A degranulation assay using Vγ9Vδ2 T cells for the rapid diagnosis of familial hemophagocytic syndromes.

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening immune disorder characterized by uncontrolled lymphocyte and macrophage activation and a subsequent cytokine storm. The timely initiation of immunosuppressive treatment is crucial for survival. Methods: Here, we harnessed Vγ9Vδ2 T cell degranulation to develop a novel functional assay for the diagnosis of HLH. We compared the novel assay with the conventional natural killer (NK) cell stimulation method in terms of efficiency, specificity, and reliability. Our analysis involved 213 samples from 182 individuals, including 23 samples from 12 patients with degranulation deficiency (10 individuals with UNC13D deficiency, 1 with STXBP2 deficiency, and 1 with RAB27A deficiency). Results: While both tests exhibited 100% sensitivity, the Vγ9Vδ2 T cell degranulation assay showed a superior specificity of 86.2% (n=70) compared to the NK cell degranulation assay, which achieved 78.9% specificity (n=213). The Vγ9Vδ2 T cell degranulation assay offered simpler technical requirements and reduced labor intensity, leading to decreased susceptibility to errors with faster processing times. Discussion: This efficiency stemmed from the sole requirement of dissolving (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) powder, contrasting with the intricate maintenance of K562 cells necessary for the NK cell degranulation assay. With its diminished susceptibility to errors, we anticipate that the assay will require fewer repetitions of analysis, rendering it particularly well-suited for testing infants. Conclusion: The Vγ9Vδ2 T cell degranulation assay is a user-friendly, efficient diagnostic tool for HLH. It offers greater specificity, reliability, and practicality than established methods. We believe that our present findings will facilitate the prompt, accurate diagnosis of HLH and thus enable rapid treatment and better patient outcomes.

The hyperinflammatory spectrum: from defects in cytotoxicity to cytokine control.

Cytotoxic lymphocytes kill target cells through polarized release of the content of cytotoxic granules towards the target cell. The importance of this cytotoxic pathway in immune regulation is evidenced by the severe and often fatal condition, known as hemophagocytic lymphohistiocytosis (HLH) that occurs in mice and humans with inborn errors of lymphocyte cytotoxic function. The clinical and preclinical data indicate that the damage seen in severe, virally triggered HLH is due to an overwhelming immune system reaction and not the direct effects of the virus per se. The main HLH-disease mechanism, which links impaired cytotoxicity to excessive release of pro-inflammatory cytokines is a prolongation of the synapse time between the cytotoxic effector cell and the target cell, which prompts the former to secrete larger amounts of cytokines (including interferon gamma) that activate macrophages. We and others have identified novel genetic HLH spectrum disorders. In the present update, we position these newly reported molecular causes, including CD48-haploinsufficiency and ZNFX1-deficiency, within the pathogenic pathways that lead to HLH. These genetic defects have consequences on the cellular level on a gradient model ranging from impaired lymphocyte cytotoxicity to intrinsic activation of macrophages and virally infected cells. Altogether, it is clear that target cells and macrophages may play an independent role and are not passive bystanders in the pathogenesis of HLH. Understanding these processes which lead to immune dysregulation may pave the way to novel ideas for medical intervention in HLH and virally triggered hypercytokinemia.

Sequence-Specific Features of Short Double-Strand, Blunt-End RNAs Have RIG-I- and Type 1 Interferon-Dependent or -Independent Anti-Viral Effects.

Pathogen-associated molecular patterns, including cytoplasmic DNA and double-strand (ds)RNA trigger the induction of interferon (IFN) and antiviral states protecting cells and organisms from pathogens. Here we discovered that the transfection of human airway cell lines or non-transformed fibroblasts with 24mer dsRNA mimicking the cellular micro-RNA (miR)29b-1* gives strong anti-viral effects against human adenovirus type 5 (AdV-C5), influenza A virus X31 (H3N2), and SARS-CoV-2. These anti-viral effects required blunt-end complementary RNA strands and were not elicited by corresponding single-strand RNAs. dsRNA miR-29b-1* but not randomized miR-29b-1* mimics induced IFN-stimulated gene expression, and downregulated cell adhesion and cell cycle genes, as indicated by transcriptomics and IFN-I responsive Mx1-promoter activity assays. The inhibition of AdV-C5 infection with miR-29b-1* mimic depended on the IFN-alpha receptor 2 (IFNAR2) and the RNA-helicase retinoic acid-inducible gene I (RIG-I) but not cytoplasmic RNA sensors MDA5 and ZNFX1 or MyD88/TRIF adaptors. The antiviral effects of miR29b-1* were independent of a central AUAU-motif inducing dsRNA bending, as mimics with disrupted AUAU-motif were anti-viral in normal but not RIG-I knock-out (KO) or IFNAR2-KO cells. The screening of a library of scrambled short dsRNA sequences identified also anti-viral mimics functioning independently of RIG-I and IFNAR2, thus exemplifying the diverse anti-viral mechanisms of short blunt-end dsRNAs.

