The Role of Interferon-γ in Autoimmune Polyendocrine Syndrome Type 1.

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood. METHODS: We examined whether APS-1 could be classified as a disease mediated by interferon-γ. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-γ using Aire-/-Ifng-/- mice and Aire-/- mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses. RESULTS: Patients with APS-1 had enhanced interferon-γ responses in blood and in all examined autoimmunity-affected tissues. Aire-/- mice had selectively increased interferon-γ production by T cells and enhanced interferon-γ, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire-/- mice normalized interferon-γ responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-γ, normalized interferon-γ and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjögren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients. CONCLUSIONS: Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-γ-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

NF-kappa-B essential modulator (NEMO) gene polymorphism in an adult woman with systemic lupus erythematosus and recurrent non-tuberculous mycobacterial disseminated infections.

Infections are among the most serious complications in patients with systemic lupus erythematosus (SLE), with bacterial and viral infections being the most common. Non-tuberculous mycobacterial (NTM) infections are quite rare and are typically seen in older patients with SLE with longstanding disease duration treated with corticosteroids. Here, we describe a 39-year-old woman with SLE and an unusual pattern of recurrent NTM disseminated infections. After excluding the presence of autoantibodies against interferon-γ, whole exome sequencing revealed a homozygous polymorphism in the NF-kappa-B essential modulator (NEMO) gene. Primary immunodeficiencies should be included in the differential diagnosis of patients with recurrent opportunistic infections, even in those with iatrogenic immunosuppression.

Dominant-negative heterozygous mutations in AIRE confer diverse autoimmune phenotypes.

Autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disease characterized by severe and childhood onset organ-specific autoimmunity caused by mutations in the autoimmune regulator (AIRE) gene. More recently, dominant-negative mutations within the PHD1, PHD2, and SAND domains have been associated with an incompletely penetrant milder phenotype with later onset familial clustering, often masquerading as organ-specific autoimmunity. Patients with immunodeficiencies or autoimmunity where genetic analyses revealed heterozygous AIRE mutations were included in the study and the dominant-negative effects of the AIRE mutations were functionally assessed in vitro. We here report additional families with phenotypes ranging from immunodeficiency, enteropathy, and vitiligo to asymptomatic carrier status. APS-1-specific autoantibodies can hint to the presence of these pathogenic AIRE variants although their absence does not rule out their presence. Our findings suggest functional studies of heterozygous AIRE variants and close follow-up of identified individuals and their families.

Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis.

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, ß-glucan-binding receptor, Dectin-1. The patient's PBMCs failed to produce TNF-α and IL-1ß in response to ß-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1ß and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1ß-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.

Antibody responses to the SARS-CoV-2 vaccine in individuals with various inborn errors of immunity.

BACKGROUND: SARS-CoV-2 vaccination is recommended in patients with inborn errors of immunity (IEIs); however, little is known about immunogenicity and safety in these patients. OBJECTIVE: We sought to evaluate the impact of genetic diagnosis, age, and treatment on antibody response to COVID-19 vaccine and related adverse events in a cohort of patients with IEIs. METHODS: Plasma was collected from 22 health care worker controls, 81 patients with IEIs, and 2 patients with thymoma; the plasma was collected before immunization, 1 to 6 days before the second dose of mRNA vaccine, and at a median of 30 days after completion of the immunization schedule with either mRNA vaccine or a single dose of Johnson & Johnson's Janssen vaccine. Anti-spike (anti-S) and anti-nucleocapsid antibody titers were measured by using a luciferase immunoprecipitation systems method. Information on T- and B-cell counts and use of immunosuppressive drugs was extracted from medical records, and information on vaccine-associated adverse events was collected after each dose. RESULTS: Anti-S antibodies were detected in 27 of 46 patients (58.7%) after 1 dose of mRNA vaccine and in 63 of 74 fully immunized patients (85.1%). A lower rate of seroconversion (7 of 11 [63.6%]) was observed in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy. Previous use of rituximab and baseline counts of less than 1000 CD3+ T cells/mL and less than 100 CD19+ B cells/mL were associated with lower anti-S IgG levels. No significant adverse events were reported. CONCLUSION: Vaccinating patients with IEIs is safe, but immunogenicity is affected by certain therapies and gene defects. These data may guide the counseling of patients with IEIs regarding prevention of SARS-CoV-2 infection and the need for subsequent boosts.

SARS-CoV-2 Spike Protein-Directed Monoclonal Antibodies May Ameliorate COVID-19 Complications in APECED Patients.

