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Background: The impact of chronic hepatic infection on antigen non-specific immune cells in circulation remains poorly understood. We reported lasting global hyperfunction of peripheral CD8 T cells in HCV-infected individuals with cirrhosis. Whether gene expression patterns in bulk CD8 T cells are associated with the severity of liver fibrosis in HCV infection is not known. Methods: RNA sequencing of blood CD8 T cells from treatment naïve, HCV-infected individuals with minimal (Metavir F0-1 ≤ 7.0 kPa) or advanced fibrosis or cirrhosis (F4 ≥ 12.5 kPa), before and after direct-acting antiviral therapy, was performed. CD8 T cell function was assessed by flow cytometry. Results: In CD8 T cells from pre-DAA patients with advanced compared to minimal fibrosis, Gene Ontology analysis and Gene Set Enrichment Analysis identified differential gene expression related to cellular function and metabolism, including upregulated Hedgehog (Hh) signaling, IFN-α, -γ, TGF-ß response genes, apoptosis, apical surface pathways, phospholipase signaling, phosphatidyl-choline/inositol activity, and second-messenger-mediated signaling. In contrast, genes in pathways associated with nuclear processes, RNA transport, cytoskeletal dynamics, cMyc/E2F regulation, oxidative phosphorylation, and mTOR signaling, were reduced. Hh signaling pathway was the top featured gene set upregulated in cirrhotics, wherein hallmark genes GLI1 and PTCH1 ranked highly. Inhibition of Smo-dependent Hh signaling ablated the expression of IFN-γ and perforin in stimulated CD8 T cells from chronic HCV-infected patients with advanced compared to minimal fibrosis. CD8 T cell gene expression profiles post-DAA remained clustered with pre-DAA profiles and disparately between advanced and minimal fibrosis, suggesting a persistent perturbation of gene expression long after viral clearance. Conclusions: This analysis of bulk CD8 T cell gene expression in chronic HCV infection suggests considerable reprogramming of the CD8 T cell pool in the cirrhotic state. Increased Hh signaling in cirrhosis may contribute to generalized CD8 T cell hyperfunction observed in chronic HCV infection. Understanding the lasting nature of immune cell dysfunction may help mitigate remaining clinical challenges after HCV clearance and more generally, improve long term outcomes for individuals with severe liver disease.
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Linfocitos T CD8-positivos , Proteínas Hedgehog , Hepatitis C Crónica , Cirrosis Hepática , Transducción de Señal , Humanos , Linfocitos T CD8-positivos/inmunología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Proteínas Hedgehog/metabolismo , Cirrosis Hepática/inmunología , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Femenino , Hepacivirus/inmunología , Adulto , Anciano , Perfilación de la Expresión Génica , Transcriptoma , Regulación de la Expresión GénicaRESUMEN
The SARS-CoV-2 pandemic highlighted the need for rapid, collaborative, and population-centric research to define health impact, develop health care policies and establish reliable diagnostic and surveillance tests. Critical for these objectives were in-depth clinical data collected in standardized fashion and large numbers of various types of human samples prior and post-viral encounter. As the pandemic evolved with the emergence of new variants of concern (VOCs), access to samples and data from infected and vaccinated individuals were needed to monitor immune durability, the possibility of increased transmissibility and virulence, and vaccine protection against new and emerging VOCs. Therefore, essential to the pandemic response is a strong laboratory and data research component, supported by effective biobanking and data sharing. Critically important to the speed of the research response is the rapid access to biobanked samples. To address critical challenges brought to light by the pandemic, the Coronavirus Variants Rapid Response Network (CoVaRR-Net), funded by the Canadian Institutes of Health Research, was established to coordinate research efforts to provide rapid evidence-based responses to emerging VOCs. The purpose of this paper is to introduce the CoVaRR-Net Biobank and define its contribution to pandemic preparedness.
