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Purpose: PI4KA-related disorder is a highly clinically variable condition characterized by neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus) and gastrointestinal (inflammatory bowel disease and multiple intestinal atresia) manifestations. Although features consistent with immunodeficiency (autoimmunity/autoinflammation and recurrent infections) have been reported in a subset of patients, the burden of B-cell deficiency and hypogammaglobulinemia has not been extensively investigated. We sought to describe the clinical presentation and manifestations of patients with PI4KA-related disorder and to investigate the metabolic consequences of biallelic PI4KA variants in B cells., Methods: Clinical data from patients with PI4KA variants were obtained. Multi-omics analyses combining transcriptome, proteome, lipidome and metabolome analyses in conjunction with functional assays were performed in EBV-transformed B cells., Results: Clinical and laboratory data of 13 patients were collected. Recurrent infections (7/13), autoimmune/autoinflammatory manifestations (5/13), B-cell deficiency (8/13) and hypogammaglobulinemia (8/13) were frequently observed. Patients' B cells frequently showed increased transitional and decreased switched memory B-cell subsets. Pathway analyses based on differentially expressed transcripts and proteins confirmed the central role of PI4KA in B cell differentiation with altered B-cell receptor (BCR) complex and signalling. By altering lipids production and tricarboxylic acid cycle regulation, and causing increased endoplasmic reticulum stress, biallelic PI4KA mutations disrupt B cell metabolism inducing mitochondrial dysfunction. As a result, B cells show hyperactive PI3K/mTOR pathway, increased autophagy and deranged cytoskeleton organization., Conclusion: By altering lipid metabolism and TCA cycle, impairing mitochondrial activity, hyperactivating mTOR pathway and increasing autophagy, PI4KA-related disorder causes a syndromic inborn error of immunity presenting with B-cell deficiency and hypogammaglobulinemia., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Common variable immune deficiency (CVID) is a heterogenous group of disorders characterized by varying degrees of hypogammaglobulinemia, recurrent infections, and autoimmunity. Currently, pathogenic variants are identified in approximately 20-30% of CVID cases. Here we report a 3-generation family with autosomal dominant Common Variable Immunodeficiency (CVID) diagnosed in 9 affected individuals. Although primary immune deficiency panels and exome sequencing were non-diagnostic, whole genome sequencing revealed a novel, pathogenic c.499C > T: p.His167Tyr variant in IKZF1, a critical regulator of B cell development. Functional testing done through pericentromeric heterochromatin localization and light shift chemiluminescent electrophoretic mobility shift assay confirmed the variant's deleterious effect via a haploinsufficiency mechanism. Our findings expand the spectrum of known IKZF1 mutations and contribute to a more comprehensive understanding of CVID's genetic heterogeneity. Furthermore, this case underscores the importance of considering whole genome sequencing for comprehensive genetic diagnosis when concern for a monogenic inborn errors of immunity is high., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.)., (Copyright © 2024 Massachusetts Medical Society.)

Heterozygous germline variants in human IKZF1 encoding for IKAROS define an inborn error of immunity with immunodeficiency, immune dysregulation and risk of malignancy with a broad phenotypic spectrum. Growing evidence of underlying pathophysiological genotype-phenotype correlations helps to improve our understanding of IKAROS-associated diseases. We describe 6 patients from 4 kindreds with two novel IKZF1 variants leading to haploinsufficiency from 3 centers in Germany. We also provide an overview of first symptoms to a final diagnosis including data from the literature., Competing Interests: Author AS and HB were employed by the company Labor Berlin Charité-Vivantes GmbH and author MF was employed by the company Hospital St. Georg GGmbH Leipzig. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (© 2024 Strauss, Körholz, Kuehn, Gil Silva, Taube, Trautmann-Grill, Stittrich, Pietzsch, Wiedemuth, Wahn, von Bernuth, Rosenzweig, Fasshauer, Krüger and Schuetz.)

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

IKAROS, encoded by IKZF1, is a tumor suppressor and a key hematopoietic transcription factor responsible for lymphoid and myeloid differentiation. IKZF1 mutations result in inborn errors of immunity presenting with increased susceptibility to infections, immune dysregulation, and malignancies. In particular, patients carrying IKZF1 gain-of-function (GOF) mutations mostly exhibit symptoms of immune dysregulation and polyclonal plasma cell proliferation. Herein, we describe seven new IKAROS GOF cases from two unrelated families, presenting with novel infectious, immune dysregulation and hematologic diseases. Two of the patients underwent allogeneic hematopoietic cell transplantation (HCT) due to poorly responsive complications. HCT was well-tolerated achieving full engraftment in both patients receiving reduced intensity, matched unrelated donor grafts, with no severe acute or chronic graft-vs-host-disease, and in remission from their diseases 2.5 and 4 years post-HCT, respectively. These results suggest that HCT is a valid and curative option in patients with IKAROS GOF disease and severe clinical manifestations., Competing Interests: Declaration of competing interest The authors have no conflicting financial interests., (Copyright © 2024. Published by Elsevier Inc.)

