Background and Aims

The genetic, molecular, and cellular analysis of autoinflammatory and immunodeficiency disorders contributes to our understanding of human immunity and leads to the development of targeted therapies. Oligoadenylate synthase 1 (OAS1) is an intracellular double-stranded RNA sensor that generates the second messenger 2'-5'-oligoadenylate to activate RNaseL as a means of antiviral defense. We analyzed four unrelated patients with a polymorphic syndrome of fever, dermatitis, pulmonary alveolar proteinosis, inflammatory bowel disease, and hypogammaglobulinemia.

Methods

We performed exome and transcriptome sequencing, RNA and protein biochemistry, heterologous and autologous molecular and cellular functional analyses, cellular and serologic immune phenotyping, and allogeneic hematopoietic stem cell transplantation.

Results

We found three de novoheterozygous OAS1variants in four unrelated patients. Variant OAS1 proteins showed double-stranded RNA-independent increased in vitro enzyme activity. They constitutively activated RNaseL resulting in increased RNA cleavage in a heterologous cell system as well as in primary monocytes and B cells. This lead to an increased interferon response, spontaneous monocyte and B cell apoptosis, impaired monocyte and B cell differentiation and function, and impaired cellular co-stimulation towards T cells. Allogeneic hematopoietic stem cell transplantation corrected the otherwise lethal phenotype.

Conclusions

Heterozygous OAS1 gain-of-function variants cause a polymorphic autoinflammatory and immunodeficiency syndrome that can be cured by allogeneic hematopoietic stem cell transplantation. OAS1 emerges as a critical regulator of monocyte and B cell human biology.

Background and Aims

Historically, hyper IgE syndromes (HIES) are characterized by a clinical triad of relapsing skin abscesses, recurrent pneumonias complicated by pneumatocoele and elevated serum levels of IgE. HIES is a multi-system disease; aside of the abovementioned triad, recurrent viral infections, eosinophilia, eczema and connective tissue abnormalities (such as retained teeth and craniosynostosis) further complicate the phenotype. With the advent of next generation sequencing, the genetic landscape of HIES is ever-expanding, providing unique opportunities to understand this complex immunodeficiency syndrome.

Methods

Here we present a Bulgarian 8-year-old girl suffering from atopic eczema, severe asthma and recurrent respiratory infections. Her father has a similar phenotype characterized by relapsing staphylococcal skin infections and severe airway infections complicated by pneumatocele and mucormycosis. Both daughter and father present with frontal bossing and supernumerary teeth, due to absent shedding. In both cases, immunological investigations reveal eosinophilia and increased levels of IgE. The father’s phenotype is additionally complicated by hypogammaglobulinemia.

Results

Genetic analysis revealed a heterozygous frameshift mutation in the gene IL6ST resulting in a premature stop (I719NfsTer2). IL6ST encodes GP130, a transmembrane protein that acts as a signal transducer for many cytokines, including IL-6 and IL-27. In two recent reports, homozygous IL6ST missense variants were identified in patients suffering from HIES with defective STAT3 signaling. Indeed, STAT3 phosphorylation is impaired in I719NfsTer2 PBMCs upon stimulation, providing first prove of the pathogenic nature of this variant.

Conclusions

In summary, the discovery of this novel variant highlights the role of IL6ST in human immunity and expands the spectrum of hyper IgE-like syndromes.

Background and Aims

Common Variable Immunodeficiency (CVID) can lead to structural airway disease (AD), interstitial lung disease (ILD), and immune dysregulation in other organs. A role for microbial dysbiosis in the respiratory tract has been hypothesised, but not been investigated.

Methods

This cross-sectional study compared the oropharyngeal microbiota of 99 patients with CVID and 49 healthy controls. qPCR was used to determine total bacterial load. Next-generation sequencing of 16S rRNA was used to characterise microbiota composition. High-resolution CT (HRCT) scans were scored for AD and ILD and correlated with microbiota composition.

Results

Bacterial load was increased in CVID patients with immune dysregulation (CVIDid) (see figure; p=0.005; median fold change=16). IgA deficiency was associated with higher alpha diversity (p=0.008), and distinct beta diversity (p=0.019). Enrichment of Alloprevotella and Prevotella was associated with CVIDid (p=0.009;p=0.027) and IgA deficiency (p=0.002;p=0.003). Radiographic AD scores were associated with increased alpha diversity (p=0.037) and correlated with Prevotella (p=0.008), Moraxella (p=0.009) and Selenomonas (p=0.01) using linear regression. Radiographic ILD scores correlated with Actinobacteria (p=0.223) and Streptococcus pneumoniae (p=0.005).