Novel Combination of Surface Markers for the Reliable and Comprehensive Identification of Human Thymic Epithelial Cells by Flow Cytometry: Quantitation and Transcriptional Characterization of Thymic Stroma in a Pediatric Cohort.

Thymic epithelial cells (TECs) are essential in supporting the development of mature T cells from hematopoietic progenitor cells and facilitate their lineage-commitment, proliferation, T-cell receptor repertoire selection and maturation. While animal model systems have greatly aided in elucidating the contribution of stromal cells to these intricate processes, human tissue has been more difficult to study, partly due to a lack of suitable surface markers comprehensively defining human TECs. Here, we conducted a flow cytometry based surface marker screen to reliably identify and quantify human TECs and delineate medullary from cortical subsets. These findings were validated by transcriptomic and histologic means. The combination of EpCAM, podoplanin (pdpn), CD49f and CD200 comprehensively identified human TECs and not only allowed their reliable distinction in medullary and cortical subsets but also their detailed quantitation. Transcriptomic profiling of each subset in comparison to fibroblasts and endothelial cells confirmed the identity of the different stromal cell subsets sorted according to the proposed strategy. Our dataset not only demonstrated transcriptional similarities between TEC and cells of mesenchymal origin but furthermore revealed a subset-specific distribution of a specific set of extracellular matrix-related genes in TECs. This indicates that TECs significantly contribute to the distinct compartmentalization - and thus function - of the human thymus. We applied the strategy to quantify TEC subsets in 31 immunologically healthy children, which revealed sex-specific differences of TEC composition early in life. As the distribution of mature CD4- or CD8-single-positive thymocytes was correspondingly altered, the composition of the thymic epithelial compartment may directly impact on the CD4-CD8-lineage choice of thymocytes. We prove that the plain, reliable strategy proposed here to comprehensively identify human TEC subpopulations by flow cytometry based on surface marker expression is suitable to determine their frequency and phenotype in health and disease and allows sorting of live cells for downstream analysis. Its use reaches from a reliable diagnostic tool for thymic biopsies to improved phenotypic characterization of thymic grafts intended for therapeutic use.

X-Linked Lymphoproliferative Disease Mimicking Multisystem Inflammatory Syndrome in Children-A Case Report.

Most children with a SARS-CoV-2 infection are asymptomatic or exhibit mild symptoms. However, a small number of children develop features of substantial inflammation temporarily related to the COVID-19 also called multisystem inflammatory syndrome in children (MIS-C) or pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS), clinically similar to Kawasaki disease, toxic shock syndrome and hemophagocytic lymphohistiocytosis (HLH). It is well-known that genetic pre-disposition plays an important role in virally-triggered diseases such as Epstein-Barr virus (EBV)-associated HLH, while this has not yet been established for patients with MIS-C. Here we describe a male patient fulfilling the diagnostic criteria of MIS-C, who was initially treated according to current consensus guidelines. Presence of hypofibrinogenemia, normal lymphocyte counts and C-reactive protein, but substantial hyperferritinemia distinguish this patient from others with MIS-C. The clinical course following initial presentation with acute respiratory distress syndrome was marked by fatal liver failure in the context of EBV-associated HLH despite treatment with steroids, intravenous immunoglobulins, interleukin (IL)-1 receptor blockade and eventually HLH-directed treatment. X-linked lymphoproliferative disease type 1 (XLP1), a subtype of primary HLH was diagnosed in this patient post-mortem. This case report highlights the importance of including HLH in the differential diagnosis in MIS-C with severe disease course to allow specific, risk-adapted treatment and genetic counseling.

Multisystem inflammation and susceptibility to viral infections in human ZNFX1 deficiency.

