Iron dysregulation and inflammatory stress erythropoiesis associates with long-term outcome of COVID-19.

Persistent symptoms following SARS-CoV-2 infection are increasingly reported, although the drivers of post-acute sequelae (PASC) of COVID-19 are unclear. Here we assessed 214 individuals infected with SARS-CoV-2, with varying disease severity, for one year from COVID-19 symptom onset to determine the early correlates of PASC. A multivariate signature detected beyond two weeks of disease, encompassing unresolving inflammation, anemia, low serum iron, altered iron-homeostasis gene expression and emerging stress erythropoiesis; differentiated those who reported PASC months later, irrespective of COVID-19 severity. A whole-blood heme-metabolism signature, enriched in hospitalized patients at month 1-3 post onset, coincided with pronounced iron-deficient reticulocytosis. Lymphopenia and low numbers of dendritic cells persisted in those with PASC, and single-cell analysis reported iron maldistribution, suggesting monocyte iron loading and increased iron demand in proliferating lymphocytes. Thus, defects in iron homeostasis, dysregulated erythropoiesis and immune dysfunction due to COVID-19 possibly contribute to inefficient oxygen transport, inflammatory disequilibrium and persisting symptomatology, and may be therapeutically tractable.

A patient-centric modeling framework captures recovery from SARS-CoV-2 infection.

The biology driving individual patient responses to severe acute respiratory syndrome coronavirus 2 infection remains ill understood. Here, we developed a patient-centric framework leveraging detailed longitudinal phenotyping data and covering a year after disease onset, from 215 infected individuals with differing disease severities. Our analyses revealed distinct 'systemic recovery' profiles, with specific progression and resolution of the inflammatory, immune cell, metabolic and clinical responses. In particular, we found a strong inter-patient and intra-patient temporal covariation of innate immune cell numbers, kynurenine metabolites and lipid metabolites, which highlighted candidate immunologic and metabolic pathways influencing the restoration of homeostasis, the risk of death and that of long COVID. Based on these data, we identified a composite signature predictive of systemic recovery, using a joint model on cellular and molecular parameters measured soon after disease onset. New predictions can be generated using the online tool http://shiny.mrc-bsu.cam.ac.uk/apps/covid-19-systemic-recovery-prediction-app , designed to test our findings prospectively.

Longitudinal analysis reveals that delayed bystander CD8+ T cell activation and early immune pathology distinguish severe COVID-19 from mild disease.

The kinetics of the immune changes in COVID-19 across severity groups have not been rigorously assessed. Using immunophenotyping, RNA sequencing, and serum cytokine analysis, we analyzed serial samples from 207 SARS-CoV2-infected individuals with a range of disease severities over 12 weeks from symptom onset. An early robust bystander CD8+ T cell immune response, without systemic inflammation, characterized asymptomatic or mild disease. Hospitalized individuals had delayed bystander responses and systemic inflammation that was already evident near symptom onset, indicating that immunopathology may be inevitable in some individuals. Viral load did not correlate with this early pathological response but did correlate with subsequent disease severity. Immune recovery is complex, with profound persistent cellular abnormalities in severe disease correlating with altered inflammatory responses, with signatures associated with increased oxidative phosphorylation replacing those driven by cytokines tumor necrosis factor (TNF) and interleukin (IL)-6. These late immunometabolic and immune defects may have clinical implications.

Age-related immune response heterogeneity to SARS-CoV-2 vaccine BNT162b2.

Although two-dose mRNA vaccination provides excellent protection against SARS-CoV-2, there is little information about vaccine efficacy against variants of concern (VOC) in individuals above eighty years of age1. Here we analysed immune responses following vaccination with the BNT162b2 mRNA vaccine2 in elderly participants and younger healthcare workers. Serum neutralization and levels of binding IgG or IgA after the first vaccine dose were lower in older individuals, with a marked drop in participants over eighty years old. Sera from participants above eighty showed lower neutralization potency against the B.1.1.7 (Alpha), B.1.351 (Beta) and P.1. (Gamma) VOC than against the wild-type virus and were more likely to lack any neutralization against VOC following the first dose. However, following the second dose, neutralization against VOC was detectable regardless of age. The frequency of SARS-CoV-2 spike-specific memory B cells was higher in elderly responders (whose serum showed neutralization activity) than in non-responders after the first dose. Elderly participants showed a clear reduction in somatic hypermutation of class-switched cells. The production of interferon-γ and interleukin-2 by SARS-CoV-2 spike-specific T cells was lower in older participants, and both cytokines were secreted primarily by CD4 T cells. We conclude that the elderly are a high-risk population and that specific measures to boost vaccine responses in this population are warranted, particularly where variants of concern are circulating.

