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 COVID-19. 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 prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two 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 COVID-19, 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.
Some individuals do not return to baseline health following SARS-CoV-2 infection, leading to a condition known as long COVID. The underlying pathophysiology of long COVID remains unknown. Given that ...autoantibodies have been found to play a role in severity of SARS-CoV-2 infection and certain other post-COVID sequelae, their potential role in long COVID is important to investigate. Here, we apply a well-established, unbiased, proteome-wide autoantibody detection technology (T7 phage-display assay with immunoprecipitation and next-generation sequencing, PhIP-Seq) to a robustly phenotyped cohort of 121 individuals with long COVID, 64 individuals with prior COVID-19 who reported full recovery, and 57 pre-COVID controls. While a distinct autoreactive signature was detected that separated individuals with prior SARS-CoV-2 infection from those never exposed to SARS-CoV-2, we did not detect patterns of autoreactivity that separated individuals with long COVID from individuals fully recovered from COVID-19. These data suggest that there are robust alterations in autoreactive antibody profiles due to infection; however, no association of autoreactive antibodies and long COVID was apparent by this assay.
Objective
Familial hemophagocytic lymphohistiocytosis (FHLH) is a complex cytokine storm syndrome caused by genetic abnormalities rendering CD8+ T cells and natural killer cells incapable of ...cytolytic killing. In murine models of FHLH, interferon‐γ (IFNγ) produced by CD8+ T cells has been identified as a critical mediator of disease, and an IFNγ‐blocking antibody (emapalumab) has recently been approved by the Food and Drug Administration. However, development of hemophagocytic lymphohistiocytosis (HLH)/macrophage activation syndrome (MAS) in patients who are genetically unresponsive to IFNγ questions the absolute necessity of IFNγ in driving disease. This study was undertaken to determine the necessity of IFNγ in driving HLH.
Methods
IFNγ−/−Prf1−/− mice were infected with lymphocytic choriomeningitis virus (LCMV), and HLH immunopathologic features, including survival, weight loss, cytopenias, cytokine profiles, and immune cell phenotypes, were assessed. Mixed bone marrow chimeras were created to determine the immune cell–intrinsic role of IFNγ receptor signaling. CD8+ T cell depletion and interleukin‐33 (IL‐33)/ST2 blockade were performed using monoclonal antibodies.
Results
LCMV infection of IFNγ−/−Prf1−/− mice resulted in severe HLH‐like disease. CD8+ T cells and the IL‐33/ST2 axis remained essential mediators of disease; however, IFNγ‐independent HLH immunopathology correlated with a 10–15‐fold increase in neutrophilia (P < 0.001) and an altered cytokine milieu dominated by IL‐6, IL‐1β, and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (P < 0.05). Furthermore, IFNγ regulated CD8+ T cell expression of GM‐CSF and neutrophil survival.
Conclusion
IFNγ is not necessary for the development of fulminant HLH, requiring physicians to consider case‐by‐case treatment strategies. Use of therapies that target upstream activators of CD8+ T cells, such as IL‐33/ST2 signaling, may be more universally applicable treatment options that ameliorate both IFNγ‐dependent and ‐independent manifestations of HLH/MAS.
X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur ...throughout the CYBB gene, resulting in absent or defective gp91phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1–13 minigene into CYBB exon 1 resulted in no detectable gp91phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2–13 minigene into exon 2 restored both gp91phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2–13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.
Sweeney et al. demonstrate TALEN- and CRISPR/Cas9-mediated targeted correction of CYBB mutations in iPSCs from X-CGD patients, and identify a key issue for the design of effective gene targeting and knockin strategies: that retention or inclusion of intronic elements may be necessary for expression from an endogenous promoter.
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).
There are five genetic forms of chronic granulomatous disease (CGD), resulting from mutations in any of five subunits of phagocyte oxidase, an enzyme complex in neutrophils, monocytes, and ...macrophages that produces microbicidal reactive oxygen species. We generated induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ hematopoietic stem cells of patients with each of five CGD genotypes. We used zinc finger nuclease (ZFN) targeting the AAVS1 safe harbor site together with CGD genotype-specific minigene plasmids with flanking AAVS1 sequence to target correction of iPSC representing each form of CGD. We achieved targeted insertion with constitutive expression of desired oxidase subunit in 70–80% of selected iPSC clones. Neutrophils and macrophages differentiated from corrected CGD iPSCs demonstrated restored oxidase activity and antimicrobial function against CGD bacterial pathogens Staphylococcus aureus and Granulibacter bethesdensis. Using a standard platform that combines iPSC generation from peripheral blood CD34+ cells and ZFN mediated AAVS1 safe harbor minigene targeting, we demonstrate efficient generation of genetically corrected iPSCs using an identical approach for all five genetic forms of CGD. This safe harbor minigene targeting platform is broadly applicable to a wide range of inherited single gene metabolic disorders.
