RESUMEN
Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/inmunología , Antígenos CD28/inmunología , Proteínas Portadoras/inmunología , Guanilato Ciclasa/inmunología , Proteína Quinasa C/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Citometría de Flujo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Análisis de Secuencia de ADN , Organismos Libres de Patógenos EspecíficosRESUMEN
Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Antígenos CD28/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Trastornos Linfoproliferativos/inmunología , Proteínas de la Membrana/inmunología , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Traslado Adoptivo , Animales , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Trastornos Linfoproliferativos/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Mutación , Fosfoproteínas/genética , Fosforilación/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Using N-ethyl-N-nitrosourea-induced mutagenesis, we established a mouse model with a novel form of neutropenia resulting from a point mutation in the transcriptional repressor Growth Factor Independence 1 (Gfi1). These mice, called Genista, had normal viability and no weight loss, in contrast to mice expressing null alleles of the Gfi1 gene. Furthermore, the Genista mutation had a very limited impact on lymphopoiesis or on T- and B-cell function. Within the bone marrow (BM), the Genista mutation resulted in a slight increase of monopoiesis and in a block of terminal granulopoiesis. This block occurred just after the metamyelocytic stage and resulted in the generation of small numbers of atypical CD11b(+) Ly-6G(int) neutrophils, the nuclear morphology of which resembled that of mature WT neutrophils. Unexpectedly, once released from the BM, these atypical neutrophils contributed to induce mild forms of autoantibody-induced arthritis and of immune complex-mediated lung alveolitis. They additionally failed to provide resistance to acute bacterial infection. Our study demonstrates that a hypomorphic mutation in the Gfi1 transcriptional repressor results in a novel form of neutropenia characterized by a split pattern of functional responses, reflecting the distinct thresholds required for eliciting neutrophil-mediated inflammatory and anti-infectious responses.
Asunto(s)
Proteínas de Unión al ADN/genética , Neutropenia/genética , Mutación Puntual , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Antígenos Ly/genética , Antígenos Ly/metabolismo , Artritis/genética , Artritis/metabolismo , Linfocitos B/metabolismo , Médula Ósea/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Proteínas de Unión al ADN/metabolismo , Etilnitrosourea , Femenino , Inflamación/genética , Inflamación/metabolismo , Linfocitos/metabolismo , Linfopoyesis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Neutropenia/inducido químicamente , Neutrófilos/metabolismo , Proteínas Represoras/metabolismo , Linfocitos T/metabolismo , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: 3D matrices are widely used as cell growth supports in basic research, regenerative medicine or cell-based drug assays. In order to genetically manipulate cells cultured within 3D matrices, two novel non-viral transfection reagents allowing preparation of matrices for in situ cell transfection were evaluated. RESULTS: Two lipidic formulations, 3D-Fect™ and 3D-FectIN™, were assessed for their ability to transfect cells cultured within 3D solid scaffolds and 3D hydrogels, respectively. These reagents showed good compatibility with the most widespread types of matrices and enabled transfection of a wide range of mammalian cells of various origins. Classical cell lines, primary cells and stem cells were thus genetically modified while colonizing their growth support. Importantly, this in situ strategy alleviated the need to manipulate cells before seeding them. CONCLUSION: Results presented here demonstrated that 3D-Fect and 3D-FectIN reagents for 3D transfection are totally compatible with cells and do not impair matrix properties. 3D-Fect and 3D-FectIN, therefore, provide valuable tools for achieving localized and sustained transgene expression and should find versatile applications in fundamental research, regenerative medicine and cell-based drug assays.
Asunto(s)
Hidrogeles , Andamios del Tejido , Transfección/métodos , Animales , Silenciador del Gen , Humanos , Hidrogeles/química , Microscopía Fluorescente , TransgenesRESUMEN
Natural killer (NK) cells are lymphocytes involved in antimicrobial and antitumoral immune responses. Using N-ethyl-N-nitrosourea mutagenesis in mice, we identified a mutant with increased resistance to viral infections because of the presence of hyperresponsive NK cells. Whole-genome sequencing and functional analysis revealed a loss-of-function mutation in the Ncr1 gene encoding the activating receptor NKp46. The down-regulation of NK cell activity by NKp46 was associated with the silencing of the Helios transcription factor in NK cells. NKp46 was critical for the subsequent development of antiviral and antibacterial T cell responses, which suggests that the regulation of NK cell function by NKp46 allows for the optimal development of adaptive immune responses. NKp46 blockade enhanced NK cell reactivity in vivo, which could enable the design of immunostimulation strategies in humans.
Asunto(s)
Antígenos Ly/fisiología , Proteínas de Unión al ADN/genética , Infecciones por Herpesviridae/inmunología , Células Asesinas Naturales/inmunología , Receptor 1 Gatillante de la Citotoxidad Natural/fisiología , Linfocitos T/inmunología , Factores de Transcripción/genética , Inmunidad Adaptativa , Sustitución de Aminoácidos , Animales , Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Ly/genética , Antígenos Ly/inmunología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Proteínas de Unión al ADN/fisiología , Regulación hacia Abajo , Prueba de Complementación Genética , Infecciones por Herpesviridae/virología , Memoria Inmunológica , Listeriosis/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muromegalovirus/fisiología , Mutagénesis , Receptor 1 Gatillante de la Citotoxidad Natural/antagonistas & inhibidores , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 1 Gatillante de la Citotoxidad Natural/inmunología , Factores de Transcripción/fisiología , Transcripción Genética , Carga ViralRESUMEN
In recent years, gene therapy has received considerable interest as a potential method for the treatment of numerous inherited and acquired diseases. However, successes have so far been hampered by several limitations, including safety issues of viral-based nucleic acid vectors and poor in vivo efficiency of nonviral vectors. Magnetofection has been introduced as a novel and powerful tool to deliver genetic material into cells. This technology is defined as the delivery of nucleic acids, either 'naked' or packaged (as complexes with lipids or polymers, and viruses) using magnetic nanoparticles under the guidance of an external magnetic field. This article first discusses the principles of the Magnetofection technology and its benefits as compared with standard transfection methods. A number of relevant examples of its use, both in vitro and in vivo, will then be highlighted. Future trends in the development of new magnetic nanoparticle formulations will also be outlined.
Asunto(s)
Tecnología Biomédica/métodos , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Campos Magnéticos , Nanopartículas del Metal/uso terapéutico , Ácidos Nucleicos/administración & dosificación , Animales , Vectores Genéticos , Humanos , Modelos Biológicos , Transfección/métodosRESUMEN
Primary neural stem cells (NSCs) can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. We describe a simple and rapid magnetofection-based method suitable for the lab bench as well as for high-throughput projects. Our method yields high transfection efficiency and can be used for deciphering the genetic control of neural cell differentiation.