Journal of Experimental Medicine

We previously reported inherited RORgammaT deficiency in seven patients from three ancestries (Chilean, Palestinian, Saudi Arabian) with mycobacterial disease and chronic mucocutaneous candidiasis (CMC). We report here five additional patients from different ancestries (Afghan, Indian, Iranian, Japanese, Sri Lankan), each homozygous for a new loss-of-function RORC variant. All but one patient, the exception receiving early prophylaxis, developed mycobacterial disease due to a near-complete depletion of innate-like adaptive T cells, including MAIT and iNKT cells, low counts of adaptive TH1* and CD8+ T cells, and impaired Mycobacterium-induced IFN-gamma production by the remaining cells of these subsets, NK cells, conventional CD4+ T, Vdelta1, and Vdelta2 gamma-delta T cells. Most patients also displayed CMC due to their low counts of TH17 and TH1* cells. One patient died from disseminated Bacille Calmette-Guerin (BCG) vaccine infection, but, unexpectedly, all the other patients are still alive and clinically stable. RORgammaT is essential for protective immunity against mycobacteria and Candida in humans.

Homozygous OTU deubiquitinase with linear linkage specificity (OTULIN) variants cause OTULIN-Related Autoinflammatory Syndrome (ORAS). This disease is characterized by early-onset autoinflammation, fever, panniculitis, diarrhea, and arthritis. In contrast, heterozygous and compound-heterozygous OTULIN variants have been associated with a phenotype defined by abscess development in different organs. Whether homozygous OTULIN variants can cause abscessing in affected patients is currently unknown. Here, we report a juvenile female patient harboring a novel homozygous OTULIN variant (Chr5:14687605G>T, p.V185F; referred to as OTULINV185F), presenting with autoinflammation and sterile abscesses in lung and skin. Through in silico analysis and functional assays, we show that OTULINV185F impairs OTULIN function, leading to compromised degradation of linear ubiquitin linkages. Notably, the patient clinically improved on anti-TNF therapy. Our findings underscore the diverse clinical manifestations of OTULIN dysfunction and call for a new classification of the disease that includes abscess formation as potential ORAS symptom.

Across the globe, 2-3% of humans carry the p.Ser132Pro single nucleotide polymorphism in MLKL, the terminal effector protein of the inflammatory form of programmed cell death, necroptosis. We show that this substitution confers a gain in necroptotic function in human cells, with more rapid accumulation of activated MLKLS132P in biological membranes and MLKLS132P overriding pharmacological and endogenous inhibition of MLKL. In mouse cells, the equivalent Mlkl S131P mutation confers a gene dosage dependent reduction in sensitivity to TNF-induced necroptosis in both hematopoietic and non-hematopoietic cells, but enhanced sensitivity to IFN-{beta} induced death in non-hematopoietic cells. In vivo, MlklS131P homozygosity reduces the capacity to clear Salmonella from major organs and retards recovery of hematopoietic stem cells. Thus, by dysregulating necroptosis, the S131P substitution impairs the return to homeostasis after systemic challenge. Present day carriers of the MLKL S132P polymorphism may be the key to understanding how MLKL and necroptosis modulate the progression of complex polygenic human disease.

Mutations in ADAR, the gene that encodes the ADAR1 RNA deaminase, cause numerous human diseases, including Aicardi-Goutieres Syndrome (AGS). ADAR1 is an essential negative regulator of the RNA sensor MDA5, and loss of ADAR1 function triggers inappropriate activation of MDA5 by self-RNAs. However, the mechanisms of MDA5-dependent disease pathogenesis in vivo remain unknown. Here, we introduce a knockin mouse that models the most common ADAR AGS mutation in humans. These Adar-mutant mice develop lethal disease that requires MDA5, the RIG-I-like receptor LGP2, type I interferons, and the eIF2 kinase PKR. We show that a small molecule inhibitor of the integrated stress response (ISR) that acts downstream of eIF2 phosphorylation prevents immunopathology and rescues the mice from mortality. These findings place PKR and the ISR as central components of immunopathology in vivo and identify new therapeutic targets for treatment of human diseases associated with the ADAR1-MDA5 axis.