BACKGROUND: Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known. OBJECTIVE: We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease. METHODS: Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids. RESULTS: Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes. CONCLUSION: ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease.

Immunological Assessment of Pediatric Multisystem Inflammatory Syndrome Related to Coronavirus Disease 2019.

BACKGROUND: Recently, cases of multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease 2019 (COVID-19) have been reported worldwide. Negative polymerase chain reaction (RT-PCR) testing associated with positive serology in most of the cases suggests a postinfectious syndrome. Because the pathophysiology of this syndrome is still poorly understood, extensive virological and immunological investigations are needed. METHODS: We report a series of 4 pediatric patients admitted to Geneva University Hospitals with persistent fever and laboratory evidence of inflammation meeting the published definition of MIS-C related to COVID-19, to whom an extensive virological and immunological workup was performed. RESULTS: RT-PCRs on multiple anatomical compartments were negative, whereas anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin A (IgA) and immunoglobulin G (IgG) were strongly positive by enzyme-linked immunosorbent assay and immunofluorescence. Both pseudoneutralization and full virus neutralization assays showed the presence of neutralizing antibodies in all children, confirming a recent infection with SARS-CoV-2. The analyses of cytokine profiles revealed an elevation in all cytokines, as reported in adults with severe COVID-19. Although differing in clinical presentation, some features of MIS-C show phenotypic overlap with hemophagocytic lymphohistiocytosis (HLH). In contrast to patients with primary HLH, our patients showed normal perforin expression and natural killer (NK) cell degranulation. The levels of soluble interleukin (IL)-2 receptor (sIL-2R) correlated with the severity of disease, reflecting recent T-cell activation. CONCLUSION: Our findings suggest that MIS-C related to COVID-19 is caused by a postinfectious inflammatory syndrome associated with an elevation in all cytokines, and markers of recent T-cell activation (sIL-2R) occurring despite a strong and specific humoral response to SARS-CoV-2. Further functional and genetic analyses are essential to better understand the mechanisms of host-pathogen interactions.

Obesity-Induced Increase in Cystatin C Alleviates Tissue Inflammation.

We recently demonstrated that removal of one kidney (uninephrectomy [UniNx]) in mice reduced high-fat diet (HFD)-induced adipose tissue inflammation, thereby improving adipose tissue and hepatic insulin sensitivity. Of note, circulating cystatin C (CysC) levels were increased in UniNx compared with sham-operated mice. Importantly, CysC may have anti-inflammatory properties, and circulating CysC levels were reported to positively correlate with obesity in humans and as shown here in HFD-fed mice. However, the causal relationship of such observation remains unclear. HFD feeding of CysC-deficient (CysC knockout [KO]) mice worsened obesity-associated adipose tissue inflammation and dysfunction, as assessed by proinflammatory macrophage accumulation. In addition, mRNA expression of proinflammatory mediators was increased, whereas markers of adipocyte differentiation were decreased. Similar to findings in adipose tissue, expression of proinflammatory cytokines was increased in liver and skeletal muscle of CysC KO mice. In line, HFD-induced hepatic insulin resistance and impairment of glucose tolerance were further aggravated in KO mice. Consistently, chow-fed CysC KO mice were more susceptible to lipopolysaccharide-induced adipose tissue inflammation. In people with obesity, circulating CysC levels correlated negatively with adipose tissue Hif1α as well as IL6 mRNA expression. Moreover, healthy (i.e., insulin-sensitive) subjects with obesity had significantly higher mRNA expression of CysC in white adipose tissue. In conclusion, CysC is upregulated under obesity conditions and thereby counteracts inflammation of peripheral insulin-sensitive tissues and, thus, obesity-associated deterioration of glucose metabolism.

Construction of a Suite of Computable Biological Network Models Focused on Mucociliary Clearance in the Respiratory Tract.

Mucociliary clearance (MCC), considered as a collaboration of mucus secreted from goblet cells, the airway surface liquid layer, and the beating of cilia of ciliated cells, is the airways' defense system against airborne contaminants. Because the process is well described at the molecular level, we gathered the available information into a suite of comprehensive causal biological network (CBN) models. The suite consists of three independent models that represent (1) cilium assembly, (2) ciliary beating, and (3) goblet cell hyperplasia/metaplasia and that were built in the Biological Expression Language, which is both human-readable and computable. The network analysis of highly connected nodes and pathways demonstrated that the relevant biology was captured in the MCC models. We also show the scoring of transcriptomic data onto these network models and demonstrate that the models capture the perturbation in each dataset accurately. This work is a continuation of our approach to use computational biological network models and mathematical algorithms that allow for the interpretation of high-throughput molecular datasets in the context of known biology. The MCC network model suite can be a valuable tool in personalized medicine to further understand heterogeneity and individual drug responses in complex respiratory diseases.

