Single cell characterization of blood and expanded regulatory T cells in autoimmune polyendocrine syndrome type 1.

Immune tolerance fails in autoimmune polyendocrine syndrome type 1 (APS-1) because of AIRE mutations. We have used single cell transcriptomics to characterize regulatory T cells (Tregs) sorted directly from blood and from in vitro expanded Tregs in APS-1 patients compared to healthy controls. We revealed only CD52 and LTB (down) and TXNIP (up) as consistently differentially expressed genes in the datasets. There were furthermore no large differences of the TCR-repertoire of expanded Tregs between the cohorts, but unique patients showed a more restricted use of specific clonotypes. We also found that in vitro expanded Tregs from APS-1 patients had similar suppressive capacity as controls in co-culture assays, despite expanding faster and having more exhausted cells. Our results suggest that APS-1 patients do not have intrinsic defects in their Treg functionality, and that their Tregs can be expanded ex vivo for potential therapeutic applications.

Rare copy number variation in autoimmune Addison's disease.

Autoimmune Addison's disease (AAD) is a rare but life-threatening endocrine disorder caused by an autoimmune destruction of the adrenal cortex. A previous genome-wide association study (GWAS) has shown that common variants near immune-related genes, which mostly encode proteins participating in the immune response, affect the risk of developing this condition. However, little is known about the contribution of copy number variations (CNVs) to AAD susceptibility. We used the genome-wide genotyping data from Norwegian and Swedish individuals (1,182 cases and 3,810 controls) to investigate the putative role of CNVs in the AAD aetiology. Although the frequency of rare CNVs was similar between cases and controls, we observed that larger deletions (>1,000 kb) were more common among patients (OR = 4.23, 95% CI 1.85-9.66, p = 0.0002). Despite this, none of the large case-deletions were conclusively pathogenic, and the clinical presentation and an AAD-polygenic risk score were similar between cases with and without the large CNVs. Among deletions exclusive to individuals with AAD, we highlight two ultra-rare deletions in the genes LRBA and BCL2L11, which we speculate might have contributed to the polygenic risk in these carriers. In conclusion, rare CNVs do not appear to be a major cause of AAD but further studies are needed to ascertain the potential contribution of rare deletions to the polygenic load of AAD susceptibility.

Interferon autoantibodies as signals of a sick thymus.

Type I interferons (IFN-I) are key immune messenger molecules that play an important role in viral defense. They act as a bridge between microbe sensing, immune function magnitude, and adaptive immunity to fight infections, and they must therefore be tightly regulated. It has become increasingly evident that thymic irregularities and mutations in immune genes affecting thymic tolerance can lead to the production of IFN-I autoantibodies (autoAbs). Whether these biomarkers affect the immune system or tissue integrity of the host is still controversial, but new data show that IFN-I autoAbs may increase susceptibility to severe disease caused by certain viruses, including SARS-CoV-2, herpes zoster, and varicella pneumonia. In this article, we will elaborate on disorders that have been identified with IFN-I autoAbs, discuss models of how tolerance to IFN-Is is lost, and explain the consequences for the host.

Regulatory T cells in autoimmune primary adrenal insufficiency.

Primary adrenal insufficiency (PAI) is most often caused by an autoimmune destruction of the adrenal cortex resulting in failure to produce cortisol and aldosterone. The aetiology is thought to be a combination of genetic and environmental risk factors, leading to breakdown of immunological tolerance. Regulatory T cells (Tregs) are deficient in many autoimmune disorders, but it is not known whether they contribute to development of PAI. We aimed to investigate the frequency and function of naive and expanded Tregs in patients with PAI and polyendocrine syndromes compared to age- and gender-matched healthy controls. Flow cytometry was used to assess the frequency and characterize functional markers of blood Tregs in PAI (N = 15). Expanded Treg suppressive abilities were assessed with a flow cytometry based suppression assay (N = 20), while bulk RNA-sequencing was used to examine transcriptomic differences (N = 16) and oxygen consumption rate was measured by a Seahorse cell metabolic assay (N = 11). Our results showed that Treg frequency and suppressive capacity were similar between patients and controls. An increased expression of killer-cell leptin-like receptors and mitochondrial genes was revealed in PAI patients, but their expanded Tregs did not display signs of mitochondrial dysfunction. Our findings do not support a clear role for Tregs in the contribution of PAI development.

