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Abstract In their natural environments, microorganisms mainly operate at suboptimal growth conditions with fluctuations in nutrient abundance. The resulting cellular adaptation is subject to conflicting tasks: growth or survival maximisation. Here, we study this adaptation by systematically measuring the impact of a nitrogen downshift to 24 nitrogen sources on cellular metabolism at the single-cell level. Saccharomyces lineages grown in rich media and exposed to a nitrogen downshift gradually differentiate to form two subpopulations of different cell sizes where one favours growth while the other favours viability with an extended chronological lifespan. This differentiation is asymmetrical with daughter cells representing the new differentiated state with increased viability. We characterise the metabolic response of the subpopulations using RNA sequencing, metabolic biosensors and a transcription factor-tagged GFP library coupled to high-throughput microscopy, imaging more than 800,000 cells. We find that the subpopulation with increased viability is associated with a dormant quiescent state displaying differences in MAPK signalling. Depending on the identity of the nitrogen source present, differentiation into the quiescent state can be actively maintained, attenuated, or aborted. These results establish amino acids as important signalling molecules for the formation of genetically identical subpopulations, involved in chronological lifespan and growth rate determination.

Abstract Background African ancestry populations have the highest burden of stroke worldwide, yet the genetic basis of stroke in these populations is obscure. The Stroke Investigative Research and Educational Network (SIREN) is a multicenter study involving 16 sites in West Africa. We conducted the first-ever genome-wide association study (GWAS) of stroke in indigenous Africans. Methods Cases were consecutively recruited consenting adults (aged > 18 years) with neuroimaging-confirmed ischemic stroke. Stroke-free controls were ascertained using a locally validated Questionnaire for Verifying Stroke-Free Status. DNA genotyping with the H3Africa array was performed, and following initial quality control, GWAS datasets were imputed into the NIH Trans-Omics for Precision Medicine (TOPMed) release2 from BioData Catalyst. Furthermore, we performed fine-mapping, trans-ethnic meta-analysis, and in silico functional characterization to identify likely causal variants with a functional interpretation. Results We observed genome-wide significant (P-value < 5.0E−8) SNPs associations near AADACL2 and miRNA (MIR5186) genes in chromosome 3 after adjusting for hypertension, diabetes, dyslipidemia, and cardiac status in the base model as covariates. SNPs near the miRNA (MIR4458) gene in chromosome 5 were also associated with stroke (P-value < 1.0E−6). The putative genes near AADACL2, MIR5186, and MIR4458 genes were protective and novel. SNPs associations with stroke in chromosome 2 were more than 77 kb from the closest gene LINC01854 and SNPs in chromosome 7 were more than 116 kb to the closest gene LINC01446 (P-value < 1.0E−6). In addition, we observed SNPs in genes STXBP5-AS1 (chromosome 6), GALTN9 (chromosome 12), FANCA (chromosome 16), and DLGAP1 (chromosome 18) (P-value < 1.0E−6). Both genomic regions near genes AADACL2 and MIR4458 remained significant following fine mapping. Conclusions Our findings identify potential roles of regulatory miRNA, intergenic non-coding DNA, and intronic non-coding RNA in the biology of ischemic stroke. These findings reveal new molecular targets that promise to help close the current gaps in accurate African ancestry-based genetic stroke’s risk prediction and development of new targeted interventions to prevent or treat stroke.

Background Baseline stroke severity is probably partly responsible for poor stroke outcomes in sub‐Saharan Africa. However, there is a paucity of information on determinants of stroke severity among indigenous Africans. We sought to identify the factors associated with stroke severity among West Africans in the SIREN (Stroke Investigative Research and Educational Networks) study. Methods and Results Stroke was diagnosed clinically and confirmed with brain neuroimaging. Severe stroke was defined as a Stroke Levity Scale score of ≤5. A multivariate logistic regression model was constructed to identify factors associated with stroke severity at 95% CI and a nominal cutoff of 5% type 1 error. A total of 3660 stroke cases were included. Overall, 50.7%% had severe stroke, including 47.6% of all ischemic strokes and 56.1% of intracerebral hemorrhage. Factors independently associated with severe stroke were meat consumption (adjusted odds ratio [aOR], 1.97 [95% CI, 1.43–2.73]), low vegetable consumption (aOR, 2.45 [95% CI, 1.93–3.12]), and lesion volume, with an aOR of 1.67 (95% CI, 1.03–2.72) for lesion volume of 10 to 30 cm3 and aOR of 3.88 (95% CI, 1.93–7.81) for lesion volume >30 cm3. Severe ischemic stroke was independently associated with total anterior circulation infarction (aOR, 3.1 [95% CI, 1.5–6.9]), posterior circulation infarction (aOR, 2.2 [95% CI, 1.1–4.2]), and partial anterior circulation infarction (aOR, 2.0 [95% CI, 1.2–3.3]) compared with lacunar stroke. Increasing age (aOR, 2.6 [95% CI, 1.3–5.2]) and lesion volume >30 cm3 (aOR, 6.2 [95% CI, 2.0–19.3]) were independently associated with severe intracerebral hemorrhage. Conclusions Severe stroke is common among indigenous West Africans, where modifiable dietary factors are independently associated with it. These factors could be targeted to reduce the burden of severe stroke.

Abstract Hypertension is one of the most important risk factors for stroke and cardiovascular diseases (CVD) globally. Understanding risk factors for hypertension among individuals with matching characteristics with stroke patients may inform primordial/primary prevention of hypertension and stroke among them. This study identified the risk factors for hypertension among community‐dwelling stroke‐free population in Ghana and Nigeria. Data for 4267 community‐dwelling stroke‐free controls subjects in the Stroke Investigative Research and Education Network (SIREN) study in Nigeria and Ghana were used. Participants were comprehensively assessed for sociodemographic, lifestyle and metabolic factors using standard methods. Hypertension was defined as a previous diagnosis by a health professional or use of an anti‐hypertensive drug or mean systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg. Logistic regression analysis was used to estimate adjusted odds ratios (aOR) of hypertension and their 95% confidence intervals (CI) at p

IntroductionGenomic research and neurobiobanking are expanding globally. Empirical evidence on the level of awareness and willingness to donate/share biological samples towards the expansion of neurobiobanking in sub-Saharan Africa is lacking.AimsTo ascertain the awareness, perspectives and predictors regarding biological sample donation, sharing and informed consent preferences among community members in Ghana and Nigeria.MethodsA questionnaire cross-sectional survey was conducted among randomly selected community members from seven communities in Ghana and Nigeria.ResultsOf the 1015 respondents with mean age 39.3 years (SD 19.5), about a third had heard of blood donation (37.2%, M: 42.4%, F: 32.0%, p = 0.001) and a quarter were aware of blood sample storage for research (24.5%; M: 29.7%, F: 19.4%, p = 0.151). Two out of ten were willing to donate brain after death (18.8%, M: 22.6%, F: 15.0%, pConclusionThere is a greater need for research attention in the area of brain banking and informed consent. Improved context-sensitive public education on neurobiobanking and informed consent, in line with the sociocultural diversities, is recommended within the African sub region.

