Search

Inclusion body myositis (IBM) is an autoimmune and degenerative disorder of skeletal muscle. The B cell infiltrates in IBM muscle tissue are predominantly fully differentiated Ab-secreting plasma cells, with scarce naive or memory B cells. The role of this infiltrate in the disease pathology is not well understood. To better define the humoral response in IBM, we used adaptive immune receptor repertoire sequencing, of human-derived specimens, to generate large BCR repertoire libraries from IBM muscle biopsies and compared them to those generated from dermatomyositis, polymyositis, and circulating CD27+ memory B cells, derived from healthy controls and Ab-secreting cells collected following vaccination. The repertoire properties of the IBM infiltrate included the following: clones that equaled or exceeded the highly clonal vaccine-associated Ab-secreting cell repertoire in size; reduced somatic mutation selection pressure in the CDRs and framework regions; and usage of class-switched IgG and IgA isotypes, with a minor population of IgM-expressing cells. The IBM IgM-expressing population revealed unique features, including an elevated somatic mutation frequency and distinct CDR3 physicochemical properties. These findings demonstrate that some of IBM muscle BCR repertoire characteristics are distinct from dermatomyositis and polymyositis and circulating Ag-experienced subsets, suggesting that it may form through selection by disease-specific Ags.

The emergence of SARS-CoV-2 variants with mutations in major neutralizing antibody-binding sites can affect humoral immunity induced by infection or vaccination(1–6). We analysed the development of anti-SARS-CoV-2 antibody and T cell responses in previously infected (recovered) or uninfected (naive) individuals that received mRNA vaccines to SARS-CoV-2. While previously infected individuals sustained higher antibody titers than uninfected individuals post-vaccination, the latter reached comparable levels of neutralization responses to the ancestral strain after the second vaccine dose. T cell activation markers measured upon spike or nucleocapsid peptide in vitro stimulation showed a progressive increase after vaccination. Comprehensive analysis of plasma neutralization using 16 authentic isolates of distinct locally circulating SARS-CoV-2 variants revealed a range of reduction in the neutralization capacity associated with specific mutations in the spike gene: lineages with E484K and N501Y/T (e.g., B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (e.g., B.1.617.2). While both groups retained neutralization capacity against all variants, plasma from previously infected vaccinated individuals displayed overall better neutralization capacity when compared to plasma from uninfected individuals that also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the impact of emerging variants on antibody neutralizing activity.

BackgroundWhile the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with qPCR, concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely-usedlytAqPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB).MethodsDuring the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple timepoints from 103 healthy adults aged 21-40 (n=34) and ≥64 (n=69) years. Following culture-enrichment, extracted DNA was tested using qPCR forpiaBandlytA. By sequencing the variable region ofrpsB(S2-typing), we identified the species of bacteria isolated from samples testinglytA-positive only.ResultsWhile 30/344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for bothpiaBandlytA, 52 (15.1%) samples testedlytA-positive only. No samples testedpiaB-positive only. Through extensive re-culture of the 32lytA-positive samples collected in 2018/2019, we isolated 23 strains (from 8 samples, from 5 individuals) that were also qPCR-positive for onlylytA. Sequencing determined thatStreptococcus mitisandStreptococcus infantiswere predominantly responsible for thislytA-positive qPCR signal.ConclusionsWe identified a comparatively large proportion of samples generating positive signals with the widely usedlytA-qPCR and identified non-pneumococcal streptococcal species responsible for this signal. This highlights the importance of testing for the presence of multiple gene targets in tandem for reliable and specific detection of pneumococcus in respiratory tract samples.

Dectin-1 is an innate immune receptor that recognizes and binds β-1,3/1,6 glucans on fungi. We evaluated Dectin-1 function in myeloid cells in a cohort of HIV-positive and HIV-negative young and older adults. Stimulation of monocytes with β-D-glucans induced a pro-inflammatory phenotype in monocytes of HIV-infected individuals that was characterized by increased levels of IL-12, TNF-α, and IL-6, with some age-associated cytokine increases also noted. Dendritic cells showed a striking HIV-associated increase in IFN-α production. These increases in cytokine production paralleled increases in Dectin-1 surface expression in both monocytes and dendritic cells that were noted with both HIV and aging. Differential gene expression analysis showed that HIV-positive older adults had a distinct gene signature compared to other cohorts characterized by a robust TNF-α and coagulation response (increased at baseline), a persistent IFN-α and IFN-γ response, and an activated dendritic cell signature/M1 macrophage signature upon Dectin-1 stimulation. Dectin-1 stimulation induced a strong upregulation of MTORC1 signaling in all cohorts, although increased in the HIV-Older cohort (stimulation and baseline). Overall, our study demonstrates that the HIV Aging population has a distinct immune signature in response to Dectin-1 stimulation. This signature may contribute to the pro-inflammatory environment that is associated with HIV and Aging.

