Jonathan J. Lyons

Early-onset lymphoproliferation and autoimmunity caused by germline STAT3 gain-of-function mutations.

Germline loss-of-function mutations in the transcription factor signal transducer and activator of transcription 3 (STAT3) cause immunodeficiency, whereas somatic gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here, we report on 13 individuals from 10 families with lymphoproliferation and early-onset solid-organ autoimmunity associated with 9 different germline heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multiorgan autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections, and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T-cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 led to clinical improvement in 1 patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly described disorder. Some patients for this study were enrolled in a trial registered at www.clinicaltrials.gov as #NCT00001350.

Loss-of-function mutations in TNFAIP3 leading to A20 haploinsufficiency cause an early-onset autoinflammatory disease

Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity. Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3, which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçets disease, which is typically considered a polygenic disorder with onset in early adulthood. A20 is a potent inhibitor of the NF-κB signaling pathway. Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB–mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB–dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

Elevated basal serum tryptase identifies a multisystem disorder associated with increased TPSAB1 copy number

Elevated basal serum tryptase levels are present in 4–6% of the general population, but the cause and relevance of such increases are unknown. Previously, we described subjects with dominantly inherited elevated basal serum tryptase levels associated with multisystem complaints including cutaneous flushing and pruritus, dysautonomia, functional gastrointestinal symptoms, chronic pain, and connective tissue abnormalities, including joint hypermobility. Here we report the identification of germline duplications and triplications in the TPSAB1 gene encoding α-tryptase that segregate with inherited increases in basal serum tryptase levels in 35 families presenting with associated multisystem complaints. Individuals harboring alleles encoding three copies of α-tryptase had higher basal serum levels of tryptase and were more symptomatic than those with alleles encoding two copies, suggesting a gene-dose effect. Further, we found in two additional cohorts (172 individuals) that elevated basal serum tryptase levels were exclusively associated with duplication of α-tryptase–encoding sequence in TPSAB1, and affected individuals reported symptom complexes seen in our initial familial cohort. Thus, our findings link duplications in TPSAB1 with irritable bowel syndrome, cutaneous complaints, connective tissue abnormalities, and dysautonomia.

Atopic Dermatitis in Children: Clinical Features, Pathophysiology, and Treatment

Atopic dermatitis (AD) is a chronic, relapsing, highly pruritic skin condition resulting from disruption of the epithelial barrier and associated immune dysregulation in the skin of genetically predisposed hosts. AD generally develops in early childhood, has a characteristic age-dependent distribution and is commonly associated with elevated IgE, peripheral eosinophilia, and other allergic diseases. Medications such as antihistamines have demonstrated poor efficacy in controlling AD-associated itch. Education of patients regarding the primary underlying defects and provision of a comprehensive skin care plan is essential for disease maintenance and management of flares.

Germline hypomorphic CARD11 mutations in severe atopic disease

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor–induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

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.

Glycans Instructing Immunity: The Emerging Role of Altered Glycosylation in Clinical Immunology

Protein glycosylation is an important epigenetic modifying process affecting expression, localization, and function of numerous proteins required for normal immune function. Recessive germline mutations in genes responsible for protein glycosylation processes result in congenital disorders of glycosylation and can have profound immunologic consequences. Genetic mutations in immune signaling pathways that affect glycosylation sites have also been shown to cause disease. Sugar supplementation and in vivo alteration of glycans by medication holds therapeutic promise for some of these disorders. Further understanding of how changes in glycosylation alter immunity may provide novel treatment approaches for allergic disease, immune dysregulation, and immunodeficiency in the future.

Distinct Cutaneous Manifestations and Cold-Induced Leukocyte Activation Associated With PLCG2 Mutations

IMPORTANCE PLCG2-associated antibody deficiency and immune dysregulation (PLAID) is a newly characterized immunodeficiency syndrome associated with distinct cutaneous features. Awareness of the cutaneous skin findings associated with PLAID may facilitate diagnosis and improve patient care. OBJECTIVES To characterize the cutaneous manifestations of PLAID and identify potential cellular mechanisms of the disease. DESIGN, SETTING, AND PARTICIPANTS In this retrospective analysis of patients with PLAID and PLAID-like disease evaluated at the National Institutes of Health from January 1, 2005, through December 31, 2014, patients with deletions in PLCG2 leading to PLAID and patients with PLAID-like disease for whom a PLAID mutation was not identified were studied. MAIN OUTCOMES AND MEASURES Characterization of cutaneous manifestations of PLAID and PLAID-like disease and analysis of PLAID immune cell activation. RESULTS Among 36 patients with PLAID and PLAID-like phenotypes, all of whom had evaporative cold urticaria, 8 patients had a history of unique neonatal-onset ulcerative and cutaneous lesions in cold-sensitive regions of the body. Granulomatous skin lesions sparing warm regions (eg, flexural surfaces and skinfolds) were identified in 4 patients. Neutrophils and monocytes from patients with PLAID exhibited enhanced baseline activation in vitro, which was potentiated by ambient temperature exposure. CONCLUSIONS AND RELEVANCE Collectively, these findings suggest that early identification of neonatal lesions may help in the diagnosis of PLAID and that leukocyte hyperactivation may underlie cutaneous lesions in patients with PLAID. Further characterization of mechanisms underlying leukocyte hyperactivation may contribute to the fundamental understanding of granuloma formation.