La pandémie de SRAS-CoV-2 a fait ressortir la nécessité de réaliser des recherches rapides, coopératives et populationnelles pour en définir les effets sur la santé, promulguer des politiques sanitaires et établir des tests diagnostiques et des tests de surveillance fiables. Pour réaliser ces objectifs, il était essentiel de colliger des données cliniques approfondies d'une manière standardisée et d'amasser un grand nombre de divers types d'échantillons humains avant et après le contact viral. Lorsque la pandémie a évolué par l'émergence de nouveaux variants préoccupants (VOC), il est devenu nécessaire d'accéder à des échantillons et à des données de personnes infectées et vaccinées pour surveiller la durabilité de l'immunité, la possibilité d'une transmissibilité et d'une virulence accrues et la protection conférée par les vaccins contre les VOC nouveaux et émergents. Ainsi, il est essentiel de disposer d'un vigoureux volet de recherches de laboratoire et de recherches à partir de données pour répondre à la pandémie, soutenu par une mise en biobanque et un partage des données efficaces. Pour assurer une réponse rapide par la recherche, il est tout aussi important d'accéder rapidement aux échantillons mis en biobanque. Afin de relever les défis cruciaux soulevés par la pandémie, le Coronavirus Variants Rapid Response Network (réseau de réponse rapide aux variants du coronavirus; CoVaRR-Net), financé par les Instituts de recherche en santé du Canada, a été créé pour coordonner les efforts de recherche afin de fournir des réponses rapides fondées sur des données probantes aux VOC en émergence. Le présent article vise à présenter la Biobanque CoVaRR-Net et à en définir la contribution à la préparation aux pandémies.
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Background: Prognostic markers for COVID-19 disease outcome are currently lacking. Plasma gelsolin (pGSN) is an actin-binding protein and an innate immune marker involved in disease pathogenesis and viral infections. Here, we demonstrate the utility of pGSN as a prognostic marker for COVID-19 disease outcome; a test performance that is significantly improved when combined with cytokines and antibodies compared to other conventional markers such as CRP and ferritin. Methods: Blood samples were longitudinally collected from hospitalized COVID-19 patients as well as COVID-19 negative controls and the levels of pGSN in µg/mL, cytokines and anti- SARS-CoV-2 spike protein antibodies assayed. Mean ± SEM values were correlated with clinical parameters to develop a prognostic platform. Results: pGSN levels were significantly reduced in COVID-19 patients compared to healthy individuals. Additionally, pGSN levels combined with plasma IL-6, IP-10 and M-CSF significantly distinguished COVID-19 patients from healthy individuals. While pGSN and anti-spike IgG titers together strongly predict COVID-19 severity and death, the combination of pGSN and IL-6 was a significant predictor of milder disease and favorable outcomes. Conclusion: Taken together, these findings suggest that multi-parameter analysis of pGSN, cytokines and antibodies could predict COVID-19 hospitalization outcomes with greater certainty compared with conventional clinical laboratory markers such as CRP and ferritin. This research will inform and improve clinical management and health system interventions in response to SARS-CoV-2 infection.
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COVID-19 , Gelsolina , Biomarcadores , Quimiocina CXCL10 , Citocinas , Ferritinas , Hospitalización , Humanos , Inmunoglobulina G , Interleucina-6 , Factor Estimulante de Colonias de Macrófagos , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
PURPOSE: To investigate the robustness and longevity of SARS-CoV-2 immune responses conferred by natural infection and vaccination among priority populations such as immunocompromised individuals and people with post-acute sequelae of COVID-19 in a prospective cohort study (Stop the Spread Ottawa-SSO) in adults living in the Ottawa region. In this paper, we describe the study design, ongoing data collection and baseline characteristics of participants. PARTICIPANTS: Since October 2020, participants who tested positive for COVID-19 (convalescents) or at high risk of exposure to the virus (under surveillance) have provided monthly blood and saliva samples over a 10-month period. As of 2 November 2021, 1026 adults had completed the baseline survey and 976 had attended baseline bloodwork. 300 participants will continue to provide bimonthly blood samples for 24 additional months (ie, total follow-up of 34 months). FINDINGS TO DATE: The median age of the baseline sample was 44 (IQR 23, range: 18-79) and just over two-thirds (n=688; 67.1%) were female. 255 participants (24.9%) had a history of COVID-19 infection confirmed by PCR and/or serology. Over 600 participants (60.0%) work in high-risk occupations (eg, healthcare, teaching and transportation). 108 participants (10.5%) reported immunocompromising conditions or treatments at baseline (eg, cancer, HIV, other immune deficiency, and/or use of immunosuppressants). FUTURE PLANS: SSO continues to yield rich research potential, given the collection of pre-vaccine baseline data and samples from the majority of participants, recruitment of diverse subgroups of interest, and a high level of participant retention and compliance with monthly sampling. The 24-month study extension will maximise opportunities to track SARS-CoV-2 immunity and vaccine efficacy, detect and characterise emerging variants, and compare subgroup humoral and cellular response robustness and persistence.