Alterations in the components of the immune system occur with aging. The introduction of combination antiretroviral therapy (ART) has dramatically improved life expectancy in human immunodeficiency virus (HIV) infected individuals by suppressing viral replication and increasing CD4

Seasonal influenza causes mild to severe respiratory infections and significant morbidity, especially in older adults. Transcriptomic analysis in populations across multiple flu seasons has provided insights into the molecular determinants of vaccine response. Still, the metabolic changes that underlie the immune response to influenza vaccination remain poorly characterized. We performed untargeted metabolomics to analyze plasma metabolites in a cohort of younger and older subjects before and after influenza vaccination to identify vaccine-induced molecular signatures. Metabolomic and transcriptomic data were combined to define networks of gene and metabolic signatures indicative of high and low antibody response in these individuals. We observed age-related differences in metabolic baselines and signatures of antibody response to influenza vaccination and the abundance of α-linolenic and linoleic acids, sterol esters, fatty-acylcarnitines, and triacylglycerol metabolism. We identified a metabolomic signature associated with age-dependent vaccine response, finding increased tryptophan and decreased polyunsaturated fatty acids (PUFAs) in young high responders (HRs), while fatty acid synthesis and cholesteryl esters accumulated in older HRs. Integrated metabolomic and transcriptomic analysis shows that depletion of PUFAs, which are building blocks for prostaglandins and other lipid immunomodulators, in young HR subjects at Day 28 is related to a robust immune response to influenza vaccination. Increased glycerophospholipid levels were associated with an inflammatory response in older HRs to flu vaccination. This multi-omics approach uncovered age-related molecular markers associated with influenza vaccine response and provides insight into vaccine-induced metabolic responses that may help guide development of more effective influenza vaccines.

The impact of coronavirus disease 2019 (COVID-19) mRNA vaccination on pregnancy and fertility has become a major topic of public interest. We investigated two of the most widely propagated claims to determine 1) whether COVID-19 mRNA vaccination of mice during early pregnancy is associated with an increased incidence of birth defects or growth abnormalities, and 2) whether COVID-19 mRNA-vaccinated human volunteers exhibit elevated levels of antibodies to the human placental protein syncytin-1. Using a mouse model, we found that intramuscular COVID-19 mRNA vaccination during early pregnancy at gestational age E7.5 did not lead to differences in fetal size by crown-rump length or weight at term, nor did we observe any gross birth defects. In contrast, injection of the TLR3 agonist and double-stranded RNA mimic polyinosinic-polycytidylic acid, or poly(I:C), impacted growth in utero leading to reduced fetal size. No overt maternal illness following either vaccination or poly(I:C) exposure was observed. We also found that term fetuses from vaccinated murine pregnancies exhibit high circulating levels of anti-Spike and anti-RBD antibodies to SARS-CoV-2 consistent with maternal antibody status, indicating transplacental transfer. Finally, we did not detect increased levels of circulating anti-syncytin-1 antibodies in a cohort of COVID-19 vaccinated adults compared to unvaccinated adults by ELISA. Our findings contradict popular claims associating COVID-19 mRNA vaccination with infertility and adverse neonatal outcomes.

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4

The emergence of SARS-CoV-2 variants with mutations in major neutralizing antibody-binding sites can affect humoral immunity induced by infection or vaccination1–6. We analysed the development of anti-SARS-CoV-2 antibody and T cell responses in previously infected (recovered) or uninfected (naive) individuals that received mRNA vaccines to SARS-CoV-2. While previously infected individuals sustained higher antibody titers than uninfected individuals post-vaccination, the latter reached comparable levels of neutralization responses to the ancestral strain than previously infected individuals 7 days after the second vaccine dose. T cell activation markers measured upon spike or nucleocapsid peptide in vitro stimulation showed a progressive increase after vaccination in the time-points analysed. Comprehensive analysis of plasma neutralization using 16 authentic isolates of distinct locally circulating SARS-CoV-2 variants revealed a range of reduction in the neutralization capacity associated with specific mutations in the spike gene: lineages with E484K and N501Y/T (e.g., B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (e.g., B.1.617.2) or with E484K (without N501Y/T). While both groups retained neutralization capacity against all variants, plasma from previously infected vaccinated individuals displayed overall better neutralization capacity when compared to plasma from uninfected individuals that also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the impact of emerging variants on antibody neutralizing activity.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). However, the exact features of antibody responses that govern COVID-19 disease outcomes remain unclear. In this study, we analyzed humoral immune responses in 229 patients with asymptomatic, mild, moderate and severe COVID-19 over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-spike (S) immunoglobulin G (IgG) levels, length of hospitalization and clinical parameters associated with worse clinical progression. Although high anti-S IgG levels correlated with worse disease severity, such correlation was time dependent. Deceased patients did not have higher overall humoral response than discharged patients. However, they mounted a robust, yet delayed, response, measured by anti-S, anti-receptor-binding domain IgG and neutralizing antibody (NAb) levels compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, although sera from 85% of patients displayed some neutralization capacity during their disease course, NAb generation before 14 d of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se but, rather, with the delayed kinetics of NAb production.

