Differential Ras signaling via the antigen receptor and IL-2 receptor in primary T lymphocytes

Marks, Reinhard; Ho, Allen W.; Rivas, Fabiola V.; Marshall, Emily L.; Janardhan, Sujit; Gajewski, Thomas F.

doi:10.1016/j.bbrc.2003.10.168

Cited by 14 publications

(17 citation statements)

References 30 publications

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“…An adenoviral vector containing the gene expression unit with a dominant negative c-Cbl coding cDNA (Ad DN-Cbl) and an adenoviral vectors without a coding cDNA (EV) were generated as described (31). Briefly, DN c-Cbl mutant was PCR-amplified from a construct containing a full-length c-Cbl cDNA with forward primer, 5' -GGGGTACCatggagcagaaactcatctctgaagaggatctggccggcaacgtgaagaag a-3'; and reverse primer, 5' -ATAGTTTAGCGGCCGCtcaatcttgaggagttggttcacataa-3' (capital letters represent restriction enzyme sequence; underlined letters represent myc-tag sequence).…”

Section: Adenoviral Vectorsmentioning

confidence: 99%

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An adenoviral vector encoding dominant negative Cbl lowers the threshold for T cell activation in post-thymic T cells

Zha

Gajewski

2007

Cellular Immunology

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Cbl family ubiquitin ligases act as key negative regulators of TCR signaling. Knockout mice lacking Cbl-b and c-Cbl show augmented T cell activation and CD28-independent IL-2 production. In order to study Cbl function directly in post-thymic T cells, a DN Cbl adenovirus was generated for transduction of T cells from Coxsackie/adenovirus receptor (CAR) transgenic (Tg) mice. We show that dominant negative (DN) Cbl-transduced CD4 + T cells exhibited enhanced IL-2 production upon TCR/CD28 engagement compared with empty adenoviral vectortransduced cells. This augmentation was reflected at both IL-2 mRNA and protein level, and correlated with increased protein phosphorylation of Vav, Akt, ERK, and p38MAPK. Our results indicate that introduction of dominant negative Cbl can potentiate activation of post-thymic CD4 + T cells, which argues for development of strategies to interfere with Cbl function as a method of immunopotentiation.

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Section: Adenoviral Vectorsmentioning

confidence: 99%

“…We have shown previously that CAR Tg T cells are readily transducible using adenoviralmediated gene transfer (28,31). For the present study, an adenoviral vector encoding a myctagged DN Cbl protein that mimics the v-Cbl oncogene (36) was generated.…”

Section: Dn Cbl Protein Is Efficiently Expressed By Car Cd4 + T Cellsmentioning

confidence: 99%

An adenoviral vector encoding dominant negative Cbl lowers the threshold for T cell activation in post-thymic T cells

Zha

Gajewski

2007

Cellular Immunology

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“…For example, the human T cell line, Jurkat, widely used for promoter analysis, lacks expression of the tumor suppressor, phosphatase and tensin homologue (PTEN), and therefore exhibits constitutive phosphatidylinositol-3-kinase signaling (Shan et al, 2000). Perhaps as a consequence of altered PTEN expression, Ras-mediated ERK activation also varies between primary T cells and Jurkat T cells (Li et al, 1999;Marks et al, 2003). Similarly, the mouse macrophage cell line RAW264.7, also widely used for promoter analysis, exhibits very different responsiveness to microbial and T cell-derived stimuli compared with primary macrophages, (Rao, 2001;Galindo et al, 2004;Rouzer et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…To circumvent this issue, transgenic mice were recently made that express the coxsackie and adenoviral receptor (CAR) under the control of the CD2 promoter (Wan et al, 2000). T cells from CAR transgenic (CAR Tg) mice are readily transduced by adenoviruses (Wan et al, 2000), and recent studies have shown that this is an efficient way to deliver dominant negative signaling molecules to interfere with signal transduction events in vitro and in vivo (Marks et al, 2003;Wan and DeGregori, 2003). Thus, recombinant adenoviruses (rAdV) are a useful tool for highly efficient DNA delivery into multiple types of immune cells.…”