AIOLOS, also known as IKZF3, is a transcription factor that is highly expressed in the lymphoid lineage and is critical for lymphocyte differentiation and development. Here, we report on 9 individuals from 3 unrelated families carrying AIOLOS variants Q402* or E82K, which led to AIOLOS haploinsufficiency through different mechanisms of action. Nonsense mutant Q402* displayed abnormal DNA binding, pericentromeric targeting, posttranscriptional modification, and transcriptome regulation. Structurally, the mutant lacked the AIOLOS zinc finger (ZF) 5-6 dimerization domain, but was still able to homodimerize with WT AIOLOS and negatively regulate DNA binding through ZF1, a previously unrecognized function for this domain. Missense mutant E82K showed overall normal AIOLOS functions; however, by affecting a redefined AIOLOS protein stability domain, it also led to haploinsufficiency. Patients with AIOLOS haploinsufficiency showed hypogammaglobulinemia, recurrent infections, autoimmunity, and allergy, but with incomplete clinical penetrance. Altogether, these data redefine the AIOLOS structure-function relationship and expand the spectrum of AIOLOS-associated diseases.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs) 1,2 , conferring a predisposition to life-threatening COVID-19 pneumonia 3 . Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52 LOF /IκBδ GOF ). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52 LOF /IκBδ LOF ) or gain-of-function of p52 (hereafter, p52 GOF /IκBδ LOF ). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases., (© 2023. The Author(s).)

Background: TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance., Objective: We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency., Methods: Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping., Results: Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating B cells but overall normal humoral immune responses., Conclusion: Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3 +/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice., (Published by Elsevier Inc.)

IKAROS/IKZF1 plays a pivotal role in lymphocyte differentiation and development. Germline mutations in IKZF1, which have been shown to be associated with primary immunodeficiency, can be classified through four different mechanisms of action depending on the protein expression and its functional defects: haploinsufficiency, dimerization defective, dominant negative, and gain of function. These different mechanisms are associated with variable degrees of susceptibility to infectious diseases, autoimmune disorders, allergic diseases, and malignancies. To date, more than 30 heterozygous IKZF1 germline variants have been reported in patients with primary immunodeficiency. Here we review recent discoveries and clinical/immunological characterization of IKAROS-associated diseases that are linked to different mechanisms of action in IKAROS function., (Published by Oxford University Press on behalf of the British Society for Immunology 2022.)

Purpose: Biallelic loss-of-function variants in IKBKB cause severe combined immunodeficiency. We describe a case of autoimmunity and autoinflammation in a male infant with a heterozygous gain-of-function (GOF) IKBKB variant., Methods: Case report and review of the literature. We performed in silico variant analysis, measurement of plasma soluble biomarkers associated with immune activation, functional stimulation of patient peripheral blood mononuclear cells, and functional validation of variants transduced in Jurkat cells., Results: A patient with two heterozygous IKBKB variants (E518K and T559M) presents with previously undescribed autoimmune cytopenias and autoinflammation. He had decreased TNF-α-induced IkBα degradation in vitro, and had increased serum biomarkers associated with macrophage recruitment and activation. Jurkat cells transduced with the IKKb T559M variant showed increased basal levels of phosphorylation of IKKα/b and p65, and higher degradation of IkBα suggesting a GOF mechanism. No significant changes were observed in Jurkat cells transduced with the E518K variant., Conclusions: A GOF variant in IKBKB may associate with autoinflammation and autoimmunity highlighting a novel clinical phenotype., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Interferon regulatory factor 4 (IRF4) is a transcription factor (TF) and key regulator of immune cell development and function. We report a recurrent heterozygous mutation in IRF4, p.T95R, causing an autosomal dominant combined immunodeficiency (CID) in seven patients from six unrelated families. The patients exhibited profound susceptibility to opportunistic infections, notably Pneumocystis jirovecii , and presented with agammaglobulinemia. Patients' B cells showed impaired maturation, decreased immunoglobulin isotype switching, and defective plasma cell differentiation, whereas their T cells contained reduced T H 17 and T FH populations and exhibited decreased cytokine production. A knock-in mouse model of heterozygous T95R showed a severe defect in antibody production both at the steady state and after immunization with different types of antigens, consistent with the CID observed in these patients. The IRF4 T95R variant maps to the TF's DNA binding domain, alters its canonical DNA binding specificities, and results in a simultaneous multimorphic combination of loss, gain, and new functions for IRF4. IRF4 T95R behaved as a gain-of-function hypermorph by binding to DNA with higher affinity than IRF4 WT . Despite this increased affinity for DNA, the transcriptional activity on IRF4 canonical genes was reduced, showcasing a hypomorphic activity of IRF4 T95R . Simultaneously, IRF4 T95R functions as a neomorph by binding to noncanonical DNA sites to alter the gene expression profile, including the transcription of genes exclusively induced by IRF4 T95R but not by IRF4 WT . This previously undescribed multimorphic IRF4 pathophysiology disrupts normal lymphocyte biology, causing human disease.