SummaryA dysregulated immune response against coronavirus-2 (SARS-CoV-2) plays a critical role in the outcome of patients with coronavirus disease 2019 (COVID-19). A significant increase in circulating plasmablasts is characteristic of COVID-19 though the underlying mechanisms and its prognostic implications are not known. Here, we demonstrate that in the acute phase of COVID-19, activated PD-1highCXCR5−CD4+ T cells, peripheral helper T cells, (Tph) are significantly increased and promote inflammatory tissue-homing plasmablasts in patients with stable but not severe COVID-19. Analysis of scRNA-seq data revealed that plasmablasts in stable patients express higher levels of tissue-homing receptors including CXCR3. The increased Tph cells exhibited “B cell help” signatures similar to that of circulating T follicular helper (cTfh) cells and promoted B cell differentiation in vitro. Compared with cTfh cells, Tph cells produced more IFNγ, inducing tissue-homing chemokine receptors on plasmablasts. Finally, expansion of activated Tph cells was correlated with the frequency of CXCR3+ plasmablasts in the acute phase of patients with stable disease. Our results demonstrate a novel role for Tph cells in acute viral immunity by inducing ectopic, antibody secreting plasmablasts.

A dysregulated immune response against the SARS-CoV-2 virus plays a critical role in severe COVID-19. However, the molecular and cellular mechanisms by which the virus causes lethal immunopathology are poorly understood. Here, we utilize multiomics single-cell analysis to probe dynamic immune responses in patients with stable or progressive manifestations of COVID-19, and assess the effects of tocilizumab, an anti-IL-6 receptor monoclonal antibody. Coordinated profiling of gene expression and cell lineage protein markers reveals a prominent type-1 interferon response across all immune cells, especially in progressive patients. An anti-inflammatory innate immune response and a pre-exhaustion phenotype in activated T cells are hallmarks of progressive disease. Skewed T cell receptor repertoires in CD8+ T cells and uniquely enriched V(D)J sequences are also identified in COVID-19 patients. B cell repertoire and somatic hypermutation analysis are consistent with a primary immune response, with possible contribution from memory B cells. Our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19, which may contribute to delayed virus clearance and has implications for therapeutic intervention.

The IL-7 receptor alpha chain (IL-7Rα or CD127) can be differentially expressed in memory CD8(+) T cells. Here we investigated whether IL-7Rα could serve as a key molecule in defining a comprehensive landscape of heterogeneity in human effector memory (EM) CD8(+) T cells using high-dimensional Cytometry by Time-Of-Flight (CyTOF) and single-cell RNA-seq (scRNA-seq). IL-7Rα had diverse, but organized, expressional relationship in EM CD8(+) T cells with molecules related to cell function and gene regulation, which rendered an immune landscape defining heterogeneous cell subsets. The differential expression of these molecules likely has biological implications as we found in vivo signatures of transcription factors and homeostasis cytokine receptors, including T-bet and IL-7Rα. Our findings indicate the existence of heterogeneity in human EM CD8(+) T cells as defined by distinct but organized expression patterns of multiple molecules in relationship to IL-7Rα and its possible biological significance in modulating downstream events.

Recent discoveries demonstrate a critical role for circadian rhythms and sleep in immune system homeostasis. Both innate and adaptive immune responses - ranging from leukocyte mobilization, trafficking, and chemotaxis to cytokine release and T cell differentiation -are mediated in a time of day-dependent manner. The National Institutes of Health (NIH) recently sponsored an interdisciplinary workshop, "Sleep Insufficiency, Circadian Misalignment, and the Immune Response," to highlight new research linking sleep and circadian biology to immune function and to identify areas of high translational potential. This Review summarizes topics discussed and highlights immediate opportunities for delineating clinically relevant connections among biological rhythms, sleep, and immune regulation.