Autoimmune primary adrenal insufficiency -current diagnostic approaches and future perspectives.

The adrenal glands are small endocrine glands located on top of each kidney, producing hormones regulating important functions in our body like metabolism and stress. There are several underlying causes for adrenal insufficiency, where an autoimmune attack by the immune system is the most common cause. A number of genes are known to confer early onset adrenal disease in monogenic inheritance patterns, usually genetic encoding enzymes of adrenal steroidogenesis. Autoimmune primary adrenal insufficiency is usually a polygenic disease where our information recently has increased due to genome association studies. In this review, we go through the physiology of the adrenals before explaining the different reasons for adrenal insufficiency with a particular focus on autoimmune primary adrenal insufficiency. We will give a clinical overview including diagnosis and current treatment, before giving an overview of the genetic causes including monogenetic reasons for adrenal insufficiency and the polygenic background and inheritance pattern in autoimmune adrenal insufficiency. We will then look at the autoimmune mechanisms underlying autoimmune adrenal insufficiency and how autoantibodies are important for diagnosis. We end with a discussion on how to move the field forward emphasizing on the clinical workup, early identification, and potential targeted treatment of autoimmune PAI.

Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Th17 Cells: Orchestrators of Mucosal Inflammation and Potential Therapeutic Targets.

T helper 17 (Th17) cells represent a specialized subgroup of effector CD4+ T cells known for their role in provoking neutrophil-driven tissue inflammation, particularly within mucosal tissues. Although they are pivotal for defending the host against extracellular bacteria and fungi, they have also been associated with development of various T cell-mediated inflammatory conditions, autoimmune diseases, and even cancer. Notably, Th17 cells exhibit a dual nature, with different Th17 cell subtypes showcasing distinct effector functions and varying capacities to incite autoimmune tissue inflammation. Furthermore, Th17 cells exhibit significant plasticity, which carries important functional implications, both in terms of their expression of cytokines typically associated with other effector T cell subsets and in their interactions with regulatory CD4+ T cells. The intricate balance of Th17 cytokines can also be a double-edged sword in inflammation, autoimmunity, and cancer. Within this article, we delve into the mechanisms that govern the differentiation, function, and adaptability of Th17 cells. We culminate with an exploration of therapeutic potentials in harnessing the power of Th17 cells and their cytokines. Targeted interventions to modulate Th17 responses are emerging as promising strategies for autoimmunity, inflammation, and cancer treatment. By precisely fine-tuning Th17-related pathways, we may unlock new avenues for personalized therapeutic approaches, aiming to restore immune balance, alleviate the challenges of these disorders, and ultimately enhance the quality of life for individuals affected by them.

Vaccination prevents severe COVID-19 outcome in patients with neutralizing type 1 interferon autoantibodies.

A hallmark of patients with autoimmune polyendocrine syndrome type 1 (APS-1) is serological neutralizing autoantibodies against type 1 interferons (IFN-I). The presence of these antibodies has been associated with severe course of COVID-19. The aims of this study were to investigate SARS-CoV-2 vaccine tolerability and immune responses in a large cohort of patients with APS-1 (N = 33) and how these vaccinated patients coped with subsequent infections. We report that adult patients with APS-1 were able to mount adequate SARS-CoV-2 spike-specific antibody responses after vaccination and observed no signs of decreased tolerability. Compared with age- and gender-matched healthy controls, patients with APS-1 had considerably lower peak antibody responses resembling elderly persons, but antibody decline was more rapid in the elderly. We demonstrate that vaccination protected patients with APS-1 from severe illness when infected with SARS-CoV-2 virus, overriding the systemic danger of IFN-I autoantibodies observed in previous studies.

