RESUMEN
How different classes of the B cell antigen receptor (BCR) sense viral antigens used in vaccination protocols is poorly understood. Here, we study antigen binding and sensing of human Ramos B cells expressing a BCR of either the IgM or IgG1 class with specificity for the CD4-binding-site of the envelope (Env) protein of the HIV-1. Both BCRs carry an identical antigen binding site derived from the broad neutralizing antibody (bnAb) CH31. We find a five times higher expression of the IgG1-BCR in comparison to the IgM-BCR on the surface of transfected Ramos B cells. The two BCR classes also differ from each other in their interaction with cognate HIV Env antigens in that the IgG1-BCR and IgM-BCR bind preferentially to polyvalent and monovalent antigens, respectively. By generating an IgM/IgG1 chimeric BCR, we found that the class-specific BCR expression and antigen-sensing behavior can be transferred with the CH1γ domain from the IgG1-BCR to the IgM-BCR. Thus, the class of CH1 domain has an impact on BCR assembly and expression as well as on antigen sensing.
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VIH-1 , Inmunoglobulina G , Inmunoglobulina M , Receptores de Antígenos de Linfocitos B , Humanos , Inmunoglobulina M/inmunología , Inmunoglobulina G/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , VIH-1/inmunología , VIH-1/genética , Linfocitos B/inmunología , Linfocitos B/metabolismo , Anticuerpos Anti-VIH/inmunología , Dominios Proteicos , Anticuerpos Neutralizantes/inmunologíaRESUMEN
HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.
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VIH-1 , Anticuerpos Neutralizantes , Antígenos Virales , Anticuerpos Anti-VIH , Inmunoglobulina M , Receptores de Antígenos de Linfocitos B/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia HumanaRESUMEN
The SARS-CoV-2 pandemic has spread to all parts of the world and can cause life-threatening pneumonia and other severe disease manifestations known as COVID-19. This health crisis has resulted in a significant effort to stop the spread of this new coronavirus. However, while propagating itself in the human population, the virus accumulates mutations and generates new variants with increased fitness and the ability to escape the human immune response. Here we describe a color-based barcoded spike flow cytometric assay (BSFA) that is particularly useful to evaluate and directly compare the humoral immune response directed against either wild type (WT) or mutant spike (S) proteins or the receptor-binding domains (RBD) of SARS-CoV-2. This assay employs the human B lymphoma cell line Ramos, transfected for stable expression of WT or mutant S proteins or a chimeric RBD-CD8 fusion protein. We find that the alpha and beta mutants are more stably expressed than the WT S protein on the Ramos B cell surface and/or bind with higher affinity to the viral entry receptor ACE2. However, we find a reduce expression of the chimeric RBD-CD8 carrying the point mutation N501Y and E484K characteristic for the alpha and beta variant, respectively. The comparison of the humoral immune response of 12 vaccinated probands with 12 COVID-19 patients shows that after the boost, the S-specific IgG class immune response in the vaccinated group is similar to that of the patient group. However, in comparison to WT the specific IgG serum antibodies bind less well to the alpha variant and only poorly to the beta variant S protein. This is in line with the notion that the beta variant is an immune escape variant of SARS-CoV-2. The IgA class immune response was more variable than the IgG response and higher in the COVID-19 patients than in the vaccinated group. In summary, we think that our BSFA represents a useful tool to evaluate the humoral immunity against emerging variants of SARS-CoV-2 and to analyze new vaccination protocols against these variants.