Biallelic deficiencies of CD27 and its ligand CD70 underlie selective susceptibility to Epstein-Barr virus (EBV) infection and its acute and chronic complications, underscoring their non-redundant roles in anti-EBV immunity. To date, 16 pathogenic CD27 variants have been reported. Here, we describe three patients from two unrelated families, homozygous for a novel loss-of-function (LOF) CD27 variant, resulting in substitution of serine 70 with proline (S70P). All three patients presented with EBV viremia and lymphoproliferative disease, with variable immune dysregulation or recurrent otosinopulmonary infections. One patient developed EBV-associated Hodgkin lymphoma. Functional studies demonstrated that the S70P variant impaired surface expression of CD27 and abolished CD70 binding, rendering complete LOF. Together, S70P represents a novel pathogenic CD27 variant causing autosomal recessive (AR) CD27 deficiency, characterized by a unified susceptibility to EBV yet variable clinical manifestations, ranging from chronic viremia to malignancy.

Acquired mutations in the UBA1 gene, occurring in myeloid cells and resulting in expression of a catalytically impaired isoform of the enzyme E1, were recently identified in patients with severe adult-onset auto-inflammatory syndrome called VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). The precise physiological and clinical impact of these mutations remains poorly defined. Here, we studied a unique prospective cohort of individuals with severe autoinflammatory disease with (VEXAS) or without (VEXAS-like) UBA1 somatic mutations and compared with low-risk myelodysplastic syndromes (MDS) and aged gender-matched healthy controls. We performed an integrated immune analysis including multiparameter phenotyping of peripheral blood leukocytes, cytokines profiling, bulk and single-cell gene expression analyses and skin tissue imaging mass cytometry. Focusing on myeloid cells, we show that monocytes from UBA1-mutated individuals were quantitatively and qualitatively impaired and displayed features of exhaustion with aberrant expression of chemokine receptors. Within affected tissues, pathological skin biopsies from VEXAS patients showed an abundant enrichment of CD16+ CD163+ monocytes adjacent to blood vessels and M1 macrophages, possibly promoting local inflammation in part through STAT3 activation. In peripheral blood from VEXAS patients, we identified a significant increase in circulating levels of many proinflammatory cytokines, including IL-1{beta} and IL-18 which reflect inflammasome activation and markers of myeloid cells dysregulation. Gene expression analysis of whole blood confirmed the role of circulating cells in the IL-1{beta} and IL-18 dysregulation in VEXAS patients and revealed a significant enrichment of TNF- and NF{kappa}B signaling pathways that could mediate cell death and inflammation. Single-cell analysis confirmed the inflammatory state of monocytes from VEXAS patients and allowed us to identify specific molecular pathways that could explain monocytopenia, especially the activation of PANoptosis and a deficiency in the TYROBP/DAP12 axis and {beta}-catenin signaling pathway. Together, these findings on monocytes from patients with UBA1 mutations provide important insights into the molecular mechanisms involving the mature myeloid commitment in VEXAS syndrome and suggest that the control of the undescribed inflammasome activation and PANoptosis could be novel therapeutic targets in this condition. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/22281005v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@10a14e4org.highwire.dtl.DTLVardef@e1b88eorg.highwire.dtl.DTLVardef@11bb0a2org.highwire.dtl.DTLVardef@e971ca_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across 3 donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

Multisystem Inflammatory Syndrome in Children (MIS-C), a hyperinflammatory syndrome associated with SARS-CoV-2 infection, shares many clinical features with toxic shock syndrome, which is triggered by bacterial superantigens. The superantigen specificity for binding different V{beta}-chains results in V{beta}-skewing, whereby T cells with specific V{beta}-chains and diverse antigen specificity are overrepresented in the TCR repertoire. Here, we characterized the TCR repertoire of MIS-C patients and found a profound expansion of TCR Beta Variable gene (TRBV)11-2. Furthermore, TRBV11-2 skewing was remarkably correlated with MIS-C severity and serum cytokine levels. Further analysis of TRBJ gene usage and CDR3 length distribution of MIS-C expanding TRBV11-2 clones revealed extensive junctional diversity, indicating a superantigen-mediated selection process for TRBV expansion. In silico modelling indicates that polyacidic residues in TCR V{beta}11-2 engage in strong interactions with the superantigen-like motif of SARS-CoV-2 spike glycoprotein. Overall, our data indicate that the immune response in MIS-C is consistent with superantigenic activation. HighlightsO_LIMultisystem Inflammatory Disease in Children (MIS-C) patients exhibit T cell receptor (TCR) repertoire skewing, with expansion of T cell Receptor Beta Variable gene (TRBV)11-2 C_LIO_LITRBV11-2 skewing correlates with MIS-C severity and cytokine storm C_LIO_LIJ gene/CDR3 diversity in MIS-C patients is compatible with a superantigen selection process C_LIO_LIIn silico modelling indicates TCR V{beta}11-2 engages in CDR3-independent interactions with the polybasic insert P681RRAR in the SAg-like motif of SARS-CoV-2 spike C_LI