Objective: To explore and define the molecular cause(s) of a multi-generational kindred affected by Bechet's-like mucocutaneous ulcerations and immune dysregulation., Methods: Whole genome sequencing and confirmatory Sanger sequencing were performed. Components of the NFκB pathway were quantified by immunoblotting, and function was assessed by cytokine production (IL-6, TNF-α, IL-1β) after lipopolysaccharide (LPS) stimulation. Detailed immunophenotyping of T-cell and B-cell subsets was performed in four patients from this cohort., Results: A novel variant in the RELA gene, p. Tyr349LeufsTer13, was identified. This variant results in premature truncation of the protein before the serine (S) 536 residue, a key phosphorylation site, resulting in enhanced degradation of the p65 protein. Immunoblotting revealed significantly decreased phosphorylated [p]p65 and pIκBα. The decrease in [p]p65 may suggest reduced heterodimer formation between p50/p65 (NFκB1/RelA). Immunophenotyping revealed decreased naïve T cells, increased memory T cells, and expanded senescent T-cell populations in one patient (P1). P1 also had substantially higher IL-6 and TNF-α levels post-stimulation compared with the other three patients., Conclusion: Family members with this novel RELA variant have a clinical phenotype similar to other reported RELA cases with predominant chronic mucocutaneous ulceration; however, the clinical phenotype broadens to include Behçet's syndrome and IBD. Here we describe the clinical, immunological and genetic evaluation of a large kindred to further expand identification of patients with autosomal dominant RELA deficiency, facilitating earlier diagnosis and intervention. The functional impairment of the canonical NFκB pathway suggests that this variant is causal for the clinical phenotype in these patients., (© The Author(s) 2022. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations., Objective: We sought to explore the pathophysiology and the underlying mechanisms of TNF-inhibitor response in these patients., Methods: We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in patients with DADA2 and studied their response to TNF-inhibitor treatment., Results: We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and nuclear factor kappa B pathways in patients' primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation toward the proinflammatory M1 macrophage subset, and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2., Conclusions: Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells, and the endothelial cell damage is rescued with anti-TNF treatment., (Published by Elsevier Inc.)

Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3 encoding Ikaros and Aiolos have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NK cells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of proinflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS-mediated epigenetic and transcriptional dysregulation., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

IKZF1/IKAROS is a key transcription factor of lymphocyte development expressed throughout hematopoiesis. Heterozygous germline IKZF1 haploinsufficient ( IKZF1 HI ) and dominant-negative ( IKZF1 DN ) variants in humans cause B cell immune deficiency and combined immunodeficiency. Here, we identified previously unidentified heterozygous IKZF1 variants (R183C/H) located in the DNA binding domain in eight individuals with inflammatory, autoimmune, allergic symptoms, and abnormal plasma cell (PC) proliferation. Leukocytes of patients exhibited specific defects including impaired IL-2 production by T cells, T helper (T H ) skewing toward T H 2, low numbers of regulatory T cells (T reg ), eosinophilia, and abnormal PC proliferation. In contrast to IKZF1 HI and IKZF1 DN , IKZF1 R183H/C proteins showed increased DNA binding associated with increased gene expression of T H 2 and PC differentiation, thus demonstrating that IKZF1 R183H/C behave as gain-of-function (GOF) alleles. In vitro treatment with lenalidomide, known to degrade IKZF1, corrected T H 2 and PC abnormalities caused by IKZF1 R183H/C . These data extend the spectrum of pathological mechanisms associated with IKZF1 deficiencies and highlight the role of IKZF1 in late lymphoid differentiation stages.

