The immunopathological landscape of human pre-TCRα deficiency: From rare to common variants.

We describe humans with rare biallelic loss-of-function PTCRA variants impairing pre-α T cell receptor (pre-TCRα) expression. Low circulating naive αß T cell counts at birth persisted over time, with normal memory αß and high γδ T cell counts. Their TCRα repertoire was biased, which suggests that noncanonical thymic differentiation pathways can rescue αß T cell development. Only a minority of these individuals were sick, with infection, lymphoproliferation, and/or autoimmunity. We also report that 1 in 4000 individuals from the Middle East and South Asia are homozygous for a common hypomorphic PTCRA variant. They had normal circulating naive αß T cell counts but high γδ T cell counts. Although residual pre-TCRα expression drove the differentiation of more αß T cells, autoimmune conditions were more frequent in these patients compared with the general population.

Rituximab and improved nodular regenerative hyperplasia-associated non-cirrhotic liver disease in common variable immunodeficiency: a case report and literature study.

Common variable immunodeficiency (CVID) associated liver disease is an underrecognized and poorly studied non-infectious complication that lacks an established treatment. We describe a CVID patient with severe multiorgan complications, including non-cirrhotic portal hypertension secondary to nodular regenerative hyperplasia leading to diuretic-refractory ascites. Remarkably, treatment with rituximab, administered for concomitant immune thrombocytopenia, resulted in the complete and sustained resolution of portal hypertension and ascites. Our case, complemented with a literature review, suggests a beneficial effect of rituximab that warrants further research.

CTLA4-Ig Effectively Controls Clinical Deterioration and Immune Condition in a Murine Model of Foxp3 Deficiency.

PURPOSE: FOXP3 deficiency results in severe multisystem autoimmunity in both mice and humans, driven by the absence of functional regulatory T cells. Patients typically present with early and severe autoimmune polyendocrinopathy, dermatitis, and severe inflammation of the gut, leading to villous atrophy and ultimately malabsorption, wasting, and failure to thrive. In the absence of successful treatment, FOXP3-deficient patients usually die within the first 2 years of life. Hematopoietic stem cell transplantation provides a curative option but first requires adequate control over the inflammatory condition. Due to the rarity of the condition, no clinical trials have been conducted, with widely unstandardized therapeutic approaches. We sought to compare the efficacy of lead therapeutic candidates rapamycin, anti-CD4 antibody, and CTLA4-Ig in controlling the physiological and immunological manifestations of Foxp3 deficiency in mice. METHOD: We generated Foxp3-deficient mice and an appropriate clinical scoring system to enable direct comparison of lead therapeutic candidates rapamycin, nondepleting anti-CD4 antibody, and CTLA4-Ig. RESULTS: We found distinct immunosuppressive profiles induced by each treatment, leading to unique protective combinations over distinct clinical manifestations. CTLA4-Ig provided superior breadth of protective outcomes, including highly efficient protection during the transplantation process. CONCLUSION: These results highlight the mechanistic diversity of pathogenic pathways initiated by regulatory T cell loss and suggest CTLA4-Ig as a potentially superior therapeutic option for FOXP3-deficient patients.

Case report: Myocarditis in congenital STAT1 gain-of function.

Autosomal dominant Signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) mutations result in an inborn error of immunity characterized by chronic mucocutaneous candidiasis, recurrent viral and bacterial infections, and diverse autoimmune manifestations. Current treatment consists of chronic antifungal therapy, antibiotics for concomitant infections, and immunosuppressive therapy in case of autoimmune diseases. More recently, treatment with Janus kinases 1 and 2 (JAK1/2) inhibitors have shown promising yet variable results. We describe a STAT1 GOF patient with an incidental finding of elevated cardiac troponins, leading to a diagnosis of a longstanding, slowly progressive idiopathic myocarditis, attributed to STAT1 GOF. Treatment with a JAK-inhibitor (baricitinib) mitigated cardiac inflammation on MRI but was unable to alter fibrosis, possibly due to the diagnostic and therapeutic delay, which finally led to fatal arrhythmia. Our case illustrates that myocarditis could be part of the heterogeneous disease spectrum of STAT1 GOF. Given the insidious presentation in our case, a low threshold for cardiac evaluation in STAT1 GOF patients seems warranted.

Homozygous DBF4 mutation as a cause of severe congenital neutropenia.

BACKGROUND: Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. OBJECTIVE: We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. METHODS: Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. RESULTS: We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. CONCLUSION: Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

Disrupted Ca2+ homeostasis and immunodeficiency in patients with functional IP3 receptor subtype 3 defects.

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca2+) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP3R), a homo- or heterotetramer of the IP3R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP3R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP3R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP3R3 in IP3R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca2+ channels and immunodeficiency and identify IP3Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca2+-associated immunodeficiency from store-operated entry to impaired Ca2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca2+ signaling.