Systemic interferon type I and B cell responses are impaired in autoimmune polyendocrine syndrome type 1.

Autoimmune polyendocrine syndrome type I (APS-1) is caused by mutations in the autoimmune regulator (AIRE) gene and characterised clinically by multiple autoimmune manifestations and serologically by autoantibodies against tissue proteins and cytokines. We here hypothesised that lack of AIRE expression in thymus affects blood immune cells and performed whole-blood microarray analysis (N = 16 APS-I patients vs 16 controls), qPCR verification, and bioinformatic deconvolution of cell subsets. We identified B cell responses as being downregulated in APS-1 patients, which was confirmed by qPCR; these results call for further studies on B cells in this disorder. The type I interferon (IFN-I) pathway was also downregulated in APS-1, and the presence of IFN antibodies is the likely reason for this mild overall downregulation of the IFN-I genes in most APS-1 patients.

Systemic Activation of the Kynurenine Pathway in Graves Disease With and Without Ophthalmopathy.

CONTEXT: Graves disease (GD) is one of the most common autoimmune disorders. Recent literature has shown an immune response involving several different inflammatory related proteins in these patients. OBJECTIVE: This work aimed to characterize the kynurenine pathway, activated during interferon-γ (IFN-γ)-mediated inflammation and cellular (T-helper type 1 [Th1] type) immunity, in GD patients with and without thyroid eye disease (TED). METHODS: We analyzed 34 biomarkers by mass spectrometry in serum samples from 100 patients with GD (36 with TED) and 100 matched healthy controls. The analytes included 10 metabolites and 3 indices from the kynurenine pathway, 6 microbiota-derived metabolites, 10 B-vitamers, and 5 serum proteins reflecting inflammation and kidney function. RESULTS: GD patients showed significantly elevated levels of 7 biomarkers compared with healthy controls (omega squared [ω2] > 0.06; P < .01). Of these 7, the 6 biomarkers with the strongest effect size were all components of the kynurenine pathway. Factor analysis showed that biomarkers related to cellular immunity and the Th1 responses (3-hydroxykynurenine, kynurenine, and quinolinic acid with the highest loading) were most strongly associated with GD. Further, a factor mainly reflecting acute phase response (C-reactive protein and serum amyloid A) showed weaker association with GD by factor analysis. There were no differences in biomarker levels between GD patients with and without TED. CONCLUSION: This study supports activation of IFN-γ inflammation and Th1 cellular immunity in GD, but also a contribution of acute-phase reactants. Our finding of no difference in systemic activation of the kynurenine pathway in GD patients with and without TED implies that the local Th1 immune response in the orbit is not reflected systemically.

Screening patients with autoimmune endocrine disorders for cytokine autoantibodies reveals monogenic immune deficiencies.

BACKGROUND: Autoantibodies against type I interferons (IFN) alpha (α) and omega (ω), and interleukins (IL) 17 and 22 are a hallmark of autoimmune polyendocrine syndrome type 1 (APS-1), caused by mutations in the autoimmune regulator (AIRE) gene. Such antibodies are also seen in a number of monogenic immunodeficiencies. OBJECTIVES: To determine whether screening for cytokine autoantibodies (anti-IFN-ω and anti-IL22) can be used to identify patients with monogenic immune disorders. METHODS: A novel ELISA assay was employed to measure IL22 autoantibodies in 675 patients with autoimmune primary adrenal insufficiency (PAI) and a radio immune assay (RIA) was used to measure autoantibodies against IFN-ω in 1778 patients with a variety of endocrine diseases, mostly of autoimmune aetiology. Positive cases were sequenced for all coding exons of the AIRE gene. If no AIRE mutations were found, we applied next generation sequencing (NGS) to search for mutations in immune related genes. RESULTS: We identified 29 patients with autoantibodies against IFN-ω and/or IL22. Of these, four new APS-1 cases with disease-causing variants in AIRE were found. In addition, we identified two patients with pathogenic heterozygous variants in CTLA4 and NFKB2, respectively. Nine rare variants in other immune genes were identified in six patients, although further studies are needed to determine their disease-causing potential. CONCLUSION: Screening of cytokine autoantibodies can efficiently identify patients with previously unknown monogenic and possible oligogenic causes of autoimmune and immune deficiency diseases. This information is crucial for providing personalised treatment and follow-up of patients and their relatives.

