REDD1 as a stress response gene in mature T cells

Regulated in development and DNA damage response 1 (REDD1) is a highly conserved stress response protein that is upregulated following many types of cellular stress including hypoxia, DNA damage, energy stress, ER stress, and nutrient deprivation. In T cells it has been shown to be upregulated following DNA damage and dexamethasone treatment and to provide protection from cell death. In the case of dexamethasone treated thymocytes, REDD1\u27s protection is mediated through the induction of autophagy. However, we know little of REDD1\u27s function in mature T cells. In this study we show that REDD1 is upregulated in mature T cells following stimulation with phytohemagglutinin (PHA) or anti-CD3 and anti-CD28 antibodies immobilized on beads. REDD1 knockout T cells exhibit a defect in proliferation and cell survival, although upregulation of the activation markers CD69 and CD25 and cytokine production appear normal. REDD1 knockdown causes a decrease in productive human immunodeficiency virus (HIV) infection in CD4 T cells. Though the mechanism is unclear, the absence of REDD1 may sensitize HIV infected cells to cell death. Collectively these findings point to a previously unappreciated protective role for REDD1 in mature T cell stress responses

REDD1 as a stress response gene in mature T cells

Regulated in development and DNA damage response 1 (REDD1) is a highly conserved stress response protein that is upregulated following many types of cellular stress including hypoxia, DNA damage, energy stress, ER stress, and nutrient deprivation. In T cells it has been shown to be upregulated following DNA damage and dexamethasone treatment and to provide protection from cell death. In the case of dexamethasone treated thymocytes, REDD1\u27s protection is mediated through the induction of autophagy. However, we know little of REDD1\u27s function in mature T cells. In this study we show that REDD1 is upregulated in mature T cells following stimulation with phytohemagglutinin (PHA) or anti-CD3 and anti-CD28 antibodies immobilized on beads. REDD1 knockout T cells exhibit a defect in proliferation and cell survival, although upregulation of the activation markers CD69 and CD25 and cytokine production appear normal. REDD1 knockdown causes a decrease in productive human immunodeficiency virus (HIV) infection in CD4 T cells. Though the mechanism is unclear, the absence of REDD1 may sensitize HIV infected cells to cell death. Collectively these findings point to a previously unappreciated protective role for REDD1 in mature T cell stress responses

REDD1 Is Essential for Optimal T Cell Proliferation and Survival.

REDD1 is a highly conserved stress response protein that is upregulated following many types of cellular stress, including hypoxia, DNA damage, energy stress, ER stress, and nutrient deprivation. Recently, REDD1 was shown to be involved in dexamethasone induced autophagy in murine thymocytes. However, we know little of REDD1's function in mature T cells. Here we show for the first time that REDD1 is upregulated following T cell stimulation with PHA or CD3/CD28 beads. REDD1 knockout T cells exhibit a defect in proliferation and cell survival, although markers of activation appear normal. These findings demonstrate a previously unappreciated role for REDD1 in T cell function

REDD1 is required for optimal T cell proliferation.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were labeled with CFSE and stimulated with 1.5 μg/ml PHA for 72 hours. <b>(A)</b> Representative flow plots of CFSE staining gated on CD4 T cells. <b>(B)</b> Average percentages of CD4 T cells that have undergone the indicated number of divisions in 72 hours. Statistically significant differences between the WT and KO populations for each division are indicated next to the legend. Statistically significant differences between 'few divisions' (divisions 0–2) and 'multiple divisions' (divisions 3–8) are also indicated. N = 8 WT; N = 8 KO. *p = 0.01 to 0.05; **p = 0.001 to 0.01; ***p < 0.001.</p

REDD1 does not affect PHA induced apoptosis.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were stimulated with 1.5 μg/ml PHA and cell survival was measured by flow cytometry with PI/Annexin V staining. <b>(A)</b> Representative flow plots of Annexin V/PI staining gated on CD4 or CD8 T cells. <b>(B)</b> Average percentages of live (PI-/Annexin V-), dead (PI+AnnexinV+), and apoptotic (Annexin V+) CD4 or CD8 T cells. N = 4 WT; N = 4 KO. *p = 0.01 to 0.05; **p = 0.001 to 0.01; ***p < 0.001.</p

REDD1 does not affect upregulation of activation markers CD69 or CD25.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were stimulated with 1.5 μg/ml PHA and CD69 and CD25 expression was measured by flow cytometry. <b>(A)</b> Representative flow plots of CD69 and CD25 staining gated on CD4 or CD8 T cells. The percentage of WT and KO cells falling within the gate are indicated in the corner of each panel. <b>(B)</b> Average percentages of CD4 or CD8 T cells expressing CD69 and CD25 after stimulation. N = 4 WT; N = 4 KO.</p

REDD1 mRNA and protein is expressed in lymphoid tissues and is upregulated during T cell activation.

<p>Primary human CD4 T cells were stimulated with 1.5 μg/ml PHA + 100 U/ml IL-2 or 3 CD3/CD28 beads/cell + 100 U/ml IL-2. REDD1 mRNA <b>(A)</b> and protein <b>(B)</b> expression was determined using qRT-PCR and immunoblot, respectively. <b>(C)</b> Mouse splenocytes were stimulated with 1.5 μg/ml PHA + 20 U/ml IL-2 and REDD1 mRNA expression was determined by qRT-PCR. All qRT-PCR data is presented as fold change compared to the unstimulated cells. qRT-PCR and immunoblot data are representative of 3 individual experiments.</p

REDD1 is required for optimal cell survival independent of stimulation.

<p>Wildtype (WT) and knockout (KO) mouse lymph node cells were stimulated with 1.5 μg/ml PHA and cell survival was measured by flow cytometry with dead cell staining. <b>(A)</b> Representative flow plots of dead cell staining, gated on CD4 or CD8 T cells. The percentage of WT and KO cells falling within the gate are indicated in the corner of each panel. <b>(B)</b> Average percentages of live CD4 or CD8 T cells. N = 8 WT; N = 8 KO. *p = 0.01 to 0.05; **p = 0.001 to 0.01; ***p < 0.001.</p

Co-Administration of Molecular Adjuvants Expressing NF-Kappa B Subunit p65/RelA or Type-1 Transactivator T-bet Enhance Antigen Specific DNA Vaccine-Induced Immunity

DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity

A novel synthetic DNA vaccine elicits protective immune responses against Powassan virus.

Powassan virus (POWV) infection is a tick-borne emerging infectious disease in the United States and North America. Like Zika virus, POWV is a member of the family Flaviviridae. POWV causes severe neurological sequalae, meningitis, encephalitis, and can cause death. Although the risk of human POWV infection is low, its incidence in the U.S. in the past 16 years has increased over 300%, urging immediate attention. Despite the disease severity and its growing potential for threatening larger populations, currently there are no licensed vaccines which provide protection against POWV. We developed a novel synthetic DNA vaccine termed POWV-SEV by focusing on the conserved portions of POWV pre-membrane and envelope (prMEnv) genes. A single immunization of POWV-SEV elicited broad T and B cell immunity in mice with minimal cross-reactivity against other flaviviruses. Antibody epitope mapping demonstrated a similarity between POWV-SEV-induced immune responses and those elicited naturally in POWV-infected patients. Finally, POWV-SEV induced immunity provided protection against POWV disease in lethal challenge experiments