RESUMO
Cell surface glycans play essential roles in diverse physiological and pathological processes and their assessment has important implications in biomedicine and biotechnology. Here we present a rapid, versatile, and single-step multicolor flow cytometry method for evaluation of cell surface glycan signatures using a panel of selected fluorochrome-conjugated lectins. This procedure allows simultaneous detection of cell surface glycans with a 10-fold reduction in the number of cells required compared with traditional multistep lectin staining methods. Interestingly, we used this one-step lectin array coupled with dimension reduction algorithms in a proof-of-concept application for discrimination among different tumor and immune cell populations. Moreover, this procedure was also able to unveil T-, B-, and myeloid cell subclusters exhibiting differential glycophenotypes. Thus, we report a rapid and versatile lectin cytometry method to simultaneously detect a particular repertoire of surface glycans on living cells that can be easily implemented in different laboratories and core facilities.
Assuntos
Corantes Fluorescentes , Lectinas , Lectinas/metabolismo , Polissacarídeos/metabolismo , Membrana Celular/metabolismoRESUMO
BACKGROUND: In spite of compelling evidence demonstrating safety and immunogenicity of adenoviral-based SARS-CoV-2 vaccines in the general population, its effects in socially vulnerable elderly individuals are poorly understood. Here we aimed to investigate the efficacy of two doses of combined vector vaccine, the Gam-COVID-Vac (Sputnik-V vaccine), at 14, 42, and 180 days after immunization, in a nursing home for underprivileged population and homeless individuals. METHODS: A phase 3, open-label clinical trial involving administration of two adenoviral vectors (Ad26-Ad5) vaccine, in elderly individuals over the ages of 60 years was performed. SARS-CoV-2 Spike RBD-specific IgG antibodies at days 21-, 42- and 180 post-vaccination was analyzed in sera of individuals receiving two doses of the Sputnik-V vaccine with an interval of 21 days. SARS-CoV-2-specific CD8+ T cell responses, measured by intracellular tumor necrosis factor (TNF) was determined by flow cytometry following antigen-specific cultures. RESULTS: A total of 72 elderly adults with a mean age of 72.6 ± 9.5 years-old was selected after applying the inclusion criteria, all corresponding to an underprivileged population. Two-doses vaccination with Sputnik-V vaccine elicited an antibody-mediated immune response (revealed by quantitative detection of SARS-CoV-2-specific IgG antibodies, CMIA) 70% at day 21, 90% at day 42, and 66.1% at day 180. Fully vaccinated individuals had robust SARS-CoV-2-specific T cell responses, evidenced by TNF production in CD4+ and CD8+ T cells in all time periods analyzed. CONCLUSION: Six months after receipt of the second dose of the Gam-COVID-Vac vaccine, SARS-CoV-2-specific IgG levels declined substantially among the tested population, whereas CD4+ and CD8+ T-cell-mediated immunity remained at high levels. These data suggest that two doses of combined adenoviral-based vaccine elicits a considerable level of SARS-CoV-2 immune responses in elderly individuals, highlighting its safety and immunogenicity in this highly vulnerable population.
RESUMO
The rapid spread of the SARS-CoV-2, the causative agent of the emergent pandemic disease COVID-19, requires the urgent commitment of the immunology community to understand the adaptive immune response developed by COVID-19 convalescent patients and individuals vaccinated with different strategies and schemes, with the ultimate goal of implementing and optimizing health care and prevention policies. Currently, assessment of SARS-CoV-2-specific immunity is mainly focused on the measurement of the antibody titers and analysis of their neutralizing capacity. However, a considerable proportion of individuals lack humoral responses or show a progressive decline of SARS-CoV-2-specific neutralizing antibodies. In order to study the cellular response of convalescent patients and vaccinated individuals, we have developed the "COVID-T Platform", an optimized strategy to study SARS-CoV-2-specific T cell responses. This platform allows assessment of the nature, magnitude and persistence of antigen-specific T-cell immunity in COVID-19-convalescent patients and vaccinated individuals. Moreover, it gives the opportunity to study cellular responses against emerging coronavirus variants and to identify individuals with cross-reactive immunity against seasonal coronaviruses.