The associations between longitudinal dynamics and the breadth of SARS‐CoV‐2 neutralizing antibody (nAb) response with various Long COVID phenotypes before vaccination are not known. The capacity of ...antibodies to cross‐neutralize a variety of viral variants may be associated with ongoing pathology and persistent symptoms. We measured longitudinal neutralizing and cross‐neutralizing antibody responses to pre‐ and post‐SARS‐CoV‐2 Omicron variants in participants infected early in the COVID‐19 pandemic, before widespread rollout of SARS‐CoV‐2 vaccines. Cross‐sectional regression models adjusted for clinical covariates and longitudinal mixed‐effects models were used to determine the impact of the breadth and rate of decay of neutralizing responses on the development of Long COVID symptoms, as well as Long COVID phenotypes. We identified several novel relationships between SARS‐CoV‐2 antibody neutralization and the presence of Long COVID symptoms. Specifically, we show that, although nAb responses to the original, infecting strain of SARS‐CoV‐2 were not associated with Long COVID in cross‐sectional analyses, cross‐neutralization ID50 levels to the Omicron BA.5 variant approximately 4 months following acute infection was independently and significantly associated with greater odds of Long COVID and with persistent gastrointestinal and neurological symptoms. Longitudinal modeling demonstrated significant associations in the overall levels and rates of decay of neutralization capacity with Long COVID phenotypes. A higher proportion of participants had antibodies capable of neutralizing Omicron BA.5 compared with BA.1 or XBB.1.5 variants. Our findings suggest that relationships between various immune responses and Long COVID are likely complex but may involve the breadth of antibody neutralization responses.
Summary
SARS‐CoV‐2‐specific antibody neutralization of Omicron BA.5 variant approximately 4 months following acute infection with wild‐type virus before vaccination was independently and significantly associated with greater odds of distinct Long COVID phenotypes.
There are 5 genetic forms of chronic granulomatous disease (CGD), resulting from mutations in one of the CYBB, CYBA, NCF1, NCF2, or NCF4 genes encoding, respectively, the subunits of the phagocyte ...oxidase (phox) gp91phox, p22phox, p47phox, p67phox and p40phox. Phox is required for blood neutrophils and monocytes to produce microbicidal superoxide and hydrogen peroxide. We previously demonstrated zinc finger nuclease (ZFN) targeting of a corrective minigene encoding gp91phox to the AAVS1 safe harbor site of iPSC from a patient with X-linked CGD (XCGD), which restored phox activity in neutrophils differentiated from the corrected iPSC (Blood 2011, 117:5561). Subsequently, we have generated iPSC from the CD34+ hematopoietic stem cells (HSC) present in small volumes of peripheral blood from additional CGD patients to include those representing the other genetic forms of CGD (Blood 2013, 121:e98). We used our previously reported gp91phox AAVS1 targeting plasmid to create CGD genotype-specific targeting plasmids for correction of the other 4 genetic forms of CGD by replacing gp91 cDNA with cDNA for each of the autosomal recessive CGD genes. Using these genotype specific targeting plasmids with AAVS1 targeting ZFNs, this approach resulted in safe harbor minigene correction of iPSC for each of the 5 genetic forms of CGD, routinely achieving minigene insertion with constitutive expression of the specific desired transgene phox subunit protein in 70-80% percent of selected iPSC clones. Neutrophils or macrophages were generated from iPSC by first differentiating in STEMdiff APEL medium for 13 days to generate CD34+CD45+ cells, which were further expanded in HSC medium for 7 days. The expanded cells were differentiated either to neutrophils with G-CSF in the presence or absence of OP9 mouse stromal cells for 7 days, or to macrophages with M-CSF for 10 to 14 days. Full restoration of oxidase activity was observed in neutrophils or macrophages derived from minigene corrected CGD iPSC representing all the 5 genetic types of CGD. Furthermore, we demonstrated significant correction of bacterial growth inhibition function in corrected CGD iPSC derived neutrophils (Staphylococci) and macrophages (Granulibacter). Of particular note, the M-CSF differentiated macrophages derived from iPSC showed a significant increase in IL-10 production (p < 0.01) in response to UV-inactivated Listeria, with comparable levels to M-CSF derived macrophages differentiated from peripheral blood monocytes. This type of iPSC gene-correction approach with ex vivo production of autologous functional neutrophils or macrophages could be used for supportive autologous cell therapy in CGD patients with infections not responding to conventional treatment. Using a universal platform approach that combines iPSC generation from small volumes of peripheral blood and ZFN mediated AAVS1 safe harbor minigene targeting, we show it is possible to efficiently go from peripheral blood sample to genetically corrected iPSC to generation of functionally corrected neutrophils and macrophages using an identical approach for all 5 genetic forms of CGD. This platform approach is broadly applicable to a wide range of inherited single gene metabolic disorders.
No relevant conflicts of interest to declare.
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