Both aging and HIV infection are associated with an enhanced pro-inflammatory environment that contributes to impaired immune responses and is mediated in part by innate immune pattern-recognition receptors. MINCLE is a C-type lectin receptor that recognizes trehalose-6,6ʹ-dimycolate or “cord factor,” the most abundant glycolipid in Mycobacterium tuberculosis. Here, we evaluated MINCLE function in monocytes in a cohort of HIV-infected and uninfected young (21–35 years) and older adults (≥60 years) via stimulation of peripheral blood mononuclear cells with trehalose-6,6-dibehenate, a synthetic analog of trehalose-6,6ʹ-dimycolate and measurement of cytokine production (interleukin [IL]-10, IL-12, IL-6, tumor necrosis factor-α) by multicolor flow cytometry. Our studies show an age- and HIV-associated increase in cytokine multifunctionality of monocytes both at the population and single cell level that was dominated by IL-12, IL-10, and IL-6. These findings provide insight into the host response to M. tuberculosis and possible sources for the pro-inflammatory environment seen in aging and HIV infection.

Recent studies have provided insights into innate and adaptive immune dynamics in coronavirus disease 2019 (COVID-19). However, the exact features of antibody responses that govern COVID-19 disease outcomes remain unclear. In this study, we analyzed humoral immune responses in 229 patients with asymptomatic, mild, moderate and severe COVID-19 over time to probe the nature of antibody responses in disease severity and mortality. We observed a correlation between anti-spike (S) immunoglobulin G (IgG) levels, length of hospitalization and clinical parameters associated with worse clinical progression. Although high anti-S IgG levels correlated with worse disease severity, such correlation was time dependent. Deceased patients did not have higher overall humoral response than discharged patients. However, they mounted a robust, yet delayed, response, measured by anti-S, anti-receptor-binding domain IgG and neutralizing antibody (NAb) levels compared to survivors. Delayed seroconversion kinetics correlated with impaired viral control in deceased patients. Finally, although sera from 85% of patients displayed some neutralization capacity during their disease course, NAb generation before 14 d of disease onset emerged as a key factor for recovery. These data indicate that COVID-19 mortality does not correlate with the cross-sectional antiviral antibody levels per se but, rather, with the delayed kinetics of NAb production.

Motivation Systems immunology leverages recent technological advancements that enable broad profiling of the immune system to better understand the response to infection and vaccination, as well as the dysregulation that occurs in disease. An increasingly common approach to gain insights from these large-scale profiling experiments involves the application of statistical learning methods to predict disease states or the immune response to perturbations. However, the goal of many systems studies is not to maximize accuracy, but rather to gain biological insights. The predictors identified using current approaches can be biologically uninterpretable or present only one of many equally predictive models, leading to a narrow understanding of the underlying biology. Results Here we show that incorporating prior biological knowledge within a logistic modeling framework by using network-level constraints on transcriptional profiling data significantly improves interpretability. Moreover, incorporating different types of biological knowledge produces models that highlight distinct aspects of the underlying biology, while maintaining predictive accuracy. We propose a new framework, Logistic Multiple Network-constrained Regression (LogMiNeR), and apply it to understand the mechanisms underlying differential responses to influenza vaccination. Although standard logistic regression approaches were predictive, they were minimally interpretable. Incorporating prior knowledge using LogMiNeR led to models that were equally predictive yet highly interpretable. In this context, B cell-specific genes and mTOR signaling were associated with an effective vaccination response in young adults. Overall, our results demonstrate a new paradigm for analyzing high-dimensional immune profiling data in which multiple networks encoding prior knowledge are incorporated to improve model interpretability. Availability and implementation The R source code described in this article is publicly available at https://bitbucket.org/kleinstein/logminer. Supplementary information Supplementary data are available at Bioinformatics online.

Summary: Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1β (IL-1β) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases β-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1β in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout. : NLRP3 inflammasome activation in macrophages and neutrophils drives painful inflammation during gout. Goldberg et al. report that ketogenic diet prevents systemic inflammation and joint damage in a rat model of gouty flare. Mechanistically, the ketone body β-hydroxybutyrate, the most abundant ketone in vivo, inhibits NLRP3/caspase-1-dependent IL-1β secretion from neutrophils. Keywords: NLRP3 inflammasome, gout, β-hydroxybutyrate, inflammation, aging, neutrophil, IL-1

