The Immunology of Multisystem Inflammatory Syndrome in Children with COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is typically very mild and often asymptomatic in children. A complication is the rare multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19, presenting 4-6 weeks after infection as high fever, organ dysfunction, and strongly elevated markers of inflammation. The pathogenesis is unclear but has overlapping features with Kawasaki disease suggestive of vasculitis and a likely autoimmune etiology. We apply systems-level analyses of blood immune cells, cytokines, and autoantibodies in healthy children, children with Kawasaki disease enrolled prior to COVID-19, children infected with SARS-CoV-2, and children presenting with MIS-C. We find that the inflammatory response in MIS-C differs from the cytokine storm of severe acute COVID-19, shares several features with Kawasaki disease, but also differs from this condition with respect to T cell subsets, interleukin (IL)-17A, and biomarkers associated with arterial damage. Finally, autoantibody profiling suggests multiple autoantibodies that could be involved in the pathogenesis of MIS-C.

Immune system adaptation during gender-affirming testosterone treatment.

Infectious, inflammatory and autoimmune conditions present differently in males and females. SARS-CoV-2 infection in naive males is associated with increased risk of death, whereas females are at increased risk of long COVID1, similar to observations in other infections2. Females respond more strongly to vaccines, and adverse reactions are more frequent3, like most autoimmune diseases4. Immunological sex differences stem from genetic, hormonal and behavioural factors5 but their relative importance is only partially understood6-8. In individuals assigned female sex at birth and undergoing gender-affirming testosterone therapy (trans men), hormone concentrations change markedly but the immunological consequences are poorly understood. Here we performed longitudinal systems-level analyses in 23 trans men and found that testosterone modulates a cross-regulated axis between type-I interferon and tumour necrosis factor. This is mediated by functional attenuation of type-I interferon responses in both plasmacytoid dendritic cells and monocytes. Conversely, testosterone potentiates monocyte responses leading to increased tumour necrosis factor, interleukin-6 and interleukin-15 production and downstream activation of nuclear factor kappa B-regulated genes and potentiation of interferon-γ responses, primarily in natural killer cells. These findings in trans men are corroborated by sex-divergent responses in public datasets and illustrate the dynamic regulation of human immunity by sex hormones, with implications for the health of individuals undergoing hormone therapy and our understanding of sex-divergent immune responses in cisgender individuals.

Inherited human RelB deficiency impairs innate and adaptive immunity to infection.

We report two unrelated adults with homozygous (P1) or compound heterozygous (P2) private loss-of-function variants of V-Rel Reticuloendotheliosis Viral Oncogene Homolog B (RELB). The resulting deficiency of functional RelB impairs the induction of NFKB2 mRNA and NF-κB2 (p100/p52) protein by lymphotoxin in the fibroblasts of the patients. These defects are rescued by transduction with wild-type RELB complementary DNA (cDNA). By contrast, the response of RelB-deficient fibroblasts to Tumor Necrosis Factor (TNF) or IL-1ß via the canonical NF-κB pathway remains intact. P1 and P2 have low proportions of naïve CD4+ and CD8+ T cells and of memory B cells. Moreover, their naïve B cells cannot differentiate into immunoglobulin G (IgG)- or immunoglobulin A (IgA)-secreting cells in response to CD40L/IL-21, and the development of IL-17A/F-producing T cells is strongly impaired in vitro. Finally, the patients produce neutralizing autoantibodies against type I interferons (IFNs), even after hematopoietic stem cell transplantation, attesting to a persistent dysfunction of thymic epithelial cells in T cell selection and central tolerance to some autoantigens. Thus, inherited human RelB deficiency disrupts the alternative NF-κB pathway, underlying a T- and B cell immunodeficiency, which, together with neutralizing autoantibodies against type I IFNs, confers a predisposition to viral, bacterial, and fungal infections.

Monitoring drug-target interactions through target engagement-mediated amplification on arrays and in situ.

Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by the dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to fixed adherent cells agreed with expectations from expression profiles of the cells. We also introduce a proximity ligation variant of TEMA to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and in tissues.

A gene-centric approach to biomarker discovery identifies transglutaminase 1 as an epidermal autoantigen.