B Cells and Autoantibodies in AIRE Deficiency.

Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare but severe monogenetic autoimmune endocrine disease caused by failure of the Autoimmune Regulator (AIRE). AIRE regulates the negative selection of T cells in the thymus, and the main pathogenic mechanisms are believed to be T cell-mediated, but little is known about the role of B cells. Here, we give an overview of the role of B cells in thymic and peripheral tolerance in APS-1 patients and different AIRE-deficient mouse models. We also look closely into which autoantibodies have been described for this disorder, and their implications. Based on what is known about B cell therapy in other autoimmune disorders, we outline the potential of B cell therapies in APS-1 and highlight the unresolved research questions to be answered.

The prospects of single-cell analysis in autoimmunity.

In the last decade, there has been a tremendous development of technologies focused on analysing various molecular attributes in single cells, with an ever-increasing number of parameters becoming available at the DNA, RNA and protein levels. Much of this progress has involved cells in suspension, but also in situ analysis of tissues has taken great leaps. Paralleling the development in the laboratory, and because of increasing complexity, the analysis of single-cell data is also constantly being updated with new algorithms and analysis platforms. Our immune system shares this complexity, and immunologists have therefore been in the forefront of this technological development. These technologies clearly open new avenues for immunology research, maybe particularly within autoimmunity where the interaction between the faulty immune system and the thymus or the target organ is important. However, the technologies currently available can seem overwhelming and daunting. The aim of this review is to remedy this by giving a balanced overview of the prospects of using single-cell analysis in basal and clinical autoimmunity research, with an emphasis on endocrine autoimmunity.

New era of therapy for endocrine autoimmune disorders.

The new era of immune and reconstitution therapy of autoimmune disorders is ongoing. However, endocrine autoimmune diseases comprise a group of elaborating pathologies where the development of new treatment strategies remains slow. Substitution of the missing hormones is still standard practice, taking care of the devastating symptoms but not the cause of disease. As our knowledge of the genetic contribution to the aetiology of endocrine disorders increases and early diagnostic tools are available, it is now possible to identify persons at risk before they acquire full-blown disease. This review summarizes current knowledge and treatment of endocrine autoimmune disorders, focusing on type 1 diabetes, Addison's disease, autoimmune thyroid diseases and primary ovarian insufficiency. We explore which new therapies might be used in the different stages of the disease, focus on legalized therapy and elaborate on the ongoing clinical studies for these diseases and the research front, before hypothesizing on the way ahead.

Altered Immune Activation and IL-23 Signaling in Response to Candida albicans in Autoimmune Polyendocrine Syndrome Type 1.