Patients with the monogenic immune dysregulatory syndrome autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which is caused by loss-of-function mutations in the autoimmune regulator (AIRE) gene, uniformly carry neutralizing autoantibodies directed against type-I interferons (IFNs) and many develop autoimmune pneumonitis, both of which place them at high risk for life-threatening COVID-19 pneumonia. Bamlanivimab and etesevimab are monoclonal antibodies (mAbs) that target the SARS-CoV-2 spike protein and block entry of SARS-CoV-2 in host cells. The use of bamlanivimab and etesevimab early during infection was associated with reduced COVID-19-associated hospitalization and death in patients at high risk for progressing to severe disease, which led the US Food and Drug Administration to issue an emergency use authorization for their administration in non-hypoxemic, non-hospitalized high-risk patients. However, the safety and efficacy of these mAbs has not been evaluated in APECED patients. We enrolled two siblings with APECED on an IRB-approved protocol (NCT01386437) and admitted them prophylactically at the NIH Clinical Center for evaluation of mild-to-moderate COVID-19. We assessed the safety and clinical effects of early treatment with bamlanivimab and etesevimab. The administration of bamlanivimab and etesevimab was well tolerated and was associated with amelioration of COVID-19 symptoms and prevention of invasive ventilatory support, admission to the intensive care, and death in both patients without affecting the production of antibodies to the nucleocapsid protein of SARS-CoV-2. If given early in the course of COVID-19 infection, bamlanivimab and etesevimab may be beneficial in APECED and other high-risk patients with neutralizing autoantibodies directed against type-I IFNs.

Heritable risk for severe anaphylaxis associated with increased α-tryptase-encoding germline copy number at TPSAB1.

BACKGROUND: An elevated basal serum tryptase level is associated with severe systemic anaphylaxis, most notably caused by Hymenoptera envenomation. Although clonal mast cell disease is the culprit in some individuals, it does not fully explain this clinical association. OBJECTIVE: Our aim was to determine the prevalence and associated impact of tryptase genotypes on anaphylaxis in humans. METHODS: Cohorts with systemic mastocytosis (SM) and venom as well as idiopathic anaphylaxis from referral centers in Italy, Slovenia, and the United States, underwent tryptase genotyping by droplet digital PCR. Associated anaphylaxis severity (Mueller scale) was subsequently examined. Healthy volunteers and controls with nonatopic disease were recruited and tryptase was genotyped by droplet digital PCR and in silico analysis of genome sequence, respectively. The effects of pooled and recombinant human tryptases, protease activated receptor 2 agonist and antagonist peptides, and a tryptase-neutralizing mAb on human umbilical vein endothelial cell permeability were assayed using a Transwell system. RESULTS: Hereditary α-tryptasemia (HαT)-a genetic trait caused by increased α-tryptase-encoding Tryptase-α/ß1 (TPSAB1) copy number resulting in elevated BST level-was common in healthy individuals (5.6% [n = 7 of 125]) and controls with nonatopic disease (5.3% [n = 21 of 398]). HαT was associated with grade IV venom anaphylaxis (relative risk = 2.0; P < .05) and more prevalent in both idiopathic anaphylaxis (n = 8 of 47; [17%; P = .006]) and SM (n = 10 of 82 [12.2%; P = .03]) relative to the controls. Among patients with SM, concomitant HαT was associated with increased risk for systemic anaphylaxis (relative risk = 9.5; P = .007). In vitro, protease-activated receptor-2-dependent vascular permeability was induced by pooled mature tryptases but not α- or ß-tryptase homotetramers. CONCLUSIONS: Risk for severe anaphylaxis in humans is associated with inherited differences in α-tryptase-encoding copies at TPSAB1.

Human TH9 differentiation is dependent on signal transducer and activator of transcription (STAT) 3 to restrain STAT1-mediated inhibition.

