RESUMEN
Research in vitro facilitates discovery, screening, and pilot experiments, often preceding research in vivo. Several technical difficulties render Dendritic Cell (DC) research particularly challenging, including the low frequency of DC in vivo, thorough isolation requirements, and the vulnerability of DC ex vivo. Critically, there is not as yet a widely accepted human or murine DC line and in vitro systems of DC research are limited. In this study, we report the generation of new murine DC lines, named MutuDC, originating from cultures of splenic CD8α conventional DC (cDC) tumors. By direct comparison to normal WT splenic cDC subsets, we describe the phenotypic and functional features of the MutuDC lines and show that they have retained all the major features of their natural counterpart in vivo, the splenic CD8α cDC. These features include expression of surface markers Clec9A, DEC205, and CD24, positive response to TLR3 and TLR9 but not TLR7 stimuli, secretion of cytokines, and chemokines upon activation, as well as cross-presentation capacity. In addition to the close resemblance to normal splenic CD8α cDC, a major advantage is the ease of derivation and maintenance of the MutuDC lines, using standard culture medium and conditions, importantly without adding supplementary growth factors or maturation-inducing stimuli to the medium. Furthermore, genetically modified MutuDC lines have been successfully obtained either by lentiviral transduction or by culture of DC tumors originating from genetically modified mice. In view of the current lack of stable and functional DC lines, these novel murine DC lines have the potential to serve as an important auxiliary tool for DC research.
RESUMEN
Plasma cells represent the end stage of B-cell development and play a key role in providing an efficient antibody response, but they are also involved in numerous pathologies. Here we show that CD93, a receptor expressed during early B-cell development, is reinduced during plasma-cell differentiation. High CD93/CD138 expression was restricted to antibody-secreting cells both in T-dependent and T-independent responses as naive, memory, and germinal-center B cells remained CD93-negative. CD93 was expressed on (pre)plasmablasts/plasma cells, including long-lived plasma cells that showed decreased cell cycle activity, high levels of isotype-switched Ig secretion, and modification of the transcriptional network. T-independent and T-dependent stimuli led to re-expression of CD93 via 2 pathways, either before or after CD138 or Blimp-1 expression. Strikingly, while humoral immune responses initially proceeded normally, CD93-deficient mice were unable to maintain antibody secretion and bone-marrow plasma-cell numbers, demonstrating that CD93 is important for the maintenance of plasma cells in bone marrow niches.
Asunto(s)
Formación de Anticuerpos/inmunología , Médula Ósea/inmunología , Glicoproteínas de Membrana/inmunología , Células Plasmáticas/inmunología , Receptores de Complemento/inmunología , Animales , Diferenciación Celular , Pollos , Factor de Transcripción Ikaros , Inmunización , Virus del Tumor Mamario del Ratón/inmunología , Ratones , Células Plasmáticas/citología , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Infecciones por Retroviridae/inmunología , Sindecano-1/inmunología , Linfocitos T/inmunología , Transactivadores/inmunología , Factores de Transcripción/inmunología , gammaglobulinas/inmunologíaRESUMEN
Division and proliferation of dendritic cells (DCs) have been proposed to contribute to homeostasis and to prolonged antigen presentation. Whether abnormal proliferation of dendritic cells causes Langerhans cell histiocytosis (LCH) is a highly debated topic. Transgenic expression of simian virus 40 (SV40) T antigens in mature DCs allowed their transformation in vivo while maintaining their phenotype, function, and maturation capacity. The transformed cells were differentiated splenic CD8 alpha-positive conventional dendritic cells with increased Langerin expression. Their selective transformation was correlated with higher steady-state cycling compared with CD8 alpha-negative DCs in wild-type and transgenic mice. Mice developed a DC disease involving the spleen, liver, bone marrow, thymus, and mesenteric lymph node. Surprisingly, lesions displayed key immunohistologic features of Langerhans cell histiocytosis, including expression of Langerin and absence of the abnormal mitoses observed in Langerhans cell sarcomas. Our results demonstrate that a transgenic mouse model with striking similarities to aggressive forms of multisystem histiocytosis, such as the Letterer-Siwe syndrome, can be obtained by transformation of conventional DCs. These findings suggest that conventional DCs may cause some human multisystem LCH. They can reveal shared molecular pathways for human histiocytosis between humans and mice.