Autoantigen discovery is a critical challenge for the understanding and diagnosis of autoimmune diseases. While autoantibody markers in current clinical use have been identified through studies focused on individual disorders, we postulated that a reverse approach starting with a putative autoantigen to explore multiple disorders might hold promise. We here targeted the epidermal protein transglutaminase 1 (TGM1) as a member of a protein family prone to autoimmune attack. By screening sera from patients with various acquired skin disorders, we identified seropositive subjects with the blistering mucocutaneous disease paraneoplastic pemphigus. Validation in further subjects confirmed TGM1 autoantibodies as a 55% sensitive and 100% specific marker for paraneoplastic pemphigus. This gene-centric approach leverages the wealth of data available for human genes and may prove generally applicable for biomarker discovery in autoimmune diseases.

Sex dimorphism in the tumor microenvironment - From bench to bedside and back.

Cancer represents a significant cause of death and suffering in both the developed and developing countries. Key underlying issues in the mortality of cancer are delayed diagnosis and resistance to treatments. However, improvements in biomarkers represent one important step that can be taken for alleviating the suffering caused by malignancy. Precision-based medicine is promising for revolutionizing diagnostic and treatment strategies for cancer patients worldwide. Contemporary methods, including various omics and systems biology approaches, as well as advanced digital imaging and artificial intelligence, allow more accurate assessment of tumor characteristics at the patient level. As a result, treatment strategies can be specifically tailored and adapted for individual and/or groups of patients that carry certain tumor characteristics. This includes immunotherapy, which is based on characterization of the immunosuppressive tumor microenvironment (TME) and, more specifically, the presence and activity of immune cell subsets. Unfortunately, while it is increasingly clear that gender strongly affects immune regulation and response, there is a knowledge gap concerning differences in sex-specific immune responses and how these contribute to the immunosuppressive TME and the response to immunotherapy. In fact, sex dimorphism is poorly understood in cancer progression and is typically ignored in current clinical practice. In this review, we aim to survey the available literature and highlight the existing knowledge gap in order to encourage further studies that would contribute to understanding both gender-biased immunosuppression in the TME and the driver of tumor progression towards invasive and metastatic disease. The review highlights the need to include sex optimized/genderized medicine as a new concept in future medicine cancer diagnostics and treatments.

Genetic and immunologic evaluation of children with inborn errors of immunity and severe or critical COVID-19.

BACKGROUND: Most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals are asymptomatic or only exhibit mild disease. In about 10% of cases, the infection leads to hypoxemic pneumonia, although it is much more rare in children. OBJECTIVE: We evaluated 31 young patients aged 0.5 to 19 years who had preexisting inborn errors of immunity (IEI) but lacked a molecular diagnosis and were later diagnosed with coronavirus disease 2019 (COVID-19) complications. METHODS: Genetic evaluation by whole-exome sequencing was performed in all patients. SARS-CoV-2-specific antibodies, autoantibodies against type I IFN (IFN-I), and inflammatory factors in plasma were measured. We also reviewed COVID-19 disease severity/outcome in reported IEI patients. RESULTS: A potential genetic cause of the IEI was identified in 28 patients (90.3%), including mutations that may affect IFN signaling, T- and B-cell function, the inflammasome, and the complement system. From tested patients 65.5% had detectable virus-specific antibodies, and 6.8% had autoantibodies neutralizing IFN-I. Five patients (16.1%) fulfilled the diagnostic criteria of multisystem inflammatory syndrome in children. Eleven patients (35.4%) died of COVID-19 complications. All together, at least 381 IEI children with COVID-19 have been reported in the literature to date. Although many patients with asymptomatic or mild disease may not have been reported, severe presentation of COVID-19 was observed in 23.6% of the published cases, and the mortality rate was 8.7%. CONCLUSIONS: Young patients with preexisting IEI may have higher mortality than children without IEI when infected with SARS-CoV-2. Elucidating the genetic basis of IEI patients with severe/critical COVID-19 may help to develop better strategies for prevention and treatment of severe COVID-19 disease and complications in pediatric patients.

Inherited IFNAR1 Deficiency in a Child with Both Critical COVID-19 Pneumonia and Multisystem Inflammatory Syndrome.