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COVID-19 , Adulto , Humanos , Femenino , Masculino , SARS-CoV-2 , Formación de Anticuerpos , Estudios Prospectivos , Anticuerpos , Vacunación , Inmunidad Celular , Anticuerpos AntiviralesRESUMEN
Signaling via the IL-7 receptor complex (IL-7Ralpha/CD127 and IL-2Rgamma/CD132) is required for T-cell development and survival. Decreased CD127 expression has been associated with persistent viral infections (e.g. HIV, HCV) and cancer. Many IL-2Rgamma-sharing (gammaC) cytokines decrease CD127 expression on CD4+ and CD8+ T cells in mice (IL-2, IL-4, IL-7, IL-15) and in humans (IL-2, IL-7), suggesting a common function. IL-4 is of particular interest as it is upregulated in HIV infection and in thyroid and colon cancers. The role of IL-4 in regulating CD127 expression and IL-7 activity in human thymocytes and mature CD8+ T cells is unknown and was therefore investigated. IL-4 decreased CD127 expression on all thymocyte subsets tested and only on naïve (CD45RA+) CD8+ T cells, without altering membrane-bound CD127 mRNA expression. Pre-treatment of thymocytes or CD8+ T cells with IL-4 inhibited IL-7-mediated phosphorylation of STAT5 and decreased proliferation of CD8+ T cells. By downregulating CD127 expression and signaling on developing thymocytes and CD8+ T cells, IL-4 is a potential contributor to impaired CD8+ T-cell function in some anti-viral and anti-tumor responses. These findings are of particular consequence to diseases such as HIV, HCV, RSV, measles and cancer, in which CD127 expression is decreased, IL-7 activity is impaired and IL-4 concentrations are elevated.
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Linfocitos T CD8-positivos/efectos de los fármacos , Interleucina-4/farmacología , Subunidad alfa del Receptor de Interleucina-7/biosíntesis , Subgrupos de Linfocitos T/efectos de los fármacos , Timo/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Técnicas de Cocultivo , Humanos , Interleucina-7/metabolismo , Subunidad alfa del Receptor de Interleucina-7/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/biosíntesis , Factor de Transcripción STAT5/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
The inflammatory and anti-inflammatory MÏs have been implicated in many diseases including rheumatoid arthritis, multiple sclerosis, and leprosy. Recent studies suggest targeting MÏ function and activation may represent a potential target to treat these diseases. Herein, we investigated the effect of second mitochondria-derived activator of caspases (SMAC) mimetics (SMs), the inhibitors of apoptosis (IAPs) proteins, on the killing of human pro- and anti-inflammatory MÏ subsets. We have shown previously that human monocytes are highly susceptible whereas differentiated MÏs (M0) are highly resistant to the cytocidal abilities of SMs. To determine whether human MÏ subsets are resistant to the cytotoxic effects of SMs, we show that M1 MÏs are highly susceptible to SM-induced cell death whereas M2a, M2b, and M2c differentiated subsets are resistant, with M2c being the most resistant. SM-induced cell death in M1 MÏs was mediated by apoptosis as well as necroptosis, activated both extrinsic and intrinsic pathways of apoptosis, and was attributed to the IFN-γ-mediated differentiation. In contrast, M2c and M0 MÏs experienced cell death through necroptosis following simultaneous blockage of the IAPs and the caspase pathways. Overall, the results suggest that survival of human MÏs is critically linked to the activation of the IAPs pathways. Moreover, agents blocking the cellular IAP1/2 and/or caspases can be exploited therapeutically to address inflammation-related diseases.
