RESUMEN
The positive and negative selection of lymphocytes by antigen is central to adaptive immunity and self-tolerance, yet how this is determined by different antigens is not completely understood. We found that thymocyte-selection-associated family member 2 (Themis2) increased the positive selection of B1 cells and germinal center B cells by self and foreign antigens. Themis2 lowered the threshold for B-cell activation by low-avidity, but not high-avidity, antigens. Themis2 constitutively bound the adaptor protein Grb2, src-kinase Lyn and signal transducer phospholipase γ2 (PLC-γ2), and increased activation of PLC-γ2 and its downstream pathways following B cell receptor stimulation. Our findings identify a unique function for Themis2 in differential signaling and provide insight into how B cells discriminate between antigens of different quantity and quality.
Asunto(s)
Linfocitos B/fisiología , Selección Clonal Mediada por Antígenos , Centro Germinal/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación de Linfocitos , Inmunidad Adaptativa , Animales , Diferenciación Celular , Linaje de la Célula , Células Cultivadas , Proteína Adaptadora GRB2/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfolipasa C gamma/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Autotolerancia , Familia-src Quinasas/metabolismoRESUMEN
Lentiviral based constructs represent a recent development in the generation of transgenic animals. The ease of use, and the fact that the same backbone vectors can be used to down-modulate endogenous gene expression and to produce transgenic animals overexpressing a gene of interest, have fuelled growing interest in this technology. In this study, we have used a lentiviral delivery system to generate transgenic mice expressing altered levels (up or downregulated) of a gene of interest. Although this lentiviral-based approach led to high levels of transgenesis and germ line transmission, a wide variation in transgene expression was observed in most first and second generation mouse lines. In particular, despite the segregation of integrants into single-copy expressing mouse lines, transgene expression appeared to be the target of epigenetic regulatory mechanism, often causing the coexistence of high and low transgene expressing cells within a given tissue such as blood peripheral lymphocytes. The establishment and analysis of large number of mouse lines may therefore be required to select a stable transgenic line with pancellular expression of a gene of interest using this lentiviral-based approach.
Asunto(s)
Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Técnicas de Transferencia de Gen , Ratones Transgénicos/genética , Animales , Western Blotting , Línea Celular , Cartilla de ADN/genética , Lentivirus , Ratones , Plásmidos/genética , Interferencia de ARN , Transgenes/genéticaRESUMEN
Promoter selection is of utmost importance for the study of in vivo gene function using transgenic models. In the present study, we have analyzed the expression of the GFP marker under the control of the composite CAG promoter in the lymphoid compartment of several transgenic mouse strains. Despite the ability of the CAG promoter to drive gene expression in almost all tissues examined to date, its activity appears to be developmentally regulated within the T lymphocyte cell lineage. In particular, CD4 and CD8-expressing, thymic immature T cells displayed lower levels of the GFP marker when compared with both bone marrow precursors and mature circulating T cells, suggesting a transient downregulation of CAG activity during T cell development. Alternative promoters may therefore be preferred for the study of T cell development in vivo using a transgenic approach.