Bacteriophage P1 Cre/loxP based systems can be used to manipulate the genomes of mice in vivo and in vitro, allowing the generation of tissue‐specific conditional mutants. Wehave generated mouse lines expressing Cre recombinase in hematopoietic tissues using the vav regulatory elements, or in lymphoid cells using the hCD2 promoter and locus control region (LCR). The R26R‐EYFP Cre reporter mouse line was used to determine the pattern of Cre expression in each line and enabled the assessment of Cre activity at a single‐cell level. Analysis showed that the vav promoter elements were able to direct Cre‐mediated recombination in all cells of the hematopoietic system. The hCD2 promoter and LCR on the other hand were able to drive Cre‐mediated recombination only in T cells and B cells, but not in other hematopoietic cell types. Furthermore, in the appropriate tissues, deletion of the floxed target was complete in all cells, thereby excluding the possibility of variegated expression of the Cre transgene. Both of these Cre‐transgenic lines will be useful in generating tissue‐specific gene deletions within all the cells of hematopoietic or lymphoid tissues.

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.

A transgenic mouse was generated expressing on most (>80%) of thymocytes and peripheral T cells a T-cell receptor isolated from a cytotoxic T-cell clone (F5). This clone is CD8+ and recognizes αα366-374 of the nucleoprotein (NP 366-374) of influenza virus (A/NT/60/68), in the context of Class ,MHC Db (Townsend et al., 1986). The receptor utilizes the Vβ11 and Vα4 gene segments for the β chain and α chain, respectively (Palmer et al., 1989). The usage of Vβ11 makes this TcR reactive to Class II IE molecules and an endogenous ligand recently identified as a product of the endogenous mammary tumour viruses (Mtv) 8, 9, and 11 (Dyson et al., 1991). Here we report the development of F5 transgenic T cells and their function in mice of the appropriate MHC (C57BL/10 H-2b, IE-) or in mice expressing Class II MHC IE (e.g., CBA/Ca H-2k and BALB/c H-2d) and the endogenous Mtv ligands. Positive selection of CD8+ T cells expressing the Vβ11 is seen in C57BL/10 transgenic mice (H-2b). Peripheral T cells from these mice are capable of killing target cells in an antigen-dependent manner after a period of in vitro culture with IL-2. In the presence of Class II MHC IE molecules and the endogenous Mtv ligand, most of the single-positive cells carrying the transgenic T-cell receptor are absent in the thymus. Unexpectedly, CD8+ peripheral T-cells in these (H-2k or H-2d) F5 mice are predominantly Vβ11 positive and also have the capacity to kill targets in an antigen-dependent manner. This is true even following backcrossing of the F5 TcR transgene to H-2d scid/scid mice, in which functional rearrangement of endogenous TcR alpha- and beta-chain genes is impaired.

Inijection of mice transgenic for a class I major histocompatibility complex-restricted T-cell receptor with a soluble peptide antigen from influenza virus nucleoprotein results in clonal depletion of double-positive Immature thymocytes in the thymus and activation of mature T cells in the periphery, accompanied by a transient up-regulation of the T-cell receptor and CD3 and CD8 coreceptor molecules.

The T lymphocyte-specific protein tyrosine kinase p56lck (Lck) is an essential component of the TCR-mediated signal transduction complex. Lck knockout mice have reduced numbers of double-positive thymocytes and very few mature single-positive cells, particularly of the CD4 lineage. Here we demonstrate the ability of a tetracycline-based tissue-specific inducible Lck transgene to restore expansion of early thymocytes and maturation of single-positive cells in Lckneg mice upon induction with doxycycline. Restoration of Lck expression is particularly important for positive selection to the CD4+ lineage but has a lesser impact on selection to the CD8+ lineage, suggesting activation of Lck is an important component of the signals involved in lineage choice during thymic differentiation.

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.