Background: CARD9 deficiency is an autosomal recessive primary immunodeficiency underlying increased susceptibility to fungal infection primarily presenting as invasive CNS Candida and/or cutaneous/invasive dermatophyte infections. More recently, a rare heterozygous dominant negative CARD9 variant c.1434 + 1G > C was reported to be protective from inflammatory bowel disease., Objective: We studied two siblings carrying homozygous CARD9 variants (c.1434 + 1G > C) and born to heterozygous asymptomatic parents. One sibling was asymptomatic and the other presented with candida esophagitis, upper respiratory infections, hypogammaglobulinemia, and low class-switched memory B cells., Methods and Results: The CARD9 c.1434 + 1G > C variant generated two mutant transcripts confirmed by mRNA and protein expression: an out-of-frame c.1358-1434 deletion/ ~ 55 kDa protein (CARD9Δex.11) and an in-frame c.1417-1434 deletion/ ~ 61 kDa protein (CARD9Δ18 nt.). Neither transcript was able to form a complete/functional CBM complex, which includes TRIM62. Based on the index patient's CVID-like phenotype, CARD9 expression was tested and detected in lymphocytes and monocytes from humans and mice. The functional impact of different CARD9 mutations and gene dosage conditions was evaluated in heterozygous and homozygous c.1434 + 1 G > C members of the index family, and in WT (two WT alleles), haploinsufficiency (one WT, one null allele), and null (two null alleles) individuals. CARD9 gene dosage impacted lymphocyte and monocyte functions including cytokine generation, MAPK activation, T-helper commitment, transcription, plasmablast differentiation, and immunoglobulin production in a differential manner., Conclusions: CARD9 exon 11 integrity is critical to CBM complex function. CARD9 is expressed and affects particular T and B cell functions in a gene dosage-dependent manner, which in turn may contribute to the phenotype of CARD9 deficiency., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: Pediatric endogenous Cushing syndrome (eCs) is mainly caused by pituitary corticotropin-producing adenomas, and most glucocorticoid-dependent effects progressively regress upon tumor removal. eCs reproduces long-term, high-dose glucocorticoid therapy, representing a clean, natural, and unbiased model in which to study glucocorticoid bona fide effects on immunity., Objective: We performed extensive immunologic studies in otherwise healthy pediatric patients with eCs before and 6 to 13 months after tumor resection, as well as in in vitro glucocorticoid-treated control cells., Methods: Flow cytometry, immunoblotting, enzyme-linked immunosorbent assay, real-time quantitative PCR, and RNA-Seq techniques were used to characterize patients' and in vitro glucocorticoid treated cells., Results: Reduced thymic output, decreased naive T cells, diminished proliferation, and increased T-cell apoptosis were detected before surgery; all these defects eventually normalized after tumor removal in patients. In vitro studies also showed increased T-cell apoptosis, with correspondingly diminished NF-κB signaling and IL-21 levels. In this setting, IL-21 addition upregulated antiapoptotic BCL2 expression and rescued T-cell apoptosis in a PI3K pathway-dependent manner. Similar and reproducible findings were confirmed in eCs patient cells as well., Conclusions: We identified decreased thymic output and lymphocyte proliferation, together with increased apoptosis, as the underlying causes to T-cell lymphopenia in eCs patients. IL-21 was decreased in both natural and in vitro long-term, high-dose glucocorticoid environments, and in vitro addition of IL-21 counteracted the proapoptotic effects of glucocorticoid therapy. Thus, our results suggest that administration of IL-21 in patients receiving long-term, high-dose glucocorticoid therapy may contribute to ameliorate lymphopenia and the complications associated to it., (Published by Elsevier Inc.)

AIOLOS/IKZF3 is a member of the IKAROS family of transcription factors. IKAROS/IKZF1 mutations have been previously associated with different forms of primary immunodeficiency. Here we describe a novel combined immunodeficiency due to an IKZF3 mutation in a family presenting with T and B cell involvement, Pneumocystis jirovecii pneumonia, and/or chronic lymphocytic leukemia. Patients carrying the AIOLOS p.N160S heterozygous variant displayed impaired humoral responses, abnormal B cell development (high percentage of CD21low B cells and negative CD23 expression), and abrogated CD40 responses. Naive T cells were increased, T cell differentiation was abnormal, and CD40L expression was dysregulated. In vitro studies demonstrated that the mutant protein failed DNA binding and pericentromeric targeting. The mutant was fully penetrant and had a dominant-negative effect over WT AIOLOS but not WT IKAROS. The human immunophenotype was recapitulated in a murine model carrying the corresponding human mutation. As demonstrated here, AIOLOS plays a key role in T and B cell development in humans, and the particular gene variant described is strongly associated with immunodeficiency and likely malignancy., Competing Interests: Disclosures:   C.M. Dutmer reported personal fees from Horizon Therapeutics and personal fees from Enzyvant Therapeutics outside the submitted work. No other disclosures were reported., (© 2021 Kuehn et al.)

Helios, a member of the Ikaros family of transcription factors, is predominantly expressed in developing thymocytes, activated T cells, and regulatory T cells (T regs ). Studies in mice have emphasized its role in maintenance of T reg immunosuppressive functions by stabilizing Foxp3 expression and silencing the Il2 locus. However, its contribution to human immune homeostasis and the precise mechanisms by which Helios regulates other T cell subsets remain unresolved. Here, we investigated a patient with recurrent respiratory infections and hypogammaglobulinemia and identified a germline homozygous missense mutation in IKZF2 encoding Helios (p.Ile325Val). We found that Helios I325V retains DNA binding and dimerization properties but loses interaction with several partners, including epigenetic remodelers. Whereas patient T regs showed increased IL-2 production, patient conventional T cells had decreased accessibility of the IL2 locus and consequently reduced IL-2 production. Reduced chromatin accessibility was not exclusive to the IL2 locus but involved a variety of genes associated with T cell activation. Single-cell RNA sequencing of peripheral blood mononuclear cells revealed gene expression signatures indicative of a shift toward a proinflammatory, effector-like status in patient CD8 + T cells. Moreover, patient CD4 + T cells exhibited a pronounced defect in proliferation with delayed expression of surface checkpoint inhibitors, suggesting an impaired onset of the T cell activation program. Collectively, we identified a previously uncharacterized, germline-encoded inborn error of immunity and uncovered a cell-specific defect in Helios-dependent epigenetic regulation. Binding of Helios with specific partners mediates this regulation, which is ultimately necessary for the transcriptional programs that enable T cell homeostasis in health and disease.