La rápida propagación del coronavirus SARS-CoV-2, agente causal de la enfermedad pandémica emergente COVID-19 y sus nuevas variantes, requiere del compromiso de la comunidad inmunológica para comprender la magnitud y naturaleza de la respuesta inmunológica adaptativa desarrollada por pacientes recuperados de COVID-19 e individuos vacunados con diferentes estrategias y protocolos, a los fines de implementar nuevas políticas sanitarias. En la actualidad, la determinación de la inmunidad contra SARS-CoV-2 se basa principalmente en la detección de anticuerpos específicos y la determinación de su actividad neutralizante. Sin embargo, a pesar de la alta sensibilidad de estos ensayos, un número considerable de pacientes e individuos vacunados carecen de respuesta humoral detectable, o evidencian una disminución rápida de la misma en el tiempo. Con el objetivo de estudiar la respuesta inmune celular desencadenada frente a SARS-CoV-2, en nuestro laboratorio desarrollamos la "Plataforma COVID-T" estrategia integral optimizada dirigida a caracterizar y monitorear la respuesta de linfocitos T específicos de SARS-CoV-2 a partir de muestras de sangre de individuos vacunados y/o recuperados de COVID-19. Esta plataforma permite evaluar la naturaleza, magnitud y persistencia de la inmunidad celular T generada tanto por la infección con SARS-CoV-2, como por distintos esquemas y protocolos de vacunación en diferentes poblaciones de individuos. Asimismo, permite evaluar la respuesta inmunológica T generada frente a nuevas variantes del virus e identificar individuos sanos resistentes a SARS-CoV-2 con inmunidad pre-existente hacia coronavirus estacionales.
Assuntos
COVID-19 , SARS-CoV-2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Linfócitos TRESUMO
Resumen La rápida propagación del coronavirus SARS-CoV-2, agente causal de la enfermedad pandémica emergente COVID-19 y sus nuevas variantes, requiere del compromiso de la comunidad inmunológica para comprender la magnitud y naturaleza de la respuesta inmunológica adaptativa desarrollada por pacientes recuperados de COVID-19 e individuos vacunados con diferentes estrategias y protocolos, a los fines de imple mentar nuevas políticas sanitarias. En la actualidad, la determinación de la inmunidad contra SARS-CoV-2 se basa principalmente en la detección de anticuerpos específicos y la determinación de su actividad neutralizante. Sin embargo, a pesar de la alta sensibilidad de estos ensayos, un número considerable de pacientes e indivi duos vacunados carecen de respuesta humoral detectable, o evidencian una disminución rápida de la misma en el tiempo. Con el objetivo de estudiar la respuesta inmune celular desencadenada frente a SARS-CoV-2, en nuestro laboratorio desarrollamos la "Plataforma COVID-T" estrategia integral optimizada dirigida a caracte rizar y monitorear la respuesta de linfocitos T específicos de SARS-CoV-2 a partir de muestras de sangre de individuos vacunados y/o recuperados de COVID-19. Esta plataforma permite evaluar la naturaleza, magnitud y persistencia de la inmunidad celular T generada tanto por la infección con SARS-CoV-2, como por distintos esquemas y protocolos de vacunación en diferentes poblaciones de individuos. Asimismo, permite evaluar la respuesta inmunológica T generada frente a nuevas variantes del virus e identificar individuos sanos resistentes a SARS-CoV-2 con inmunidad pre-existente hacia coronavirus estacionales.
Abstract The rapid spread of the SARS-CoV-2, the caus ative agent of the emergent pandemic disease COVID-19, requires the urgent commitment of the immunology community to understand the adaptive immune response developed by COVID-19 convalescent patients and individuals vaccinated with different strategies and schemes, with the ultimate goal of implementing and optimizing health care and prevention policies. Currently, assessment of SARS-CoV-2-specific immunity is mainly focused on the measurement of the antibody titers and analysis of their neutralizing capacity. However, a considerable proportion of individuals lack humoral responses or show a progressive decline of SARS-CoV-2-specific neutral izing antibodies. In order to study the cellular response of convalescent patients and vaccinated individuals, we have developed the 'COVID-T Platform', an optimized strategy to study SARS-CoV-2-specific T cell responses. This platform allows assessment of the nature, magnitude and persistence of antigen-specific T-cell immunity in COVID-19-convalescent patients and vaccinated individuals. Moreover, it gives the opportunity to study cellular responses against emerging coronavirus variants and to identify individuals with cross-reactive immunity against seasonal coronaviruses.
Assuntos
Humanos , SARS-CoV-2 , COVID-19 , Linfócitos T , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
Diverse immunoregulatory circuits operate to preserve intestinal homeostasis and prevent inflammation. Galectin-1 (Gal1), a ß-galactoside-binding protein, promotes homeostasis by reprogramming innate and adaptive immunity. Here, we identify a glycosylation-dependent "on-off" circuit driven by Gal1 and its glycosylated ligands that controls intestinal immunopathology by targeting activated CD8+ T cells and shaping the cytokine profile. In patients with inflammatory bowel disease (IBD), augmented Gal1 was associated with dysregulated expression of core 2 ß6-N-acetylglucosaminyltransferase 1 (C2GNT1) and α(2,6)-sialyltransferase 1 (ST6GAL1), glycosyltransferases responsible for creating or masking Gal1 ligands. Mice lacking Gal1 exhibited exacerbated colitis and augmented mucosal CD8+ T cell activation in response to 2,4,6-trinitrobenzenesulfonic acid; this phenotype was partially ameliorated by treatment with recombinant Gal1. While C2gnt1-/- mice exhibited aggravated colitis, St6gal1-/- mice showed attenuated inflammation. These effects were associated with intrinsic T cell glycosylation. Thus, Gal1 and its glycosylated ligands act to preserve intestinal homeostasis by recalibrating T cell immunity.