Asunto(s)
Antígenos CD8/inmunología , Células Dendríticas/inmunología , Histiocitosis de Células de Langerhans/inmunología , Activación de Linfocitos/inmunología , Animales , Antígeno CD11c/genética , Cartilla de ADN , Marcadores Genéticos , Proteínas Fluorescentes Verdes/genética , Ratones , Ratones Endogámicos , Ratones Transgénicos , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Development of Peyer's patches and lymph nodes requires the interaction between CD4+ CD3- IL-7Ralpha+ lymphoid-tissue inducer (LTi) and VCAM-1+ organizer cells. Here we showed that by promoting their survival, enhanced expression of interleukin-7 (IL-7) in transgenic mice resulted in accumulation of LTi cells. With increased IL-7 availability, de novo formation of VCAM-1+ Peyer's patch anlagen occurred along the entire fetal gut resulting in a 5-fold increase in Peyer's patch numbers. IL-7 overexpression also led to formation of multiple organized ectopic lymph nodes and cecal patches. After immunization, ectopic lymph nodes developed normal T cell-dependent B cell responses and germinal centers. Mice overexpressing IL-7 but lacking either RORgamma, a factor required for LTi cell generation, or lymphotoxin alpha1beta2 had neither Peyer's patches nor ectopic lymph nodes. Therefore, by controlling LTi cell numbers, IL-7 can regulate the formation of both normal and ectopic lymphoid organs.
Asunto(s)
Diferenciación Celular/inmunología , Interleucina-7/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Supervivencia Celular , Células Cultivadas , Feto/metabolismo , Regulación de la Expresión Génica , Interleucina-7/genética , Tejido Linfoide/inmunología , Ratones , Ratones Transgénicos , Células Madre/citología , Células Madre/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Molécula 1 de Adhesión Celular Vascular/metabolismoAsunto(s)
Genes MHC Clase II , Antígenos HLA-D/genética , Mutagénesis Insercional , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Transactivadores/deficiencia , Transactivadores/genética , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular Tumoral , Clonación Molecular , Prueba de Complementación Genética , Antígenos HLA-D/biosíntesis , Historia del Siglo XX , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/biosíntesis , Empalme del ARN , Transactivadores/biosíntesisRESUMEN
CIITA is a master regulatory factor for the expression of MHC class II (MHC-II) and accessory genes involved in Ag presentation. It has recently been suggested that CIITA also regulates numerous other genes having diverse functions within and outside the immune system. To determine whether these genes are indeed relevant targets of CIITA in vivo, we studied their expression in CIITA-transgenic and CIITA-deficient mice. In contrast to the decisive control of MHC-II and related genes by CIITA, nine putative non-MHC target genes (Eif3s2, Kpna6, Tap1, Yars, Col1a2, Ctse, Ptprr, Tnfsf6 and Plxna1) were found to be CIITA independent in all cell types examined. Two other target genes, encoding IL-4 and IFN-gamma, were indeed found to be up- and down-regulated, respectively, in CIITA-transgenic CD4(+) T cells. However, there was no correlation between MHC-II expression and this Th2 bias at the level of individual transgenic T cells, indicating an indirect control by CIITA. These results show that MHC-II-restricted Ag presentation, and its indirect influences on T cells, remains the only pathway under direct control by CIITA in vivo. They also imply that precisely regulated MHC-II expression is essential for maintaining a proper Th1-Th2 balance.
