Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19.

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

Epigenetic programming underpins B cell dysfunction in human SLE.

Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naive cells. Although these cells express distinct markers, their epigenetic architecture and how it contributes to SLE remain poorly understood. To address this, we determined the DNA methylomes, chromatin accessibility profiles and transcriptomes from five human B cell subsets, including a newly defined effector B cell subset, from subjects with SLE and healthy controls. Our data define a differentiation hierarchy for the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Importantly, an SLE molecular signature was already established in resting naive cells and was dominated by enrichment of accessible chromatin in motifs for AP-1 and EGR transcription factors. Together, these factors acted in synergy with T-BET to shape the epigenome of expanded SLE effector B cell subsets. Thus, our data define the molecular foundation of pathogenic B cell dysfunction in SLE.

Hotspot shelters stimulate frog resistance to chytridiomycosis.

Many threats to biodiversity cannot be eliminated; for example, invasive pathogens may be ubiquitous. Chytridiomycosis is a fungal disease that has spread worldwide, driving at least 90 amphibian species to extinction, and severely affecting hundreds of others1-4. Once the disease spreads to a new environment, it is likely to become a permanent part of that ecosystem. To enable coexistence with chytridiomycosis in the field, we devised an intervention that exploits host defences and pathogen vulnerabilities. Here we show that sunlight-heated artificial refugia attract endangered frogs and enable body temperatures high enough to clear infections, and that having recovered in this way, frogs are subsequently resistant to chytridiomycosis even under cool conditions that are optimal for fungal growth. Our results provide a simple, inexpensive and widely applicable strategy to buffer frogs against chytridiomycosis in nature. The refugia are immediately useful for the endangered species we tested and will have broader utility for amphibian species with similar ecologies. Furthermore, our concept could be applied to other wildlife diseases in which differences in host and pathogen physiologies can be exploited. The refugia are made from cheap and readily available materials and therefore could be rapidly adopted by wildlife managers and the public. In summary, habitat protection alone cannot protect species that are affected by invasive diseases, but simple manipulations to microhabitat structure could spell the difference between the extinction and the persistence of endangered amphibians.

Dysregulated naive B cells and de novo autoreactivity in severe COVID-19.

Severe SARS-CoV-2 infection1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic2-5. More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential6-10, although their origins and resolution have remained unclear11. Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity12,13, as a dominant feature of severe and critical COVID-19 (refs. 14-18). Here, using single-cell B cell repertoire analysis of patients with mild and severe disease, we identify the expansion of a naive-derived, low-mutation IgG1 population of antibody-secreting cells (ASCs) reflecting features of low selective pressure. These features correlate with progressive, broad, clinically relevant autoreactivity, particularly directed against nuclear antigens and carbamylated proteins, emerging 10-15 days after the onset of symptoms. Detailed analysis of the low-selection compartment shows a high frequency of clonotypes specific for both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against the glomerular basement membrane. We further identify the contraction of this pathway on recovery, re-establishment of tolerance standards and concomitant loss of acute-derived ASCs irrespective of antigen specificity. However, serological autoreactivity persists in a subset of patients with postacute sequelae, raising important questions as to the contribution of emerging autoreactivity to continuing symptomology on recovery. In summary, this study demonstrates the origins, breadth and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae.

Diversity, cellular origin and autoreactivity of antibody-secreting cell population expansions in acute systemic lupus erythematosus.

Acute systemic lupus erythematosus (SLE) courses with surges of antibody-secreting cells (ASCs) whose origin, diversity and contribution to serum autoantibodies remain unknown. Here, deep sequencing, proteomic profiling of autoantibodies and single-cell analysis demonstrated highly diversified ASCs punctuated by clones expressing the variable heavy-chain region VH4-34 that produced dominant serum autoantibodies. A fraction of ASC clones contained autoantibodies without mutation, a finding consistent with differentiation outside the germinal centers. A substantial ASC segment was derived from a distinct subset of newly activated naive cells of considerable clonality that persisted in the circulation for several months. Thus, selection of SLE autoreactivities occurred during polyclonal activation, with prolonged recruitment of recently activated naive B cells. Our findings shed light on the pathogenesis of SLE, help explain the benefit of agents that target B cells and should facilitate the design of future therapies.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

Patients taking benralizumab, dupilumab, or mepolizumab have lower postvaccination SARS-CoV-2 immunity.

