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.

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.

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.

CCL5 is a potential bridge between type 1 and type 2 inflammation in asthma.

BACKGROUND: Type 1 (T1) inflammation (marked by IFN-γ expression) is now consistently identified in subsets of asthma cohorts, but how it contributes to disease remains unclear. OBJECTIVE: We sought to understand the role of CCL5 in asthmatic T1 inflammation and how it interacts with both T1 and type 2 (T2) inflammation. METHODS: CCL5, CXCL9, and CXCL10 messenger RNA expression from sputum bulk RNA sequencing, as well as clinical and inflammatory data were obtained from the Severe Asthma Research Program III (SARP III). CCL5 and IFNG expression from bronchoalveolar lavage cell bulk RNA sequencing was obtained from the Immune Mechanisms in Severe Asthma (IMSA) cohort and expression related to previously identified immune cell profiles. The role of CCL5 in tissue-resident memory T-cell (TRM) reactivation was evaluated in a T1high murine severe asthma model. RESULTS: Sputum CCL5 expression strongly correlated with T1 chemokines (P < .001 for CXCL9 and CXCL10), consistent with a role in T1 inflammation. CCL5high participants had greater fractional exhaled nitric oxide (P = .009), blood eosinophils (P < .001), and sputum eosinophils (P = .001) in addition to sputum neutrophils (P = .001). Increased CCL5 bronchoalveolar lavage expression was unique to a previously described T1high/T2variable/lymphocytic patient group in the IMSA cohort, with IFNG trending with worsening lung obstruction only in this group (P = .083). In a murine model, high expression of the CCL5 receptor CCR5 was observed in TRMs and was consistent with a T1 signature. A role for CCL5 in TRM activation was supported by the ability of the CCR5 inhibitor maraviroc to blunt reactivation. CONCLUSION: CCL5 appears to contribute to TRM-related T1 neutrophilic inflammation in asthma while paradoxically also correlating with T2 inflammation and with sputum eosinophilia.

Bridging the Gap between Innate and Adaptive Immunity in the Lung: Summary of the Aspen Lung Conference 2022.

Although significant strides have been made in the understanding of pulmonary immunology, much work remains to be done to comprehensively explain coordinated immune responses in the lung. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic only served to highlight the inadequacy of current models of host-pathogen interactions and reinforced the need for current and future generations of immunologists to unravel complex biological questions. As part of that effort, the 64th Annual Thomas L. Petty Aspen Lung Conference was themed "Bridging the Gap between Innate and Adaptive Immunity in the Lung" and featured exciting work from renowned immunologists. This report summarizes the proceedings of the 2022 Aspen Lung Conference, which was convened to discuss the roles played by innate and adaptive immunity in disease pathogenesis, evaluate the interface between the innate and adaptive immune responses, assess the role of adaptive immunity in the development of autoimmunity and autoimmune lung disease, discuss lessons learned from immunologic cancer treatments and approaches, and define new paradigms to harness the immune system to prevent and treat lung diseases.

Detection of Newly Secreted Antibodies Predicts Nonrecurrence in Primary Clostridioides difficile Infection.

Within 8 weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no clear clinical correlates or validated biomarkers that can predict recurrence during primary infection. This study demonstrated the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence. Forty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Samples of serum and a novel matrix from circulating plasmablasts called "medium-enriched for newly synthesized antibodies" (MENSA) were collected during weeks 1, 2, and 4. Antibodies specific for 10 C. difficile antigens were measured in each sample. Among the 46 C. difficile-infected patients, 9 (19.5%) experienced recurrence within 8 weeks of primary infection. Among the 37 nonrecurrent patients, 23 (62%; 23/37) had anti-C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred early (within the first 12 days post-symptom onset), including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (51%; 19/37). In contrast, none (0%; 0/9) of the patients who subsequently recurred after hospitalization produced antibodies specific for any of the three C. difficile toxin antigens. Thus, patients with a negative early MENSA response against all three C. difficile toxin antigens had a 19-fold greater relative risk of recurrence. MENSA and serum levels of immunoglobulin A (IgA) and/or IgG antibodies for three C. difficile toxins have prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence thereby providing a biomarker of protection from recurrent CDI. Patients who are positive by this immunoassay are unlikely to suffer a recurrence. Early identification of patients at risk for recurrence by negative MENSA creates opportunities for targeted prophylactic strategies that can reduce the incidence, cost, and morbidity due to recurrent CDI.

