Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4 + CD8 + double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-γ1 (PLCγ1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCγ1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCγ1 and the adaptor molecule Src homology 2 domain–containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.
Bad is a distant relative of Bcl-2 and acts to promote cell death. Here, we show that Bad expression levels are greatly increased in thymocytes during apoptosis. We generated bad transgenic mice to study the action of upregulated Bad expression on T cell apoptosis. The T cells from these mice are highly sensitive to apoptotic stimuli, including anti-CD95. The numbers of T cells are greatly depleted and the processes of T cell development and selection are perturbed. We show that the proapoptotic function of Bad in primary T cells is regulated by Akt kinase and that Bad overexpression enhances both cell cycle progression and interleukin 2 production after T cell activation. These data suggest that Bad can act as a key regulator of T cell apoptosis and that this is a consequence of its upregulation after exposure to death stimuli.
T cell development and selection in the thymus are shaped by the induction of apoptosis. However, a direct role in T cell development and selection for any of the molecules known to regulate apoptosis has remained controversial. We have studied the effect of bax and bcl-2 transgenes in recombination activation gene 1–deficient (RAG-1 −/− ) mice transgenic for the major histocompatibility complex class I–restricted F5 T cell receptor. Overexpression of a bax transgene in the thymus seriously impairs the production of mature T cells, whereas bcl-2 overexpression greatly promotes it. The effect of bax and bcl-2 overexpression on antigen-induced negative selection was studied using fetal thymic organ cultures. This analysis showed that Bcl-2 strongly inhibits negative selection, whereas Bax does not affect it. Our data directly show that Bcl-2 family members have specific roles in T cell selection and also lend support to the hypothesis that Bax and Bcl-2 can antagonize each other's action in a certain apoptosis pathway while in another they can be functionally nonreciprocal.