BACKGROUND: Inborn errors of immunity (IEI) and autoantibodies to type I interferons (IFNs) underlie critical COVID-19 pneumonia in at least 15% of the patients, while the causes of multisystem inflammatory syndrome in children (MIS-C) remain elusive. OBJECTIVES: To detect causal genetic variants in very rare cases with concomitant critical COVID-19 pneumonia and MIS-C. METHODS: Whole exome sequencing was performed, and the impact of candidate gene variants was investigated. Plasma levels of cytokines, specific antibodies against the virus, and autoantibodies against type I IFNs were also measured. RESULTS: We report a 3-year-old child who died on day 56 of SARS-CoV-2 infection with an unusual clinical presentation, combining both critical COVID-19 pneumonia and MIS-C. We identified a large, homozygous loss-of-function deletion in IFNAR1, underlying autosomal recessive IFNAR1 deficiency. CONCLUSIONS: Our findings confirm that impaired type I IFN immunity can underlie critical COVID-19 pneumonia, while suggesting that it can also unexpectedly underlie concomitant MIS-C. Our report further raises the possibility that inherited or acquired dysregulation of type I IFN immunity might contribute to MIS-C in other patients.

X-Linked TLR7 Deficiency Underlies Critical COVID-19 Pneumonia in a Male Patient with Ataxia-Telangiectasia.

BACKGROUND: Coronavirus disease 2019 (COVID-19) exhibits a wide spectrum of clinical manifestations, ranging from asymptomatic to critical conditions. Understanding the mechanism underlying life-threatening COVID-19 is instrumental for disease prevention and treatment in individuals with a high risk. OBJECTIVES: We aimed to identify the genetic cause for critical COVID-19 pneumonia in a patient with a preexisting inborn error of immunity (IEI). METHODS: Serum levels of specific antibodies against the virus and autoantibodies against type I interferons (IFNs) were measured. Whole exome sequencing was performed, and the impacts of candidate gene variants were investigated. We also evaluated 247 ataxia-telangiectasia (A-T) patients in the Iranian IEI registry. RESULTS: We report a 7-year-old Iranian boy with a preexisting hyper IgM syndrome who developed critical COVID-19 pneumonia. IgM only specific COVID-19 immune response was detected but no autoantibodies against type I IFN were observed. A homozygous deleterious mutation in the ATM gene was identified, which together with his antibody deficiency, radiosensitivity, and neurological signs, established a diagnosis of A-T. Among the 247 A-T patients evaluated, 36 had SARS-CoV-2 infection, but all had mild symptoms or were asymptomatic except the index patient. A hemizygous deleterious mutation in the TLR7 gene was subsequently identified in the patient. CONCLUSIONS: We report a unique IEI patient with combined ATM and TLR7 deficiencies. The two genetic defects underlie A-T and critical COVID-19 in this patient, respectively.

Screening for autoantibody targets in post-vaccination narcolepsy using proteome arrays.

Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a specific loss of hypocretin-producing neurons. The incidence of NT1 increased in Sweden, Finland and Norway following Pandemrix®-vaccination, initiated to prevent the 2009 influenza pandemic. The pathogenesis of NT1 is poorly understood, and causal links to vaccination are yet to be clarified. The strong association with Human leukocyte antigen (HLA) DQB1*06:02 suggests an autoimmune pathogenesis, but proposed autoantigens remain controversial. We used a two-step approach to identify autoantigens in patients that acquired NT1 after Pandemrix®-vaccination. Using arrays of more than 9000 full-length human proteins, we screened the sera of 10 patients and 24 healthy subjects for autoantibodies. Identified candidate antigens were expressed in vitro to enable validation studies with radiobinding assays (RBA). The validation cohort included NT1 patients (n = 39), their first-degree relatives (FDR) (n = 66), population controls (n = 188), and disease controls representing multiple sclerosis (n = 100) and FDR to type 1 diabetes patients (n = 41). Reactivity towards previously suggested NT1 autoantigen candidates including Tribbles homolog 2, Prostaglandin D2 receptor, Hypocretin receptor 2 and α-MSH/proopiomelanocortin was not replicated in the protein array screen. By comparing case to control signals, three novel candidate autoantigens were identified in the protein array screen; LOC401464, PARP3 and FAM63B. However, the RBA did not confirm elevated reactivity towards either of these proteins. In summary, three putative autoantigens in NT1 were identified by protein array screening. Autoantibodies against these candidates could not be verified with independent methods. Further studies are warranted to identify hypothetical autoantigens related to the pathogenesis of Pandemrix®-induced NT1.