Accumulation of human CD21 low B cells in peripheral blood is a hallmark of chronic activation of the adaptive immune system in certain infections and autoimmune disorders. The molecular pathways underpinning the development, function, and fate of these CD21 low B cells remain incompletely characterized. Here, combined transcriptomic and chromatin accessibility analyses supported a prominent role for the transcription factor T-bet in the transcriptional regulation of these T-bet high CD21 low B cells. Investigating essential signals for generating these cells in vitro established that B cell receptor (BCR)/interferon-γ receptor (IFNγR) costimulation induced the highest levels of T-bet expression and enabled their differentiation during cell cultures with Toll-like receptor (TLR) ligand or CD40L/interleukin-21 (IL-21) stimulation. Low proportions of CD21 low B cells in peripheral blood from patients with defined inborn errors of immunity (IEI), because of mutations affecting canonical NF-κB, CD40, and IL-21 receptor or IL-12/IFNγ/IFNγ receptor/signal transducer and activator of transcription 1 (STAT1) signaling, substantiated the essential roles of BCR- and certain T cell–derived signals in the in vivo expansion of T-bet high CD21 low B cells. Disturbed TLR signaling due to MyD88 or IRAK4 deficiency was not associated with reduced CD21 low B cell proportions. The expansion of human T-bet high CD21 low B cells correlated with an expansion of circulating T follicular helper 1 (cTfh1) and T peripheral helper (Tph) cells, identifying potential sources of CD40L, IL-21, and IFNγ signals. Thus, we identified important pathways to target autoreactive T-bet high CD21 low B cells in human autoimmune conditions, where these cells are linked to pathogenesis and disease progression.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA-corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1-/- and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival., (© 2021 by The American Society of Hematology.)

Purpose: IKAROS, encoded by IKZF1, is a member of the IKAROS family of zinc-finger transcription factors playing critical roles in lymphocyte development, differentiation, and tumor suppression. Several studies demonstrated that IKZF1 mutations affecting DNA binding or homo-/hetero-dimerization are mostly associated with common variable immunodeficiency, combined immunodeficiency, or hematologic manifestations. Herein we report a likely de novo, nonsense IKZF1 mutation (p.C182*) in a baby with low T cell receptor excision circles (TREC) identified by newborn screening testing for severe combined immunodeficiency. The patient also presented a profound B cell deficiency at birth., Methods: Genetic, functional, immunologic, and clinical outcome data associated with this patient and her mutation were evaluated., Results: Mutant p.C182* was detected in the cytoplasm of the patient's primary cells, in contrast to wild type (WT) IKAROS protein, only detected in the nucleus. Functional in vitro assessments revealed that p.C182* was less stable than WT IKAROS protein and failed to bind to its target DNA binding sequence and dimerize with WT IKAROS protein, resulting in impaired pericentromeric targeting and transcriptional repression by means of haploinsufficiency. During follow-up, while a spontaneous recovery of TREC and T cells was observed, B cells improved but not to sustained normal ranges., Conclusions: Patients with IKAROS-associated diseases can present with SCID-like TREC values through newborn screening testing. IKZF1 mutations should be added to the low TREC differential, although spontaneous recovery has to be considered., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

The normal expression of Ikaros (IKZF1) is important for the proper functioning of both the human and murine immune systems. Whilst our understanding of IKZF1 in the immune system has been greatly enhanced by the study of mice carrying mutations in Ikzf1 , analyses of human patients carrying germline IKZF1 mutations have been instrumental in understanding its biological role within the human immune system and its effect on human disease. A myriad of different mutations in IKZF1 have been identified, spanning across the entire gene causing differential clinical outcomes in patients including immunodeficiency, immune dysregulation, and cancer. The majority of mutations in humans leading to IKAROS-associated diseases are single amino acid heterozygous substitutions that affect the overall function of the protein. The majority of mutations studied in mice however, affect the expression of the protein rather than its function. Murine studies would suggest that the complete absence of IKZF1 expression leads to severe and sometimes catastrophic outcomes, yet these extreme phenotypes are not commonly observed in patients carrying IKZF1 heterozygous mutations. It is unknown whether this discrepancy is simply due to differences in zygosity, the role and regulation of IKZF1 in the murine and human immune systems, or simply due to a lack of similar controls across both groups. This review will focus its analysis on the current literature surrounding what is known about germline IKZF1 defects in both the human and the murine immune systems, and whether existing mice models are indeed accurate tools to study the effects of IKZF1-associated diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Boast, Nunes-Santos, Kuehn and Rosenzweig.)

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.

IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous Ikzf1 mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in IKZF1 are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through N -ethyl- N -nitrosourea mutagenesis, we have discovered a novel Ikzf1 mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation ( Ikzf1 L132P ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1 L132P overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1 L132P Furthermore, Ikzf1 +/L132P mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of Hsf1 expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in Ikzf1 L132P/L132P mice., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Objective: Mutations in IKZF1, which encodes Ikaros family zinc finger 1 (IKAROS) transcription factor, are associated with recurrent infections, cytopenia, autoimmune diseases, and hematologic malignancies. Diverse clinical phenotypes resulting from IKZF1 mutations include pulmonary fungal infections, cytopenia, autoimmune hemolytic anemia (AIHA), and malignancies. In this study, we aimed to assess the DNA-binding ability and pericentromeric (PC) localization of a variant of IKZF discovered in a patient., Materials and Methods: DNA-binding ability of a pathogenic IKZF variant was tested using electrophoretic mobility shift assay and PC localization of the variant was assessed by immunofluorescent microscopy in NIH3T3 cells., Results: Clinical features of a 3-month-old male infant who underwent hematopoietic stem cell transplantation because of an IKZF1 mutation-associated common variable immunodeficiency, AIHA, and pancytopenia are described. DNA studies revealed a heterozygous missense variant (IKZF1 NM_006060 c.427C>T; p.R143W). Cotransfection studies revealed that mutant R143W has a partial dominant-negative effect over PC targeting and DNA binding., Conclusions: IKZF1 mutation must be kept in mind if neonatal AIHA, common variable immunodeficiency, and pancytopenia are observed., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction : The transcription factor IKAROS and IKAROS family members are critical for the development of lymphocyte and other blood cell lineages. Germline heterozygous IKZF1 mutations have been described in primary immunodeficiency as well as in human hematologic malignancies, affecting both B and T cells. Depending on the allelic variants of IKZF1 mutations (haploinsufficiency and dominant negative) clinical phenotypes vary from bacterial, viral, or fungal infection to autoimmune disease and malignancy. Areas covered : In this review, the authors provide an overview of genotype-phenotype correlation and clinical manifestations in patients with IKZF1 mutations. The importance of accurate diagnosis and monitoring immunological changes is also discussed for the management of these complex and rare diseases. IKZF1/IKAROS, immunodeficiency, and CVID were used as the search terms in PubMed and Google Scholar. Expert opinion : Over the past 5 years both genetic and molecular studies have unveiled surprisingly diverse roles of IKZF1 mutations in primary immunodeficiency. While an increasing number of novel IKZF1 variants are being reported, limited, and complex laboratory testing is necessary to verify the mutation's pathogenicity. Therefore, the combination of understanding mechanistic concepts and clinical and immunological follow-up is necessary to increase our knowledge of IKAROS-associated diseases.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that emerged recently and has created a global pandemic. Symptomatic SARS-CoV-2 infection, termed coronavirus disease 2019 (COVID-19), has been associated with a host of symptoms affecting numerous organ systems, including the lungs, cardiovascular system, kidney, central nervous system, gastrointestinal tract, and skin, among others., Objective: Although several risk factors have been identified as related to complications from and severity of COVID-19, much about the virus remains unknown. The host immune response appears to affect the outcome of disease. It is not surprising that patients with intrinsic or secondary immune compromise might be particularly susceptible to complications from SARS-CoV-2 infection. Pathogenic loss-of-function or gain-of-function heterozygous variants in nuclear factor-κB2 have been reported to be associated with either a combined immunodeficiency or common variable immunodeficiency phenotype., Methods: We evaluated the functional consequence and immunologic phenotype of a novel NFKB2 loss of function variant in a 17-year-old male patient and describe the clinical management of SARS-CoV-2 infection in this context., Results: This patient required a 2-week hospitalization for SARS-CoV-2 infection, including 7 days of mechanical ventilation. We used biologic therapies to avert potentially fatal acute respiratory distress syndrome and treat hyperinflammatory responses. The patient had an immunologic phenotype of B-cell dysregulation with decreased switched memory B cells. Despite the underlying immune dysfunction, he recovered from the infection with intense management., Conclusions: This clinical case exemplifies some of the practical challenges in management of patients with SARS-CoV-2 infection, especially in the context of underlying immune dysregulation., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

In 2014, we reported two siblings with a rare congenital disorder of glycosylation due to mutations in mannosyl-oligosaccharide glucosidase (MOGS). The glycan alteration derived from this disease resulted in an in vitro infection resistance to particular enveloped, N-glycosylation-dependent viruses as influenza and HIV. As part of the global effort to find safe and effective antiviral therapies for Covid-19, we assessed the in vitro activity of the FDA-approved α-glucosidase inhibitor miglustat against SARS-CoV-2. Expression plasmids encoding SARS-CoV-2 spike (S) and human ACE2 glycoproteins (GP) were tested to evaluate N-glycan modifications induced by α-glucosidase inhibition. Immunoprecipitation was used to assess binding between these two GP. Cell-to-cell fusion was assessed by immunofluorescence of cocultures of SARS-CoV-2 S and ACE2-expressing cells. Miglustat effect on immune response was tested by measuring cytokine release from PBMC exposed to purified SARS-CoV-2 S. In our overexpression system, miglustat successfully and specifically modified N-glycans in both SARS-CoV-2 S and its main receptor ACE2. Binding between these two GP was not affected by glycan modifications. A surrogate marker for viral cytopathic effect, measured as receptor-dependent SARS-CoV-2 S-driven cell-to-cell fusion, was not disrupted by miglustat treatment. This observation was further confirmed in MOGS-null transfected cells. Miglustat produced no statistically significant effects on cytokine production following SARS-CoV-2 S glycoprotein stimulation of PBMC. Our work shows that despite clear N-glycan alteration in the presence of miglustat, the functions of the Covid-19-related glycoproteins studied were not affected, making it unlikely that miglustat can change the natural course of the disease.