Effect of Anti-IL5, Anti-IL5R, Anti-IL13 Therapy on Asthma Exacerbations: A Network Meta-analysis.

BACKGROUND: Several new treatments for severe asthma have become available in the last decade; yet, little data exist to guide their use in specific patient populations. OBJECTIVE: A network meta-analysis was conducted comparing the efficacy of FDA-approved monoclonal antibody therapies in preventing exacerbations in patients with severe eosinophilic asthma. METHODS: PubMed and Ovid were searched from inception until July 2019 for randomized controlled trials that studied the efficacy of benralizumab, dupilumab, mepolizumab, and reslizumab, in preventing acute exacerbations of asthma. Studies were included if they reported data for patients with severe eosinophilic asthma (defined in this meta-analysis as absolute eosinophil count ≥ 250 cells/µL). Annualized rate ratios for asthma exacerbations (during treatment) were calculated and converted to log rate ratios. Direct and indirect treatment estimates (for inter-drug differences) were analyzed using frequentist network meta-analysis methodology in R and treatments were ranked based on P-scores. RESULTS: In total, nine studies were included in the final analysis. Network meta-analysis revealed that all drugs were superior to placebo in preventing rates of asthma exacerbation in the study population and no inter-drug differences existed. Dupilumab was found to have the greatest magnitudes of effect on decreasing log rate ratio of asthma exacerbation based on P-score (0.83). CONCLUSION: Benralizumab, dupilumab, mepolizumab, and reslizumab are all associated with decreased asthma exacerbations in patients with eosinophilic asthma, with no significant inter-drug differences.

Single-cell analysis of human nasal mucosal IgE antibody secreting cells reveals a newly minted phenotype.

Immunoglobulin (Ig) E is central to the pathogenesis of allergic conditions, including allergic fungal rhinosinusitis. However, little is known about IgE antibody secreting cells (ASCs). We performed single-cell RNA sequencing from cluster of differentiation (CD)19+ and CD19- ASCs of nasal polyps from patients with allergic fungal rhinosinusitis (n = 3). Nasal polyps were highly enriched in CD19+ ASCs. Class-switched IgG and IgA ASCs were dominant (95.8%), whereas IgE ASCs were rare (2%) and found only in the CD19+ compartment. Through Ig gene repertoire analysis, IgE ASCs shared clones with IgD-CD27- "double-negative" B cells, IgD+CD27+ unswitched memory B cells, and IgD-CD27+ switched memory B cells, suggesting ontogeny from both IgD+ and memory B cells. Transcriptionally, mucosal IgE ASCs upregulate pathways related to antigen presentation, chemotaxis, B cell receptor stimulation, and survival compared with non-IgE ASCs. Additionally, IgE ASCs have a higher expression of genes encoding lysosomal-associated protein transmembrane 5 (LAPTM5) and CD23, as well as upregulation of CD74 (receptor for macrophage inhibitory factor), store-operated Calcium entry-associated regulatory factor (SARAF), and B cell activating factor receptor (BAFFR), which resemble an early minted ASC phenotype. Overall, these findings reinforce the paradigm that human ex vivo mucosal IgE ASCs have a more immature plasma cell phenotype than other class-switched mucosal ASCs and suggest unique functional roles for mucosal IgE ASCs in concert with Ig secretion.

Transcriptional reprogramming of infiltrating neutrophils drives lung pathology in severe COVID-19 despite low viral load.

Troubling disparities in COVID-19-associated mortality emerged early, with nearly 70% of deaths confined to Black/African American (AA) patients in some areas. However, targeted studies on this vulnerable population are scarce. Here, we applied multiomics single-cell analyses of immune profiles from matching airways and blood samples of Black/AA patients during acute SARS-CoV-2 infection. Transcriptional reprogramming of infiltrating IFITM2+/S100A12+ mature neutrophils, likely recruited via the IL-8/CXCR2 axis, leads to persistent and self-sustaining pulmonary neutrophilia with advanced features of acute respiratory distress syndrome (ARDS) despite low viral load in the airways. In addition, exacerbated neutrophil production of IL-8, IL-1ß, IL-6, and CCL3/4, along with elevated levels of neutrophil elastase and myeloperoxidase, were the hallmarks of transcriptionally active and pathogenic airway neutrophilia. Although our analysis was limited to Black/AA patients and was not designed as a comparative study across different ethnicities, we present an unprecedented in-depth analysis of the immunopathology that leads to acute respiratory distress syndrome in a well-defined patient population disproportionally affected by severe COVID-19.