Autoantibodies Targeting a Collecting Duct-Specific Water Channel in Tubulointerstitial Nephritis.

Tubulointerstitial nephritis is a common cause of kidney failure and may have diverse etiologies. This form of nephritis is sometimes associated with autoimmune disease, but the role of autoimmune mechanisms in disease development is not well understood. Here, we present the cases of three patients with autoimmune polyendocrine syndrome type 1 who developed tubulointerstitial nephritis and ESRD in association with autoantibodies against kidney collecting duct cells. One of the patients developed autoantibodies targeting the collecting duct-specific water channel aquaporin 2, whereas autoantibodies of the two other patients reacted against the HOXB7 or NFAT5 transcription factors, which regulate the aquaporin 2 promoter. Our findings suggest that tubulointerstitial nephritis developed in these patients as a result of an autoimmune insult on the kidney collecting duct cells.

No link between type I interferon autoantibody positivity and adverse reactions to COVID-19 vaccines.

Type I interferons act as gatekeepers against viral infection, and autoantibodies that neutralize these signaling molecules have been associated with COVID-19 severity and adverse reactions to the live-attenuated yellow fever vaccine. On this background, we sought to examine whether autoantibodies against type I interferons were associated with adverse events following COVID-19 vaccination. Our nationwide analysis suggests that type I interferon autoantibodies were not associated with adverse events after mRNA or viral-vector COVID-19 vaccines.

Autoantibodies to protein S may explain rare cases of coagulopathy following COVID-19 vaccination.

While Coronavirus disease 2019 (COVID-19) vaccines have proven to be both effective and generally safe, rare but severe adverse events following immunization (AEFIs) are described. Autoantibodies to platelet factor-4 are associated with catastrophic thrombotic AEFIs, but comprehensive investigations of other autoantibodies are lacking. We aimed to detect and describe autoantibodies targeting coagulation-related proteins in a population-wide cohort (SWEDEGENE) including AEFIs attributed to COVID-19 vaccines in Sweden. Subjects were recruited from December 2020 to October 2022 and were stratified based on diagnosis and COVID-19 exposure. Screening was carried out in two phases, with a multiplex bead-based assay in the first subset (until September 2021) and with targeted assays for the second (until October 2022). Positivity was defined based on absolute, relative, and biological/technical thresholds. Patients with coagulation-related AEFIs were older and the Vaxzevria vaccine was overrepresented in this group. Two cases had antiphospholipid antibodies but none had PF4 antibodies. We identified six positives for protein S autoantibodies. Protein S concentrations were negatively correlated with autoantibody response in patients with immunoreactivity and functional analysis revealed low protein S activity in three subjects. Our population-wide analysis reveals cases with autoantibodies against protein S which possibly underlie coagulopathic AEFIs.

Neutralizing IFN-γ autoantibodies are rare and pathogenic in HLA-DRB1*15:02 or 16:02 individuals.