Limonium vulgare Mill. is a plant growing widely in harsh environments, such as salt marshes, for which a chemical profile is still unknown, although some interesting bioactivities were already reported. So, this halophyte chemical profile must be established to find the possible bioactive compounds, valorize the species, and contribute to the salt marsh’s exploitation. This work set the chemical profile of L. vulgare’s aerial parts (leaves and inflorescences) using UHPLC-DAD-ESI/MS2 and GC-MS analysis. The lipophilic profile showed a richness in fatty acids, alkanes, and terpenoids, β-sitosterol being the major compound in inflorescences in the fruiting stage (0.822 ± 0.015 mg/g of the dry plant) and leaves (0.534 ± 0.017 mg/g of the dry plant). In contrast, in the inflorescences in the flowering stage, the major compound is nonacosane (0.228 ± 0.001 mg/g of the dry plant). The polyphenolic profile demonstrates that L. vulgare produces several flavonoids from which quercetin and myricetin can be highlighted; in particular, myricetin derivatives are prevalent in all extracts. Amongst the flavonoids, myricetin 3-rhamnoside is the most abundant in the inflorescences in the flowering stage (6.35 ± 0.05 mg/g of the dry plant), myricetin in leaves (9.69 ± 0.11 mg/g of the dry plant), and in the inflorescences in the fruiting stage baicalin presents the highest amount (5.15 ± 0.07 mg/g of the dry plant). This is the first report on L. vulgare’s chemical profile and the results indicate that this species is an exciting source of bioactive compounds, suggesting it has a use to produce nutraceuticals and/or pharmaceuticals.

INTRODUCTION: The prevalence of non-communicable diseases (NCDs) is increasing in African countries. This study aimed to estimate the prevalence and correlates of multiple NCD risk factors (NCDRF) among the adult population in Zambia. METHODS: Nationally representative cross-sectional data from 4,302 individuals aged 18-69 years of the “2017 Zambia STEPS survey” were analysed. RESULTS: The prevalence of insufficient fruit and vegetable consumption was 90.4%, followed by overweight/obesity (24.4%), low physical activity (19.5%), hypertension (18.9%), daily tobacco use (10.7%), sedentary behaviour (8.9%), suicidal behaviour (8.5%), alcohol dependence (7.4%), raised total cholesterol (7.4%), and diabetes (6.2%). The distribution of NCDRF was 41.5% 0-1 NCDRF, 48.2% 2-3, 10.4% 4-10, and 26.7% 3-10 NCDRF. In adjusted ordinal logistic regression analysis, compared to persons aged 18-34 years, individuals aged 50-69 years were 3.58 times (AOR: 3.58, 95% CI: 3.95-4.49) more likely to have a higher number of NCDRF. Women were 24% (AOR: 1.24, 95% CI: 1.03-1.49) more likely than men to have a higher number of NCDRF. Persons living in urban locations were 71% (AOR: 1.74, 95% CI: 1.43-2.16) more likely than persons living in rural locations to have a higher number of NCDRF, and compared to individuals with lower than primary education, persons with more than primary education were 20% (AOR: 0.80, 95% CI: 0.65-0.98) less likely to have a higher number of NCDRF. CONCLUSION: More than one in four study participants had three to ten NCDRF and several associated factors were found that can aid to target interventions.

The ethical, legal, and social implications (ELSI) of emerging neurobiobanks and data resources are unclear in an African scientific landscape with unique cultural, linguistic, and belief systems. The overarching goal of the African Neurobiobank for Precision Stroke Medicine–—ELSI Project is to identify, examine, and develop novel approaches to address ELSI issues of biobanking and stroke genomic research in sub-Saharan Africa (SSA). To accomplish the goal we will (1) explore knowledge, attitude, perceptions, barriers, and facilitators influencing ELSI issues related to biobanking and stroke genomic research; (2) use information obtained to craft a community intervention program focused on ELSI issues; and (3) build capacity and careers related to genomics and biobanking for effective client/community engagement while enhancing regulatory, governance, and implementation competences in biobanking science in SSA. A community-based participatory research and mixed-methodological approach, focused on various levels of the social ecological model, will be used to identify and examine relevant ELSI issues. Contextual intervention tools, platforms, and practices will be developed to enhance community understanding and participation in stroke biobanking and genomics research activities while facilitating enduring trust, and equitable and fair utilization of biobanking resources for genetic and trans-omics research. A concurrent capacity building program related to genetic counseling and biobanking will be implemented for early career researchers. The huge potential for neurobiobanking and genomics research in Africa to advance precision medicine applicable to stroke and other neurological disorders requires addressing ELSI challenges while building sustainable research, career, and regulatory capacities in trans-omics and biobanking science.

Chronic lymphocytic leukaemia is a slow-growing leukaemia of developing B-lymphocytes, which may transform to an aggressive lymphoma known as Richter’s syndrome. While Richter’s syndrome can present in untreated or relapsed-refractory cases, it may occur upon the commencement of less intensity treatment regimens. We present a case of Richter’s syndrome following treatment with chlorambucil and obinutuzumab and review of available literature on the topic.

Summary: Background: Sub-Saharan Africa has the highest incidence, prevalence, and fatality from stroke globally. Yet, only little information about context-specific risk factors for prioritising interventions to reduce the stroke burden in sub-Saharan Africa is available. We aimed to identify and characterise the effect of the top modifiable risk factors for stroke in sub-Saharan Africa. Methods: The Stroke Investigative Research and Educational Network (SIREN) study is a multicentre, case-control study done at 15 sites in Nigeria and Ghana. Cases were adults (aged ≥18 years) with stroke confirmed by CT or MRI. Controls were age-matched and gender-matched stroke-free adults (aged ≥18 years) recruited from the communities in catchment areas of cases. Comprehensive assessment for vascular, lifestyle, and psychosocial factors was done using standard instruments. We used conditional logistic regression to estimate odds ratios (ORs) and population-attributable risks (PARs) with 95% CIs. Findings: Between Aug 28, 2014, and June 15, 2017, we enrolled 2118 case-control pairs (1192 [56%] men) with mean ages of 59·0 years (SD 13·8) for cases and 57·8 years (13·7) for controls. 1430 (68%) had ischaemic stoke, 682 (32%) had haemorrhagic stroke, and six (

INTRODUCTION:Stroke is a prominent cause of death, disability, and dementia in sub-Saharan Africa (SSA). The Stroke Investigative Research and Education Network works collaboratively with stroke survivors and individuals serving as community controls to comprehensively characterize the genomic, sociocultural, economic and behavioral risk factors for stroke in SSA. PURPOSE:In this paper, we aim to: i) explore the attitudes, beliefs, and practices related to stroke in Ghana and Nigeria using the process of qualitative description; and ii) propose actions for future research and community-based participation and education. METHODS:Stroke survivors, their caregivers, health care professionals, and community representatives and faith-based leaders participated in one of twenty-six focus groups, which qualitatively explored community beliefs, attitudes and practices related to stroke in Ghana and Nigeria. Arthur Kleinman's Explanatory Model of Illness and the Social Ecological Model guided the questions and/or thematic analysis of the qualitative data. We hereby describe our focus group methods and analyses of qualitative data, as well as the findings and suggestions for improving stroke outcomes. RESULTS AND DISCUSSION:The major findings illustrate the fears, causes, chief problems, treatment, and recommendations related to stroke through the views of the participants, as well as recommendations for working effectively with the SIREN communities. Findings are compared to SIREN quantitative data and other qualitative studies in Africa. As far as we are aware, this is the first paper to qualitatively explore and contrast community beliefs, attitudes, and practices among stroke survivors and their caregivers, community and faith-based leaders, and health professionals in multiple communities within Nigeria and Ghana.