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.

Systemic hypersensitivity reaction mimicking anaphylaxis after first filgrastim administration in a healthy donor

Recombinant human granulocyte–colony-stimulating factor (rHuG-CSF) is approved for use to accelerate neutrophil recovery after myelosuppressive therapy and to mobilize hematopoietic progenitor cells from the marrow into the blood to facilitate peripheral blood stem cell (PBSC) collection. Commercially available forms of G-CSF include filgrastim (Neupogen, Amgen, Thousand Oaks, CA) and lenograstim (Granocyte, Chugai, Tokyo, Japan), both of which are manufactured by recombinant DNA technology. Filgrastim is a nonglycosylated Escherichia coli–derived 175-amino-acid glycoprotein with an extra N-terminal methionine group, and lenograstim is a Chinese hamster ovary–derived 174-amino-acid glycoprotein with 4% carbohydrate, indistinguishable from native G-CSF. Modest and reversible side effects of rHuG-CSF are common, including bone pain, myalgias, and headache. More severe adverse effects, including splenic rupture, have been described in a handful of healthy donors.1 Anaphylactoid episodes associated with a first dose of rHuG-CSF in healthy donors have only been reported twice, once after filgrastim and once after lenograstim,2,3 although additional reports of hypersensitivity reactions in patients receiving myeloablative therapy are described.4,5 In these latter cases, confounding effects of multiple concomitant drugs made attribution of causality uncertain. We describe a second case of severe systemic hypersensitivity reaction mimicking anaphylaxis after filgrastim administration in a healthy donor. A 17-year-old healthy Haitian female PBSC donor presented to the outpatient clinic to receive her first dose of filgrastim. She had no history of allergies and met all institutional criteria for sibling allogeneic stem cell donation. She received 720 mg (10 mg/kg) of filgrastim subcutaneously and remained in the clinic, feeling well, for 75 minutes. Fifteen minutes after leaving (90 min after the filgrastim injection), she developed difficulty breathing and throat tightness. She was transported back to clinic by wheelchair by her mother. Upon arrival, she complained of severe cramping abdominal pain and developed diarrhea with lightheadedness and extreme diaphoresis. She was tachycardic (pulse, 144) and hypotensive (80/40), with normal oxygen saturation and no rash. She rapidly became confused, then unresponsive, with an undetectable blood pressure. Intramuscular epinephrine and intravenous diphenhydramine, ranitidine, methylprednisolone, and saline were given. Her symptoms and vital signs stabilized rapidly and she was admitted for over-night observation, continuing to receive steroids and H1 and H2 receptor blockers every 8 hours. Serum tryptase, measured within 3 hours of the event, and 24-hour urinary N-methylhistamine were both within the reference range. She was discharged 30 hours after the incident without further sequelae. A severe anaphylactoid reaction to filgrastim in a healthy allogeneic donor has only been reported once in the past, 50 minutes after a first drug dose in a 16-year-old granulocyte donor whose symptoms and response to therapy were identical to those in our donor.2 Similar reactions have been reported in patients with malignancies after a first or subsequent filgrastim dose, but confounding conditions and medications rendered causality problematic.4,5 Prior sensitization to E. coli derivatives has been assumed to be the cause of these events, but one case of anaphylaxis after lenograstim has also been reported in a healthy donor,3 so that the inciting immunogen is uncertain. The cause of the reaction could be rHuG-CSF itself or another component of the drug preparation such as polysorbate 80 (Tween 80), which has been implicated in hypersensitivity reactions.6 The clinical course and response to therapy in our donor were consistent with an anaphylactoid event. However, laboratory testing with serum tryptase and urinary N-methylhistamine provided strong objective data that the reaction was not due to mast cell or basophil activation and degranulation. Our case serves as a reminder that filgrastim administration may be associated with severe hypersensitivity reactions mimicking anaphylaxis and that such reactions should be treated promptly with antianaphylaxis medications. Initial doses of filgrastim should be given in a monitored medical setting, followed by at least an hour of observation.