BACKGROUNDWeakly virulent environmental mycobacteria (EM) can cause severe disease in HLA-DRB1*15:02 or 16:02 adults harboring neutralizing anti-IFN-γ autoantibodies (nAIGAs). The overall prevalence of nAIGAs in the general population is unknown, as are the penetrance of nAIGAs in HLA-DRB1*15:02 or 16:02 individuals and the proportion of patients with unexplained, adult-onset EM infections carrying nAIGAs.METHODSThis study analyzed the detection and neutralization of anti-IFN-γ autoantibodies (auto-Abs) from 8,430 healthy individuals of the general population, 257 HLA-DRB1*15:02 or 16:02 carriers, 1,063 patients with autoimmune disease, and 497 patients with unexplained severe disease due to EM.RESULTSWe found that anti-IFN-γ auto-Abs detected in 4,148 of 8,430 healthy individuals (49.2%) from the general population of an unknown HLA-DRB1 genotype were not neutralizing. Moreover, we did not find nAIGAs in 257 individuals carrying HLA-DRB1* 15:02 or 16:02. Additionally, nAIGAs were absent in 1,063 patients with an autoimmune disease. Finally, 7 of 497 patients (1.4%) with unexplained severe disease due to EM harbored nAIGAs.CONCLUSIONThese findings suggest that nAIGAs are isolated and that their penetrance in HLA-DRB1*15:02 or 16:02 individuals is low, implying that they may be triggered by rare germline or somatic variants. In contrast, the risk of mycobacterial disease in patients with nAIGAs is high, confirming that these nAIGAs are the cause of EM disease.FUNDINGThe Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the National Institutes of Health (NIH) (R01AI095983 and U19AIN1625568), the National Center for Advancing Translational Sciences (NCATS), the NIH Clinical and Translational Science Award (CTSA) program (UL1 TR001866), the French National Research Agency (ANR) under the "Investments for the Future" program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), ANR-GENMSMD (ANR-16-CE17-0005-01), ANR-MAFMACRO (ANR-22-CE92-0008), ANRSECTZ170784, the French Foundation for Medical Research (FRM) (EQU201903007798), the ANRS-COV05, ANR GENVIR (ANR-20-CE93-003), and ANR AI2D (ANR-22-CE15-0046) projects, the ANR-RHU program (ANR-21-RHUS-08-COVIFERON), the European Union's Horizon 2020 research and innovation program under grant agreement no. 824110 (EASI-genomics), the Square Foundation, Grandir - Fonds de solidarité pour l'enfance, the Fondation du Souffle, the SCOR Corporate Foundation for Science, the Battersea & Bowery Advisory Group, William E. Ford, General Atlantic's Chairman and Chief Executive Officer, Gabriel Caillaux, General Atlantic's Co-President, Managing Director, and Head of business in EMEA, and the General Atlantic Foundation, Institut National de la Santé et de la Recherche Médicale (INSERM) and of Paris Cité University. JR was supported by the INSERM PhD program for doctors of pharmacy (poste d'accueil INSERM). JR and TLV were supported by the Bettencourt-Schueller Foundation and the MD-PhD program of the Imagine Institute. MO was supported by the David Rockefeller Graduate Program, the Funai Foundation for Information Technology (FFIT), the Honjo International Scholarship Foundation (HISF), and the New York Hideyo Noguchi Memorial Society (HNMS).

Relation between HLA and copy number variation of steroid 21-hydroxylase in a Swedish cohort of patients with autoimmune Addison's disease.

OBJECTIVE: Autoantibodies against the adrenal enzyme 21-hydroxylase is a hallmark manifestation in autoimmune Addison's disease (AAD). Steroid 21-hydroxylase is encoded by CYP21A2, which is located in the human leucocyte antigen (HLA) region together with the highly similar pseudogene CYP21A1P. A high level of copy number variation is seen for the 2 genes, and therefore, we asked whether genetic variation of the CYP21 genes is associated with AAD. DESIGN: Case-control study on patients with AAD and healthy controls. METHODS: Using next-generation DNA sequencing, we estimated the copy number of CYP21A2 and CYP21A1P, together with HLA alleles, in 479 Swedish patients with AAD and autoantibodies against 21-hydroxylase and in 1393 healthy controls. RESULTS: With 95% of individuals carrying 2 functional 21-hydroxylase genes, no difference in CYP21A2 copy number was found when comparing patients and controls. In contrast, we discovered a lower copy number of the pseudogene CYP21A1P among AAD patients (P = 5 × 10-44), together with associations of additional nucleotide variants, in the CYP21 region. However, the strongest association was found for HLA-DQB1*02:01 (P = 9 × 10-63), which, in combination with the DRB1*04:04-DQB1*03:02 haplotype, imposed the greatest risk of AAD. CONCLUSIONS: We identified strong associations between copy number variants in the CYP21 region and risk of AAD, although these associations most likely are due to linkage disequilibrium with disease-associated HLA class II alleles.

Autoantibodies and SARS-CoV2 infection: The spectrum from association to clinical implication: Report of the 15th Dresden Symposium on Autoantibodies.

The relation between infections and autoimmune diseases has been extensively investigated. Multiple studies suggest a causal relation between these two entities with molecular mimicry, hyperstimulation and dysregulation of the immune system as plausible mechanisms. The recent pandemic with a new virus, i.e., SARS-CoV-2, has resulted in numerous studies addressing the potential of this virus to induce autoimmunity and, eventually, autoimmune disease. In addition, it has also revealed that pre-existing auto-immunity (auto-Abs neutralizing type I IFNs) could cause life-threatening disease. Therefore, the topic of the 15th Dresden Symposium on Autoantibodies was focused on autoimmunity in the SARS-CoV-2 era. This report is a collection and distillation of the topics presented at this meeting.

Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq.

Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.