RESUMEN
T follicular helper (Tfh) cells are a specialized T cell subset that regulates the long-lived production of highly specific Abs by B cells during the germinal center (GC) reaction. However, the transcriptional network sustaining the Tfh cell phenotype and function is still incompletely understood. In this study, we identify the transcription factor Bach2 as a central negative regulator of Tfh cells. Ectopic overexpression of Bach2 in murine Tfh cells resulted in a rapid loss of their phenotype and subsequent breakdown of the GC response. Low Bach2 expression levels are required to maintain high expression of the signature cytokine IL-21, the coinhibitory receptor TIGIT and the transcriptional repressor Bcl-6. In stark contrast to the regulatory network in GC B cells, Bach2 in Tfh cells is not coexpressed with Bcl-6 at high levels to inhibit the antagonizing factor Blimp-1, but suppresses Bcl-6 by direct binding to the promoter. These data reveal that by replacing an activating complex of Batf and Irf-4 at the Bcl-6 promoter, Bach2 regulates the transcriptional network of Tfh cells in a different way, as in GC B cells.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Regulación de la Expresión Génica/inmunología , Centro Germinal/inmunología , Proteínas Proto-Oncogénicas c-bcl-6 , Linfocitos T Reguladores/inmunología , Transcripción Genética/inmunología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/inmunología , Centro Germinal/citología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/inmunología , Interleucinas/genética , Interleucinas/inmunología , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/inmunología , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/inmunología , Linfocitos T Reguladores/citologíaRESUMEN
Group 2 innate lymphoid cells (ILC2s) are innate effectors playing an important role in the defense against helminthic infections and in the pathogenesis of allergic inflammation. Cytokines have been identified as the major stimuli driving ILC2 activation and expansion. Conversely, it is unclear whether costimulatory molecules contribute to regulation of ILC2 functions. ILC2s display high expression of inducible T-cell costimulator (ICOS), which belongs to the CD28 superfamily, and which has been shown to control late effector T-cell functions, and is of utmost importance for the humoral immune response. However, the biological function of ICOS expression on ILC2s is unknown. Here, we show that ICOS signaling in mice regulates ILC2 homeostasis independently of T cells and B cells, by promoting proliferation and accumulation of mature ILC2s in lung and intestine. In a model of IL-33-induced airway inflammation, ICOS controls ILC2 activation and eosinophil infiltration in the lung. Our data identify a role of ICOS in innate immunity and indicate that not only cytokines, but also costimulatory pathways such as those involving ICOS, can contribute to regulate the ILC2 pool. Thus, ICOS costimulation blockade, which is currently under clinical evaluation for inhibiting the humoral immune response, could also target innate inflammatory circuits.
Asunto(s)
Homeostasis/inmunología , Inmunidad Humoral/fisiología , Inmunidad Innata/fisiología , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Intestinos/inmunología , Pulmón/inmunología , Animales , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Inflamación/genética , Inflamación/inmunología , Interleucina-33/genética , Interleucina-33/inmunología , Intestinos/citología , Pulmón/citología , Ratones , Ratones Mutantes , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Follicular helper T (Tfh) cells play a key role in B cell activation and differentiation. Within recent years, distinct subsets of follicular T cells, including regulatory and cytotoxic T cells, have been identified. Apart from classical Tfh cells in secondary lymphoid organs, Tfh-like cells are found in chronically inflamed nonlymphoid tissues. Here, we provide protocols to identify different follicular T cell subsets in murine and human tissues by flow cytometry. This chapter also contains an immunization protocol for the induction of large numbers of Tfh cells in mice.
Asunto(s)
Citometría de Flujo , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Animales , Biomarcadores/metabolismo , Centro Germinal/citología , Humanos , Inmunización , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Receptores de Antígenos de Linfocitos T/metabolismo , Proyectos de Investigación , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/trasplante , Flujo de TrabajoRESUMEN
Isolation of cells from organs and tissues represents a challenge for many researchers. Cell yield, purity, and cell death are common problems associated with it, which greatly affect experimental results in terms of reproducibility and biological observations. Here, we describe state-of-the-art protocols of how to isolate CD4+ T cells from both human and murine organs and tissues, reducing at minimum cell death, while increasing at the same time cell yield and purity.
Asunto(s)
Separación Celular , Citometría de Flujo , Tejido Linfoide/citología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Biomarcadores/metabolismo , Supervivencia Celular , Enzimas/metabolismo , Humanos , Separación Inmunomagnética , Ratones Endogámicos C57BL , Fenotipo , Proyectos de Investigación , Linfocitos T Colaboradores-Inductores/metabolismo , Flujo de TrabajoRESUMEN
RORγt+ group 3 innate lymphoid cells (ILC3s) maintain intestinal homeostasis through secretion of type 3 cytokines such as interleukin (IL)-17 and IL-22. However, CCR6- ILC3s additionally co-express T-bet allowing for the acquisition of type 1 effector functions. While T-bet controls the type 1 programming of ILC3s, the molecular mechanisms governing T-bet are undefined. Here, we identify c-Maf as a crucial negative regulator of murine T-bet+ CCR6- ILC3s. Phenotypic and transcriptomic profiling of c-Maf-deficient CCR6- ILC3s revealed a hyper type 1 differentiation status, characterized by overexpression of ILC1/NK cell-related genes and downregulation of type 3 signature genes. On the molecular level, c-Maf directly restrained T-bet expression. Conversely, c-Maf expression was dependent on T-bet and regulated by IL-1ß, IL-18 and Notch signals. Thus, we define c-Maf as a crucial cell-intrinsic brake in the type 1 effector acquisition which forms a negative feedback loop with T-bet to preserve the identity of CCR6- ILC3s.
Asunto(s)
Reprogramación Celular/fisiología , Inmunidad Innata , Linfocitos/metabolismo , Proteínas Proto-Oncogénicas c-maf/fisiología , Receptores CCR6/metabolismo , Proteínas de Dominio T Box/fisiología , Animales , Linaje de la Célula , Interleucina-18/fisiología , Interleucina-1beta/fisiología , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Regiones Promotoras Genéticas , Receptores Notch/metabolismo , Transducción de Señal , Proteínas de Dominio T Box/genéticaRESUMEN
The co-stimulators ICOS (inducible T cell co-stimulator) and CD28 are both important for T follicular helper (TFH) cells, yet their individual contributions are unclear. Here, we show that each molecule plays an exclusive role at different stages of TFH cell development. While CD28 regulated early expression of the master transcription factor Bcl-6, ICOS co-stimulation was essential to maintain the phenotype by regulating the novel TFH transcription factor Klf2 via Foxo1. Klf2 directly binds to Cxcr5, Ccr7, Psgl-1, and S1pr1, and low levels of Klf2 were essential to maintain this typical TFH homing receptor pattern. Blocking ICOS resulted in relocation of fully developed TFH cells back to the T cell zone and reversion of their phenotype to non-TFH effector cells, which ultimately resulted in breakdown of the germinal center response. Our study describes for the first time the exclusive role of ICOS and its downstream signaling in the maintenance of TFH cells by controlling their anatomical localization in the B cell follicle.