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COVID-19/inmunología , Separación Celular/métodos , Citometría de Flujo/métodos , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Anticuerpos Antivirales/metabolismo , Formación de Anticuerpos , Femenino , Humanos , Inmunización Secundaria , Inmunoglobulina A/metabolismo , Inmunoglobulina G/metabolismo , Masculino , Persona de Mediana Edad , Mutación/genética , Glicoproteína de la Espiga del Coronavirus/genética , VacunaciónRESUMEN
Multiple sclerosis is an autoimmune disease that is caused by the interplay of genetic, particularly the HLA-DR15 haplotype, and environmental risk factors. How these etiologic factors contribute to generating an autoreactive CD4+ T cell repertoire is not clear. Here, we demonstrate that self-reactivity, defined as "autoproliferation" of peripheral Th1 cells, is elevated in patients carrying the HLA-DR15 haplotype. Autoproliferation is mediated by memory B cells in a HLA-DR-dependent manner. Depletion of B cells in vitro and therapeutically in vivo by anti-CD20 effectively reduces T cell autoproliferation. T cell receptor deep sequencing showed that in vitro autoproliferating T cells are enriched for brain-homing T cells. Using an unbiased epitope discovery approach, we identified RASGRP2 as target autoantigen that is expressed in the brain and B cells. These findings will be instrumental to address important questions regarding pathogenic B-T cell interactions in multiple sclerosis and possibly also to develop novel therapies.
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Linfocitos B/patología , Subtipos Serológicos HLA-DR/inmunología , Esclerosis Múltiple/inmunología , Autoantígenos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/fisiopatología , Linfocitos B/metabolismo , Encéfalo/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Subtipos Serológicos HLA-DR/genética , Humanos , Esclerosis Múltiple/genética , Esclerosis Múltiple/fisiopatología , Receptores de Antígenos de Linfocitos T , Células TH1/fisiologíaRESUMEN
OBJECTIVE: Multiple sclerosis (MS) is a disease of the central nervous system with marked heterogeneity in several aspects including pathological processes. Based on infiltrating immune cells, deposition of humoral factors and loss of oligodendrocytes and/or myelin proteins, four lesion patterns have been described. Pattern II is characterized by antibody and complement deposition in addition to T-cell infiltration. MS is considered a T-cell-mediated disease, but until now the study of pathogenic T cells has encountered major challenges, most importantly the limited access of brain-infiltrating T cells. Our objective was to identify, isolate, and characterize brain-infiltrating clonally expanded T cells in pattern II MS lesions. METHODS: We used next-generation sequencing to identify clonally expanded T cells in demyelinating pattern II brain autopsy lesions, subsequently isolated these as T-cell clones from autologous cerebrospinal fluid and functionally characterized them. RESULTS: We identified clonally expanded CD8(+) but also CD4(+) T cells in demyelinating pattern II lesions and for the first time were able to isolate these as live T-cell clones. The functional characterization shows that T cells releasing Th2 cytokines and able to provide B cell help dominate the T-cell infiltrate in pattern II brain lesions. INTERPRETATION: Our data provide the first functional evidence for a putative role of Th2/Tc2 cells in pattern II MS supporting the existence of this pathogenic phenotype and questioning the protective role that is generally ascribed to Th2 cells. Our observations are important to consider for future treatments of pattern II MS patients.