Figure: Bacterial load- and 16S copy number-corrected 16S rRNA sequencing results. HC=healthy controls, CVIDio=CVID with infection only, CVIDid=CVID with immune dysregulation. MW-U test on total bacterial load per sample.

Conclusions

IgA-associated microbial dysbiosis in the upper respiratory tract, with increased bacterial load and diversity, correlated with radiographic lung disease In CVID patients. Prevotella spp were identified here as immune dysregulation-associated bacteria. This is the first study describing respiratory microbiota in humoral immunodeficiency, and illustrates the importance of IgA in the maintenance of respiratory microbiota homeostasis.

Background and Aims

We evaluated two Finnish patients, a mother and her son, presenting with early-onset autoimmunity and combined immunodeficiency. The mother suffered from recurrent respiratory tract infections, atopic eczema, and oligoarthritis since childhood and later developed chronic diarrhea and persistent EBV-viremia. Her son had growth retardation, autoimmune enteropathy, eczema, and interstitial lung disease. Both patients displayed hypogammaglobulinemia and impaired regulatory T cell function. The son died at the age of 10 due to pulmonary complications following allogeneic hematopoietic stem cell transplantation. 

Whole-exome sequencing of these patients revealed a heterozygous variant (p.G307D) in the target of myb1 membrane trafficking protein (TOM1) -gene. TOM1 is known to be involved in autophagy, endosomal trafficking, and receptor recycling, all of which have been implicated in monogenic autoimmune diseases. 

Our objective was to elucidate the mechanisms by which this mutation causes the phenotype seen in these patients.

Methods

Fluorescence microscopy and western blotting were utilized to study autophagy and innate immune signaling in primary skin fibroblasts and lymphoblasts from the patients carrying TOM1 mutation.

Results

TOM1 mutant cells displayed a higher level of autophagy compared to wild-type cells. Amino acid starvation led to a significantly higher amount of autophagosomes compared to healthy control cells. When returned in complete culture media, patient cells showed slower autophagosome clearance, suggesting delayed degradation rather than enhanced autophagosome formation.

Conclusions

TOM1 mutation carried by the patients causes defective autophagosome clearance and is likely to contribute to the clinical phenotype of these patients.

Background and Aims

Gaucher Disease(GD) is a congenital disorder secondary to GBA1-gene mutations causing the impairment of the β-glucocerebrosidase(GBA) enzyme with progressive accumulation of its substrate(glucocerebroside, GC) in monocyte/macrophage system and various organs. Bone infiltration, hepatosplenomegaly and cytopenia represent the most common clinical features which might overlap with Autoimmune Lymphoproliferative Sindrome(ALPS) ones.GD patients also show hyperinflammatory features -secondary to machrophages activation- and an aspecific impaiment involving B,T,and NK-cells. The aim of this study is to evaluate ALPS-like immunological and serological pattern in a cohort patients with GD and to test apoptosis function in those showing ALPS-like features.

Methods

Lymphocytes subsets and ALPS-related serum biomarkers (Double-Negative T-cells,B220+DNTs,CD27+,T-regs/HLA-DR ratio,IL-10,IL-18,VitB12) were analyzed in GD patients followed-up at Istituto Gaslini. Furthermore, in those patients showing an ALPS-like phenotype, apoptosis function was analyzed after FAS–induced stimulation of EBV-immortalized B-cells and Western-blot analysis of CASP10,CASP8, and PARP proteins.

Results

41 patients(8 naive,33 treated)were studied. 10/41(24%) patients showed an ALPS-like immunological pattern which was more frequent in naïve patients(p=0.003) and in patients with an earlier onset of the diesease(p=0.010). EBV-immortalized B-cells of 7/10 patients were further studied and all of them showed a defective apoptosis and caspases activation(Fig1).

Conclusions

This study shows for the first time that untreated subjects with early-onset GD present an immune-dysregulation pattern secondary to an apoptosis defect as seen in ALPS patients. These results suggest that diagnostic work-up of both diseases must consider this clinical/biochemical overlap. Further studies are needed to evaluate the potential role of cell-membrane lipids impairment on FAS receptor structure as a cause of such defect.

Background and Aims

Patients with gain-of-function (GOF) STAT1 mutations display a variety of infectious and non-infectious manifestations. Some of them present severe infections and/or autoimmunity, resulting in poor prognosis. Therefore, it is imperative to establish effective treatments based on a precise understanding of the molecular mechanisms of this disorder. To tackle this problem and establish a disease mouse model, we generated GOF-Stat1 knock-in (GOF-Stat1^R274Q) mice. Since a decrease in Th17 cells has been already correlated with the development of chronic mucocutaneous candidiasis in patients, we focused on the small intestine, which contains abundant Th17 cells in mice.