The seasonal influenza vaccine is an important public health tool but is only effective in a subset of individuals. The identification of molecular signatures provides a mechanism to understand the drivers of vaccine-induced immunity. Most previously reported molecular signatures of influenza vaccination were derived from a single age group or season, ignoring the effects of immunosenescence or vaccine composition. Thus, it remains unclear how immune signatures of vaccine response change with age across multiple seasons. Here we profile the transcriptional landscape of young and older adults over five consecutive vaccination seasons to identify shared signatures of vaccine response as well as marked seasonal differences. Along with substantial variability in vaccine-induced signatures across seasons, we uncovered a common transcriptional signature 28 days post-vaccination in both young and older adults. However, gene expression patterns associated with vaccine-induced antibody responses were distinct in young and older adults; for example, increased expression of Killer Cell Lectin Like Receptor B1 (KLRB1;CD161) 28 days post-vaccination positively and negatively predicted vaccine-induced antibody responses in young and older adults, respectively. These findings contribute new insights for developing more effective influenza vaccines, particularly in older adults.

Leptospirosis is the leading cause of zoonotic morbidity and mortality globally, yet little is known about the immune mechanisms that may contribute to pathogenesis and severe disease. Although neutrophils are a key component of early immune responses to infection, they have been associated with tissue damage and inflammation in some febrile infections. To assess whether neutrophils contribute to the pathogenesis observed in severe leptospirosis, we quantitated levels of neutrophil activation markers in patients with varying disease severities. Hospitalized leptospirosis patients had significantly higher levels of toll-like receptors 2 and 4 (TLR2 and TLR4, respectively) on peripheral neutrophils than healthy controls, with the highest levels detected in patients with organ dysfunction. We observed no significant differences in other neutrophil baseline activation markers (CD62L and CD11b) or activation capacity (CD62L and CD11b levels following stimulation), regardless of disease severity. Our results provide preliminary evidence supporting the hypothesis that higher initial bacterial loads or inadequate or delayed neutrophil responses, rather than TLR-driven inflammation, may drive severe disease outcomes.

Here, we investigated the relationship of the age-associated expansion of IL-7 receptor alpha low (IL-7Rαlow ) effector memory (EM) CD8+ T cells with the global transcriptomic profile of peripheral blood cells in humans. We found 231 aging signature genes of IL-7Rαlow EM CD8+ T cells that corresponded to 15% of the age-associated genes (231/1,497) reported by a meta-analysis study on human peripheral whole blood from approximately 15,000 individuals, having high correlation with chronological age. These aging signature genes were the target genes of several transcription factors including MYC, SATB1, and BATF, which also belonged to the 231 genes, supporting the upstream regulatory role of these transcription factors in altering the gene expression profile of peripheral blood cells with aging. We validated the differential expression of these transcription factors between IL-7Rαlow and high EM CD8+ T cells as well as in peripheral blood mononuclear cells (PBMCs) of young and older adults. Finally, we found a significant association with influenza vaccine responses in older adults, suggesting the possible biological significance of the aging signature genes of IL-7Rαlow EM CD8+ T cells. The results of our study support the relationship of the expansion of IL-7Rαlow EM CD8+ T cells with the age-associated changes in the gene expression profile of peripheral blood cells and its possible biological implications.

Here, we investigated the relationship of the age‐associated expansion of IL‐7 receptor alpha low (IL‐7Rαlow) effector memory (EM) CD8+ T cells with the global transcriptomic profile of peripheral blood cells in humans. We found 231 aging signature genes of IL‐7Rαlow EM CD8+ T cells that corresponded to 15% of the age‐associated genes (231/1,497) reported by a meta‐analysis study on human peripheral whole blood from approximately 15,000 individuals, having high correlation with chronological age. These aging signature genes were the target genes of several transcription factors including MYC, SATB1, and BATF, which also belonged to the 231 genes, supporting the upstream regulatory role of these transcription factors in altering the gene expression profile of peripheral blood cells with aging. We validated the differential expression of these transcription factors between IL‐7Rαlow and high EM CD8+ T cells as well as in peripheral blood mononuclear cells (PBMCs) of young and older adults. Finally, we found a significant association with influenza vaccine responses in older adults, suggesting the possible biological significance of the aging signature genes of IL‐7Rαlow EM CD8+ T cells. The results of our study support the relationship of the expansion of IL‐7Rαlow EM CD8+ T cells with the age‐associated changes in the gene expression profile of peripheral blood cells and its possible biological implications.