RESUMEN
Allospecific memory T cells are a barrier against long-term graft survival. Production of multiple cytokines by a single T cell is considered a sign of an active ongoing immune response, the presence of these polyfunctional cells has not been addressed in transplanted patients accordingly to graft outcome. Memory phenotype, based on the expression of CD45RO and CD27, and polyfunctional T cells were evaluated in long-term graft survival patients (LTS), short-term survival patients (STS), chronic rejection patients (ChrRx), dialysis patients (DIAL) and healthy controls (Ctrls). Memory T cells were quantified ex vivo, after allogeneic and anti-CD3 plus anti-CD28 stimulation, in cells proliferating or not to these stimuli. The percentages of cells producing IFNγ, IL-2 and/or TNFα after allogeneic stimulation and the memory phenotype of single cytokine producing cells were evaluated. Ex vivo CD8+CD45RO-CD27- effector cells were decreased in transplanted patients compared to non-transplanted individuals. After allogeneic stimulation, CD4+CD45RO+CD27+, central memory cells in LTS and CD4+CD45RO-CD27- effector cells in Dial were augmented compared to Ctrls and ChrRx, and CD8+CD45RO-CD27- effector cells were increased in ChrRx. There were no differences in the percentage of single cytokine producing cells among the groups. IFNγ+TNFα+CD4 and CD8 cells were detected in Ctrls, STS and ChrRx and no cells positive for the three cytokines were found. The phenotype of cytokine producing cells was mainly effector memory. Interestingly, in LTS there was an increase in effector cells producing IFNγ and IL-2. Changes in subpopulation distribution in patients with different outcomes may be a reflection of the graft acceptance or rejection status.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/inmunología , Trasplante de Riñón , Adulto , Anciano , Femenino , Supervivencia de Injerto , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Adulto JovenRESUMEN
Transplant patients with long-term graft survival (LTS) may have developed mechanisms that prevent rejection and allow graft function under low or no immunosuppressive therapy. In murine models, T cell tolerance is associated with alterations in the expression/activation of proteins involved in T cell signaling. These alterations have not been reported in transplanted patients with different outcomes. This study aimed to evaluate calcium mobilization, the phosphorylation of different proteins involved in T cell signaling and the expression of molecules associated with anergy, in T cells from kidney transplant patients. No differences were observed in calcium mobilization, although transplanted patients had a tendency toward augmented calcium flux. Chronic rejection patients (ChrRx) displayed lower Lck basal phosphorylation levels compared with LTS patients, and the phosphorylation profile of proteins evaluated was different. Among the groups, phosphorylation of Zap-70 was higher in LTS patients compared with ChrRx, and LAT phosphorylation was lower in LTS and ChrRx patients compared with healthy controls. The expression of molecules related to the anergic phenotype was similar among the study groups. Results suggest that phosphorylation patterns, rather than phosphorylation levels, may correlate with transplant outcome and that anergy may not be the main mechanism mediating LTS.
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Enfermedades Renales/cirugía , Trasplante de Riñón/mortalidad , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Adulto , Western Blotting , Calcio/metabolismo , Estudios de Casos y Controles , Anergia Clonal , Femenino , Estudios de Seguimiento , Supervivencia de Injerto , Humanos , Tolerancia Inmunológica , Masculino , Persona de Mediana Edad , Fosforilación , Pronóstico , Transducción de Señal , Tasa de Supervivencia , Adulto JovenRESUMEN
El informe contiene: INTRODUCCIÓN, CAPÍTULO I: OBJETIVO ESPECÍFICO 1: DIAGNÓSTICO DE LA SITUACIÓN EN SALUD; CAPÍTULO II: OBJETIVO ESPECÍFICO 2: PARTICIPACIÓN ACTIVA DE ORGANIZACIONES; CAPÍTULO III: OBJETIVO ESPECÍFICO 3: MECANISMOS DE ATENCIÓN EN SALUD; CAPÍTULO IV: OBJETIVO ESPECÍFICO 4 Y 5: CAPACITACIÓN EN FORMULACIÓN DE PROYECTOS Y GESTIÓN SANITARIA; CAPÍTULO V CONCLUSIONES: LOGROS Y LIMITACIONES; ANEXOS.
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Atención Integral de Salud , Gestión en Salud , Integración de Sistemas , Planes Regionales AmbientalesRESUMEN
El contar con un area geograficamente definida y una poblacion determinada es un elemento importante para medir el buen funcionamiento de un servicio de salud de primer nivel. La APS continua siendo estrategia valida para brindar una atencion efectiva en un primer nievel de atencion. La calidad de atencion dependera de la organizacion de los servicios de salud, la capacitacion tecnica de los recursos humanos y la actitud del personal de salud hacia la comunidad. La participacion comunitaria se constituye en una linea estrategica importante para lograr un funcionamiento optimo. La articulacion de los servicios depende de la capacidad del equipo de distrito y del apoyo tecnico y administrativo que pueda otorgar el equipo a los servicios de salud...