Vaccination with self- and foreign peptides induces weak and strong expansion of antigen-specific CD4 + T cells, respectively, but the mechanism is not known. In the present study, we used computational analysis of the entire mouse major histocompatibility complex class II peptidome to test how much of the naive CD4 + T cell repertoire specific for self-antigens was shaped by negative selection in the thymus and found that negative selection only partially explained the difference between responses to self and foreign. In naive uninfected and unimmunized mice, we identified higher expression of programmed cell death protein 1 (PD-1) and CD73 mRNA and protein on self-specific CD4 + T cells compared with foreign-specific CD4 + T cells. Pharmacological or genetic blockade of PD-1 and CD73 significantly increased the vaccine-induced expansion of self-specific CD4 + T cells and their transcriptomes were similar to those of foreign-specific CD4 + T cells. We concluded that PD-1 and CD73 synergistically limited CD4 + T cell responses to self. These observations have implications for the development of tolerogenic vaccines and cancer immunotherapy., Competing Interests: Competing interests: K.L. is founder and co-owner of Atherovax, Inc. He receives no compensation from Atherovax. No Atherovax funds were used in the present study. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Human cytomegalovirus (HCMV) causes severe birth defects, lifelong health complications, and $4 billion in annual costs in the United States alone. A major challenge in vaccine design is the incomplete understanding of the diverse protein complexes the virus uses to infect cells. In Herpesviridae , the gH/gL glycoprotein heterodimer is expected to be a basal element of virion cell entry machinery. For HCMV, gH/gL forms a "trimer" with gO and a "pentamer" with UL128, UL130, and UL131A, with each complex binding distinct receptors to enter varied cell types. Here, we reveal a third glycoprotein complex, abundant in HCMV virions, which significantly enhances infection of endothelial cells. In this "3-mer" complex, gH, without gL, associates with UL116 and UL141, an immunoevasin previously known to function in an intracellular role. Cryo-EM reveals the virion-surface 3-mer is structurally unique among Herpesviridae gH complexes, with gH-only scaffolding, UL141-mediated dimerization and a heavily glycosylated UL116 cap. Given that antibodies directed at gH and UL141 each can restrict HCMV replication, our work highlights this virion surface complex as a new target for vaccines and antiviral therapies., Competing Interests: Competing interests. The authors declare no competing interests.

Methanotrophic bacteria play a vital role in the biogeochemical carbon cycle due to their unique ability to use CH 4 as a carbon and energy source. Evidence suggests that some methanotrophs, including Methylococcus capsulatus , can also use CO 2 as a carbon source, making these bacteria promising candidates for developing biotechnologies targeting greenhouse gas capture and mitigation. However, a deeper understanding of the dual CH 4 and CO 2 metabolism is needed to guide methanotroph strain improvements and realize their industrial utility. In this study, we show that M. capsulatus expresses five carbonic anhydrase (CA) isoforms, one α-CA, one γ-CA, and three β-CAs, that play a role in its inorganic carbon metabolism and CO 2 -dependent growth. The CA isoforms are differentially expressed, and transcription of all isoform genes is induced in response to CO 2 limitation. CA null mutant strains exhibited markedly impaired growth compared to an isogenic wild-type control, suggesting that the CA isoforms have independent, non-redundant roles in M. capsulatus metabolism and physiology. Overexpression of some, but not all, CA isoforms improved bacterial growth kinetics and decreased CO 2 evolution from CH 4 -consuming cultures. Notably, we developed an engineered methanotrophic biocatalyst overexpressing the native α-CA and β-CA with a 2.5-fold improvement in the conversion of CH 4 to biomass. Given that product yield is a significant cost driver of methanotroph-based bioprocesses, the engineered strain developed here could improve the economics of CH 4 biocatalysis, including the production of single-cell protein from natural gas or anaerobic digestion-derived biogas.IMPORTANCEMethanotrophs transform CH 4 into CO 2 and multi-carbon compounds, so they play a critical role in the global carbon cycle and are of interest for biotechnology applications. Some methanotrophs, including Methylococcus capsulatus , can also use CO 2 as a carbon source, but this dual one-carbon metabolism is incompletely understood. In this study, we show that M. capsulatus carbonic anhydrases are critical for this bacterium to optimally utilize CO 2 . We developed an engineered strain with improved CO 2 utilization capacity that increased the overall carbon conversion to cell biomass. The improvements to methanotroph-based product yields observed here are expected to reduce costs associated with CH 4 conversion bioprocesses., Competing Interests: Some aspects of this work are included as part of a U.S. patent application (PCT/US2024/034678).