OBJECTIVE: Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare, childhood onset disease caused by mutations in the autoimmune regulator (AIRE) gene. Chronic mucocutaneous candidiasis (CMC) is one of the three major disease components and is, to date, mainly explained by the presence of neutralizing auto-antibodies against cytokines [interleukin (IL)-17A, IL-17F, and IL-22] from T helper 17 cells, which are critical for the protection against fungal infections. However, patients without current auto-antibodies also present CMC and we, therefore, hypothesized that other immune mechanisms contribute to CMC in APS-1. METHODS: Whole blood was stimulated with Candida albicans (C. albicans) in a standardized assay, and immune activation was investigated by analyzing 46 secreted immune mediators. Then, peripheral blood mononuclear cells were stimulated with curdlan, a Dectin-1 agonist and IL-23 inducer, and the IL-23p19 response in monocytes was analyzed by flow cytometry. RESULTS: We found an altered immune response in APS-1 patients compared with healthy controls. Patients fail to increase the essential ILs, such as IL-2, IL-17A, IL-22, and IL-23, when stimulating whole blood with C. albicans. A significantly altered IL-23p19 response was detected in patients' monocytes upon stimulation with curdlan. CONCLUSION: APS-1 patients have an altered immune response to C. albicans including a dysregulation of IL-23p19 production in monocytes. This probably contributes to the selective susceptibility to CMC found in the majority of patients.

Expanding the Phenotypic and Genotypic Landscape of Autoimmune Polyendocrine Syndrome Type 1.

Context: Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare monogenic autoimmune disease caused by mutations in the autoimmune regulator (AIRE) gene and characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and primary adrenal insufficiency. Comprehensive characterizations of large patient cohorts are rare. Objective: To perform an extensive clinical, immunological, and genetic characterization of a large nationwide Russian APS-1 cohort. Subjects and Methods: Clinical components were mapped by systematic investigations, sera were screened for autoantibodies associated with APS-1, and AIRE mutations were characterized by Sanger sequencing. Results: We identified 112 patients with APS-1, which is, to the best of our knowledge, the largest cohort described to date. Careful phenotyping revealed several additional and uncommon phenotypes such as cerebellar ataxia with pseudotumor, ptosis, and retinitis pigmentosa. Neutralizing autoantibodies to interferon-ω were found in all patients except for one. The major Finnish mutation c.769C>T (p.R257*) was the most frequent and was present in 72% of the alleles. Altogether, 19 different mutations were found, of which 9 were unknown: c.38T>C (p.L13P), c.173C>T (p.A58V), c.280C>T (p.Q94*), c.554C>G (p.S185*), c.661A>T (p.K221*), c.821del (p.Gly274Afs*104), c.1195G>C (p.A399P), c.1302C>A (p.C434*), and c.1497del (p.A500Pfs*21). Conclusions: The spectrum of phenotypes and AIRE mutation in APS-1 has been expanded. The Finnish major mutation is the most common mutation in Russia and is almost as common as in Finland. Assay of interferon antibodies is a robust screening tool for APS-1.

Autoantibody Repertoire in APECED Patients Targets Two Distinct Subgroups of Proteins.

High titer autoantibodies produced by B lymphocytes are clinically important features of many common autoimmune diseases. APECED patients with deficient autoimmune regulator (AIRE) gene collectively display a broad repertoire of high titer autoantibodies, including some which are pathognomonic for major autoimmune diseases. AIRE deficiency severely reduces thymic expression of gene-products ordinarily restricted to discrete peripheral tissues, and developing T cells reactive to those gene-products are not inactivated during their development. However, the extent of the autoantibody repertoire in APECED and its relation to thymic expression of self-antigens are unclear. We here undertook a broad protein array approach to assess autoantibody repertoire in APECED patients. Our results show that in addition to shared autoantigen reactivities, APECED patients display high inter-individual variation in their autoantigen profiles, which collectively are enriched in evolutionarily conserved, cytosolic and nuclear phosphoproteins. The APECED autoantigens have two major origins; proteins expressed in thymic medullary epithelial cells and proteins expressed in lymphoid cells. These findings support the hypothesis that specific protein properties strongly contribute to the etiology of B cell autoimmunity.

Impaired salivary gland activity in patients with autoimmune polyendocrine syndrome type I.