BACKGROUND: Biologic therapies inhibiting the IL-4 or IL-5 pathways are very effective in the treatment of asthma and other related conditions. However, the cytokines IL-4 and IL-5 also play a role in the generation of adaptive immune responses. Although these biologics do not cause overt immunosuppression, their effect in primary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunization has not been studied completely. OBJECTIVE: Our aim was to evaluate the antibody and cellular immunity after SARS-CoV-2 mRNA vaccination in patients on biologics (PoBs). METHODS: Patients with severe asthma or atopic dermatitis who were taking benralizumab, dupilumab, or mepolizumab and had received the initial dose of the 2-dose adult SARS-CoV-2 mRNA vaccine were enrolled in a prospective, observational study. As our control group, we used a cohort of immunologically healthy subjects (with no significant immunosuppression) who were not taking biologics (NBs). We used a multiplexed immunoassay to measure antibody levels, neutralization assays to assess antibody function, and flow cytometry to quantitate Spike-specific lymphocytes. RESULTS: We analyzed blood from 57 patients in the PoB group and 46 control subjects from the NB group. The patients in the PoB group had lower levels of SARS-CoV-2 antibodies, pseudovirus neutralization, live virus neutralization, and frequencies of Spike-specific B and CD8 T cells at 6 months after vaccination. In subgroup analyses, patients with asthma who were taking biologics had significantly lower pseudovirus neutralization than did subjects with asthma who were not taking biologics. CONCLUSION: The patients in the PoB group had reduced SARS-CoV-2-specific antibody titers, neutralizing activity, and virus-specific B- and CD8 T-cell counts. These results have implications when considering development of a more individualized immunization strategy in patients who receive biologic medications blocking IL-4 or IL-5 pathways.

Plasma cell survival: The intrinsic drivers, migratory signals, and extrinsic regulators.

Antibody-secreting cells (ASC) are the effectors of protective humoral immunity and the only cell type that produces antibodies or immunoglobulins in mammals. In addition to their formidable capacity to secrete massive quantities of proteins, ASC are terminally differentiated and have unique features to become long-lived plasma cells (LLPC). Upon antigen encounter, B cells are activated through a complex multistep process to undergo fundamental morphological, subcellular, and molecular transformation to become an efficient protein factory with lifelong potential. The ASC survival potential is determined by factors at the time of induction, capacity to migration from induction to survival sites, and ability to mature in the specialized bone marrow microenvironments. In the past decade, considerable progress has been made in identifying factors regulating ASC longevity. Here, we review the intrinsic drivers, trafficking signals, and extrinsic regulators with particular focus on how they impact the survival potential to become a LLPC.

Long-Lived Plasma Cells Are Contained within the CD19(-)CD38(hi)CD138(+) Subset in Human Bone Marrow.

Antibody responses to viral infections are sustained for decades by long-lived plasma cells (LLPCs). However, LLPCs have yet to be characterized in humans. Here we used CD19, CD38, and CD138 to identify four PC subsets in human bone marrow (BM). We found that the CD19(-)CD38(hi)CD138(+) subset was morphologically distinct, differentially expressed PC-associated genes, and exclusively contained PCs specific for viral antigens to which the subjects had not been exposed for more than 40 years. Protein sequences of measles- and mumps-specific circulating antibodies were encoded for by CD19(-)CD38(hi)CD138(+) PCs in the BM. Finally, we found that CD19(-)CD38(hi)CD138(+) PCs had a distinct RNA transcriptome signature and human immunoglobulin heavy chain (VH) repertoire that was relatively uncoupled from other BM PC subsets and probably represents the B cell response's "historical record" of antigenic exposure. Thus, our studies define human LLPCs and provide a mechanism for the life-long maintenance of anti-viral antibodies in the serum.