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Apoptosis , Inhibidores de Caspasas/farmacología , Polaridad Celular , Macrófagos/citología , Oligopéptidos/farmacología , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Polaridad Celular/efectos de los fármacos , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Quinasas Janus/metabolismo , Cinética , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Ratones , Necroptosis/efectos de los fármacos , Fenotipo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Chronic hepatitis C virus (HCV) infection disrupts immune functions, including that of cytotoxic CD8+ T-cells which are important mediators of immune response. While HCV cure aims to eliminate long term sequelae of infection, whether direct-acting antiviral (DAA) treatment results in immune reconstitution remains unclear. We and others have reported generalized CD8+ T-cell dysfunction in chronic HCV infection and our research suggests that the degree of liver damage is a factor in this process. Our recent research indicates that liver fibrosis is not readily reversed after DAA-mediated clearance of chronic HCV infection. We therefore examined the function of circulating CD8+ T-cell subsets in chronic HCV infection in the context of liver fibrosis severity, determined by ultrasound elastography and Metavir F-score system. We observed progressive shifts in CD8+ T-cell subset distribution in HCV-infected individuals with advanced liver fibrosis (F4) compared to minimal fibrosis (F0-1) or uninfected controls, and this remained unchanged after viral cure. Impaired CD8+ T-cell function was observed as a reduced proportion of CD107+ and perforin+ late effector memory cells in HCV+(F4) and HCV+(F0-1) individuals, respectively. In HCV+(F4) individuals, nearly all CD8+ T-cell subsets had an elevated proportion of perforin+ cells while naïve cells had increased proportions of IFN-γ+ and CD107+ cells. These exaggerated CD8+ T-cell activities were not resolved when evaluated 24 weeks after completion of DAA therapy and HCV clearance. This was further supported by sustained, high levels of cell proliferation and cytolytic activity. Furthermore, DAA therapy had no effect on elevated concentrations of systemic inflammatory cytokines and decreased levels of inhibitory TGF-ß in the plasma of HCV+(F4) individuals, suggesting HCV infection and advanced liver disease result in a long-lasting immune activating microenvironment. These data demonstrate that in chronic HCV infection, liver fibrosis severity is associated with generalized hyperfunctional CD8+ T-cells, particularly with perforin production and cytotoxicity, and this persists after viral clearance. Whether DAA therapy will eliminate other related long-term sequelae in HCV+(F4) individuals remains an important research question.
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Antivirales/uso terapéutico , Linfocitos T CD8-positivos/inmunología , Hepatitis C/inmunología , Cirrosis Hepática/inmunología , Cirrosis Hepática/virología , Anciano , Linfocitos T CD8-positivos/efectos de los fármacos , Degranulación de la Célula , Células Cultivadas , Citocinas/sangre , Femenino , Hepatitis C/tratamiento farmacológico , Humanos , Memoria Inmunológica , Interferón gamma/metabolismo , Masculino , Persona de Mediana Edad , Perforina/metabolismoRESUMEN
IL-7 plays an important role in T cell survival, function, and memory cell development, yet the role of cytokine signaling pathways in these processes has not been fully elucidated. Moreover, the underlying mechanisms for the observed impairment of IL-7 activity in diseases, such as HIV infection, breast cancer, and autoimmunity, are not well understood. It was therefore hypothesized that IL-7-induced signaling molecules could be linked with distinct IL-7-associated activities. To address this, the activation and functional associations of IL-7-induced signaling pathways, specifically antigen-independent activities that are relevant to T cell homeostasis, were examined. Low concentrations of IL-7 (100 pg/ml) are capable of activating the Jak-STAT and PI3K signaling pathways, whereas higher concentrations (500-1000 pg/ml) were required to induce Bcl-2 production and glucose uptake. Even higher concentrations of IL-7 (10,000 pg/ml) were needed to induce cell proliferation and intracellular accumulation of perforin. Inhibition of Jak activation reduced IL-7-induced Bcl-2 and perforin production, whereas inhibition of Jak/STAT or PI3K pathways reduced glucose uptake and proliferation. This study suggests a complex control of IL-7-associated activities in the absence of antigen stimulation. These data may provide insights into mechanisms of impaired IL-7 signaling and function in disease and could be relevant for the study of IL-7-based immunotherapeutics. Specifically, this study has linked STAT5 and PI3K activation to shared and distinct IL-7-associated activities in human CD8+ T cells.
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Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Interleucina-7/farmacología , Quinasas Janus/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Activación Enzimática/efectos de los fármacos , Glucosa/metabolismo , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Perforina/biosíntesis , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Decreased expression of interleukin (IL)-7 receptor α (CD127) on CD8(+) T cells in progressive HIV disease suggests a role for CD127 regulation in HIV immunopathogenesis. The direct effect of HIV on CD127 expression has not been explored to explain these in vivo findings. Peripheral blood mononuclear cells (PBMCs) or isolated CD8(+) T cells from healthy individuals were cultured with either X4 (HIV-1(IIIB)), R5 (HIV-1(BaL)), dual tropic (HIV-1(CS204)), or replication-incompetent (HIV(8E5)) strains of HIV. Both X4 and R5 strains transiently decreased CD127 expression on CD8(+) T cells in PBMC cultures but had no effect on isolated CD8(+) T cell cultures. Isolated CD8(+) T cells exposed to either (1) PBMCs incubated with HIV and cultured in a transwell or (2) supernatants from PBMCs incubated with HIV resulted in decreased CD127 expression. Under no conditions did the replication-incompetent HIV strain affect CD127 expression. As observed in vivo, infection of PBMCs with HIV in vitro results in the downregulation of CD127 surface expression on CD8(+) T cells. Collectively, these data indicate that soluble factor(s) released as a result of HIV infection regulate CD127 expression. Further elucidation of the mechanism(s) of CD127 downregulation will provide important insights into the immunopathogenesis of HIV disease.