Conventional antiviral memory CD4 T cells typically arise during the first two weeks of acute infection. Unlike most viruses, cytomegalovirus (CMV) exhibits an extended persistent replication phase followed by lifelong latency accompanied with some gene expression. We show that during mouse CMV (MCMV) infection, CD4 T cells recognizing an epitope derived from the viral M09 protein only develop after conventional memory T cells have already peaked and contracted. Ablating these CD4 T cells by mutating the M09 genomic epitope in the MCMV Smith strain, or inducing them by introducing the epitope into the K181 strain, resulted in delayed or enhanced control of viral persistence, respectively. These cells were shown to be unique compared to their conventional memory counterparts; producing higher IFNγ and IL-2 and lower IL-10 levels. RNAseq analyses revealed them to express distinct subsets of effector genes as compared to classical CD4 T cells. Additionally, when M09 cells were induced by epitope vaccination they significantly enhanced protection when compared to conventional CD4 T cells alone. These data show that late-rising CD4 T cells are a unique memory subset with excellent protective capacities that display a development program strongly differing from the majority of memory T cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Brunel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Histamine-releasing factor (HRF) is implicated in allergic diseases. We previously showed its pathogenic role in murine models of asthma., Objective: We aim to present data analysis from 3 separate human samples (sera samples from asthmatic patients, nasal washings from rhinovirus [RV]-infected individuals, and sera samples from patients with RV-induced asthma exacerbation) and 1 mouse sample to investigate correlates of HRF function in asthma and virus-induced asthma exacerbations., Methods: Total IgE and HRF-reactive IgE/IgG as well as HRF in sera from patients with mild/moderate asthma or severe asthma (SA) and healthy controls (HCs) were quantified by ELISA. HRF secretion in culture media from RV-infected adenovirus-12 SV40 hybrid virus transformed human bronchial epithelial cells and in nasal washings from experimentally RV-infected subjects was analyzed by Western blotting. HRF-reactive IgE/IgG levels in longitudinal serum samples from patients with asthma exacerbations were also quantified., Results: HRF-reactive IgE and total IgE levels were higher in patients with SA than in HCs, whereas HRF-reactive IgG (and IgG 1 ) level was lower in asthmatic patients versus HCs. In comparison with HRF-reactive IgE low asthmatic patients, HRF-reactive IgE high asthmatic patients had a tendency to release more tryptase and prostaglandin D 2 on anti-IgE stimulation of bronchoalveolar lavage cells. RV infection induced HRF secretion from adenovirus-12 SV40 hybrid virus transformed bronchial epithelial cells, and intranasal RV infection of human subjects induced increased HRF secretion in nasal washes. Asthmatic patients had higher levels of HRF-reactive IgE at the time of asthma exacerbations associated with RV infection, compared with those after the resolution. This phenomenon was not seen in asthma exacerbations without viral infections., Conclusions: HRF-reactive IgE is higher in patients with SA. RV infection induces HRF secretion from respiratory epithelial cells both in vitro and in vivo. These results suggest the role of HRF in asthma severity and RV-induced asthma exacerbation., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Although mucosal-associated invariant T (MAIT) cells provide rapid, innate-like responses, they are not pre-set, and memory-like responses have been described for MAIT cells following infections. The importance of metabolism for controlling these responses, however, is unknown. Here, following pulmonary immunization with a Salmonella vaccine strain, mouse MAIT cells expanded as separate CD127 - Klrg1 + and CD127 + Klrg1 - antigen-adapted populations that differed in terms of their transcriptome, function and localization in lung tissue. These populations remained altered from steady state for months as stable, separate MAIT cell lineages with enhanced effector programmes and divergent metabolism. CD127 + MAIT cells engaged in an energetic, mitochondrial metabolic programme, which was critical for their maintenance and IL-17A synthesis. This programme was supported by high fatty acid uptake and mitochondrial oxidation and relied on highly polarized mitochondria and autophagy. After vaccination, CD127 + MAIT cells protected mice against Streptococcus pneumoniae infection. In contrast, Klrg1 + MAIT cells had dormant but ready-to-respond mitochondria and depended instead on Hif1a-driven glycolysis to survive and produce IFN-γ. They responded antigen independently and participated in protection from influenza virus. These metabolic dependencies may enable tuning of memory-like MAIT cell responses for vaccination and immunotherapies., (© 2023. The Author(s).)

Despite the prevalence and medical significance of human cytomegalovirus (HCMV) infections, a systematic analysis of the targets of T cell recognition in humans that spans the entire genome and includes recently described potential novel open reading frames (ORFs) is not available. Here, we screened a library of epitopes predicted to bind HLA class II that spans over 350 different HCMV ORFs and includes ∼150 previously described and ∼200 recently described potential novel ORFs by using an ex vivo gamma interferon (IFN-γ) FluoroSpot assay. We identified 235 unique HCMV-specific epitopes derived from 100 ORFs, some previously described as immunodominant and others that were not previously described to be immunogenic. Of those, 41 belong to the set of recently reported novel ORFs, thus providing evidence that at least some of these are actually expressed in vivo in humans. These data reveal that the breadth of the human T cell response to HCMV is much greater than previously thought. The ORFs and epitopes identified will help elucidate how T cell immunity relates to HCMV pathogenesis and instruct ongoing HCMV vaccine research. IMPORTANCE To understand the crucial role of adaptive immunity in controlling cytomegalovirus infection and disease, we systematically analyzed the CMV "ORFeome" to identify new CMV epitopes targeted primarily by CD4 T cells in humans. Our study identified >200 new T cell epitopes derived from both canonical and novel ORFs, highlighting the substantial breadth of the anti-CMV T cell response and providing new targets for vaccine design.

Background: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4 + T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B 100 (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T H 1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4 + T cells with an atheroprotective, regulatory T cell (T reg ) phenotype in healthy individuals. Yet, the function of apoB-reactive T regs and their relationship with pathogenic T H 1 cells remain unknown., Methods: To interrogate the function of autoreactive CD4 + T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B 978-993 (apoB + ) at the single-cell level., Results: We found that apoB + T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T reg -like transcriptome, although only 21% of all apoB + T cells expressed the T reg transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB + T cells formed several clusters with mixed T H signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T H 1, T helper cell type 2 (T H 2), and T helper cell type 17 (T H 17), and of follicular-helper T cells. ApoB + T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T H 1/T H 17-like cells with proinflammatory properties and only a residual T reg transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T H 1/T H 17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB + T regs in lineage tracing of hyperlipidemic Apoe -/- mice. In adoptive transfer experiments, converting apoB + T regs failed to protect from atherosclerosis., Conclusions: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T regs as a novel cellular target in atherosclerosis.

Mechanisms linking immune sensing of DNA danger signals in the extracellular environment to innate pathways in the cytosol are poorly understood. Here, we identify a previously unidentified immune-metabolic axis by which cells respond to purine nucleosides and trigger a type I interferon-β (IFN-β) response. We find that depletion of ADA2, an ectoenzyme that catabolizes extracellular dAdo to dIno, or supplementation of dAdo or dIno stimulates IFN-β. Under conditions of reduced ADA2 enzyme activity, dAdo is transported into cells and undergoes catabolysis by the cytosolic isoenzyme ADA1, driving intracellular accumulation of dIno. dIno is a functional immunometabolite that interferes with the cellular methionine cycle by inhibiting SAM synthetase activity. Inhibition of SAM-dependent transmethylation drives epigenomic hypomethylation and overexpression of immune-stimulatory endogenous retroviral elements that engage cytosolic dsRNA sensors and induce IFN-β. We uncovered a previously unknown cellular signaling pathway that responds to extracellular DNA-derived metabolites, coupling nucleoside catabolism by adenosine deaminases to cellular IFN-β production., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Cytomegalovirus (CMV) establishes a lifelong infection facilitated, in part, by circumventing immune defenses mediated by tumor necrosis factor (TNF)-family cytokines. An example of this is the mouse CMV (MCMV) m166 protein, which restricts expression of the TNF-related apoptosis-inducing ligand (TRAIL) death receptors, promoting early-phase replication. We show here that replication of an MCMV mutant lacking m166 is also severely attenuated during viral persistence in the salivary glands (SG). Depleting group I innate lymphoid cells (ILCs) or infecting Trail -/- mice completely restored persistent replication of this mutant. Group I ILCs are comprised of two subsets, conventional natural killer cells (cNK) and tissue-resident cells often referred to as innate lymphoid type I cells (ILC1). Using recently identified phenotypic markers to discriminate between these two cell types, their relative expression of TRAIL and gamma interferon (IFN-γ) was assessed during both early and persistent infection. ILC1 were found to be the major TRAIL expressers during both of these infection phases, with cNK expressing very little, indicating that it is ILC1 that curtail replication via TRAIL in the absence of m166-imposed countermeasures. Notably, despite high TRAIL expression by SG-resident ILC1, IFN-γ production by both ILC1 and cNK was minimal at this site of viral persistence. Together these results highlight TRAIL as a key ILC1-utilized effector molecule that can operate in defense against persistent infection at times when other innate control mechanisms may be muted and highlight the importance for the evolution of virus-employed countermeasures. IMPORTANCE Cytomegalovirus (a betaherpesvirus) is a master at manipulating immune responses to promote its lifelong persistence, a result of millions of years of coevolution with its host. Using a one-of-a-kind MCMV mutant unable to restrict expression of the TNF-related apoptosis-inducing ligand death receptors (TRAIL-DR), we show that TRAIL-DR signaling significantly restricts both early and persistent viral replication. Our results also reveal that these defenses are employed by TRAIL-expressing innate lymphoid type I cells (ILC1) but not conventional NK cells. Overall, our results are significant because they show the key importance of viral counterstrategies specifically neutralizing TRAIL effector functions mediated by a specific, tissue-resident subset of group I ILCs., (Copyright © 2019 American Society for Microbiology.)