Activation of T lineage cells through the TCR by peptide-MHC complexes on APC is critically dependent on rearrangement of the actin cytoskeleton. Vav1 is a guanine nucleotide exchange factor for members of the Rho/Rac family of GTPases which is activated following TCR stimulation, suggesting that it may transduce TCR signals to the activation of some or all actin-controlled processes. We show that Vav1-deficient double-positive thymocytes are less efficient at forming conjugates with APC presenting agonist peptide than wild-type cells are. Furthermore we demonstrate that Vav1 is required for TCR-induced activation of the integrin LFA-1, which is likely to explain the defect in conjugate formation. However, once Vav1-deficient cells form a conjugate, the assembly of proteins into an immunological synapse at the conjugate interface is normal. In contrast, thymocyte polarization is defective in the absence of Vav1, as judged by the relocalization of the microtubule-organizing center. These data demonstrate that Vav1 transduces signals to only a subset of cytoskeletondependent events at the immunological synapse.

Vav1 is a signaling protein required for both positive and negative selection of CD4 ؉ CD8 ؉ double positive thymocytes. Activation of the ERK MAPK pathway is also required for positive selection. Previous work has shown that Vav1 transduces T cell receptor (TCR) signals leading to an intracellular calcium flux. We now show that in double positive thymocytes Vav1 is required for TCR-induced activation of the ERK1 and ERK2 kinases via a pathway involving the Ras GTPase, and B-Raf, MEK1, and MEK2 kinases. Furthermore, we show that Vav1 transduces TCR signals to Ras by controlling the membrane recruitment of two guanine nucleotide exchange factors. First, Vav1 transduces signals via phospholipase C␥1 leading to the membrane recruitment of RasGRP1. Second, Vav1 is required for recruitment of Sos1 and -2 to the transmembrane adapter protein LAT. Finally, we show that Vav1 is required for TCR-induced LAT phosphorylation, a key event for the activation of both phospholipase C␥1 and Sos1/2. We propose that reduced LAT phosphorylation is the key reason for defective TCR-induced calcium flux and ERK activation in Vav1-deficient cells. Signals from the T cell receptor (TCR)1 play a critical role in the positive and negative selection of immature CD4 ϩ CD8 ϩ double positive (DP) thymocytes, which lead to the development of mature T cells. The precise outcome of these selection events is determined by signals generated following interactions between the TCR on DP thymocytes and peptides presented by MHC class I and class II molecules on thymic stromal cells (1). Cells whose TCR binds with no or low avidity to peptide-MHC complexes undergo apoptosis in a process termed death by neglect. Cells bearing a TCR with moderate avidity for a peptide-MHC complex undergo positive selection and differentiate into mature CD4 ϩ CD8 Ϫ or CD4 Ϫ CD8 ϩ single positive thymocytes, and eventually emigrate to the periphery as mature CD4 ϩ or CD8 ϩ T cells. Finally, DP thymocytes whose TCR binds with high avidity to peptide/MHC undergo negative selection and are eliminated by apoptosis. Much interest has focused on elucidating the signal transduction pathways that regulate positive and negative selection, respectively.Vav1 is a 95-kDa cytoplasmic protein that is rapidly tyrosine-phosphorylated following TCR stimulation (2, 3). Sequence analysis showed that Vav1 contained a number of domains typical of signal transducing proteins (4, 5). In particular, Vav1 has a Dbl homology domain that is characteristic of guanine nucleotide exchange factors (GEFs) for Rho family GTPases. Vav1 has been shown to function as a GEF for Rac1, Rac2, and RhoG and to be activated by tyrosine phosphorylation (6 -8). The importance of Vav1 in TCR signaling was demonstrated by defective TCR-induced interleukin-2 secretion and proliferation in Vav1-deficient T cells (9 -11). Furthermore, Vav1 was shown to be required for both positive and negative selection in DP thymocytes (12, 13). Analysis of signaling pathways showed that in CD4 ϩ T cells, Vav1 transduced TCR signals t...