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Linfocitos T CD8-positivos/inmunología , Seropositividad para VIH/inmunología , VIH-1 , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Células Cultivadas , Regulación hacia Abajo , Humanos , Memoria Inmunológica , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/inmunología , Carga Viral , Replicación ViralRESUMEN
This study tests the hypothesis that IL-7 signaling and activity of CD8(+) T cells are impaired in HIV infection. IL-7 is necessary for optimal CTL activity and T cell survival and proliferation. Defects in IL-7R signaling may contribute to impaired activity of IL-7 observed in progressive HIV disease. A decreased proportion of CD8(+) T cells expressing the IL-7Rα chain (CD127) in progressive HIV disease would be expected to affect IL-7 activity. Alternatively, disease-associated defects of remaining CD8(+)CD127(+) T cells may influence IL-7 responsiveness. Therefore, the IL-7 responsiveness of CD8(+)CD127(+) T cells from HIV(-) and untreated or treated HIV(+) individuals was investigated. Blood was collected from HIV(-) and untreated or effectively treated HIV(+) (<50 viral copies/ml for >1 year) individuals, and CD8(+)CD127(+) T cells were isolated and cultured with IL-7. Indicators of IL-7 signaling (P-STAT5) and activity (Bcl-2 and proliferation) were evaluated by flow cytometry. Isolated CD8(+)CD127(+) T cells from untreated HIV(+) individuals expressed significantly less P-STAT5 in response to IL-7 compared with CD8(+)CD127(+) T cells from HIV(-) individuals. In effectively treated HIV(+) individuals, CD8(+)CD127(+) T cells also expressed significantly lower levels of P-STAT5 compared with HIV(-) individuals. IL-7-dependent proliferation of CD8(+)CD127(+) T cells from untreated HIV(+) individuals was similarly impaired. In contrast, IL-7-induced Bcl-2 expression was not impaired in CD8(+)CD127(+) T cells from HIV(+) individuals. These data demonstrate that IL-7/IL-7R dysfunction in HIV infection may contribute to IL-7-specific signaling defects. Decreased, IL-7-dependent activation of STAT5 and impaired proliferation may negatively impact the maintenance of CD8(+) T cell responsiveness in HIV infection.
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Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/patogenicidad , Interleucina-7/farmacología , Activación de Linfocitos , Factor de Transcripción STAT5/metabolismo , Adulto , Estudios Transversales , Femenino , Citometría de Flujo , Humanos , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Interleucina-7/metabolismo , Transducción de SeñalRESUMEN
IL-7 receptor alpha (CD127) signaling is essential for T-cell development and regulation of naive and memory T-cell homeostasis. Fewer CD8(+) T cells from HIV-infected patients express CD127 compared with healthy individuals, suggesting that specific host and/or viral factors regulate IL-7 receptor expression. Factors relevant to HIV infection that could potentially decrease CD127 expression on human CD8(+) T cells and the mechanisms by which this occurs were therefore evaluated. IL-7, but not HIV gp120, IL-1-beta, IL-6, IL-10, IL-13, transforming growth factor-beta or tumor necrosis factor-alpha, reduced CD127-surface expression and did so without altering CD127 mRNA expression. Furthermore, IL-7 did not increase the amount of cytoplasmic CD127 in CD8(+) T cells. Interestingly, IL-7 induced the shedding of CD127 from CD8(+) T cells, suggesting a mechanism that may contribute to the increased concentration of CD127 in the plasma of HIV(+) individuals, a novel finding reported here. Naive CD8(+) T cells are more sensitive to IL-7 that mediated the down-regulation of CD127, suggesting that these effects may have particular significance early in T-cell life cycle. Since CD127 down-regulation may be an important contributor to HIV-associated T-cell dysfunction, determining the mechanism thereof may prove to be of considerable significance.