Background Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). Methods We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues. Findings From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (, Context and Significance Frequent testing is critical to limit SARS-CoV-2 transmission. In response to this need, we developed SalivaDirect, a sensitive, simplified, and flexible testing framework, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). We tested saliva collected from a hospital cohort and showed a high positive agreement (94%) as compared to paired nasopharyngeal swabs tested with a commercial diagnostic kit. Then, we partnered with the National Basketball Association (NBA) to test a large cohort of mostly healthy individuals, and we detected low rates of invalid (0.3%) and false-positive (0.03%–0.05%) results. Our study shows that SalivaDirect can help to increase testing capacity by providing access to an affordable framework that is less prone to supply chain shortages., Graphical Abstract, Highlights SalivaDirect is a simplified saliva-based test for detection of SARS-CoV-2 The testing framework is flexible to minimize the risk of supply chain issues SalivaDirect is sensitive, with low rates of invalid and false-positive results Laboratories can be designated to use SalivaDirect to increase testing capacity, SalivaDirect is a sensitive saliva-based COVID-19 diagnostic test, which received Emergency Use Authorization from the U.S. FDA. With the ability to designate other laboratories, the flexible and simplified framework helps to increase the capacity of existing laboratory infrastructure for SARS-CoV-2 testing.

Flu immunity shows its age As we age, our immune systems change; in many ways not for the better. For instance, the elderly account for 90% of influenza deaths annually. Pillai et al. now report that influenza-infected human monocytes, a type of immune cell, exhibit reduced antiviral activity. In influenza-infected mice, two innate immune sensing pathways work together to promote antiviral immunity to influenza. Mice lacking antiviral immunity (similar to the situation in elderly people) had elevated bacterial burdens in their lungs and increased inflammatory responses, which both contributed to their increased susceptibility to influenza. Science , this issue p. 463

The IL-7 receptor alpha chain (IL-7Rα or CD127) can be differentially expressed in memory CD8+ T cells. Here we investigated whether IL-7Rα could serve as a key molecule in defining a comprehensive landscape of heterogeneity in human effector memory (EM) CD8+ T cells using high-dimensional Cytometry by Time-Of-Flight (CyTOF) and single-cell RNA-seq (scRNA-seq). IL-7Rα had diverse, but organized, expressional relationship in EM CD8+ T cells with molecules related to cell function and gene regulation, which rendered an immune landscape defining heterogeneous cell subsets. The differential expression of these molecules likely has biological implications as we found in vivo signatures of transcription factors and homeostasis cytokine receptors, including T-bet and IL-7Rα. Our findings indicate the existence of heterogeneity in human EM CD8+ T cells as defined by distinct but organized expression patterns of multiple molecules in relationship to IL-7Rα and its possible biological significance in modulating downstream events.

Pneumocephalus, or air within the cranium, can be caused by trauma, intracranial infections, or tumors, and can also occur as a complication of neurosurgery and lumbar puncture. Continuous positive airway pressure (CPAP) therapy can precipitate or worsen pneumocephalus in cases of known head trauma. However, nontraumatic pneumocephalus being caused by CPAP is a highly unexpected clinical event. We describe a case of a patient who presented with meningitis due to an atypical organism that usually resides in the oral cavity, and who developed nontraumatic pneumocephalus in the hospital due to CPAP therapy. The underlying cause, a cerebrospinal fluid leak, was likely the mediator for both pathologies. In the setting of the increasing prevalence of obstructive sleep apnea, physicians can benefit from being aware of this atypical presentation of meningitis and atypical complication of CPAP therapy. CITATION: Charokopos A, Card ME, Manes RP, Shaw A. Iatrogenic nontraumatic CPAP-induced pneumocephalus in a patient with meningitis. J Clin Sleep Med. 2019;15(5):781–783.

We investigated the effect of aging on the multi-dimensional characteristics and heterogeneity of human peripheral CD8(+) T cells defined by the expression of a set of molecules at the single cell level using the recently developed mass cytometry or Cytometry by Time-Of-Flight (CyTOF) and computational algorithms. CD8(+) T cells of young and older adults had differential expression of molecules, especially those related to cell activation and migration, permitting the clustering of young and older adults through an unbiased approach. The changes in the expression of individual molecules were collectively reflected in the altered high-dimensional profiles of CD8(+) T cells in older adults as visualized by the dimensionality reduction analysis tools principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE). A combination of PhenoGraph clustering and t-SNE analysis revealed heterogeneous subsets of CD8(+) T cells that altered with aging. Furthermore, intermolecular quantitative relationships in CD8(+) T cells appeared to change with age as determined by the computational algorithm conditional-Density Resampled Estimate of Mutual Information (DREMI). The results of our study showed that heterogeneity, multidimensional characteristics, and intermolecular quantitative relationships in human CD8(+) T cells altered with age, distinctively clustering young and older adults through an unbiased approach.