Viral infections causing asthma exacerbations in the age of biologics and the COVID-19 pandemic.

PURPOSE OF REVIEW: Asthma exacerbations are associated with substantial symptom burden and healthcare costs. Viral infections are the most common identified cause of asthma exacerbations. The epidemiology of viral respiratory infections has undergone a significant evolution during the COVID-19 pandemic. The relationship between viruses and asthmatic hosts has long been recognized but it is still incompletely understood. The use of newly approved asthma biologics has helped us understand this interaction better. RECENT FINDINGS: We review recent updates on the interaction between asthma and respiratory viruses, and we address how biologics and immunotherapies could affect this relationship by altering the respiratory mucosa cytokine milieu. By exploring the evolving epidemiological landscape of viral infections during the different phases of the COVID-19 pandemic, we emphasize the early post-pandemic stage, where a resurgence of pre-pandemic viruses with atypical seasonality patterns occurred. Finally, we discuss the newly developed RSV and SARS-CoV-2 vaccines and how they reduce respiratory infections. SUMMARY: Characterizing how respiratory viruses interact with asthmatic hosts will allow us to identify tailored therapies to reduce the burden of asthma exacerbations. New vaccination strategies are likely to shape the future viral asthma exacerbation landscape.

Safety and feasibility pilot study of continuous low-dose maternal supplemental oxygen in fetal single ventricle heart disease.

OBJECTIVES: Fetuses with single ventricle physiology (SVP) exhibit reductions in fetal cerebral oxygenation with associated delays in fetal brain growth and neurodevelopmental outcomes. Maternal supplemental oxygen (MSO) has been proposed to improve fetal brain growth but current evidence on dosing, candidacy, and outcomes are limited. In this pilot study, we evaluated the safety and feasibility of continuous low-dose MSO in the setting of SVP. METHODS: This single-centre, open-label, pilot phase 1 safety and feasibility clinical trial included 25 pregnant individuals with a fetal diagnosis of SVP. Participants self-administered continuous supplemental oxygen using medical-grade oxygen concentrators for up to 24 hours per day from the second half of gestation until delivery. The primary aim was the evaluation of the safety profile and feasibility of MSO. A secondary preliminary analysis was performed to assess the impact of MSO on the fetal circulation by echocardiography and late-gestational cardiovascular magnetic resonance, early outcomes including brain growth and pre-operative brain injury, and 18-month neurodevelopmental outcomes by the Bayley Scales of Infant and Toddler Development 3rd Edition compared to a contemporary fetal SVP cohort that received standard of care (SOC). RESULTS: Among 25 participants, the average maternal age at conception was 35 years, and fetal SVP diagnoses included 16 right ventricle dominant, 8 left ventricle dominant, and 1 indeterminant ventricular morphology. Participants started the trial at approximately 29.3 gestational weeks and took MSO for a median 16.1 hours per day for 63 days, cumulating a median 1029 hours of oxygen intake from enrollment until delivery. The only treatment-associated adverse events were nasal complications that were typically resolved by attaching a humidifier unit to the oxygen concentrator. No premature closure of the ductus arteriosus or unexpected fetal demise was observed. In the secondary analysis, MSO was not associated with any changes in fetal growth, middle cerebral artery pulsatility index, cerebroplacental ratio, nor head circumference to abdominal circumference ratio Z-scores over gestation compared to SOC. Although MSO was associated with changes in umbilical artery pulsatility index Z-score over gestation compared to SOC (p=0.02), this was likely due to initial baseline differences in placental resistance. At late-gestational cardiovascular magnetic resonance, MSO was not associated with any significant increase in umbilical vein oxygen saturation, fetal oxygen delivery, or fetal cerebral oxygen delivery. Similarly, we observed no differences in newborn outcomes including brain volume and pre-operative brain injury, nor mortality by 18 months of age, nor neurodevelopmental outcomes at 18 months of age. CONCLUSIONS: This pilot phase 1 clinical trial indicates low-dose maternal supplemental oxygen therapy is safe and well tolerated in pregnancies diagnosed with fetal SVP. However, our protocol was not associated with any significant changes in fetal circulatory physiology or improvements in early neurologic or neurodevelopmental outcomes. This article is protected by copyright. All rights reserved.