Asunto(s)
Antígenos de Histocompatibilidad Clase II/genética , Proteínas Nucleares/genética , Transactivadores/genética , Animales , Femenino , Citometría de Flujo , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/inmunología , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-4/biosíntesis , Interleucina-4/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Especificidad por Sustrato , Células TH1/inmunología , Células Th2/inmunología , Transactivadores/biosíntesis , Transactivadores/inmunologíaRESUMEN
The class II transactivator (CIITA) has been referred to as the "master control factor" for the expression of MHC class II (MHCII) genes. As our knowledge on the specificity and function of CIITA grows, it is becoming increasingly evident that this sobriquet is entirely justified. First, despite extensive investigations, the major target genes of CIITA remain those implicated in the presentation of antigenic peptides by MHCII molecules. Although other putative target genes have been reported, the contribution of CIITA to their expression remains indirect, controversial or comparatively minor relative to its decisive role as a regulator of MHCII and related genes. Second, the most important parameter dictating MHCII expression is by far the expression pattern of the gene encoding CIITA (MHC2TA). The vast majority of signals that activate or repress MHCII expression under physiological and pathological situations converge on one or more of the three alternative promoters that drive transcription of the MHC2TA gene. In short, with respect to its specificity and its exquisitely controlled pattern of expression, CIITA is by a long stretch the single most important transcription factor for the regulation of genes required for MHCII-restricted antigen-presentation.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Proteínas Nucleares/inmunología , Transactivadores/inmunología , Animales , Presentación de Antígeno/inmunología , Genes MHC Clase II/inmunología , Humanos , Ratones , Ratones Noqueados , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Transactivadores/biosíntesis , Transactivadores/genética , Factores de Transcripción/inmunología , Activación Transcripcional/inmunologíaRESUMEN
Notch1 proteins are involved in binary cell fate decisions. To determine the role of Notch1 in the differentiation of CD4(+) Th1 versus Th2 cells, we have compared T helper polarization in vitro in naive CD4(+) T cells isolated from mice in which the N1 gene is specifically inactivated in all mature T cells. Following activation, Notch1-deficient CD4(+) T cells transcribed and secreted IFN-gamma under Th1 conditions and IL-4 under Th2 conditions at levels similar to that of control CD4(+) T cells. These results show that Notch1 is dispensable for the development of Th1 and Th2 phenotypes in vitro. The requirement for Notch1 in Th1 differentiation in vivo was analyzed following inoculation of Leishmania major in mice with a T cell-specific inactivation of the Notch1 gene. Following infection, these mice controlled parasite growth at the site of infection and healed their lesions. The mice developed a protective Th1 immune response characterized by high levels of IFN-gamma mRNA and protein and low levels of IL-4 mRNA with no IL-4 protein in their lymph node cells. Taken together, these results indicate that Notch1 is not critically involved in CD4(+) T helper 1 differentiation and in resolution of lesions following infection with L. major.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/inmunología , Receptores de Superficie Celular/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Transcripción , Animales , Anticuerpos Antiprotozoarios/sangre , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Femenino , Interferón gamma/genética , Interferón gamma/inmunología , Interleucina-4/genética , Interleucina-4/inmunología , Leishmania major/inmunología , Leishmaniasis/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN/química , ARN/genética , Receptor Notch1 , Receptores de Superficie Celular/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Organismos Libres de Patógenos Específicos , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
Activation of dendritic cells (DC) by microbial products via Toll-like receptors (TLR) is instrumental in the induction of immunity. In particular, TLR signaling plays a major role in the instruction of Th1 responses. The development of Th2 responses has been proposed to be independent of the adapter molecule myeloid differentiation factor 88 (MyD88) involved in signal transduction by TLRs. In this study we show that flagellin, the bacterial stimulus for TLR5, drives MyD88-dependent Th2-type immunity in mice. Flagellin promotes the secretion of IL-4 and IL-13 by Ag-specific CD4(+) T cells as well as IgG1 responses. The Th2-biased responses are associated with the maturation of DCs, which are shown to express TLR5. Flagellin-mediated DC activation requires MyD88 and induces NF-kappaB-dependent transcription and the production of low levels of proinflammatory cytokines. In addition, the flagellin-specific response is characterized by the lack of secretion of the Th1-promoting cytokine IL-12 p70. In conclusion, this study suggests that flagellin and, more generally, TLR ligands can control Th2 responses in a MyD88-dependent manner.