Adults older than 65 account for most of the deaths caused by respiratory influenza A virus (IAV) infections, but the underlying mechanisms for this susceptibility are poorly understood. IAV RNA is detected by the cytosolic sensor retinoic acid–inducible gene I (RIG-I), which induces the production of type I interferons (IFNs) that curtail the spread of the virus and promote the elimination of infected cells. We have previously identified a marked defect in the IAV-inducible secretion of type I IFNs, but not proinflammatory cytokines, in monocytes from older (>65 years) healthy human donors. We found that monocytes from older adults exhibited decreased abundance of the adaptor protein TRAF3 (tumor necrosis factor receptor–associated factor 3) because of its increased proteasomal degradation with age, thereby impairing the primary RIG-I signaling pathway for the induction of type I IFNs. We determined that monocytes from older adults also failed to effectively stimulate the production of the IFN regulatory transcription factor IRF8, which compromised IFN induction through secondary RIG-I signaling. IRF8 played a central role in IFN induction in monocytes, because knocking down IRF8 in monocytes from younger adults was sufficient to replicate the IFN defects observed in monocytes from older adults, whereas restoring IRF8 expression in older adult monocytes was sufficient to restore RIG-I–induced IFN responses. Aging thus compromises both the primary and secondary RIG-I signaling pathways that govern expression of type I IFN genes, thereby impairing antiviral resistance to IAV.

DNA methylation is an epigenetic mechanism that modulates gene expression in mammalian cells including T cells. Memory T cells are heterogeneous populations. Human effector memory (EM) CD8+ T cells in peripheral blood contain two cell subsets with distinct traits that express low and high levels of the IL-7Rα. However, epigenetic mechanisms involved in defining such cellular traits are largely unknown. In this study, we use genome-wide DNA methylation and individual gene expression to show the possible role of DNA methylation in conferring distinct traits of chemotaxis and inflammatory responses in human IL-7Rαlow and IL-7Rαhigh EM CD8+ T cells. In particular, IL-7Rαlow EM CD8+ T cells had increased expression of CX3CR1 along with decreased DNA methylation in the CX3CR1 gene promoter compared with IL-7Rαhigh EM CD8+ T cells. Altering the DNA methylation status of the CX3CR1 gene promoter changed its activity and gene expression. IL-7Rαlow EM CD8+ T cells had an increased migratory capacity to the CX3CR1 ligand fractalkine compared with IL-7Rαhigh EM CD8+ T cells, suggesting an important biological outcome of the differential expression of CX3CR1. Moreover, IL-7Rαlow EM CD8+ T cells induced fractalkine expression on endothelial cells by producing IFN-γ and TNF-α, forming an autocrine amplification loop. Overall, our study shows the role of DNA methylation in generating unique cellular traits in human IL-7Rαlow and IL-7Rαhigh EM CD8+ T cells, including differential expression of CX3CR1, as well as potential biological implications of this differential expression.

To elucidate gene expression pathways underlying age-associated impairment in influenza vaccine response, we screened young (age 21-30) and older (age ≥65) adults receiving influenza vaccine in two consecutive seasons and identified those with strong or absent response to vaccine, including a subset of older adults meeting criteria for frailty. PBMCs obtained prior to vaccination (Day 0) and at day 2 or 4, day 7 and day 28 post-vaccine were subjected to gene expression microarray analysis. We defined a response signature and also detected induction of a type I interferon response at day 2 and a plasma cell signature at day 7 post-vaccine in young responders. The response signature was dysregulated in older adults, with the plasma cell signature induced at day 2, and was never induced in frail subjects (who were all non-responders). We also identified a mitochondrial signature in young vaccine responders containing genes mediating mitochondrial biogenesis and oxidative phosphorylation that was consistent in two different vaccine seasons and verified by analyses of mitochondrial content and protein expression. These results represent the first genome-wide transcriptional profiling analysis of age-associated dynamics following influenza vaccination, and implicate changes in mitochondrial biogenesis and function as a critical factor in human vaccine responsiveness.

Rationale: Cytokine receptors can be markers defining different T-cell subsets and considered as therapeutic targets. The association of IL-6 and IL-6 receptor α (IL-6Rα) with asthma was reported, suggesting their involvement in asthma. Objectives: To determine whether and how IL-6Rα defines a distinct effector memory (EM) CD8+ T-cell population in health and disease. Methods: EM CD8+ T cells expressing IL-6Rα (IL-6Rαhigh) were identified in human peripheral blood and analyzed for function, gene, and transcription factor expression. The relationship of these cells with asthma was determined using blood and sputum. Measurements and Main Results: A unique population of IL-6Rαhigh EM CD8+ T cells was found in peripheral blood. These cells that potently proliferated, survived, and produced high levels of the Th2-type cytokines IL-5 and IL-13 had increased levels of GATA3 and decreased levels of T-bet and Blimp-1 in comparison with other EM CD8+ T cells. In fact, GATA3 was required for IL-6Rα expression. Patients with asthma had an increased frequency of IL-6Rαhigh EM CD8+ T cells in peripheral blood compared with healthy control subjects. Also, IL-6Rαhigh EM CD8+ T cells exclusively produced IL-5 and IL-13 in response to asthma-associated respiratory syncytial virus and bacterial superantigens. Conclusions: Human IL-6Rαhigh EM CD8+ T cells is a unique cell subset that may serve as a reservoir for effector CD8+ T cells, particularly the ones producing Th2-type cytokines, and expand in asthma.