RESUMO
Type-2 diabetes mellitus (T2DM) is an expanding global health problem, involving defective insulin secretion by pancreatic ß-cells and peripheral insulin resistance, leading to impaired glucose regulation. Galectin-1-an endogenous lectin with affinity for N-acetyllactosamine (LacNAc)-containing glycans-has emerged as a regulator of inflammatory and metabolic disorders. However, the role of galectin-1 in glucose homeostasis and pancreatic ß-cell function, independently of hypercaloric diets, has not been explored. Here, we identified a phenotype compatible with T2DM, involving alterations in glucose metabolism and pancreatic insulin release, in female but not male mice lacking galectin-1 (Lgals1-/-). Compared with age-matched controls, Lgals1-/- female mice exhibited higher body weight and increased food intake ad libitum as well as after fasting and acute re-feeding. Although fasted serum insulin levels and insulin sensitivity were similar in both genotypes, Lgals1-/- female mice presented altered glucose tolerance and higher basal glucose levels depending on the fasting period. Insulin response to glucose overload was impaired, while pancreatic insulin content was enhanced in the absence of galectin-1. Accordingly, recombinant galectin-1 enhanced glucose-stimulated insulin release in vitro. Our study identifies a role for galectin-1 in regulating glucose metabolism through modulation of pancreatic insulin secretion, highlighting novel opportunities to control T2DM.
Assuntos
Resistência à Insulina , Insulina , Animais , Feminino , Galectina 1/genética , Galectina 1/metabolismo , Glucose/metabolismo , Homeostase , Insulina/metabolismo , Secreção de Insulina , Masculino , CamundongosRESUMO
The relevance of glycan-binding proteins in immune tolerance and inflammation has been well established, mainly by studies of C-type lectins, siglecs and galectins, both in experimental models and patient samples. Galectins, a family of evolutionarily conserved lectins, are characterized by sequence homology in the carbohydrate-recognition domain, atypical secretion via an endoplasmic reticulum-Golgi-independent pathway and by the ability to recognize ß-galactoside-containing saccharides. Galectin-1 (Gal-1), a prototype member of this family, displays mainly anti-inflammatory and immunosuppressive activities, although, similar to many cytokines and growth factors, it may also trigger paradoxical pro-inflammatory effects under certain circumstances. These dual effects could be associated to tissue-, time- or context-dependent regulation of galectin expression and function, including particular pathophysiologic settings and/or environmental conditions influencing the structure of this lectin, as well as the availability of glycosylated ligands in immune cells during the course of inflammatory responses. Here, we discuss the tissue-specific role of Gal-1 as a master regulator of inflammatory responses across different pathophysiologic settings, highlighting its potential role as a therapeutic target. Further studies designed at analyzing the intrinsic and extrinsic pathways that control Gal-1 expression and function in different tissue microenvironments may contribute to delineate tailored therapeutic strategies aimed at positively or negatively modulating this glycan-binding protein in pathologic inflammatory conditions.
Assuntos
Galectina 1 , Galectinas , Carboidratos , Galectina 1/genética , Galectinas/metabolismo , Humanos , Inflamação/metabolismo , Polissacarídeos/metabolismoRESUMO
Aging elicits quantitative and qualitative changes in different immune components, leading to disruption of tolerogenic circuits and development of autoimmune disorders. Galectin-1 (Gal1), an endogenous glycan-binding protein, has emerged as a regulator of immune cell homeostasis by shaping the fate of myeloid and lymphoid cells. Here, we demonstrate that aged Gal1-null mutant (Lgals1-/- ) mice develop a spontaneous inflammatory process in salivary glands that resembles Sjögren's syndrome. This spontaneous autoimmune phenotype was recapitulated in mice lacking ß1,6N-acetylglucosaminyltransferase V (Mgat5), an enzyme responsible for generating ß1,6-branched complex N-glycans, which serve as a major ligand for this lectin. Lack of Gal1 resulted in CD11c+ dendritic cells (DCs) with higher immunogenic potential, lower frequency of Foxp3+ regulatory T cells (Tregs), and increased number of CD8+ T cells with greater effector capacity. Supporting its tolerogenic activity, Gal1 expression decreased with age in autoimmunity-prone nonobese diabetic (NOD) mice. Treatment with recombinant Gal1 restored tolerogenic mechanisms and reduced salivary gland inflammation. Accordingly, labial biopsies from primary Sjögren's syndrome patients showed reduced Gal1 expression concomitant with higher number of infiltrating CD8+ T cells. Thus, endogenous Gal1 serves as a homeostatic rheostat that safeguards immune tolerance and prevents age-dependent development of spontaneous autoimmunity.