Chronic inflammation, neutrophil activity, and autoreactivity splits long COVID.

While immunologic correlates of COVID-19 have been widely reported, their associations with post-acute sequelae of COVID-19 (PASC) remain less clear. Due to the wide array of PASC presentations, understanding if specific disease features associate with discrete immune processes and therapeutic opportunities is important. Here we profile patients in the recovery phase of COVID-19 via proteomics screening and machine learning to find signatures of ongoing antiviral B cell development, immune-mediated fibrosis, and markers of cell death in PASC patients but not in controls with uncomplicated recovery. Plasma and immune cell profiling further allow the stratification of PASC into inflammatory and non-inflammatory types. Inflammatory PASC, identifiable through a refined set of 12 blood markers, displays evidence of ongoing neutrophil activity, B cell memory alterations, and building autoreactivity more than a year post COVID-19. Our work thus helps refine PASC categorization to aid in both therapeutic targeting and epidemiological investigation of PASC.

Inactivation of SARS-CoV-2 and COVID-19 Patient Samples for Contemporary Immunology and Metabolomics Studies.

Due to the severity of COVID-19 disease, the U.S. Centers for Disease Control and Prevention and World Health Organization recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level (BSL)3 laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred to lower containment levels (BSL2), while maintaining the fidelity of complex downstream assays to expedite the development of medical countermeasures. In this study, we demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with commonly used reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and Ab detection, heat inactivation following cDNA amplification for droplet-based single-cell mRNA sequencing, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of viral inactivation protocols for downstream contemporary assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Checklists Improve Team Performance During Simulated Extracorporeal Membrane Oxygenation Emergencies: A Randomized Trial.

Extracorporeal membrane oxygenation-related complications are potentially catastrophic if not addressed quickly. Because complications are rare, high-fidelity simulation is recommended as part of the training regimen for extracorporeal membrane oxygenation specialists. We hypothesized that the use of standardized checklists would improve team performance during simulated extracorporeal membrane oxygenation emergencies. DESIGN: Randomized simulation-based trial. SETTING: A quaternary-care academic hospital with a regional extracorporeal membrane oxygenation referral program. SUBJECTS: Extracorporeal membrane oxygenation specialists and other healthcare providers. INTERVENTIONS: We designed six read-do checklists for use during extracorporeal membrane oxygenation emergencies using a modified Delphi process. Teams of two to three providers were randomized to receive the checklists or not. All teams then completed four simulated extracorporeal membrane oxygenation emergencies. MEASUREMENTS AND MAIN RESULTS: Simulation sessions were video-recorded, and the number of critical tasks performed and time-to-completion were compared between groups. A survey instrument was administered before and after simulations to assess participants' attitudes toward the simulations and checklists. We recruited 36 subjects from a single institution, randomly assigned to 15 groups. The groups with checklists completed more critical tasks than participants in the control groups (90% vs 75%; p < 0.001). The groups with checklists performed a higher proportion of both nontechnical tasks (71% vs 44%; p < 0.001) and extracorporeal membrane oxygenation-specific technical tasks (94% vs 86%; p < 0.001). Both groups reported an increase in reported self-efficacy after the simulations (p = 0.003). After adjusting for multiple comparisons, none of the time-to-completion measures achieved statistical significance. CONCLUSIONS: The use of checklists resulted in better team performance during simulated extracorporeal membrane oxygenation emergencies. As extracorporeal membrane oxygenation use continues to expand, checklists may be an attractive low-cost intervention for centers looking to reduce errors and improve response to crisis situations.

Longitudinal trends using a point-of-care gelatin-based model for ultrasound-guided central venous catheter insertion.