The NHR1-1 of Prs1 and the pentameric motif 284KKCPK288 of Prs3 permit multi-functionality of the PRPP synthetase in Saccharomyces cerevisiae.

The five-membered PRS gene family of Saccharomyces cerevisiae is an example of gene duplication allowing the acquisition of novel functions. Each of the five Prs polypeptides is theoretically capable of synthesising PRPP but at least one of the following heterodimers is required for survival: Prs1/Prs3, Prs2/Prs5 and Prs4/Prs5. Prs3 contains a pentameric motif 284KKCPK288 found only in nuclear proteins. Deletion of 284KKCPK288 destabilises the Prs1/Prs3 complex resulting in a cascade of events, including reduction in PRPP synthetase activity and altered cell wall integrity (CWI) as measured by caffeine sensitivity and Rlm1 expression. Prs3 also interacts with the kinetochore-associated protein, Nuf2. Following the possibility of 284KKCPK288-mediated transport of the Prs1/Prs3 complex to the nucleus, it may interact with Nuf2 and phosphorylated Slt2 permitting activation of Rlm1. This scenario explains the breakdown of CWI encountered in mutants lacking PRS3 or deleted for 284KKCPK288. However, removal of NHR1-1 from Prs1 does not disrupt the Prs1/Prs3 interaction as shown by increased PRPP synthetase activity. This is evidence for the separation of the two metabolic functions of the PRPP-synthesising machinery: provision of PRPP and maintenance of CWI and is an example of evolutionary development when multiple copies of a gene were present in the ancestral organism.

Epithelial proliferation in inflammatory skin disease is regulated by tetratricopeptide repeat domain 7 (Ttc7) in fibroblasts and lymphocytes.

BACKGROUND: Mutations in tetratricopeptide repeat domain 7A (TTC7A) and its mouse orthologue, Ttc7, result in a multisystemic disease, mostly affecting the epithelial barriers and immune system. Despite successful hematopoietic stem cell transplantation, ongoing progression of gastrointestinal manifestations can be life-threatening in TTC7A-deficient patients. OBJECTIVE: We sought to identify whether TTC7A mutations dysregulate epithelial cells only or whether a cell-intrinsic defect in lymphocytes or other cells contributes to disease manifestations. METHODS: Ttc7-mutated (Ttc7fsn/fsn) mice were crossed to generate double-mutant (Rag2-/-Ttc7fsn/fsn) and triple-mutant (Rag2-/-IL2rg-/-Ttc7fsn/fsn) mice. These models, together with bone marrow chimeras, were used to explore the role of adaptive and innate lymphocytes in the flaky skin phenotype. The effect of the Ttc7fsn/fsn mutation on stromal cells was tested in a xenograft model in conjunction with transcriptomic analysis of Ttc7fsn/fsn fibroblasts. RESULTS: We observed that the severity of epithelial hyperproliferation was accentuated by lymphocytes, whereas the phenotype was not induced by transfer of Ttc7-mutated hematopoietic cells. Furthermore, mice completely lacking the lymphocytic compartment were not protected from epithelial hyperproliferation. Ttc7-mutated mouse fibroblasts expressed increased transcript levels of insulin-like growth factor 1 (Igf1) and the antimicrobial protein regenerating islet-derived protein 3γ (Reg3γ). In a xenograft model Ttc7-mutated fibroblasts markedly increased epithelial proliferation of keratinocytes. Thus Ttc7-mutated fibroblasts were identified as potent instigators of epithelial hyperproliferation. CONCLUSION: Our results reveal a previously unsuspected fundamental cell-extrinsic role of Ttc7. We have identified potential candidates for molecularly targeted treatment strategies that will need to be evaluated in future preclinical studies.

Life-Threatening Primary Varicella Zoster Virus Infection With Hemophagocytic Lymphohistiocytosis-Like Disease in GATA2 Haploinsufficiency Accompanied by Expansion of Double Negative T-Lymphocytes.