Ribosome biogenesis defects are increasingly recognized in hematologic disorders, yet their contribution to human combined immunodeficiency (CID) remains largely unexplored. Here, we identify compound heterozygous mutations in GTF3A, encoding transcription factor IIIA (TFIIIA), in a patient with CID presenting with profound T-cell lymphopenia, diminished thymic output, and humoral failure. The patient-derived TFIIIA variants, I267S and L364Yfs*37, disrupted 5S rRNA transcription, impaired RNA-binding capacity, and compromised protein stability. Patient T and B lymphocytes exhibited intrinsic proliferative and differentiation defects, as well as increased apoptotic susceptibility in T cells, recapitulating the clinical phenotype. To model TFIIIA dosage, we generated heterozygous and progressively depleted Jurkat cell clones via sequential CRISPR editing; complete TFIIIA loss was lethal, whereas graded reduction impaired proliferation in a dose-dependent manner, fully rescued by wild-type GTF3A reconstitution. Collectively, these findings establish deficiency of GTF3A resulting in combined immunodeficiency (DoGCID) within the spectrum of ribosomopathies, highlighting the exquisite sensitivity of lymphocyte fitness to disruptions in ribosome biogenesis.

OSMR{beta} (Oncostatin M receptor beta), a member of the IL-6 superfamily of cell surface receptors, binds OSM and IL-31 and plays a critical role in human immunity. We identified probands from four kindreds with biallelic damaging variants in OSMR, which encodes OSMR{beta}. Patients had a unifying phenotype for severe widespread, early-onset atopic dermatitis, peripheral eosinophilia, and elevated serum IgE. Patient OSMR{beta} variants were not appropriately expressed on the cell surface compared to OSMR{beta}WT. Patient OSMR variants showed significantly reduced OSM-mediated activation of STAT1, STAT3, and STAT5 and distinct transcriptional changes in primary dermal fibroblasts, including loss of interferon and inflammatory signatures. These defects were rescued upon lentiviral transduction of WT-OSMR. Together, these data establish that human germline biallelic loss-of-function OSMR variants cause severe allergic disease. We anticipate that this discovery will facilitate the recognition of additional affected individuals and the full definition of this novel primary atopic disorder.

