Use of Cre-adenovirus and CAR transgenic mice for efficient deletion of genes in post-thymic T cells.

Conditional gene deletion using lineage-specific transgenic expression of Cre has been useful for defining the role of specific gene products in mice in vivo. However, this technology has had limitations for studies of peripheral T cell biology, since the T-lineage promoters commonly used are active early in thymic development. As such, T cell development can be altered by the resulting genetic alterations, thus limiting the interpretation of the data in post-thymic T cell studies. Thus, new strategies are needed to delete targeted genes directly in peripheral T lymphocytes. The availability of transgenic mice expressing the CAR in the T cell compartment enabled testing of Cre-mediated recombination using an adenoviral vector in naïve peripheral T cells in vitro, even without cellular activation. Using Rosa26R reporterxCAR transgenic mice, we describe conditions by which Cre-mediated deletion of targeted genes can be achieved with primary T cells in vitro. These cells can also be adoptively transferred into defined recipient mice for study in vivo. We use conditional PTEN-deficient mice as proof of concept to confirm the value of this technique for deleting a negative regulator of T cell activation. This technology should be broadly applicable for studies of T cell-specific gene deletion to gain understanding of function in the post-thymic T cell compartment.

Specific glucose-induced control of insulin receptor substrate-2 expression is mediated via Ca2+-dependent calcineurin/NFAT signaling in primary pancreatic islet ß-cells.

OBJECTIVE: Insulin receptor substrate-2 (IRS-2) plays an essential role in pancreatic islet ß-cells by promoting growth and survival. IRS-2 turnover is rapid in primary ß-cells, but its expression is highly regulated at the transcriptional level, especially by glucose. The aim was to investigate the molecular mechanism on how glucose regulates IRS-2 gene expression in ß-cells. RESEARCH DESIGN AND METHODS: Rat islets were exposed to inhibitors or subjected to adenoviral vector-mediated gene manipulations and then to glucose-induced IRS-2 expression analyzed by real-time PCR and immunoblotting. Transcription factor nuclear factor of activated T cells (NFAT) interaction with IRS-2 promoter was analyzed by chromatin immunoprecipitation assay and glucose-induced NFAT translocation by immunohistochemistry. RESULTS: Glucose-induced IRS-2 expression occurred in pancreatic islet ß-cells in vivo but not in liver. Modulating rat islet ß-cell Ca(2+) influx with nifedipine or depolarization demonstrated that glucose-induced IRS-2 gene expression was dependent on a rise in intracellular calcium concentration derived from extracellular sources. Calcineurin inhibitors (FK506, cyclosporin A, and a peptide calcineurin inhibitor [CAIN]) abolished glucose-induced IRS-2 mRNA and protein levels, whereas expression of a constitutively active calcineurin increased them. Specific inhibition of NFAT with the peptide inhibitor VIVIT prevented a glucose-induced IRS-2 transcription. NFATc1 translocation to the nucleus in response to glucose and association of NFATc1 to conserved NFAT binding sites in the IRS-2 promoter were demonstrated. CONCLUSIONS: The mechanism behind glucose-induced transcriptional control of IRS-2 gene expression specific to the islet ß-cell is mediated by the Ca(2+)/calcineurin/NFAT pathway. This insight into the IRS-2 regulation could provide novel therapeutic means in type 2 diabetes to maintain an adequate functional mass.

Involvement of Per-Arnt-Sim Kinase and extracellular-regulated kinases-1/2 in palmitate inhibition of insulin gene expression in pancreatic beta-cells.

OBJECTIVE: Prolonged exposure of pancreatic beta-cells to simultaneously elevated levels of fatty acids and glucose (glucolipotoxicity) impairs insulin gene transcription. However, the intracellular signaling pathways mediating these effects are mostly unknown. This study aimed to ascertain the role of extracellular-regulated kinases (ERKs)1/2, protein kinase B (PKB), and Per-Arnt-Sim kinase (PASK) in palmitate inhibition of insulin gene expression in pancreatic beta-cells. RESEARCH DESIGN AND METHODS: MIN6 cells and isolated rat islets were cultured in the presence of elevated glucose, with or without palmitate or ceramide. ERK1/2 phosphorylation, PKB phosphorylation, and PASK expression were examined by immunoblotting and real-time PCR. The role of these kinases in insulin gene expression was assessed using pharmacological and molecular approaches. RESULTS: Exposure of MIN6 cells and islets to elevated glucose induced ERK1/2 and PKB phosphorylation, which was further enhanced by palmitate. Inhibition of ERK1/2, but not of PKB, partially prevented the inhibition of insulin gene expression in the presence of palmitate or ceramide. Glucose-induced expression of PASK mRNA and protein levels was reduced in the presence of palmitate. Overexpression of wild-type PASK increased insulin and pancreatic duodenal homeobox-1 gene expression in MIN6 cells and rat islets incubated with glucose and palmitate, whereas overexpression of a kinase-dead PASK mutant in rat islets decreased expression of insulin and pancreatic duodenal homeobox-1 and increased C/EBPbeta expression. CONCLUSIONS: Both the PASK and ERK1/2 signaling pathways mediate palmitate inhibition of insulin gene expression. These findings identify PASK as a novel mediator of glucolipotoxicity on the insulin gene in pancreatic beta-cells.