DNA replication-associated inborn errors of immunity.

Inborn errors of immunity are a heterogeneous group of monogenic immunologic disorders caused by mutations in genes with critical roles in the development, maintenance, or function of the immune system. The genetic basis is frequently a mutation in a gene with restricted expression and/or function in immune cells, leading to an immune disorder. Several classes of inborn errors of immunity, however, result from mutation in genes that are ubiquitously expressed. Despite the genes participating in cellular processes conserved between cell types, immune cells are disproportionally affected, leading to inborn errors of immunity. Mutations in DNA replication, DNA repair, or DNA damage response factors can result in monogenic human disease, some of which are classified as inborn errors of immunity. Genetic defects in the DNA repair machinery are a well-known cause of T-B-NK+ severe combined immunodeficiency. An emerging class of inborn errors of immunity is those caused by mutations in DNA replication factors. Considerable heterogeneity exists within the DNA replication-associated inborn errors of immunity, with diverse immunologic defects and clinical manifestations observed. These differences are suggestive for differential sensitivity of certain leukocyte subsets to deficiencies in specific DNA replication factors. Here, we provide an overview of DNA replication-associated inborn errors of immunity and discuss the emerging mechanistic insights that can explain the observed immunologic heterogeneity.

Common variable immunodeficiency in two kindreds with heterogeneous phenotypes caused by novel heterozygous NFKB1 mutations.

NFKB1 haploinsufficiengcy was first described in 2015 in three families with common variable immunodeficiency (CVID), presenting heterogeneously with symptoms of increased infectious susceptibility, skin lesions, malignant lymphoproliferation and autoimmunity. The described mutations all led to a rapid degradation of the mutant protein, resulting in a p50 haploinsufficient state. Since then, more than 50 other mutations have been reported, located throughout different domains of NFKB1 with the majority situated in the N-terminal Rel homology domain (RHD). The clinical spectrum has also expanded with possible disease manifestations in almost any organ system. In silico prediction tools are often used to estimate the pathogenicity of NFKB1 variants but to prove causality between disease and genetic findings, further downstream functional validation is required. In this report, we studied 2 families with CVID and two novel variants in NFKB1 (c.1638-2A>G and c.787G>C). Both mutations affected mRNA and/or protein expression of NFKB1 and resulted in excessive NLRP3 inflammasome activation in patient macrophages and upregulated interferon stimulated gene expression. Protein-protein interaction analysis demonstrated a loss of interaction with NFKB1 interaction partners for the p.V263L mutation. In conclusion, we proved pathogenicity of two novel variants in NFKB1 in two families with CVID characterized by variable and incomplete penetrance.

A Novel Homozygous Stop Mutation in IL23R Causes Mendelian Susceptibility to Mycobacterial Disease.

PURPOSE: Mendelian susceptibility to mycobacterial disease (MSMD) is caused by inborn errors of IFN-γ immunity. The most frequent genetic defects are found in IL12 or a subunit of its receptor. IL23R deficiency in MSMD has only been reported once, in two pediatric patients from the same kindred with isolated disseminated Bacille Calmette-Guérin disease. We evaluated the impact of a homozygous stop mutation in IL23R (R381X), identified by whole exome sequencing, in an adult patient with disseminated non-tuberculous mycobacterial disease. METHODS: We performed functional validation of the R381X mutation by evaluating IL23R expression and IL-23 signaling (STAT3 phosphorylation, IFN-γ production) in primary cells (PBMCs, EBV-B cells) and cell lines (HeLa) with or without back-complementation of wild-type IL23R. RESULTS: We report on a 48-year-old male with disseminated non-tuberculous mycobacterial disease. We identified and characterized a homozygous loss-of-function stop mutation underlying IL23R deficiency, resulting in near absent expression of membrane bound IL23R. IL23R deficiency was characterized by impaired IL-23-mediated IFN-γ secretion in CD4+, CD8+ T, and mucosal-associated invariant T (MAIT) cells, and low frequencies of circulating Th17 (CD3+CD45RA-CCR4+CXCR3-RORγT+), Th1* (CD45RA-CCR4-CXCR3+RORγT+), and MAIT (CD3+CD8+Vα7.2+CD161+) cells. Although the patient did not have a history of recurrent fungal infections, impaired Th17 differentiation and blunted IL-23-mediated IL-17 secretion in PBMCs were observed. CONCLUSION: We demonstrate that impaired IL-23 immunity caused by a homozygous R381X mutation in IL23R underlies MSMD, corroborating earlier findings with a homozygous p.C115Y IL23R mutation. Our report further supports a model of redundant contribution of IL-23- to IL-17-mediated anti-fungal immunity.1.

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal α-toxin.

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor κB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor α-toxin. Naturally elicited antibodies against α-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to α-toxin in nonleukocytic cells.