TNF is a potent proinflammatory cytokine that can induce cell death by activating the kinase RIPK1. The adaptor proteins TANK and AZI2 protect against cell death by recruiting TBK1 to the TNF receptor signaling complex, thereby inhibiting RIPK1. While deficiency of either adaptor alone is well tolerated, combined loss of TANK and AZI2 results in partial embryonic lethality and severe TNF- and RIPK1-driven autoinflammation. Here, we show that TANK/AZI2-deficient mice exhibit a striking expansion of regulatory T cells (Tregs), most of which display an effector phenotype with high expression of immunosuppressive genes. Although thymic Treg generation is modestly increased, Tregs arise predominantly in the periphery through a largely cell-intrinsic mechanism. The marked accumulation of effector Tregs suggested that the T-cell compartment may limit TNF-driven pathology in this model. Supporting this, T cell ablation in TANK/AZI2-deficient mice markedly exacerbates disease progression and enhances TNF-driven, RIPK1-mediated inflammation. Similarly, T cells protect against acute TNF-induced systemic inflammatory response syndrome by limiting RIPK1-mediated cell death. Together, our findings identify TANK and AZI2 as negative regulators of Treg formation, demonstrate that T cells restrain TNF-driven inflammation by limiting RIPK1-dependent cell death, and suggest that this protective effect is mediated primarily by Tregs.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibodies raised against nuclear antigens and whose production is promoted by autoreactive T follicular helper (TFH) cells. Basophils, by accumulating in secondary lymphoid organs (SLO), amplify autoantibody production and disease progression through mechanisms to be defined. Here, we demonstrate that a functional relationship between TFH cells and basophils occurs in SLO during lupus pathogenesis. On SLE patient blood basophils, PD-L1 expression was upregulated and associated with TFH and TFH2 cell expansions and with disease activity. In two distinct lupus-like mouse models, TFH cell pathogenic accumulation, maintenance and function, and disease activity were dependent on basophils and their expressions of PD-L1 and IL-4. Our study establishes a direct link between basophils and TFH cells in the SLE context that promotes autoreactive IgG production and lupus nephritis pathogenesis. Altering the basophil/TFH cell axis in the SLE context may represent a promising innovative intervention strategy in SLE.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease, with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3 - a negative regulator of cytokine and growth factor receptor signaling - harbors rare coding variants in over 5% of SLE patients. Here we show that unlike the variant found exclusively in healthy controls, most SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles. Generation of two mouse lines carrying variants orthologous to those found in patients revealed SH2B3 is important to limit the numbers of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk. SummaryZhang et al. reveal a role for hypomorphic SH2B3 in lupus risk. The study shows rare and damaging variants identified in lupus patients enable breach of B cell immune tolerance checkpoints and suggests involvement for dysregulated IL-4R signaling and BAFF-R expression.

Infection directly influences adult hematopoietic stem cell (HSC) function and differentiation, but much less is known about the fetal hematopoietic response to infection during pregnancy. Here, we investigated the fetal hematopoietic response to maternal infection with Toxoplasma gondii (T. gondii), an intracellular parasite that elicits Type II IFN{gamma}-mediated maternal immunity. The effects of maternal IFN{gamma} on developing HSCs and the signals that mediate these interactions have not been investigated. Our investigation reveals that the fetal HSCs respond to T. gondii infection with virulence-dependent changes in proliferation, self-renewal potential, and lineage output. We demonstrate that maternal IFN{gamma} crosses the fetal-maternal interface and is perceived by fetal HSCs. By comparing the effects of maternal IFN{gamma} injection with maternal T. gondii infection, our observations reveal that IFN{gamma} mimics aspects of the fetal HSC response to infection. Moreover, our data disentangle the role of infection-induced inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors. Our findings illuminate that the fetal HSC response to prenatal infection is distinct from the adult HSC response to IFN{gamma}-induced inflammation.

Dysregulated STAT3 signalling is correlated with antibody-mediated autoimmunity and B- cell neoplasia, but its effect on B cells is underexplored. Here we address this in children with STAT3 gain-of-function (GOF) syndrome and in mice with STAT3T716M, the most common STAT3 GOF syndrome human mutation, or STAT3K658N, a dimerization interface mutation responsible for STAT3 GOF syndrome in two children. The main B cell consequence of overactive STAT3 was accumulation of CD19high CD21low atypical memory B cells in humans and of CD21low CD23low B cells in mice resembling age-associated B cells expressing T-bet, CD11c and plasma cell differentiation genes. Overactive STAT3 within B cells increased expression of many genes in the B cell receptor and T cell help pathways, increased the tolerogenic receptor CD22, but opposed B cell tolerance checkpoints and increased formation of T-bet+ B cells upon BCR and CD40 stimulation. These results reveal overactive STAT3 as a central driver of a key class of disease- associated B-lymphocytes in humans and mice.

