RESUMO
Survival of naive T cells requires engagement of TCR with self-peptide major histocompatibility Ags. The signaling pathways required to transmit this survival signal are poorly understood. In this study, we asked whether the tyrosine kinase Zap70 is required to transmit survival signals in naive CD8 T cells. In the absence of Zap70 expression, thymic development is completely blocked. Using a tetracycline-inducible Zap70 transgene (TetZap70), thymic development of Zap70-deficient TCR transgenic F5 mice was restored. Feeding mice doxycycline to induce Zap70 expression resulted in repopulation of the peripheral naive compartment. Zap70 transgene expression was then ablated by withdrawal of doxycycline. Survival of Zap70-deficient naive CD8 T cells depended on host environment. In hosts with a replete T cell compartment, naive T cells died rapidly in the absence of Zap70 expression. In lymphopenic hosts, Zap70-deficient T cells survived far longer, in an IL-7-dependent manner, but failed to undergo lymphopenia-induced proliferation. Analyzing mixed bone marrow chimeras revealed that intact Zap70-dependent signaling was important for integration of recent thymic emigrants into the mature naive compartment. Finally, we asked whether adaptor function conferred by Zap70 tyrosines 315 and 319 was necessary for transmission of homeostatic TCR signals. This was done by analyzing F5 mice expressing mutant Zap70 in which these residues had been mutated to alanines (Zap70(YYAA)). Inducible Zap70 expression rescued thymic development in F5 TetZap70 Zap70(YYAA) mice. However, in the absence of wild-type Zap70 expression, the Zap70(YYAA) mutant failed to transmit either survival or proliferative homeostatic signals.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfopoese/genética , Receptores de Antígenos de Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/genética , Animais , Proliferação de Células , Sobrevivência Celular/genética , Doxiciclina/farmacologia , Técnicas de Introdução de Genes , Proteínas de Homeodomínio/genética , Receptores de Hialuronatos/biossíntese , Receptores de Hialuronatos/genética , Interleucina-7/imunologia , Linfopenia/imunologia , Camundongos , Camundongos Knockout , Receptores de Interleucina-7/biossíntese , Receptores de Interleucina-7/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transgenes/genética , Proteína-Tirosina Quinase ZAP-70/biossíntese , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here, we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.