[This corrects the article DOI: 10.1371/journal.ppat.1005943.].

Annual influenza vaccinations are currently recommended for all individuals 6 months and older. Antibodies induced by vaccination are an important mechanism of protection against infection. Despite the overall public health success of influenza vaccination, many individuals fail to induce a substantial antibody response. Systems-level immune profiling studies have discerned associations between transcriptional and cell subset signatures with the success of antibody responses. However, existing signatures have relied on small cohorts and have not been validated in large independent studies. We leveraged multiple influenza vaccination cohorts spanning distinct geographical locations and seasons from the Human Immunology Project Consortium (HIPC) and the Center for Human Immunology (CHI) to identify baseline (i.e., before vaccination) predictive transcriptional signatures of influenza vaccination responses. Our multicohort analysis of HIPC data identified nine genes (RAB24, GRB2, DPP3, ACTB, MVP, DPP7, ARPC4, PLEKHB2, and ARRB1) and three gene modules that were significantly associated with the magnitude of the antibody response, and these associations were validated in the independent CHI cohort. These signatures were specific to young individuals, suggesting that distinct mechanisms underlie the lower vaccine response in older individuals. We found an inverse correlation between the effect size of signatures in young and older individuals. Although the presence of an inflammatory gene signature, for example, was associated with better antibody responses in young individuals, it was associated with worse responses in older individuals. These results point to the prospect of predicting antibody responses before vaccination and provide insights into the biological mechanisms underlying successful vaccination responses.

Influenza remains a significant cause of morbidity and mortality in older adults, with 90% of annual influenza-related deaths occurring in individuals older than 65 years [1]. Although the seasonal inactivated vaccine is recommended for prevention, a meta-analysis of 64 studies of influenza vaccine response in older adults (>65 years) revealed an efficacy of 23% for the prevention of influenzalike illness [2]. This poor vaccine response is a result in part of age-associated alterations in immune function [3]. Immunosenescence affects the adaptive immune system, resulting in impairments in B- and T-cell development, signaling, and antigen receptor repertoire [4–8]. The human innate immune system is also affected by aging, with chronic inflammation arising from sources including age-associated herpesvirus reactivation and increased non–cell-associated DNA reflected in elevated levels of cytokines and acute phase reactants; such inflammation contributes to dysregulated innate immune function, particularly of pattern recognition receptors such as Toll-like receptors (TLRs) [9]. However, how aging affects human innate immunity to influenza vaccination remains incompletely understood. Here, we evaluated monocyte function at baseline and after immunization with the inactivated influenza vaccine in a cohort of young and older adults.

HIV infection is associated with a chronic inflammatory state arising from multiple factors, including innate immune recognition of HIV, increased microbial translocation, and release of endogenous ligands from damaged cells (such as CD4 T cells). In many respects, this heightened pro-inflammatory environment resembles that associated with aging in the absence of HIV infection, and evidence of dysregulated innate immune responses can be found in not only older HIV-negative adults, but also adults with HIV infection. While the study of innate immune aging in HIV infection is still in its early stages, it seems likely that at least additive, or potentially synergistic effects of aging and HIV infection will be found.

IL-15 is involved in regulating host defense and inflammation. Monocytes produce the biologically active cell surface IL-15 in response to IFN-γ. Although aging can alter the immune system, little is known about whether and how aging affects IFN-γ-mediated IL-15 production in human monocytes. We showed that monocytes of healthy older adults (age ≥ 65) had increased cell surface IL-15 expression in response to IFN-γ compared to those of healthy young adults (age ≤ 40). This finding stems in part from increased IFN-γ receptor (R)1/2 expression on monocytes in older adults, leading to enhanced STAT1 activation and interferon regulatory factor 1 synthesis with increased IL15 gene expression. Our study suggests that with aging the IFN-γ-mediated IL-15 production pathway in human monocytes is uncompromised, but rather augmented, and could be considered as a therapeutic target point to modulate host defense and inflammation in older adults.