IKAROS is a transcription factor forming homo- and heterodimers and regulating lymphocyte development and function. Germline mutations affecting the IKAROS N-terminal DNA binding domain, acting in a haploinsufficient or dominant-negative manner, cause immunodeficiency. Herein, we describe 4 germline heterozygous IKAROS variants affecting its C-terminal dimerization domain, via haploinsufficiency, in 4 unrelated families. Index patients presented with hematologic disease consisting of cytopenias (thrombocytopenia, anemia, neutropenia)/Evans syndrome and malignancies (T-cell acute lymphoblastic leukemia, Burkitt lymphoma). These dimerization defective mutants disrupt homo- and heterodimerization in a complete or partial manner, but they do not affect the wild-type allele function. Moreover, they alter key mechanisms of IKAROS gene regulation, including sumoylation, protein stability, and the recruitment of the nucleosome remodeling and deacetylase complex; none affected in N-terminal DNA binding defects. These C-terminal dimerization mutations are largely associated with hematologic disorders, display dimerization haploinsufficiency and incomplete clinical penetrance, and differ from previously reported allelic variants in their mechanism of action. Dimerization mutants contribute to the growing spectrum of IKAROS-associated diseases displaying a genotype-phenotype correlation.

IKAROS, encoded by IKZF1, is a zinc finger transcription factor and a critical regulator of hematopoiesis. Mutations in IKZF1 have been implicated in immune deficiency, autoimmunity, and malignancy in humans. Somatic IKZF1 loss-of-function mutations and deletions have been shown to increase predisposition to the development of B cell acute lymphoblastic leukemia (B-ALL) and associated with poor prognosis. In the last 4 years, germline heterozygous IKZF1 mutations have been reported in primary immune deficiency/inborn errors of immunity. These allelic variants, acting by either haploinsufficiency or dominant negative mechanisms affecting particular functions of IKAROS, are associated with common variable immunodeficiency, combined immunodeficiency, or primarily hematologic phenotypes in affected patients. In this review, we provide an overview of genetic, clinical, and immunological manifestations in patients with IKZF1 mutations, and the molecular and cellular mechanisms that contribute to their disease as a consequence of IKAROS dysfunction.

The noncanonical NF-κB pathway is implicated in diverse biological and immunological processes. Monoallelic C-terminus loss-of-function and gain-of-function mutations of NFKB2 have been recently identified as a cause of immunodeficiency manifesting with common variable immunodeficiency (CVID) or combined immunodeficiency (CID) phenotypes. Herein we report a family carrying a heterozygous nonsense mutation in NFKB2 (c.809G > A, p.W270*). This variant is associated with increased mRNA decay and no mutant NFKB2 protein expression, leading to NFKB2 haploinsufficiency. Our findings demonstrate that bona fide NFKB2 haploinsufficiency, likely caused by mutant mRNA decay and protein instability leading to the transcription and expression of only the wild-type allele, is associated with clinical immunodeficiency, although with incomplete clinical penetrance. Abnormal B cell development, hypogammaglobulinemia, poor antibody response, and abnormal noncanonical (but normal canonical) NF-κB pathway signaling are the immunologic hallmarks of this disease. This adds a third allelic variant to the pathophysiology of NFKB2-mediated immunodeficiency disorders.

We report the case of a patient with X-linked severe combined immunodeficiency (X-SCID) who survived for over 20 years without hematopoietic stem cell transplantation (HSCT) because of a somatic reversion mutation. An important feature of this rare case included the strategy to validate the pathogenicity of a variant of the IL2RG gene when the T and B cell lineages comprised only revertant cells. We studied the X-inactivation of sorted T cells from the mother to show that the pathogenic variant was indeed the cause of his SCID. One interesting feature was a progressive loss of B cells over 20 years. CyTOF (cytometry time of flight) analysis of bone marrow offered a potential explanation of the B cell failure, with expansions of progenitor populations that suggest a developmental block. Another interesting feature was that the patient bore extensive granulomatous disease and skin cancers that contained T cells, despite severe T cell lymphopenia in the blood. Finally, the patient had a few hundred T cells on presentation but his TCRs comprised a very limited repertoire, supporting the important conclusion that repertoire size trumps numbers of T cells.