Human cytomegalovirus (HCMV) is a β-herpesvirus that has co-evolved with the host immune system to establish lifelong persistence. HCMV encodes many immunomodulatory molecules, including the glycoprotein UL144. UL144 is a structural mimic of the tumor necrosis factor receptor superfamily member HVEM (herpesvirus entry mediator), which binds to the various ligands LIGHT, LTα, BTLA, CD160, and gD. However, in contrast to HVEM, UL144 only binds BTLA, inhibiting T-cell activation. Here, we report the crystal structure of the UL144-BTLA complex, revealing that UL144 utilizes residues from its N-terminal cysteine-rich domain 1 (CRD1) to interact uniquely with BTLA. The shorter CRD2 loop of UL144 also alters the relative orientation of BTLA binding with both N-terminal CRDs. By employing structure-guided mutagenesis, we have identified a mutant of BTLA (L123A) that interferes with HVEM binding but preserves UL144 interactions. Furthermore, our results illuminate structural differences between UL144 and HVEM that explain its binding selectivity and highlight it as a suitable scaffold for designing superior, immune inhibitory BTLA agonists., (© 2019 Bitra et al.)

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus. While HCMV infection is generally asymptomatic in the immunocompetent, it can have devastating consequences in those with compromised or underdeveloped immune systems, including transplant recipients and neonates. Galectins are a widely expressed protein family that have been demonstrated to modulate both antiviral immunity and regulate direct host-virus interactions. The potential for galectins to directly modulate HCMV infection has not previously been studied, and our results reveal that galectin-9 (Gal-9) can potently inhibit HCMV infection. Gal-9-mediated inhibition of HCMV was dependent upon its carbohydrate recognition domains and thus dependent on glycan interactions. Temperature shift studies revealed that Gal-9 specific inhibition was mediated primarily at the level of virus-cell fusion and not binding. Additionally, we found that during reactivation of HCMV in hematopoietic stem cell transplant (HSCT) patients soluble Gal-9 is upregulated. This study provides the first evidence for Gal-9 functioning as a potent antiviral defense effector molecule against HCMV infection and identifies it as a potential clinical candidate to restrict HCMV infections. IMPORTANCE Human cytomegalovirus (HCMV) continues to cause serious and often life-threatening disease in those with impaired or underdeveloped immune systems. This virus is able to infect and replicate in a wide range of human cell types, which enables the virus to spread to other individuals in a number of settings. Current antiviral drugs are associated with a significant toxicity profile, and there is no vaccine; these factors highlight a need to identify additional targets for the development of anti-HCMV therapies. We demonstrate for the first time that secretion of a member of the galectin family of proteins, galectin-9 (Gal-9), is upregulated during natural HCMV-reactivated infection and that this soluble cellular protein possesses a potent capacity to block HCMV infection by inhibiting virus entry into the host cell. Our findings support the possibility of harnessing the antiviral properties of Gal-9 to prevent HCMV infection and disease., (Copyright © 2019 American Society for Microbiology.)

Innate immune cells quickly infiltrate the site of pathogen entry and not only stave off infection but also initiate antigen presentation and promote adaptive immunity. The recruitment of innate leukocytes has been well studied in the context of extracellular bacterial and fungal infection but less during viral infections. We have recently shown that the understudied cytokine Interleukin (IL)-17D can mediate neutrophil, natural killer (NK) cell and monocyte infiltration in sterile inflammation and cancer. Herein, we show that early immune cell accumulation at the peritoneal site of infection by mouse cytomegalovirus (MCMV) is mediated by IL-17D. Mice deficient in IL-17D or the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an inducer of IL-17D, featured an early decreased number of innate immune cells at the point of viral entry and were more susceptible to MCMV infection. Interestingly, we were able to artificially induce innate leukocyte infiltration by applying the Nrf2 activator tert-butylhydroquinone (tBHQ), which rendered mice less susceptible to MCMV infection. Our results implicate the Nrf2/IL-17D axis as a sensor of viral infection and suggest therapeutic benefit in boosting this pathway to promote innate antiviral responses.

The role of IL-27 in antiviral immunity is still incompletely understood, especially in the context of chronic viruses that induce a unique environment in their infected host. Cytomegalovirus (CMV) establishes a persistent, tissue localized infection followed by lifelong latency. CMV infects the majority of people and although asymptomatic in healthy individuals, can cause serious disease or death in those with naïve or compromised immune systems. Therefore, there is an urgent need to develop a protective CMV vaccine for people at-risk and identifying key regulators of the protective immune response towards CMV will be crucial. Here we studied mouse CMV (MCMV) in IL-27 receptor deficient animals (Il27ra-/-) to assess the role of IL-27 in regulating CMV immunity. We found that IL-27 enhanced the number of antiviral CD4 T cells upon infection. However, in contrast to a well-established role for CD4 T cells in controlling persistent replication and a positive effect of IL-27 on their numbers, IL-27 promoted MCMV persistence in the salivary gland. This coincided with IL-27 mediated induction of IL-10 production in CD4 T cells. Moreover, IL-27 reduced expression of the transcription factor T-bet and restricted a cytotoxic phenotype in antiviral CD4 T cells. This is a highly intriguing result given the profound cytotoxic phenotype of CMV-specific CD4 T cells seen in humans and we established that dendritic cell derived IL-27 was responsible for this effect. Together, these data show that IL-27 regulates the number and effector functions of MCMV-specific CD4 T cells and could be targeted to enhance control of persistent/latent infection., Competing Interests: The authors have declared that no competing interests exist.

Systems-based based approaches have begun to shed light on extrinsic factors that contribute to immune system variation. Among these, CMV (HHV-5, a β-herpesvirus) imposes a surprisingly profound impact. Most of the world's population is CMV + , and the virus goes through three distinct infection phases en route to establishing lifelong détente with its host. Immune control of CMV in each phase recruits unique arms of host defense, and in turn the virus employs multiple immune-modulatory strategies that help facilitate the establishment of lifelong persistence. In this review, we explain how CMV shapes immunity and discuss the impact it may have on overall health., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Upon infection with an intracellular pathogen, cytotoxic CD8 + T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy., (© 2018 Delpoux et al.)