Serine protease inhibitor 6 protects cytotoxic T cells from self-inflicted injury by ensuring the integrity of cytotoxic granules.

How cytotoxic T lymphocytes (CTLs) kill intracellular pathogens without killing themselves has been a recurring question ever since their discovery. By using mice deficient in Serine Protease Inhibitor 6 (Spi6), we show that by inhibiting granzyme B (GrB), Spi6 protects CTLs from self-inflicted injury. Infection with either Lymphocytic Choriomeningitis virus (LCMV) or Listeria monocytogenes (LM) revealed increased apoptosis and diminished survival of Spi6 knockout (KO) CTLs, which was cell autonomous and could be corrected by GrB deficiency. Spi6 KO mice in turn were impaired in their ability to clear LCMV infection. Spi6 KO CTLs revealed a breakdown in the integrity of cytotoxic granules, increased cytoplasmic GrB, and ensuing apoptosis. We conclude that Spi6 protects CTLs from suicide caused by GrB-mediated breakdown of cytotoxic granules.

A role for the granzyme B inhibitor serine protease inhibitor 6 in CD8+ memory cell homeostasis.

Generation and maintenance of protective immunological memory is the goal of vaccination programs. It has recently become clear that CD8+ memory T cells are derived directly from CTLs. The mechanisms underlying this transformation and the subsequent survival of memory cells are not completely understood. However, some effector molecules required by CTLs to eliminate infected cells have also been shown to control the number of Ag-specific cells. We report that memory cells express high levels of serine protease inhibitor (Spi) 6, an inhibitor of the effector molecule granzyme B, and that Spi6 can protect T cells from granzyme B-mediated apoptosis. In mouse models, both elevated expression of Spi6 and the complete absence of granzyme B in CD8+ T cells led to an increase in memory cells after infection with lymphocytic choriomeningitis virus. This was not the result of increased levels of antilymphocytic choriomeningitis virus CD8+ T cells during the expansion or contraction phases, but rather transgenic Spi6 directly influenced the survival of CD8+ memory T cells. We propose that expression of protective molecules, like Spi6, serves to shield metabolically active CD8+ memory T cells from their own effector molecules.

Serine protease inhibitor 2A is a protective factor for memory T cell development.

An essential event in the development of memory CD8(+) T lymphocytes is the escape of progenitors from programmed cell death, but how this is mediated is unclear. Here we report that the gene encoding serine protease inhibitor 2A (Spi2A), an inhibitor of lysosomal executioner proteases dependent on transcription factor NF-kappaB, is upregulated in memory cell precursors. Spi2A upregulation protected lymphocytic choriomeningitis virus-specific memory progenitors from programmed cell death. Thus, Spi2A promotes the survival of cytotoxic T lymphocytes, allowing them to differentiate into memory CD8 T cells. These findings suggest a model in which commitment to the memory lineage is facilitated by the upregulation of protective genes.

NF-kappaB protects from the lysosomal pathway of cell death.

The programme of gene expression induced by RelA/NF-kappaB transcription factors is critical to the control of cell survival. Ligation of 'death receptors' such as tumor necrosis factor receptor 1 (TNF-R1) triggers apoptosis, as well as NF-kappaB, which counteracts this process by activating the transcription of anti-apoptotic genes. In addition to activating caspases, TNF-R1 stimulation causes the release of cathepsins, most notably cathepsin B, from the lysosome into the cytoplasm where they induce apoptosis. Here we report a mechanism by which NF-kappaB protects cells against TNF-alpha-induced apoptosis: inhibition of the lysosomal pathway of apoptosis. NF-kappaB can protect cells from death after TNF-R1 stimulation, by extinguishing cathepsin B activity in the cytosol. This activity of NF-kappaB is mediated, at least in part, by the upregulation of Serine protease inhibitor 2A (Spi2A), a potent inhibitor of cathepsin B. Indeed, Spi2A can substitute for NF-kappaB in suppressing the induction of cathepsin B activity in the cytosol. Thus, inhibition of cathepsin B by Spi2A is a mechanism by which NF-kappaB protects cells from lysosome-mediated apoptosis.