Background: Chronic active Epstein-Barr virus (CAEBV) presents with high levels of viral genomes in blood and tissue infiltration with Epstein-Barr virus (EBV)-positive lymphocytes. The pathogenesis of CAEBV is poorly understood., Methods: We evaluated 2 patients with natural killer (NK) cell CAEBV and studied their NK cell phenotype and signaling pathways in cells., Results: Both patients had increased numbers of NK cells, EBV predominantly in NK cells, and immature NK cells in the blood. Both patients had increased phosphorylation of Akt, S6, and STAT1 in NK cells, and increased total STAT1. Treatment of 1 patient with sirolimus reduced phosphorylation of S6 in T and B cells, but not in NK cells and did not reduce levels of NK cells or EBV DNA in the blood. Treatment of both patients' cells with JAK inhibitors in vitro reduced phosphorylated STAT1 to normal. Patients with T- or B-cell CAEBV had increased phosphorylation of Akt and S6 in NK cells, but no increase in total STAT1., Conclusions: The increase in phosphorylated Akt, S6, and STAT1, as well as immature NK cells describe a new phenotype for NK cell CAEBV. The reduction of STAT1 phosphorylation in their NK cells with JAK inhibitors suggests a novel approach to therapy., (Published by Oxford University Press for the Infectious Diseases Society of America 2020.)

Ikaros zinc finger 1 (IKZF1 or Ikaros) is a hematopoietic zinc finger DNA-binding transcription factor that acts as a critical regulator of lymphocyte and myeloid differentiation. Loss-of-function germline heterozygous mutations in IKZF1 affecting DNA-binding were described as causative of 2 distinct primary immunodeficiency (PID)/inborn error of immunity diseases. Mutations acting by haploinsufficiency present with a common variable immune deficiency-like phenotype mainly characterized by increased susceptibility to infections. Mutations acting in a dominant negative fashion present with a combined immunodeficiency phenotype with high prevalence of Pneumocystis jirovecii pneumonia. Pathophysiology and manifestations of IKAROS-associated diseases in patients with PID are reviewed here., Competing Interests: Disclosure of potential conflict of interest The authors have no relevant conflicts of interest., (Published by Elsevier Inc.)

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage 1-7 . The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis 8 . Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1 D325A mutant mouse strain. Whereas Ripk1 -/- mice died postnatally from systemic inflammation, Ripk1 D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1 D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1 D325A/D325A and Ripk1 D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1 D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

IL-6 excess is central to the pathogenesis of multiple inflammatory conditions and is targeted in clinical practice by immunotherapy that blocks the IL-6 receptor encoded by IL6R We describe two patients with homozygous mutations in IL6R who presented with recurrent infections, abnormal acute-phase responses, elevated IgE, eczema, and eosinophilia. This study identifies a novel primary immunodeficiency, clarifying the contribution of IL-6 to the phenotype of patients with mutations in IL6ST, STAT3 , and ZNF341, genes encoding different components of the IL-6 signaling pathway, and alerts us to the potential toxicity of drugs targeting the IL-6R., (© 2019 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

Signal transducer and activator of transcription (STAT1)1 gain of function (GOF) pathogenic variants have been associated with increased levels of phosphorylated STAT1 and STAT1-dependent cellular responses. Delayed dephosphorylation was proposed as the underlying mechanism leading to the characteristically raised pSTAT1 levels. We examined the levels of STAT1 protein and message as well as rates of STAT1 phosphorylation, dephosphorylation, and degradation associated with STAT1 GOF pathogenic variants. Fresh peripheral blood mononuclear cells (PBMC) from 14 STAT1 GOF patients carrying 10 different pathogenic variants in the coiled-coil, DNA binding, and SH2 domains and healthy donors were used to study STAT1 levels and phosphorylation (pSTAT1) following IFNγ and IFNα stimulation. STAT1 protein levels were measured by flow cytometry and immunoblot. STAT1 mRNA levels were measured using quantitative reverse transcription PCR. STAT1 protein degradation was studied using cycloheximide. Patient IFNγ and IFNα induced peak pSTAT1 was higher than in healthy controls. The velocity of pSTAT1 dephosphorylation after treatment of IFNγ stimulated CD14 + monocytes with the Janus Kinase (JAK)-inhibitor ruxolitinib was significantly faster in patient cells. STAT1 protein levels in patient CD14 + monocytes and CD3 + T cells were higher than in healthy donors. There was a strong and positive correlation between CD14 + STAT1 protein levels and peak pSTAT1 levels. Patient fresh PBMC STAT1 mRNA levels were increased at rest and after 16 h of incubation. STAT1 protein degradation was similar in patient and healthy volunteer cells. Patient IFNγ receptors 1 and 2 and JAK2 levels were normal. One patient in our cohort was treated with the oral JAK inhibitor ruxolitinib. Treatment was associated with normalization of both STAT1 protein and peak pSTAT1 levels. After JAK inhibitor treatment was stopped the patient's CD14 + monocyte STAT1 protein and peak phosphorylation levels increased proportionally. These findings suggest that patients with STAT1 GOF mutations have higher levels of total STAT1 protein, leading to high levels of pSTAT1 after stimulation, despite rapid STAT1 dephosphorylation and normal degradation.