Small Interfering RNA Mediated Messenger RNA Knockdown in the Amphibian Pathogen Batrachochytrium dendrobatidis.

RNA interference (RNAi) has not been tested in the pandemic amphibian pathogen, Batrachochytrium dendrobatidis, but developing this technology could be useful to elucidate virulence mechanisms, identify therapeutic targets, and may present a novel antifungal treatment option for chytridiomycosis. To manipulate and decipher gene function, rationally designed small interfering RNA (siRNA) can initiate the destruction of homologous messenger RNA (mRNA), resulting in the "knockdown" of target gene expression. Here, we investigate whether siRNA can be used to manipulate gene expression in B. dendrobatidis via RNAi using differing siRNA strategies to target genes involved in glutathione and ornithine synthesis. To determine the extent and duration of mRNA knockdown, target mRNA levels were monitored for 24-48 h after delivery of siRNA targeting glutamate-cysteine ligase, with a maximum of ~56% reduction in target transcripts occurring at 36 h. A second siRNA design targeting glutamate-cysteine ligase also resulted in ~53% knockdown at this time point. siRNA directed toward a different gene target, ornithine decarboxylase, achieved 17% reduction in target transcripts. Although no phenotypic effects were observed, these results suggest that RNAi is possible in B. dendrobatidis, and that gene expression can be manipulated in this pathogen. We outline ideas for further optimization steps to increase knockdown efficiency to better harness RNAi techniques for control of B. dendrobatidis.

One Health Approach to Globalizing, Accelerating, and Focusing Amphibian and Reptile Disease Research-Reflections and Opinions from the First Global Amphibian and Reptile Disease Conference.

The world's reptiles and amphibians are experiencing dramatic and ongoing losses in biodiversity, changes that can have substantial effects on ecosystems and human health. In 2022, the first Global Amphibian and Reptile Disease Conference was held, using One Health as a guiding principle. The conference showcased knowledge on numerous reptile and amphibian pathogens from several standpoints, including epidemiology, host immune defenses, wild population effects, and mitigation. The conference also provided field experts the opportunity to discuss and identify the most urgent herpetofaunal disease research directions necessary to address current and future threats to reptile and amphibian biodiversity.

Evidence for sodium-rich alkaline water in the Tagish Lake parent body and implications for amino acid synthesis and racemization.

Understanding the timing and mechanisms of amino acid synthesis and racemization on asteroidal parent bodies is key to demonstrating how amino acids evolved to be mostly left-handed in living organisms on Earth. It has been postulated that racemization can occur rapidly dependent on several factors, including the pH of the aqueous solution. Here, we conduct nanoscale geochemical analysis of a framboidal magnetite grain within the Tagish Lake carbonaceous chondrite to demonstrate that the interlocking crystal arrangement formed within a sodium-rich, alkaline fluid environment. Notably, we report on the discovery of Na-enriched subgrain boundaries and nanometer-scale Ca and Mg layers surrounding individual framboids. These interstitial coatings would yield a surface charge state of zero in more-alkaline fluids and prevent assimilation of the individual framboids into a single grain. This basic solution would support rapid synthesis and racemization rates on the order of years, suggesting that the low abundances of amino acids in Tagish Lake cannot be ascribed to fluid chemistry.