4-1BB (CD137) is a TNF receptor superfamily (TNFRSF) member that is thought to undergo receptor trimerization upon binding to its trimeric TNF superfamily ligand (4-1BBL) to stimulate immune responses. 4-1BB also can bind to the tandem repeat-type lectin galectin-9 (Gal-9), and signaling through mouse (m)4-1BB is reduced in galectin-9 (Gal-9)-deficient mice, suggesting a pivotal role of Gal-9 in m4-1BB activation. Here, using sulfur-SAD phasing, we determined the crystal structure of m4-1BB to 2.2-Å resolution. We found that similar to other TNFRSFs, m4-1BB has four cysteine-rich domains (CRDs). However, the organization of CRD1 and the orientation of CRD3 and CRD4 with respect to CRD2 in the m4-1BB structure distinctly differed from those of other TNFRSFs. Moreover, we mapped two Asn residues within CRD4 that are N -linked glycosylated and mediate m4-1BB binding to Gal-9. Kinetics studies of m4-1BB disclosed a very tight nanomolar binding affinity to m4-1BBL with an unexpectedly strong avidity effect. Both N- and C-terminal domains of Gal-9 bound m4-1BB, but with lower affinity compared with m4-1BBL. Although the TNF homology domain (THD) of human (h)4-1BBL forms non-covalent trimers, we found that m4-1BBL formed a covalent dimer via 2 cysteines absent in h4-1BBL. As multimerization and clustering is a prerequisite for TNFR intracellular signaling, and as m4-1BBL can only recruit two m4-1BB monomers, we hypothesize that m4-1BBL and Gal-9 act together to aid aggregation of m4-1BB monomers to efficiently initiate m4-1BB signaling., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recognition of pathogen-associated molecular patterns and danger-associated molecular patterns by host cells is an important step in innate immune activation. The DNA sensor cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) binds to DNA and produces cGAMP, which in turn binds to stimulator of interferon genes (STING) to activate IFN-I. Here we show that cGAMP has a noncanonical function in inflammasome activation in human and mouse cells. Inflammasome activation requires two signals, both of which are activated by cGAMP. cGAMP alone enhances expression of inflammasome components through IFN-I, providing the priming signal. Additionally, when combined with a priming signal, cGAMP activates the inflammasome through an AIM2, NLRP3, ASC, and caspase-1 dependent process. These two cGAMP-mediated functions, priming and activation, have differential requirements for STING. Temporally, cGAMP induction of IFN-I precedes inflammasome activation, which then occurs when IFN-I is waning. In mice, cGAS/cGAMP amplify both inflammasome and IFN-I to control murine cytomegalovirus. Thus, cGAMP activates the inflammasome in addition to IFN-I, and activation of both is needed to control infection by a DNA virus., (© 2017 Swanson et al.)

Human cytomegalovirus (HCMV) encodes numerous proteins and microRNAs that function to evade the immune response and allow the virus to replicate and disseminate in the face of a competent innate and acquired immune system. The establishment of a latent infection by CMV, which if completely quiescent at the level of viral gene expression would represent an ultimate in immune evasion strategies, is not sufficient for lifelong persistence and dissemination of the virus. CMV needs to reactivate and replicate in a lytic cycle of infection in order to disseminate further, which occurs in the face of a fully primed secondary immune response. Without reactivation, latency itself would be redundant for the virus. It is also becoming clear that latency is not a totally quiescent state, but is characterized by limited viral gene expression. Therefore, the virus also needs immune evasion strategies during latency. An effective immune response to CMV is required or viral replication will cause morbidity and ultimately mortality in the host. There is clearly a complex balance between virus immune evasion and host immune recognition over a lifetime. This poses the important question of whether long-term evasion or manipulation of the immune response driven by CMV is detrimental to health. In this meeting report, three groups used the murine model of CMV (MCMV) to examine if the contribution of the virus to immune senescence is set by the (i) initial viral inoculum, (ii) inflation of T cell responses, (iii) or the balance between functionally distinct effector CD4+ T cells. The work of other groups studying the CMV response in humans is discussed. Their work asks whether the ability to make immune responses to new antigens is compromised by (i) age and HCMV carriage, (ii) long-term exposure to HCMV giving rise to an overall immunosuppressive environment and increased levels of latent virus, or (iii) adapted virus mutants (used as potential vaccines) that have the capacity to elicit conventional and unconventional T cell responses.

APO2L/TRAIL (TNF-related apoptosis-inducing ligand) induces death of tumor cells through two agonist receptors, TRAIL-R1 and TRAIL-R2. We demonstrate here that N-linked glycosylation (N-glyc) plays also an important regulatory role for TRAIL-R1-mediated and mouse TRAIL receptor (mTRAIL-R)-mediated apoptosis, but not for TRAIL-R2, which is devoid of N-glycans. Cells expressing N-glyc-defective mutants of TRAIL-R1 and mouse TRAIL-R were less sensitive to TRAIL than their wild-type counterparts. Defective apoptotic signaling by N-glyc-deficient TRAIL receptors was associated with lower TRAIL receptor aggregation and reduced DISC formation, but not with reduced TRAIL-binding affinity. Our results also indicate that TRAIL receptor N-glyc impacts immune evasion strategies. The cytomegalovirus (CMV) UL141 protein, which restricts cell-surface expression of human TRAIL death receptors, binds with significant higher affinity TRAIL-R1 lacking N-glyc, suggesting that this sugar modification may have evolved as a counterstrategy to prevent receptor inhibition by UL141. Altogether our findings demonstrate that N-glyc of TRAIL-R1 promotes TRAIL signaling and restricts virus-mediated inhibition.