Two unrelated patients with GATA2-haploinsufficiency developed a hemophagocytic lymphohistiocytosis (HLH)-like disease during a varicella zoster virus (VZV) infection. High copy numbers of VZV were detected in the blood, and the patients were successfully treated with acyclovir and intravenous immunoglobulins. After treatment with corticosteroids for the HLH, both patients made a full recovery. Although the mechanisms leading to this disease constellation have yet to be characterized, we hypothesize that impairment of the immunoregulatory role of NK cells in GATA2-haploinsufficiency may have accentuated the patients' susceptibility to HLH. Expansion of a double negative T-lymphocytic population identified with CyTOF could be a further factor contributing to HLH in these patients. This is the first report of VZV-triggered HLH-like disease in a primary immunodeficiency and the third report of HLH in GATA2-haploinsufficiency. Since HLH was part of the presentation in one of our patients, GATA2-haploinsufficiency represents a potential differential diagnosis in patients presenting with the clinical features of HLH-especially in cases of persisting cytopenia after recovery from HLH.

Lymphadenopathy driven by TCR-Vγ8Vδ1 T-cell expansion in FAS-related autoimmune lymphoproliferative syndrome.

FAS-dependent apoptosis in Vδ1 T cells makes the latter possible culprits for the lymphadenopathy observed in patients with FAS mutations.Rapamycin and methylprednisolone resistance should prompt clinicians to look for Vδ1 T cell proliferation in ALPS-FAS patients.

Organelle acidification negatively regulates vacuole membrane fusion in vivo.

The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector.

Mouse chronic social stress increases blood and brain kynurenine pathway activity and fear behaviour: Both effects are reversed by inhibition of indoleamine 2,3-dioxygenase.

Psychosocial stress is a major risk factor for mood and anxiety disorders, in which excessive reactivity to aversive events/stimuli is a major psychopathology. In terms of pathophysiology, immune-inflammation is an important candidate, including high blood and brain levels of metabolites belonging to the kynurenine pathway. Animal models are needed to study causality between psychosocial stress, immune-inflammation and hyper-reactivity to aversive stimuli. The present mouse study investigated effects of psychosocial stress as chronic social defeat (CSD) versus control-handling (CON) on: Pavlovian tone-shock fear conditioning, activation of the kynurenine pathway, and efficacy of a specific inhibitor (IDOInh) of the tryptophan-kynurenine catabolising enzyme indoleamine 2,3-dioxygenase (IDO1), in reversing CSD effects on the kynurenine pathway and fear. CSD led to excessive fear learning and memory, whilst repeated oral escitalopram (antidepressant and anxiolytic) reversed excessive fear memory, indicating predictive validity of the model. CSD led to higher blood levels of TNF-α, IFN-γ, kynurenine (KYN), 3-hydroxykynurenine (3-HK) and kynurenic acid, and higher KYN and 3-HK in amygdala and hippocampus. CSD was without effect on IDO1 gene or protein expression in spleen, ileum and liver, whilst increasing liver TDO2 gene expression. Nonetheless, oral IDOInh reduced blood and brain levels of KYN and 3-HK in CSD mice to CON levels, and we therefore infer that CSD increases IDO1 activity by increasing its post-translational activation. Furthermore, repeated oral IDOInh reversed excessive fear memory in CSD mice to CON levels. IDOInh reversal of CSD-induced hyper-activity in the kynurenine pathway and fear system contributes significantly to the evidence for a causal pathway between psychosocial stress, immune-inflammation and the excessive fearfulness that is a major psychopathology in stress-related neuropsychiatric disorders.

A new life for an old pump: V-ATPase and neurotransmitter release.

Neurons fire by releasing neurotransmitters via fusion of synaptic vesicles with the plasma membrane. Fusion can be evoked by an incoming signal from a preceding neuron or can occur spontaneously. Synaptic vesicle fusion requires the formation of trans complexes between SNAREs as well as Ca(2+) ions. Wang et al. (2014. J. Cell Biol. http://dx.doi.org/jcb.201312109) now find that the Ca(2+)-binding protein Calmodulin promotes spontaneous release and SNARE complex formation via its interaction with the V0 sector of the V-ATPase.

Dynamic regulation of Ero1α and peroxiredoxin 4 localization in the secretory pathway.

In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H2O2 as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H2O2 to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.