The potential role of antibodies against minor blood group antigens in renal transplantation.

Blood group antigens are red blood cell (RBC) surface markers comprising specific carbohydrate moieties attached to the glycolipids and glycoproteins within the membrane. In addition to the major ABO blood group antigens, at least 35 minor blood group antigens have been defined to date. These antigens have immunogenic potential and may cause a transfusion reaction. There is evidence for renal expression of antigens from the Kidd, MNS, Duffy and Lewis groups and therefore the potential for antibodies directed against these antigens to cross-react in a transplanted kidney. In individuals lacking a specific RBC antigen, antibodies may develop after de novo exposure to that antigen, in addition to the potential presence of pre-existing innate antibodies. Relatively little attention has been paid to non-ABO system antibodies, with most reports in the literature focusing on transfusion reactions rather than on any putative role in allograft rejection. Here, we review each of these antigens in the context of renal transplantation and what limited evidence there is on how such immunological risk may be assessed and managed.

Human B Cell Receptor Repertoire Sequencing.

Next-generation sequencing has the potential to uncover the complex nature of B cell immunity by revealing the full complexity of B cell receptor (BCR) repertoires in health and disease. However, there are drawbacks which can compromise the validity of the repertoire analysis caused by quantitative bias and accumulation of sequencing errors during the library preparation and sequencing. Here, we provide an optimized protocol designed to minimize bias for reproducible and accurate preparation of human BCR repertoire libraries for high-throughput sequencing.

Genetically and Functionally Distinct Immunoglobulin Heavy Chain Locus Duplication in Bats.

The genetic locus encoding immunoglobulin heavy chains (IgH) is critical for vertebrate humoral immune responses and diverse antibody repertoires. Immunoglobulin and T cell receptor loci of most bat species have not been annotated, despite the recurrent role of bats as viral reservoirs and sources of zoonotic pathogens. We investigated the genetic structure and function of IgH loci across the largest bat family, Vespertilionidae, focusing on big brown bats (Eptesicus fuscus ). We discovered that E. fuscus and ten other species within Vespertilionidae have two complete, functional, and distinct immunoglobulin heavy chain loci on separate chromosomes. This locus organization is previously unknown in mammals, but is reminiscent of more limited duplicated loci in teleost fish. Single cell transcriptomic data validate functional rearrangement and expression of immunoglobulin heavy chains of both loci in the expressed repertoire of Eptesicus fuscus , with maintenance of allelic exclusion, bias of usage toward the smaller and more compact IgH locus, and evidence of differential selection of antigen-experienced B cells and plasma cells varying by IgH locus use. This represents a unique mechanism for mammalian humoral immunity and may contribute to bat resistance to viral pathogenesis.

Regulation of immune responses in primary biliary cholangitis: a transcriptomic analysis of peripheral immune cells.

BACKGROUND AIMS: In patients with primary biliary cholangitis (PBC), the serum liver biochemistry measured during treatment with ursodeoxycholic acid-the UDCA response-accurately predicts long-term outcome. Molecular characterization of patients stratified by UDCA response can improve biological understanding of the high-risk disease, thereby helping to identify alternative approaches to disease-modifying therapy. In this study, we sought to characterize the immunobiology of the UDCA response using transcriptional profiling of peripheral blood mononuclear cell subsets. METHODS: We performed bulk RNA-sequencing of monocytes and TH1, TH17, TREG, and B cells isolated from the peripheral blood of 15 PBC patients with adequate UDCA response ("responders"), 16 PBC patients with inadequate UDCA response ("nonresponders"), and 15 matched controls. We used the Weighted Gene Co-expression Network Analysis to identify networks of co-expressed genes ("modules") associated with response status and the most highly connected genes ("hub genes") within them. Finally, we performed a Multi-Omics Factor Analysis of the Weighted Gene Co-expression Network Analysis modules to identify the principal axes of biological variation ("latent factors") across all peripheral blood mononuclear cell subsets. RESULTS: Using the Weighted Gene Co-expression Network Analysis, we identified modules associated with response and/or disease status (q<0.05) in each peripheral blood mononuclear cell subset. Hub genes and functional annotations suggested that monocytes are proinflammatory in nonresponders, but antiinflammatory in responders; TH1 and TH17 cells are activated in all PBC cases but better regulated in responders; and TREG cells are activated-but also kept in check-in responders. Using the Multi-Omics Factor Analysis, we found that antiinflammatory activity in monocytes, regulation of TH1 cells, and activation of TREG cells are interrelated and more prominent in responders. CONCLUSIONS: We provide evidence that adaptive immune responses are better regulated in patients with PBC with adequate UDCA response.