Lack of NKG2D in MAGT1-deficient patients is caused by hypoglycosylation.

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG.

Monogenic Adult-Onset Inborn Errors of Immunity.

Inborn errors of immunity (IEI) are a heterogenous group of disorders driven by genetic defects that functionally impact the development and/or function of the innate and/or adaptive immune system. The majority of these disorders are thought to have polygenic background. However, the use of next-generation sequencing in patients with IEI has led to an increasing identification of monogenic causes, unravelling the exact pathophysiology of the disease and allowing the development of more targeted treatments. Monogenic IEI are not only seen in a pediatric population but also in adulthood, either due to the lack of awareness preventing childhood diagnosis or due to a delayed onset where (epi)genetic or environmental factors can play a role. In this review, we discuss the mechanisms accounting for adult-onset presentations and provide an overview of monogenic causes associated with adult-onset IEI.

Unstable regulatory T cells, enriched for naïve and Nrp1neg cells, are purged after fate challenge.

Regulatory T cells (Tregs) are indispensable for the control of immune homeostasis and have clinical potential as a cell therapy for treating autoimmunity. Tregs can lose expression of the lineage-defining Foxp3 transcription factor and acquire effector T cell (Teff) characteristics, a process referred to as Treg plasticity. The extent and reversibility of such plasticity during immune responses remain unknown. Here, using a murine genetic fate-mapping system, we show that Treg stability is maintained even during exposure to a complex microbial/antigenic environment. Furthermore, we demonstrate that the observed plasticity of Tregs after adoptive transfer into a lymphopenic environment is a property limited to only a subset of the Treg population, with the nonconverting majority of Tregs being resistant to plasticity upon secondary stability challenge. The unstable Treg fraction is a complex mixture of phenotypically distinct Tregs, enriched for naïve and neuropilin-1-negative Tregs, and includes peripherally induced Tregs and recent thymic emigrant Tregs These results suggest that a "purging" process can be used to purify stable Tregs that are capable of robust fate retention, with potential implications for improving cell transfer therapy.

Case Report: VEXAS Syndrome: From Mild Symptoms to Life-Threatening Macrophage Activation Syndrome.

Recently, a novel disorder coined VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome was identified in patients with adult-onset inflammatory syndromes, often accompanied by myelodysplastic syndrome1. All patients had myeloid lineage-restricted somatic mutations in UBA1 affecting the Met41 residue of the protein and resulting in decreased cellular ubiquitylation activity and hyperinflammation. We here describe the clinical disease course of two VEXAS syndrome patients with somatic UBA1 mutations of which one with a mild phenotype characterized by recurrent rash and symmetric polyarthritis, and another who was initially diagnosed with idiopathic multicentric Castleman disease and developed macrophage activation syndrome as a complication of the VEXAS syndrome. The latter patients was treated with anti-IL6 therapy (siltuximab) leading to a resolution of systemic symptoms and reduction of transfusion requirements.

Systemic autoinflammatory disease in adults.

Systemic autoinflammatory disorders comprise an expanding group of rare conditions. They are mediated by dysfunction of the innate immune system and share a core of phenotypic manifestations including recurrent attacks of fever, cutaneous signs, chest or abdominal pain, lymphadenopathy, vasculopathy, and musculoskeletal symptoms. Diagnosis is often established in childhood, but a growing number of adult patients are being recognized with systemic autoinflammatory disorders, including adult-onset disease. In this review, we provide a concise update on the pathophysiology, clinical presentation, and diagnostic approach of systemic autoinflammatory disorders with an emphasis on the adult patient population. Despite the recent advances in genetic testing, the diagnosis of autoinflammatory disease in adult patients is often based on a thorough knowledge of the clinical phenotype. Becoming acquainted with the clinical features of these rare disorders may assist in developing a high index of suspicion for autoinflammatory disease in patients presenting with unexplained episodes of fever or inflammation.

Adult-Onset ANCA-Associated Vasculitis in SAVI: Extension of the Phenotypic Spectrum, Case Report and Review of the Literature.

STING-associated vasculopathy with onset in infancy (SAVI) is an autosomal dominant disorder due to gain-of-function mutations in STING1, also known as TMEM173, encoding for STING. It was reported as a vasculopathy of infancy. However, since its description a wider spectrum of associated manifestations and disease-onset has been observed. We report a kindred with a heterozygous STING mutation (p.V155M) in which the 19-year-old proband suffered from isolated adult-onset ANCA-associated vasculitis. His father suffered from childhood-onset pulmonary fibrosis and renal failure attributed to ANCA-associated vasculitis, and died at the age of 30 years due to respiratory failure. In addition, an overview of the phenotypic spectrum of SAVI is provided highlighting (a) a high phenotypic variability with in some cases isolated manifestations, (b) the potential of adult-onset disease, and (c) a novel manifestation with ANCA-associated vasculitis.