Unlabelled: Several innate sensing pathways contribute to the control of early cytomegalovirus (CMV) infection, leading to a multiphasic type I interferon (IFN-I) response that limits viral replication and promotes host defenses. Toll-like receptor (TLR)-dependent pathways induce IFN-I production in CMV-infected plasmacytoid dendritic cells; however, the initial burst of IFN-I that occurs within the first few hours in vivo is TLR independent and emanates from stromal cells. Here we show that primary human endothelial cells mount robust IFN-I responses to human CMV that are dependent upon cyclic GMP-AMP synthase (cGAS), STING, and interferon regulatory factor 3 (IRF3) signaling. Disruption of STING expression in endothelial cells by clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 revealed that it is essential for the induction of IFN-I and restriction of CMV replication. Consistently, STING was necessary to mount the first phase of IFN-I production and curb CMV replication in infected mice. Thus, DNA sensing through STING is critical for primary detection of both human and mouse CMV in nonhematopoietic cells and drives the initial wave of IFN-I that is key for controlling early viral replication in vivo ., Importance: Cytomegalovirus (CMV) is one of the most common viral pathogens, with the majority of people contracting the virus in their lifetime. Although acute infection is mostly asymptomatic in healthy persons, significant pathology is observed in immunocompromised individuals, and chronic CMV infection may exacerbate a myriad of inflammatory conditions. Here we show that primary human endothelial cells mount robust IFN-I responses against CMV via a cGAS/STING/IRF3 pathway. Disruption of STING expression by CRISPRs revealed an essential role in eliciting IFN-I responses and restricting CMV replication. Consistently, in mice, STING is necessary for the first phase of IFN-I production that limits early CMV replication. Our results demonstrate a pivotal role for the cGAS-STING pathway in the initial detection of CMV infection., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: CD4 T cells provide protection against cytomegalovirus (CMV) and other persistent viruses, and the ability to quantify and characterize epitope-specific responses is essential to gain a more precise understanding of their effector roles in this regard. Here, we report the first two I-A(d)-restricted CD4 T cell responses specific for mouse CMV (MCMV) epitopes and use a major histocompatibility complex class II (MHC-II) tetramer to characterize their phenotypes and functions. We demonstrate that MCMV-specific CD4 T cells can express high levels of granzyme B and kill target cells in an epitope- and organ-specific manner. In addition, CD4 T cell epitope vaccination of immunocompetent mice reduced MCMV replication in the same organs where CD4 cytotoxic T lymphocyte (CTL) activity was observed. Together, our studies show that MCMV epitope-specific CD4 T cells have the potential to mediate antiviral defense by multiple effector mechanisms in vivo., Importance: CD4 T cells mediate immune protection by using their T cell receptors to recognize specific portions of viral proteins, called epitopes, that are presented by major histocompatibility complex class II (MHC-II) molecules on the surfaces of professional antigen-presenting cells (APCs). In this study, we discovered the first two epitopes derived from mouse cytomegalovirus (MCMV) that are recognized by CD4 T cells in BALB/c mice, a mouse strain commonly used to study the pathogenesis of this virus infection. Here, we report the sequences of these epitopes, characterize the CD4 T cells that recognize them to fight off MCMV infection, and show that we can use the epitopes to vaccinate mice and protect against MCMV., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Cardif, also known as IPS-1, VISA, and MAVS, is an intracellular adaptor protein that functions downstream of the retinoic acid-inducible gene I family of pattern recognition receptors. Cardif is required for the production of type I IFNs and other inflammatory cytokines after retinoic acid-inducible gene I-like receptors recognize intracellular antigenic RNA. Studies have recently shown that Cardif may have other roles in the immune system in addition to its role in viral immunity. In this study, we find that the absence of Cardif alters normal NK cell development and maturation. Cardif(-/-) mice have a 35% loss of mature CD27(-)CD11b(+) NK cells in the periphery. In addition, Cardif(-/-) NK cells have altered surface marker expression, lower cytotoxicity, decreased intracellular STAT1 levels, increased apoptosis, and decreased proliferation compared with wild-type NK cells. Mixed chimeric mice revealed that the defective maturation and increased apoptotic rate of peripheral Cardif(-/-) NK cells is cell intrinsic. However, Cardif(-/-) mice showed enhanced control of mouse CMV (a DNA β-herpesvirus) by NK cells, commensurate with increased activation and IFN-γ production by these immature NK cell subsets. These results indicate that the skewed differentiation and altered STAT expression of Cardif(-/-) NK cells can result in their hyperresponsiveness in some settings and support recent findings that Cardif-dependent signaling can regulate aspects of immune cell development and/or function distinct from its well-characterized role in mediating cell-intrinsic defense to RNA viruses., (Copyright © 2015 by The American Association of Immunologists, Inc.)

TNF-related apoptosis inducing ligand (TRAIL) death receptors (DR) regulate apoptosis and inflammation, but their role in antiviral defense is poorly understood. Cytomegaloviruses (CMV) encode many immune-modulatory genes that shape host immunity, and they utilize multiple strategies to target the TNF-family cytokines. Here we show that the m166 open reading frame (orf) of mouse CMV (MCMV) is strictly required to inhibit expression of TRAIL-DR in infected cells. An MCMV mutant lacking m166 expression (m166stop) is severely compromised for replication in vivo, most notably in the liver, and depleting natural killer (NK) cells, or infecting TRAIL-DR-/- mice, restored MCMV-m166stop replication completely. These results highlight the critical importance for CMV to have evolved a strategy to inhibit TRAIL-DR signaling to thwart NK-mediated defenses.

NK cells are key regulators of innate defense against mouse CMV (MCMV). Like NK cells, NKT cells also produce high levels of IFN-γ rapidly after MCMV infection. However, whether similar mechanisms govern activation of these two cell types, as well as the significance of NKT cells for host resistance, remain unknown. In this article, we show that, although both NKT and NK cells are activated via cytokines, their particular cytokine requirements differ significantly in vitro and in vivo. IL-12 is required for NKT cell activation in vitro but is not sufficient, whereas NK cells have the capacity to be activated more promiscuously in response to individual cytokines from innate cells. In line with these results, GM-CSF-derived dendritic cells activated only NK cells upon MCMV infection, consistent with their virtual lack of IL-12 production, whereas Flt3 ligand-derived dendritic cells produced IL-12 and activated both NK and NKT cells. In vivo, NKT cell activation was abolished in IL-12(-/-) mice infected with MCMV, whereas NK cells were still activated. In turn, splenic NK cell activation was more IL-18 dependent. The differential requirements for IL-12 and IL-18 correlated with the levels of cytokine receptor expression by NK and NKT cells. Finally, mice lacking NKT cells showed reduced control of MCMV, and depleting NK cells further enhanced viral replication. Taken together, our results show that NKT and NK cells have differing requirements for cytokine-mediated activation, and both can contribute nonredundantly to MCMV defense, revealing that these two innate lymphocyte subsets function together to fine-tune antiviral responses.

During primary infection, murine cytomegalovirus (MCMV) spreads systemically, resulting in virus replication and pathology in multiple organs. This disseminated infection is ultimately controlled, but the underlying immune defense mechanisms are unclear. Investigating the role of the cytokine IL-22 in MCMV infection, we discovered an unanticipated function for neutrophils as potent antiviral effector cells that restrict viral replication and associated pathogenesis in peripheral organs. NK-, NKT-, and T cell-secreted IL-22 orchestrated antiviral neutrophil-mediated responses via induction in stromal nonhematopoietic tissue of the neutrophil-recruiting chemokine CXCL1. The antiviral effector properties of infiltrating neutrophils were directly linked to the expression of TNF-related apoptosis-inducing ligand (TRAIL). Our data identify a role for neutrophils in antiviral defense, and establish a functional link between IL-22 and the control of antiviral neutrophil responses that prevents pathogenic herpesvirus infection in peripheral organs., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Lymphocyte activation is regulated by costimulatory and inhibitory receptors, of which both B and T lymphocyte attenuator (BTLA) and CD160 engage herpesvirus entry mediator (HVEM). Notably, it remains unclear how HVEM functions with each of its ligands during immune responses. In this study, we show that HVEM specifically activates CD160 on effector NK cells challenged with virus-infected cells. Human CD56(dim) NK cells were costimulated specifically by HVEM but not by other receptors that share the HVEM ligands LIGHT, Lymphotoxin-α, or BTLA. HVEM enhanced human NK cell activation by type I IFN and IL-2, resulting in increased IFN-γ and TNF-α secretion, and tumor cell-expressed HVEM activated CD160 in a human NK cell line, causing rapid hyperphosphorylation of serine kinases ERK1/2 and AKT and enhanced cytolysis of target cells. In contrast, HVEM activation of BTLA reduced cytolysis of target cells. Together, our results demonstrate that HVEM functions as a regulator of immune function that activates NK cells via CD160 and limits lymphocyte-induced inflammation via association with BTLA.