Regional differences in human ependymal and subventricular zone cytoarchitecture are unchanged in neuropsychiatric disease.

Much work has focused on the possible contribution of adult hippocampal neurogenesis to neuropsychiatric diseases. The hippocampal subgranular zone and the other stem cell-containing neurogenic niche, the subventricular zone (SVZ), share several cytological features and are regulated by some of the same molecular mechanisms. However, very little is known about the SVZ in neuropsychiatric disorders. This is important since it surrounds the lateral ventricles and in schizophrenia ventricular enlargement frequently follows forebrain nuclei shrinkage. Also, adult neurogenesis has been implicated in pharmacotherapy for affective disorders and many of the molecules associated with neuropsychiatric disorders affect SVZ biology. To assess the neurogenic niche, we examined material from 60 humans (Stanley Collection) and characterized the cytoarchitecture of the SVZ and ependymal layer in age-, sex- and post mortem interval-matched controls, and patients diagnosed with schizophrenia, bipolar illness, and depression (n = 15 each). There is a paucity of post mortem brains available for study in these diseases, so to maximize the number of possible parameters examined here, we quantified individual sections rather than a large series. Previous work showed that multiple sclerosis is associated with increased width of the hypocellular gap, a cell-sparse region that typifies the human SVZ. Statistically there were no differences between disease groups and controls in the width of the hypocellular gap or in the density of cells in the hypocellular gap. Because ventricular enlargement in schizophrenia may disrupt ependymal cells, we quantified them, but observed no difference between diagnostic groups and controls. There are significant differences in the prevalence of neuropsychiatric illness between the sexes. Therefore, we looked for male versus female differences, but did not observe any in the parameters quantified. We next turned to a finer spatial resolution and asked if there were differences amongst the disease groups in dorsal ventral subdivisions of the SVZ. Similar to when we treated the SVZ as a whole, we did not find such differences. However, compared to the dorsal SVZ, the ventral SVZ had a wider hypocellular gap and more ependymal cells in all four groups. In contrast, cell density was similar in dorsal ventral subregions of the SVZ hypocellular gap. These results show that though there are regional differences in the SVZ in humans, neuropsychiatric disorders do not seem to alter several fundamental histological features of this adult neurogenic zone.

The impact of hypoxia on B cells in COVID-19.

BACKGROUND: Prominent early features of COVID-19 include severe, often clinically silent, hypoxia and a pronounced reduction in B cells, the latter important in defence against SARS-CoV-2. This presentation resembles the phenotype of mice with VHL-deficient B cells, in which Hypoxia-Inducible Factors are constitutively active, suggesting hypoxia might drive B cell abnormalities in COVID-19. METHODS: Detailed B cell phenotyping was undertaken by flow-cytometry on longitudinal samples from patients with COVID-19 across a range of severities (NIHR Cambridge BioResource). The impact of hypoxia on the transcriptome was assessed by single-cell and whole blood RNA sequencing analysis. The direct effect of hypoxia on B cells was determined through immunisation studies in genetically modified and hypoxia-exposed mice. FINDINGS: We demonstrate the breadth of early and persistent defects in B cell subsets in moderate/severe COVID-19, including reduced marginal zone-like, memory and transitional B cells, changes also observed in B cell VHL-deficient mice. These findings were associated with hypoxia-related transcriptional changes in COVID-19 patient B cells, and similar B cell abnormalities were seen in mice kept in hypoxic conditions. INTERPRETATION: Hypoxia may contribute to the pronounced and persistent B cell pathology observed in acute COVID-19 pneumonia. Assessment of the impact of early oxygen therapy on these immune defects should be considered, as their correction could contribute to improved outcomes. FUNDING: Evelyn Trust, Addenbrooke's Charitable Trust, UKRI/NIHR, Wellcome Trust.

B cell receptor repertoire kinetics after SARS-CoV-2 infection and vaccination.

B cells are important in immunity to both severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and vaccination, but B cell receptor (BCR) repertoire development in these contexts has not been compared. We analyze serial samples from 171 SARS-CoV-2-infected individuals and 63 vaccine recipients and find the global BCR repertoire differs between them. Following infection, immunoglobulin (Ig)G1/3 and IgA1 BCRs increase, somatic hypermutation (SHM) decreases, and, in severe disease, IgM and IgA clones are expanded. In contrast, after vaccination, the proportion of IgD/M BCRs increase, SHM is unchanged, and expansion of IgG clones is prominent. VH1-24, which targets the N-terminal domain (NTD) and contributes to neutralization, is expanded post infection except in the most severe disease. Infection generates a broad distribution of SARS-CoV-2-specific clones predicted to target the spike protein, while a more focused response after vaccination mainly targets the spike's receptor-binding domain. Thus, the nature of SARS-CoV-2 exposure differentially affects BCR repertoire development, potentially informing vaccine strategies.