Ultrasound (US)-guided central venous catheter (CVC) insertion is a procedure that carries the risk of significant complications. Simulation provides a safe learning atmosphere, but most CVC simulators are not available outside of simulation centers. To explore longitudinal trends in US-guided CVC insertion competency in internal medicine (IM) interns, we studied the use of a low-fidelity, gelatin-based, US-guided CVC insertion simulation model combined with a simulation curriculum. This prospective observational study of IM interns was performed over the course of one academic year. Interns (n = 56) underwent model-based, US-guided procedure simulation training program and a repeated training course prior to their intensive care unit (ICU) rotation. CVC insertion competency at different timepoints was recorded. Survey data about intern experience and attitudes were also collected. Out of the 56 interns initially trained, 40 were included in the final analysis. Across all outcomes, interns experienced skill atrophy between initial training and the beginning of their ICU month. However, by the end of the month, there was a significant improvement in competency as compared to initial procedural training, which then waned by the end of the intern year. Attitudes toward the model were generally positive and self-reported confidence improved throughout the course of the year and correlated with objective measures of competency. Over the course of their intern year, which included simulation training using a gelatin-based model, interns demonstrated consistent competency trends. The use of a gelatin-based CVC insertion simulation model warrants further study as an adjunctive aid to existing simulation training.

Diagnosis of Streptococcus pneumoniae infection using circulating antibody secreting cells.

BACKGROUND: Streptococcus pneumoniae infections cause morbidity and mortality worldwide. A rapid, simple diagnostic method could reduce the time needed to introduce definitive therapy potentially improving patient outcomes. METHODS: We introduce two new methods for diagnosing S. pneumoniae infections by measuring the presence of newly activated, pathogen-specific, circulating Antibody Secreting Cells (ASC). First, ASC were detected by ELISpot assays that measure cells secreting antibodies specific for signature antigens. Second, the antibodies secreted by isolated ASC were collected in vitro in a novel matrix, MENSA (media enriched with newly synthesized antibodies) and antibodies against S. pneumoniae antigens were measured using Luminex immunoassays. Each assay was evaluated using blood from S. pneumoniae and non-S. pneumoniae-infected adult patients. RESULTS: We enrolled 23 patients with culture-confirmed S. pneumoniae infections and 24 controls consisting of 12 non-S. pneumoniae infections, 10 healthy donors and two colonized with S. pneumoniae. By ELISpot assays, twenty-one of 23 infected patients were positive, and all 24 controls were negative. Using MENSA samples, four of five S. pneumoniae-infected patients were positive by Luminex immunoassays while all five non-S. pneumoniae-infected patients were negative. CONCLUSION: Specific antibodies produced by activated ASC may provide a simple diagnostic for ongoing S. pneumoniae infections. This method has the potential to diagnose acute bacterial infections.

Relaxed peripheral tolerance drives broad de novo autoreactivity in severe COVID-19.

An emerging feature of COVID-19 is the identification of autoreactivity in patients with severe disease that may contribute to disease pathology, however the origin and resolution of these responses remain unclear. Previously, we identified strong extrafollicular B cell activation as a shared immune response feature between both severe COVID-19 and patients with advanced rheumatic disease. In autoimmune settings, this pathway is associated with relaxed peripheral tolerance in the antibody secreting cell compartment and the generation of de novo autoreactive responses. Investigating these responses in COVID-19, we performed single-cell repertoire analysis on 7 patients with severe disease. In these patients, we identify the expansion of a low-mutation IgG1 fraction of the antibody secreting cell compartment that are not memory derived, display low levels of selective pressure, and are enriched for autoreactivity-prone IGHV4-34 expression. Within this compartment, we identify B cell lineages that display specificity to both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against glomerular basement membrane, and describe progressive, broad, clinically relevant autoreactivity within these patients correlated with disease severity. Importantly, we identify anti-carbamylated protein responses as a common hallmark and candidate biomarker of broken peripheral tolerance in severe COVID-19. Finally, we identify the contraction of this pathway upon recovery, and re-establishment of tolerance standards coupled with a concomitant loss of acute-derived ASCs irrespective of antigen specificity. In total, this study reveals the origins, breadth, and resolution of acute-phase autoreactivity in severe COVID-19, with significant implications in both early interventions and potential treatment of patients with post-COVID sequelae.