Asunto(s)
Antígenos de Diferenciación/fisiología , Flagelina/farmacología , Receptores Inmunológicos/fisiología , Células Th2/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/fisiología , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos , Factor 88 de Diferenciación Mieloide , Ovalbúmina/inmunología , Subunidades de Proteína/biosíntesis , Receptores de Superficie Celular/fisiología , Receptor Toll-Like 5 , Receptores Toll-LikeRESUMEN
The MHC class II (MHC-II) transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC-II-restricted Ag presentation. Fine tuning of CIITA gene expression determines the cell type-specific expression of MHC-II genes. This regulation is achieved by the selective usage of multiple CIITA promoters. It has recently been suggested that CIITA also contributes to Th cell differentiation by suppressing IL-4 expression in Th1 cells. In this study, we show that endogenous CIITA is expressed at low levels in activated mouse T cells. Importantly CIITA is not regulated differentially in murine and human Th1 and Th2 cells. Ectopic expression of a CIITA transgene in multiple mouse cell types including T cells, does not interfere with normal development of CD4(+) T cells. However, upon TCR activation the CIITA transgenic CD4(+) T cells preferentially differentiate into IL-4-secreting Th2-type cells. These results imply that CIITA is not a direct Th1-specific repressor of the IL-4 gene and that tight control over the expression of CIITA and MHC-II is required to maintain the normal balance between Th1 and Th2 responses.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Regulación de la Expresión Génica/inmunología , Genes MHC Clase II/genética , Proteínas Nucleares , Células Th2/inmunología , Células Th2/metabolismo , Transactivadores/biosíntesis , Transactivadores/deficiencia , Adulto , Animales , Linfocitos T CD4-Positivos/citología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citocinas/biosíntesis , Humanos , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Especificidad de la Especie , Células TH1/inmunología , Células TH1/metabolismo , Transactivadores/genéticaRESUMEN
Six monogenic forms of maturity-onset diabetes of the young (MODY) have been identified to date. Except for MODY2 (glucokinase), all other MODY subtypes have been linked to transcription factors. We have established a MODY3 transgenic model through the beta-cell-targeted expression of dominant-negative HNF-1alpha either constitutively (rat insulin II promoter) or conditionally (Tet-On system). The animals display either overt diabetes or glucose intolerance. Decreased insulin secretion and reduced pancreatic insulin content contribute to the hyperglycemic state. The conditional approach in INS-1 cells helped to define new molecular targets of hepatocyte nuclear factor (HNF)-1alpha. In the cellular system, nutrient-induced insulin secretion was abolished because of impaired glucose metabolism. Conditional suppression of HNF-4alpha, the MODY1 gene, showed a similar phenotype in INS-1 cells to HNF-1alpha. The existence of a regulatory circuit between HNF-4alpha and HNF-1alpha is confirmed in these cell models. The MODY4 gene, IPF-1 (insulin promoter factor-1)/PDX-1 (pancreas duodenum homeobox-1), controls not only the transcription of insulin but also expression of enzymes involved in its processing. Suppression of Pdx-1 function in INS-1 cells does not alter glucose metabolism but rather inhibits insulin release by impairing steps distal to the generation of mitochondrial coupling factors. The presented experimental models are important tools for the elucidation of the beta-cell pathogenesis in MODY syndromes.
Asunto(s)
Proteínas de Unión al ADN , Diabetes Mellitus Tipo 2/fisiopatología , Proteínas Nucleares , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Mutación/fisiología , Factores de Transcripción/genéticaRESUMEN
The recognition of microbial pathogens based on their molecular patterns is essential for host defense. Recently, Toll-like receptors have been shown not only to recognize viruses as well as bacteria and fungi, but also to trigger an efficient immune response. A recent publication proposed that the retrovirus mouse mammary tumor virus exploits the pattern-recognition receptor Toll-like receptor 4 to achieve more efficient infection.