Efficacy, toxicities, and prognostic factors of stereotactic body radiotherapy for unresectable liver metastases.

INTRODUCTION: This study aims to determine the outcomes of stereotactic body radiotherapy (SBRT) for liver metastases in patients not eligible for surgery. METHODS: This study included 31 consecutive patients with unresectable liver metastases who received SBRT between January 2012 and December 2017; 22 patients had primary colorectal cancer and nine patients had primary non-colorectal cancer. Treatments ranged from 24 Gy to 48 Gy in 3 to 6 fractions over 1 to 2 weeks. Survival, response rates, toxicities, clinical characteristics, and dosimetric parameters were evaluated. Multivariate analysis was performed to identify significant prognostic factors for survival. RESULTS: Among these 31 patients, 65% had received at least one prior regimen of systemic therapy for metastatic disease, whereas 29% had received chemotherapy for disease progression or immediately after SBRT. The median follow-up interval was 18.9 months; actuarial in-field local control rates at 1, 2, and 3 years after SBRT were 94%, 55%, and 42%, respectively. The median survival duration was 32.9 months; 1-year, 2-year, and 3-year actuarial survival rates were 89.6%, 57.1%, and 46.2%, respectively. The median time to progression was 10.9 months. Stereotactic body radiotherapy was well-tolerated, with grade 1 toxicities of fatigue (19%) and nausea (10%). Patients who received post-SBRT chemotherapy had significant longer overall survival (P=0.039 for all patients and P=0.001 for patients with primary colorectal cancer). CONCLUSION: Stereotactic body radiotherapy can be safely administered to patients with unresectable liver metastases, and it may delay the need for chemotherapy. This treatment should be considered for selected patients with unresectable liver metastases.

Extrafollicular responses in humans and SLE.

Chronic autoimmune diseases, and in particular Systemic Lupus Erythematosus (SLE), are endowed with a long-standing autoreactive B-cell compartment that is presumed to reactivate periodically leading to the generation of new bursts of pathogenic antibody-secreting cells (ASC). Moreover, pathogenic autoantibodies are typically characterized by a high load of somatic hypermutation and in some cases are highly stable even in the context of prolonged B-cell depletion. Long-lived, highly mutated antibodies are typically generated through T-cell-dependent germinal center (GC) reactions. Accordingly, an important role for GC reactions in the generation of pathogenic autoreactivity has been postulated in SLE. Nevertheless, pathogenic autoantibodies and autoimmune disease can be generated through B-cell extrafollicular (EF) reactions in multiple mouse models and human SLE flares are characterized by the expansion of naive-derived activated effector B cells of extrafollicular phenotype. In this review, we will discuss the properties of the EF B-cell pathway, its relationship to other effector B-cell populations, its role in autoimmune diseases, and its contribution to human SLE. Furthermore, we discuss the relationship of EF B cells with Age-Associated B cells (ABCs), a TLR-7-driven B-cell population that mediates murine autoimmune and antiviral responses.

Understanding and measuring human B-cell tolerance and its breakdown in autoimmune disease.

The maintenance of immunological tolerance of B lymphocytes is a complex and critical process that must be implemented as to avoid the detrimental development of autoreactivity and possible autoimmunity. Murine models have been invaluable to elucidate many of the key components in B-cell tolerance; however, translation to human homeostatic and pathogenic immune states can be difficult to assess. Functional autoreactive, flow cytometric, and single-cell cloning assays have proven to be critical in deciphering breaks in B-cell tolerance within autoimmunity; however, newer approaches to assess human B-cell tolerance may prove to be vital in the further exploration of underlying tolerance defects. In this review, we supply a comprehensive overview of human immune tolerance checkpoints with associated mechanisms of enforcement, and highlight current and future methodologies which are likely to benefit future studies into the mechanisms that become defective in human autoimmune conditions.