Methods

We generated GOF-Stat1^R274Q mice by introducing the human R274Q mutation. Small intestinal lymphocytes from these mice were analyzed at 3 weeks after Candida albicans infection by flow cytometry, qPCR and RNA-seq.

Results

Stat1^R274Q/R274Q mice showed excess phosphorylation of STAT1, with broad upregulation of IFN-stimulated genes, in CD4⁺ T cells upon IFN-γ stimulation, consistent with the phenotype of patients. Stat1^R274Q/R274Q mice infected with C. albicans elicited decreased IL-17-producing CD4⁺RORγt⁺ cells and excreted larger amounts of C. albicans DNA in their feces compared to controls. Under these conditions, there was upregulation of T-bet in CD4⁺ T cells in Stat1^R274Q/R274Q mice, which may play an inhibitory role in the regulation of RORγt and Th17 differentiation.

Conclusions

The GOF-Stat1^R274Q mice generated in the current study turned out to be a reasonable model of patients with GOF STAT1 mutations and is expected to be a useful tool not only to pursue the molecular mechanism, but also to develop therapeutic strategies, of this disorder.

Background and Aims

We retrospectively reviewed 21 lymphoid neoplasms occurring in the context of CVID and CVID-like disorders.

Methods

We correlated clinical, germline genetic and histopathological findings.

Results

Median age at diagnosis of lymphoma was 38 years. Median time between onset of first symptoms attributed to CVID and lymphoma was 14 years. Non-infectious inflammatory and autoimmune manifestations had a higher prevalence in CVID patients with lymphoid malignancies compared to the CVID population in general. Next-generation sequencing revealed three CTLA4 mutation carriers, one TNFRSF13B variant, one BACH2 mutation, one STAT1 mutation and one NFKB2 variant of unknown significance. The most prevalent B-cell lymphoma subtypes were marginal zone lymphoma (MZL) and diffuse-large B-cell lymphoma (DLBCL). The majority of Hodgkin lymphomas occurred in germline CTLA4 mutation carriers and was EBV+ mixed cellularity classical subtype. T-cell lymphomas were less frequent and mostly T-cell large granular lymphocytic leukemia (T-LGLL). EBV association of DLBCL was more frequent compared to the general population and was accompanied by expression of PDL1 and PD1 suggesting a tolerogenic tumor environment. The prognosis of the CVID patients was predominantly determined by the underlying lymphoid neoplasm while toxicity of treatment had a minor impact.

Conclusions

Our data illustrate that an inflammatory environment facilitates the development of a distinct spectrum of lymphoid neoplasms in CVID. Hodgkin lymphoma occurring in the context of antibody deficiency should raise the suspicion of a CTLA4 mutation carrier status. Future studies will have to dissect the complex interplay between host and environmental factors facilitating the development of lymphoid neoplasms in CVID and CVID-like disorders.

Background and Aims

PFAPA syndrome (the syndrome of Periodic Fever, Aphthous stomatitis, Pharyngitis and cervical Adenitis) is an idiopathic autoinflammatory condition. The current pharmacological treatment includes episodic application of corticosteroids and many other options for the prophylaxis of flares (e.g. anakinra, ketofifen, oral probiotics). We present our group of patients and the efficacy of different prophylactic options.

Methods

We present our group of patients with PFAPA syndrome, which were diagnosed according to valid clinical criteria in our Centre for Periodic Fever Syndromes in Martin, Slovakia.

Results

144 patients with this diagnosis were identified, 74 boys and 70 girls, aged 4.01±2.78 years. The mean age of symptoms’ onset was 2.26±1.92 years. Positive family history was found in 21.36% of patients. During attacks 100% of patients had fever; pharyngitis and cervical adenitis was present in 96% of patients, 50% of patients had aphthous stomatitis. All of our patients yielded elevation of inflammatory parameters (CRP, serum amyloid A, IL-6) during attacks. All of the patients were treated episodically with prednisone with excellent clinical response. For the prohylaxis of febrile episode we used ketotifen with positive effect observed in 63% of patients. Specific oral probiotic strain (Streptococcus salivarius K12) was used in 22 patients with recurrent aphthous stomatitis with good outcome in 5 of 22 patients. Tonsillectomy was indicated in 14% of patients with excellent clinical response.

Conclusions

In our cohort of PFAPA patients we showed unique characteristics of this patients and confirmed the positive effect of new treatment possibilities, e.g. ketotifen or oral probiotic strain.