Section: Introductionmentioning

confidence: 99%

An adenoviral vector for probing promoter activity in primary immune cells

Tripathi

Madan

Chougnet

et al. 2006

Journal of Immunological Methods

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Functional analysis of the DNA regulatory regions that control gene expression has largely been performed through transient transfection of promoter-reporter constructs into transformed cells. However, transformed cells are often poor models of primary cells. To directly analyze DNA regulatory regions in primary cells, we generated a novel adenoviral luciferase reporter vector, pShuttle-luciferase-GFP (pSLUG) that contains a promoterless luciferase cassette (with an upstream cloning site) for probing promoter activity, and a GFP expression cassette that allows for the identification of transduced cells. Recombinant adenoviruses generated from this vector can transduce a wide range of primary immune cells with high efficiency, including human macrophages, dendritic cells and T cells; and mouse T cells transgenic for the coxsackie and adenoviral receptor (CAR). In primary T cells, we show inducible nuclear factor of activated T cells (NF-AT) activity using a recombinant pSLUG adenovirus containing a consensus NF-AT promoter. We further show inducible IL-12/23 p40 promoter activity in primary macrophages and dendritic cells using a recombinant pSLUG adenovirus containing the proximal human IL-12/23 p40 promoter. The pSLUG system promises to be a powerful tool for the analysis of DNA regulatory regions in diverse types of primary immune cells.

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“…STAT5A-CA, STAT5B-CA and GFP adenoviral constructs kindly developed and provided by Dr. Andrew Brooks (UQ Diamantina Institute, Woolloongabba, Queensland, Australia) were added at 1:40 dilution in each well containing C57BL/6 SKPs in 3ml culture medium. The crerecombinase adenoviral construct (Marks et al, 2003;Zha et al, 2008) …”

Section: Cell Culture and Viral Transductionmentioning

confidence: 99%

Mesenchymal-epidermal interactions in hair follicle cycling and regeneration

Legrand¹

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The hair follicle is an important skin appendage that arises during development from complex reciprocal signalling between the epidermis and dermis. The multiple epidermal layers of the hair follicle are known to be under the control of the mesenchymal component, the dermal papilla (DP), which has been described as the key regulator of hair follicle induction and maintenance. The hair follicle has elicited interest as a model system for the investigation of tissue and organ regeneration due to its ability to regenerate completely throughout the life of a mammal. This characteristic is attributed to the reservoir of stem cells within the hair follicle, which is modulated by the DP.Although it is known that the DP is central to regulation of the hair follicle, mediators of its signalling are incompletely understood. Potential mediators of DP activity continue to emerge, two of which include the SoxF transcription factor, Sox18, and the transcription factor, STAT5. Sox18 has been implicated in hair type specification by the DP; however, its expression and function remains unclear. STAT5 has been shown in microarray analyses of the DP to be specifically expressed and upregulated, while studies involving STAT5 genetic knockouts display a hair phenotype. More recently, STAT5 has been implicated in autoimmune conditions affecting the hair follicle such as alopecia areata.To investigate further the role of STAT5 in the DP, I have hypothesised that STAT5 plays a key role in the DP's hair follicle inductive properties and regulation of cycling.STAT5 activation in the DP was characterised through immunofluorescence staining of mouse back skin at various stages of hair follicle development and cycling. Back skin was obtained from Topflash transgenic mice, a canonical Wnt reporter transgenic mouse driving luciferase expression, using β-catenin activity to monitor hair cycling via live bioluminescence imaging. While embryonic skin did not display any activation of STAT5, during postnatal hair follicle development (P0-P19), the majority of phospho-STAT5 positive DP were present within telogen phase, to a lesser extent within catagen and not at all during anagen. Adult skin showed phospho-STAT5 activation beginning in late catagen and persisting through to early anagen.Characterisation of SOCS2 by immunofluorescence, a downstream STAT5 transcriptional target, showed expression corresponding to the chronology of phospho-STAT5 activity.Hair regeneration assays were performed with DP cells using SKP culture with STAT5 activity modified through adenovirus constructs. SKPs derived from C57BL/6 neonates and STAT5 lox/lox neonates were transduced with STAT5A/B constitutively-active and crerecombinase constructs, respectively. These transduced SKPs were then used in patch assay experiments and DP formation was quantified using a green fluorescent protein ii (GFP) reporter. SKPs transduced with constitutively-active STAT5 were significantly better at forming hair follicle DP than control SKPs transduced with GFP alone. SKPs with...

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Differential Ras signaling via the antigen receptor and IL-2 receptor in primary T lymphocytes

Cited by 14 publications

References 30 publications

An adenoviral vector encoding dominant negative Cbl lowers the threshold for T cell activation in post-thymic T cells

An adenoviral vector encoding dominant negative Cbl lowers the threshold for T cell activation in post-thymic T cells

An adenoviral vector for probing promoter activity in primary immune cells

Mesenchymal-epidermal interactions in hair follicle cycling and regeneration

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