During mouse cytomegalovirus (MCMV) infection, the first wave of type I interferon (IFN-I) production peaks at ≈ 8 h. This IFN-I emanates from splenic stromal cells located in the marginal zone (MZ) and requires B cells that express lymphotoxin. The amount of IFN-I produced at these initial times is at least equivalent in magnitude to that produced later by dendritic cells (≈ 36 to 48 h), but the relative roles of these two IFN-I sources in regulating MCMV defense remain unclear. Here we show that IFN-I produced by MZ stromal cells dramatically restricts the first measurable burst of viral production, which occurs at ≈ 32 h. This primary innate control by IFN-I is partially mediated through the activation of natural killer (NK) cells, which produce gamma interferon in an IFN-I-dependent fashion, and is independent of Ly49H. Strikingly, MCMV production in the spleens of immunocompetent mice never increases at times after 32 h. These results highlight the critical importance of lymphoid-tissue stromal cells in orchestrating the earliest phase of innate defense to MCMV infection, capping replication levels, and blocking spread until infection is ultimately controlled.

Cytomegaloviruses (CMVs) establish lifelong infections that are controlled in part by CD4(+) and CD8(+) T cells. To promote persistence, CMVs utilize multiple strategies to evade host immunity, including modulation of costimulatory molecules on infected antigen-presenting cells. In humans, CMV-specific memory T cells are characterized by the loss of CD27 expression, which suggests a critical role of the costimulatory receptor-ligand pair CD27-CD70 for the development of CMV-specific T cell immunity. In this study, the in vivo role of CD27-CD70 costimulation during mouse CMV infection was examined. During the acute phase of infection, the magnitudes of CMV-specific CD4(+) and CD8(+) T cell responses were decreased in mice with abrogated CD27-CD70 costimulation. Moreover, the accumulation of inflationary memory T cells during the persistent phase of infection and the ability to undergo secondary expansion required CD27-CD70 interactions. The downmodulation of CD27 expression, however, which occurs gradually and exclusively on inflationary memory T cells, is ligand independent. Furthermore, the IL-2 production in both noninflationary and inflationary CMV-specific T cells was dependent on CD27-CD70 costimulation. Collectively, these results highlight the importance of the CD27-CD70 costimulation pathway for the development of CMV-specific T cell immunity during acute and persistent infection.

It is generally accepted that, following primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in CD34(+) progenitor cells and other derivative cells of the myeloid lineage. In this study, we show that the viral UL144 gene is expressed during latent infection in two cell types of the myeloid lineage, CD34(+) and CD14(+) monocytes, and that the UL144 protein is functional in latently infected monocytes. However, this latency-associated expression of UL144 occurs only in certain isolates of HCMV and depends on the presence of functional GATA-2 transcription factor binding sites in the UL144 promoter, in contrast to the viral latency-associated gene LUNA, which we also show is regulated by GATA-2 but expressed uniformly during latent infection independent of the virus isolate. Taken together, these data suggest that the HCMV latency-associated transcriptome may be virus isolate specific and dependent on the repertoire of transcription factor binding sites in the promoters of latency-associated genes.

Death receptors (DRs) of the TNFR superfamily contribute to antiviral immunity by promoting apoptosis and regulating immune homeostasis during infection, and viral inhibition of DR signaling can alter immune defenses. Here we identify the human cytomegalovirus (HCMV) UL141 glycoprotein as necessary and sufficient to restrict TRAIL DR function. Despite showing no primary sequence homology to TNF family cytokines, UL141 binds the ectodomains of both human TRAIL DRs with affinities comparable to the natural ligand TRAIL. UL141 binding promotes intracellular retention of the DRs, thus protecting virus infected cells from TRAIL and TRAIL-dependent NK cell-mediated killing. The identification of UL141 as a herpesvirus modulator of the TRAIL DRs strongly implicates this pathway as a regulator of host defense to HCMV and highlights UL141 as a pleiotropic inhibitor of NK cell effector function., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The TRAIL (TNF-related apoptosis inducing ligand) death receptors (DRs) of the tumor necrosis factor receptor superfamily (TNFRSF) can promote apoptosis and regulate antiviral immunity by maintaining immune homeostasis during infection. In turn, human cytomegalovirus (HCMV) expresses immunomodulatory proteins that down-regulate cell surface expression of TNFRSF members as well as poliovirus receptor-related proteins in an effort to inhibit host immune effector pathways that would lead to viral clearance. The UL141 glycoprotein of human cytomegalovirus inhibits host defenses by blocking cell surface expression of TRAIL DRs (by retention in ER) and poliovirus receptor CD155, a nectin-like Ig-fold molecule. Here we show that the immunomodulatory function of HCMV UL141 is associated with its ability to bind diverse proteins, while utilizing at least two distinct binding sites to selectively engage TRAIL DRs or CD155. Binding studies revealed high affinity interaction of UL141 with both TRAIL-R2 and CD155 and low affinity binding to TRAIL-R1. We determined the crystal structure of UL141 bound to TRAIL-R2 at 2.1 Å resolution, which revealed that UL141 forms a homodimer that engages two TRAIL-R2 monomers 90° apart to form a heterotetrameric complex. Our structural and biochemical data reveal that UL141 utilizes its Ig-domain to facilitate non-canonical death receptor interactions while UL141 partially mimics the binding site of TRAIL on TRAIL-R2, which we found to be distinct from that of CD155. Moreover, UL141 also binds to an additional surface patch on TRAIL-R2 that is distinct from the TRAIL binding site. Therefore, the breadth of UL141-mediated effects indicates that HCMV has evolved sophisticated strategies to evade the immune system by modulating multiple effector pathways.

Inhibitors of tumour necrosis factor (TNF) are among the most successful protein-based drugs (biologics) and have proven to be clinically efficacious at reducing inflammation associated with several autoimmune diseases. As a result, attention is focusing on the therapeutic potential of additional members of the TNF superfamily of structurally related cytokines. Many of these TNF-related cytokines or their cognate receptors are now in preclinical or clinical development as possible targets for modulating inflammatory diseases and cancer as well as other indications. This Review focuses on the biologics that are currently in clinical trials for immune-related diseases and other syndromes, discusses the successes and failures to date as well as the expanding therapeutic potential of modulating the activity of this superfamily of molecules.