Monocyte-derived macrophages aggravate pulmonary vasculitis via cGAS/STING/IFN-mediated nucleic acid sensing.

Autoimmune vasculitis is a group of life-threatening diseases, whose underlying pathogenic mechanisms are incompletely understood, hampering development of targeted therapies. Here, we demonstrate that patients suffering from anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) showed increased levels of cGAMP and enhanced IFN-I signature. To identify disease mechanisms and potential therapeutic targets, we developed a mouse model for pulmonary AAV that mimics severe disease in patients. Immunogenic DNA accumulated during disease onset, triggering cGAS/STING/IRF3-dependent IFN-I release that promoted endothelial damage, pulmonary hemorrhages, and lung dysfunction. Macrophage subsets played dichotomic roles in disease. While recruited monocyte-derived macrophages were major disease drivers by producing most IFN-ß, resident alveolar macrophages contributed to tissue homeostasis by clearing red blood cells and limiting infiltration of IFN-ß-producing macrophages. Moreover, pharmacological inhibition of STING, IFNAR-I, or its downstream JAK/STAT signaling reduced disease severity and accelerated recovery. Our study unveils the importance of STING/IFN-I axis in promoting pulmonary AAV progression and identifies cellular and molecular targets to ameliorate disease outcomes.

Coagulation factor V is a T-cell inhibitor expressed by leukocytes in COVID-19.

Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

Immunodeficiency, autoimmunity, and increased risk of B cell malignancy in humans with TRAF3 mutations.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is a central regulator of immunity. TRAF3 is often somatically mutated in B cell malignancies, but its role in human immunity is not defined. Here, in five unrelated families, we describe an immune dysregulation syndrome of recurrent bacterial infections, autoimmunity, systemic inflammation, B cell lymphoproliferation, and hypergammaglobulinemia. Affected individuals each had monoallelic mutations in TRAF3 that reduced TRAF3 expression. Immunophenotyping showed that patients' B cells were dysregulated, exhibiting increased nuclear factor-κB 2 activation, elevated mitochondrial respiration, and heightened inflammatory responses. Patients had mild CD4+ T cell lymphopenia, with a reduced proportion of naïve T cells but increased regulatory T cells and circulating T follicular helper cells. Guided by this clinical phenotype, targeted analyses demonstrated that common genetic variants, which also reduce TRAF3 expression, are associated with an increased risk of B cell malignancies, systemic lupus erythematosus, higher immunoglobulin levels, and bacterial infections in the wider population. Reduced TRAF3 conveys disease risks by driving B cell hyperactivity via intrinsic activation of multiple intracellular proinflammatory pathways and increased mitochondrial respiration, with a likely contribution from dysregulated T cell help. Thus, we define monogenic TRAF3 haploinsufficiency syndrome and demonstrate how common TRAF3 variants affect a range of human diseases.

Subventricular zone cytoarchitecture changes in autism.

Autism is thought to be a neurodevelopmental disorder with symptoms developing during neonatal neurogenesis in the subventricular zone (SVZ). Autism associated genes alter SVZ proliferation and cytoarchitecture, yet the response of the human SVZ in autism is unknown. Epilepsy drives neurogenesis in rodents, but it is unclear how epilepsy interacts with autism in SVZ responses. The striatal and septal SVZ derive from separate lineages in rodents and generate different interneuron types. Yet it is unclear if autism unevenly regulates the striatal and septal SVZ. The human SVZ was immunohistochemically examined post-mortem from individuals with autism (n = 11) and controls (n = 11). Autism showed a lower cell density in the septal, but not striatal, SVZ hypocellular gap only in the absence of epilepsy. There was a decline in septal hypocellular gap cells with age in autism, but no correlation with age in controls. In contrast, PCNA+ cell numbers increased only in autism with epilepsy both in the hypocellular gap and in the ependymal layer on the septal but not striatal side. Ependymal cells also became GFAP immunoreactive in autism irrespective of epilepsy co-morbidity; however, this only occurred on the striatal side. In examining these questions we also discovered a subset of ependymal, astrocyte ribbon and RMS cells which express PCNA and Ki67, PLP, and α-tubulin. These results are the first example of a neuropsychiatric disease differentially affecting the septal and striatal SVZ. Altered cell density in the hypocellular gap and proliferation marker expression suggest individuals with autism may follow a different growth-trajectory.