Autoimmune polyendocrine syndrome type I (APS-I) is a severe disease caused by mutations in the autoimmune regulator (AIRE) gene. We hypothesized that salivary gland dysfunction could be a possible unexplored component of these patients and here aimed to investigate salivary and lachrymal symptoms in the Norwegian cohort of APS-I patients (N = 41) and the aetiology behind it. Sicca symptoms and possible corresponding underlying factors were assessed by subjective reports combined with objective measures of saliva and tear flow, serological testing, immune fluorescence microscopy, ultrasonography and searching for putative autoantibodies in the salivary glands. In addition, defensin and anti-defensin levels were analysed in patients and compared with healthy controls. Our results indicate mild salivary and/or lachrymal gland dysfunction manifesting in low saliva or tear flow in a total of 62% of APS-I patients. Serum IgG from 9 of 12 patients bound to targets in salivary gland biopsy slides, although the specificity and pattern of binding varied. There was no reactivity against known Sjögren-associated autoantigens in sera from APS-I patients using quantitative methods, but 11% were ANA positive by immunofluorescence microscopy. We identified several putative autoantigens in one patient, although none of these were verified as APS-I specific. We conclude that impaired salivary gland activity is part of the clinical picture of APS-I and our findings could indicate an autoimmune aetiology. We further show that APS-I patients have an altered antimicrobial signature in both sera and saliva, which requires further investigations.

AIRE-Deficient Patients Harbor Unique High-Affinity Disease-Ameliorating Autoantibodies.

APS1/APECED patients are defined by defects in the autoimmune regulator (AIRE) that mediates central T cell tolerance to many self-antigens. AIRE deficiency also affects B cell tolerance, but this is incompletely understood. Here we show that most APS1/APECED patients displayed B cell autoreactivity toward unique sets of approximately 100 self-proteins. Thereby, autoantibodies from 81 patients collectively detected many thousands of human proteins. The loss of B cell tolerance seemingly occurred during antibody affinity maturation, an obligatorily T cell-dependent step. Consistent with this, many APS1/APECED patients harbored extremely high-affinity, neutralizing autoantibodies, particularly against specific cytokines. Such antibodies were biologically active in vitro and in vivo, and those neutralizing type I interferons (IFNs) showed a striking inverse correlation with type I diabetes, not shown by other anti-cytokine antibodies. Thus, naturally occurring human autoantibodies may actively limit disease and be of therapeutic utility.

Hypomagnesemia and functional hypoparathyroidism due to novel mutations in the Mg-channel TRPM6.

Primary hypomagnesemia with secondary hypocalcemia (HSH) is an autosomal recessive disorder characterized by neuromuscular symptoms in infancy due to extremely low levels of serum magnesium and moderate to severe hypocalcemia. Homozygous mutations in the magnesium transporter gene transient receptor potential cation channel member 6 (TRPM6) cause the disease. HSH can be misdiagnosed as primary hypoparathyroidism. The aim of this study was to describe the genetic, clinical and biochemical features of patients clinically diagnosed with HSH in a Norwegian cohort. Five patients in four families with clinical features of HSH were identified, including one during a national survey of hypoparathyroidism. The clinical history of the patients and their families were reviewed and gene analyses of TRPM6 performed. Four of five patients presented with generalized seizures in infancy and extremely low levels of serum magnesium accompanied by moderate hypocalcemia. Two of the patients had an older sibling who died in infancy. Four novel mutations and one large deletion in TRPM6 were identified. In one patient two linked homozygous mutations were located in exon 22 (p.F978L) and exon 23 (p.G1042V). Two families had an identical mutation in exon 25 (p.E1155X). The fourth patient had a missense mutation in exon 4 (p.H61N) combined with a large deletion in the C-terminal end of the gene. HSH is a potentially lethal condition that can be misdiagnosed as primary hypoparathyroidism. The diagnosis is easily made if serum magnesium is measured. When treated appropriately with high doses of oral magnesium supplementation, severe hypomagnesemia is uncommon and the long-term prognosis seems to be good.