Acute respiratory distress syndrome (ARDS) is a devastating complication of respiratory infections; however, the biological mechanisms that initiate its onset are poorly defined. Here we show that TNFRSF13B polymorphisms increase the risk of ARDS following SARS-CoV-2 infection up to 7.4-fold compared to the WT genotype. The increased risk was not due to immune-deficiency or impaired virus neutralization. On the contrary, TNFRSF13B mutant subjects mounted better antibody neutralization compared to subjects with WT TNFRSF13B. However, IgG from subjects expressing TNFRSF13B variants had less sialic acid, terminal galactose, and fucose than IgG from subjects with a WT genotype. Moreover, IgG from TNFRSF13B mutant subjects exhibited increased recruitment of complement factors. Thus, besides well-known actions governing plasma cell differentiation, TNFRSF13B impacts both affinity maturation and effector functions of IgG in ways that independently govern complement activation controlling inflammatory responses known to trigger ARDS.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). Yet, the exact feature of antibody responses that governs COVID-19 disease outcomes remain unclear. Here, we analysed humoral immune responses in 209 asymptomatic, mild, moderate and severe COVID-19 patients over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-Spike (S) IgG levels, length of hospitalization and clinical parameters associated with worse clinical progression. While high anti-S IgG levels correlated with worse disease severity, such correlation was time-dependent. Deceased patients did not have higher overall humoral response than live discharged patients. However, they mounted a robust, yet delayed response, measured by anti-S, anti-RBD IgG, and neutralizing antibody (NAb) levels, compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, while sera from 89% of patients displayed some neutralization capacity during their disease course, NAb generation prior to 14 days of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se, but rather with the delayed kinetics of NAb production.

In response to antigens, B cells undergo affinity maturation and class switching mediated by activation-induced cytidine deaminase (AID) in germinal centers (GCs) of secondary lymphoid organs, but uncontrolled AID activity can precipitate autoimmunity and cancer. The regulation of GC antibody diversification is of fundamental importance but not well understood. We found that autoimmune regulator (AIRE), the molecule essential for T cell tolerance, is expressed in GC B cells in a CD40-dependent manner, interacts with AID and negatively regulates antibody affinity maturation and class switching by inhibiting AID function. AIRE deficiency in B cells caused altered antibody repertoire, increased somatic hypermutations, elevated autoantibodies to T helper 17 effector cytokines and defective control of skin Candida albicans. These results define a GC B cell checkpoint of humoral immunity and illuminate new approaches of generating high-affinity neutralizing antibodies for immunotherapy.

Nucleic acids are powerful triggers of innate immunity and can adopt the unusual Z-conformation. The p150 isoform of adenosine deaminase acting on RNA 1 (ADAR1) prevents aberrant interferon (IFN) induction and contains a Z-nucleic acid binding (Z) domain. We report that knock-in mice bearing two point mutations in the Z domain of ADAR1, which abolish binding to Z-form nucleic acids, spontaneously induced type I IFNs and IFN-stimulated genes (ISGs) in multiple organs. This included the lung where both stromal and haematopoietic cells displayed ISG induction in Adar1mZ/mZ mice. Concomitantly, Adar1mZ/mZ mice showed improved control of influenza A virus. The spontaneous IFN response in Adar1mZ/mZ mice required MAVS, implicating cytosolic RNA sensing. Finally, analysis of A-to-I changes revealed a specific requirement of ADAR1s Z domain in editing of a subset of RNAs. In summary, our results reveal that endogenous RNAs in Z-conformation have immunostimulatory potential that is curtailed by ADAR1.

STAT3 function in hematopoietic stem and progenitor cells (HSPCs) has been difficult to discern as Stat3 deficiency in the hematopoietic system induces systemic inflammation, which can impact HSPC activity. To address this, we established mixed bone marrow (BM) chimeric mice with CreER-mediated Stat3 deletion in 20% of the hematopoietic compartment. Stat3-deficient HSPCs had impaired hematopoietic activity and failed to undergo expansion in BM in contrast to Stat3-sufficient (CreER) controls. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells revealed altered transcriptional responses in Stat3-deficient hematopoietic stem cells (HSCs) and multipotent progenitors, including intrinsic activation of cell cycle, stress response, and interferon signaling pathways. Consistent with their deregulation, Stat3-deficient Lin-ckit+Sca1+ cells accumulated {gamma}H2AX over time. Following secondary BM transplantation, Stat3-deficient HSPCs failed to reconstitute peripheral blood effectively, indicating a severe functional defect in the HSC compartment. Our results reveal essential roles for STAT3 in HSCs and suggest the potential for using targeted synthetic lethal approaches with STAT3 inhibition to remove defective or diseased HSPCs. Key PointsO_LISTAT3 is critical for hematopoietic activity and hematopoietic stem cell maintenance in non-inflammatory conditions C_LIO_LISTAT3 has a cell-intrinsic role in the suppression of interferon signaling and myeloid-skewed transcription in hematopoietic stem cells C_LI