficient Tregs efficiently contained Th1- and Th17-type effector differentiation in vivo, they failed to restrain Th2 effector responses that drive allergic intestinal inflammation. Loss of WASP was phenotypically associated with increased GATA3 expression in effector memory FOXP3+ Tregs, but not in naive-like FOXP3+ Tregs, an effect that occurred independently of increased IL-4 signaling. Our results reveal a Treg-specific role for WASP that is required for prevention of Th2 effector cell differentiation and allergic sensitization to dietary antigens.

T-cell biology in immunotherapy

OBJECTIVE This review discusses the current state of immunotherapy and how the CD4 T-cell response is pivotal in altering the allergic response. DATA SOURCES PubMed literature review. STUDY SELECTIONS Articles pertaining to subcutaneous, sublingual, and oral immunotherapies, with specific emphasis on those describing the T-cell response. RESULTS Although many drugs are available that help ameliorate allergic symptoms, the only intervention that has proved to provide long-term benefit and modulation of disease is immunotherapy. Many routes of immunotherapy are being pursued, including subcutaneous, sublingual, and oral immunotherapies; however, subcutaneous immunotherapy has the historical record of leading to immune changes that alter the immune response at subsequent allergen exposure. These changes are mediated by the induction of peripherally derived T-regulatory cells and appear to occur only after high-dose therapy for 3 to 5 years. Newer methods of sublingual and oral immunotherapies are currently being investigated, but their efficacy is not yet on par with subcutaneous immunotherapy. CONCLUSION The primary cells ultimately responsible for successful immunomodulation are CD4 T cells, specifically peripherally derived T-regulatory cells.

ERBIN deficiency links STAT3 and TGF-β pathway defects with atopy in humans

Nonimmunological connective tissue phenotypes in humans are common among some congenital and acquired allergic diseases. Several of these congenital disorders have been associated with either increased TGF-&bgr; activity or impaired STAT3 activation, suggesting that these pathways might intersect and that their disruption may contribute to atopy. In this study, we show that STAT3 negatively regulates TGF-&bgr; signaling via ERBB2-interacting protein (ERBIN), a SMAD anchor for receptor activation and SMAD2/3 binding protein. Individuals with dominant-negative STAT3 mutations (STAT3mut) or a loss-of-function mutation in ERBB2IP (ERBB2IPmut) have evidence of deregulated TGF-&bgr; signaling with increased regulatory T cells and total FOXP3 expression. These naturally occurring mutations, recapitulated in vitro, impair STAT3–ERBIN–SMAD2/3 complex formation and fail to constrain nuclear pSMAD2/3 in response to TGF-&bgr;. In turn, cell-intrinsic deregulation of TGF-&bgr; signaling is associated with increased functional IL-4R&agr; expression on naive lymphocytes and can induce expression and activation of the IL-4/IL-4R&agr;/GATA3 axis in vitro. These findings link increased TGF-&bgr; pathway activation in ERBB2IPmut and STAT3mut patient lymphocytes with increased T helper type 2 cytokine expression and elevated IgE.