BACKGROUND: Patients with loss-of-function (LOF) signal transducer and activator of transcription 3 (STAT3) mutations have dermatitis, enhanced IgE production despite a relative lack of immediate hypersensitivity, recurrent infection, and an increased rate of lymphoma in addition to a number of skeletal and connective tissue abnormalities. Patients with STAT1 gain-of-function (GOF) mutations also have susceptibility to candidiasis and sinopulmonary infection, as well as autoimmunity and squamous cell carcinoma, in addition to even more broad phenotypes. OBJECTIVE: Because of the link between TH9 cells and allergic inflammation, autoimmunity, and antitumor surveillance and because evidence shows a role for either STAT3 or STAT1 in TH9 differentiation conflicts, we sought to determine the status on this lineage of STAT1 GOF and STAT3 LOF mutations in human subjects. METHODS: We detected IL-9 levels and TH9 differentiation in patients with STAT3 LOF and STAT1 GOF mutations, together with TH9 transcript factors, and partially rescued their deficiency in vitro by adding cytokines they lacked or transfecting key molecules. RESULTS: We found that PBMCs or sorted naive CD4+ T cells from patients with STAT3 LOF and STAT1 GOF mutations had impaired TH9 generation/differentiation. STAT3 inhibition in normal TH9 cultures diminished early IL-21 induction and late IL-9 production, whereas exogenous IL-21 enhanced TH9 differentiation, even with STAT3 inhibition, by restoring suppressor of cytokine signaling 3 expression and thus inhibiting excessive phosphorylated signal transducer and activator of transcription (p-STAT) 1 activation. Furthermore, exogenous expression of suppressor of cytokine signaling 3 or either T-bet or STAT1 RNA interference in STAT3 LOF cells partially rescued IL-9 differentiation. CONCLUSION: Collectively, these results suggest that human TH9 differentiation depends on normal p-STAT3 and IL-21 production to suppress p-STAT1 activation and T-bet transcription.

Loss-of-function mutations in caspase recruitment domain-containing protein 14 (CARD14) are associated with a severe variant of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a highly prevalent chronic inflammatory skin disease that is known to be, at least in part, genetically determined. Mutations in caspase recruitment domain-containing protein 14 (CARD14) have been shown to result in various forms of psoriasis and related disorders. OBJECTIVE: We aimed to identify rare DNA variants conferring a significant risk for AD through genetic and functional studies in a cohort of patients affected with severe AD. METHODS: Whole-exome and direct gene sequencing, immunohistochemistry, real-time PCR, ELISA, and functional assays in human keratinocytes were used. RESULTS: In a cohort of patients referred with severe AD, DNA sequencing revealed in 4 patients 2 rare heterozygous missense mutations in the gene encoding CARD14, a major regulator of nuclear factor κB (NF-κB). A dual luciferase reporter assay demonstrated that both mutations exert a dominant loss-of-function effect and result in decreased NF-κB signaling. Accordingly, immunohistochemistry staining showed decreased expression of CARD14 in patients' skin, as well as decreased levels of activated p65, a surrogate marker for NF-κB activity. CARD14-deficient or mutant-expressing keratinocytes displayed abnormal secretion of key mediators of innate immunity. CONCLUSIONS: Although dominant gain-of-function mutations in CARD14 are associated with psoriasis and related diseases, loss-of-function mutations in the same gene are associated with a severe variant of AD.

Identification and analysis of peanut-specific effector T and regulatory T cells in children allergic and tolerant to peanut.

BACKGROUND: Peanut allergy (PA) is potentially life-threatening and generally persists for life. Recent data suggest the skin might be an important route of initial sensitization to peanut, whereas early oral exposure to peanut is protective. In mice regulatory T (Treg) cells are central to the development of food tolerance, but their contribution to the pathogenesis of food allergy in human subjects is less clear. OBJECTIVE: We sought to quantify and phenotype CD4+ peanut-specific effector T (ps-Teff) cells and peanut-specific regulatory T (ps-Treg) cells in children with and without PA or PS. METHODS: ps-Teff and ps-Treg cells were identified from peripheral blood of children with PA, children with PS, and nonsensitized/nonallergic (NA) school-aged children and 1-year-old infants based on upregulation of CD154 or CD137, respectively, after stimulation with peanut extract. Expression of cytokines and homing receptors was evaluated by using flow cytometry. Methylation at the forkhead box protein 3 (FOXP3) locus was measured as a marker of Treg cell stability. RESULTS: Differential upregulation of CD154 and CD137 efficiently distinguished ps-Teff and ps-Treg cells. A greater percentage of ps-Teff cells from infants with PA and infants with PS expressed the skin-homing molecule cutaneous lymphocyte antigen, suggesting activation after exposure through the skin, compared with NA infants. Although ps-Teff cells in both school-aged and infant children with PA produced primarily TH2 cytokines, a TH1-skewed antipeanut response was seen only in NA school-aged children. The frequency, homing receptor expression, and stability of ps-Treg cells in infants and school-aged children were similar, regardless of allergic status. CONCLUSIONS: Exposure to peanut through the skin can prime the development of TH2 ps-Teff cells, which promote sensitization to peanut, despite the presence of normal numbers of ps-Treg cells.