D. B. W. received a commercial research grant from Inovio, Touchlight, Oncosec, and AERAS; has received honoraria from the speaker’s bureaus of Pfizer, Merck, Novartis, Roche; has ownership interest, and is a consultant and advisory board member of Inovio. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: ELR KM DBW ZM THF. Performed the experiments: ELR JFW DKS. Analyzed the data: ELR JFW ZM THF. Contributed reagents/materials/analysis tools: THF. Wrote the paper: ELR JFW DKS KM DBW ZM THF.
Current address: Department of Pediatrics, Nemours Children’s Hospital, Orlando, Florida, United States of America
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.
Regulated in development and DNA damage response 1 (REDD1), also referred to as Dig2, RTP801, and DDIT4, is a highly conserved stress response gene that is upregulated following many types of cellular stress. It was first identified as being upregulated by hypoxia [
Significantly, REDD1 has been shown to function as an inhibitor of mammalian target of rapamycin (mTOR), specifically mTOR complex 1 (mTORC1) in several cell types [
Though REDD1 has been shown to be upregulated in thymocytes in response to treatment with dexamethasone, a stress hormone, very little is known about REDD1 in mature T cells. Upon activation via the T cell receptor and costimulatory molecules, mature naive T cells undergo massive metabolic changes in order to proliferate and perform effector functions such as cytokine secretion [
All mouse work was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Children’s Hospital of Philadelphia Institutional Animal Care and Use Committee (protocol number: 449). De-identified human primary T cells were obtained from the University of Pennsylvania's Human Immunology Core. Secondary use of de-identified human specimens by individual Core users is not considered human subjects research by the National Institutes of Health or the University of Pennsylvania's Institutional Review Board.
REDD1 knockout mice [
Primary human CD4 T cells were obtained from the University of Pennsylvania’s Human Immunology Core. Human CD4 T cells were maintained in RPMI (Gibco) with 10% FBS (Benchmark), 100 U/ml Penicillin-Streptomycin (Gibco), and 2 mM L-glutamine (Gibco). Mouse cells were maintained in Click's media (Irvine Scientific) with 10% FBS, 100 U/ml Penicillin-Streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate (Cellgro), 1x MEM-NEAA (Gibco) and 50 μM 2-mercaptoethanol (BioRad).
Cells were plated at 1 million cells/ml in media, and then stimulated with the indicated concentrations of Human T Activator CD3/CD28 beads (Dynal) or PHA (Remel) and IL-2 (NIH AIDS Research and Reference Reagent Program). For proliferation assays, cells were resuspended at 2 million cells/ml in phosphate buffered saline (PBS) (Gibco) with 0.1% FBS. A 20 μM solution of 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma) in PBS with 0.1% FBS was mixed 1:1 with the cell suspension, and incubated for 15 minutes at room temperature in the dark. The labeling was stopped by adding 5 volumes of cold media and incubating on ice for 5 minutes. Cells were then stimulated with PHA and IL-2 as indicated. For cytokine production assays, T cells were first purified from total lymph node cells using the EasySep Negative Selection Mouse T Cell Isolation Kit (StemCell Tech) as directed, then stimulated as in the activation and cell survival assays.
Flow cytometry data was collected on a FACS Fortessa (BD) and analyzed using FlowJo (Treestar). The following antibodies and reagents were used: LIVE/DEAD Fixable Dead Cell Stain (Invitrogen), CD3-BV421 (BioLegend), CD4-BV711 (BioLegend), CD8-BV605 (BioLegend), CD19-FITC (BD Pharmingen), CD69-PE-Cy7 (BD Pharmingen), CD25-PE-CF594, (BD Horizon), Annexin V-APC (BD Pharmingen), and propidium iodide (PI) (BD Pharmingen). For proliferation and activation assays, cells were washed once with PBS, and then incubated with LIVE/DEAD Fixable Dead Cell Stain in PBS for 30 minutes at room temperature in the dark. After washing with PBS with 1% FBS, cells were incubated with surface marker antibodies in PBS with 1% FBS for 20 minutes at room temperature in the dark. The cells were then washed and resuspended in PBS with 1% FBS, and the samples were run on the flow cytometer. For Annexin V and PI staining, cells were washed once with binding buffer (eBioscience), then incubated with Annexin V and cell surface markers in binding buffer for 15 minutes at room temperature in the dark. After washing with binding buffer, cells were resuspended in binding buffer with PI, and the samples were analyzed by flow cytometry.
Total RNA was extracted using the RNA/DNA/Protein Purification Kit (Norgen). cDNA was reverse transcribed from 0.1 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed in 20 μl reactions containing 1 μl of cDNA, 1 μl of the appropriate primer/probe set, 8 μl of TaqMan Universal Mastermix, No AmpErase UNG (Applied Biosystems), and 10 μl of PCR-grade water (Fisher). All samples were run in triplicate. The following primer/probe sets were purchased from Applied Biosystems: human REDD1 (Hs01111686_g1), mouse REDD1 (Mm00512504_g1), and RN18S1 (Hs03928990_g1). qPCR reactions were run on an SDS 7500 Real-time PCR system (Applied Biosystems) with the following protocol: 1 cycle of 95°C for 10 minutes; 40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. REDD1 expression levels were normalized to the endogenous control, RN18S1. RN18S1 was identified as stably expressed in our system from among the other commonly used housekeeping genes RPL13A, TBP, B2M, and GAPDH using GeNorm, a Visual Basic Application for Excel developed by Vandesompele
Protein was extracted using the RNA/DNA/Protetin Purification Kit (Norgen). Protein concentration was measured using Coomassie Plus (Pierce). Equal amounts of total protein were mixed with sample buffer (Invitrogen) and reducing agent (Invitrogen). Samples were heated at 70°C for 10 minutes and loaded onto a 4–12% Bis-Tris NuPAGE gel (Invitrogen). Gels were run on a NuPAGE electophoresis system (Invitrogen) at 200 V for 50 minutes in MOPS running buffer. Samples were then transferred onto an Immobilon-FL PVDF membrane (Millipore) at 30 V for 1 hour and blocked with Odyssey blocking buffer (LiCore). The following antibodies were used: rabbit-anti-REDD1 (Proteintech), mouse-anti-actin (Sigma), goat-anti-rabbit-IRDye 800CW (LiCor) and goat-anti-mouse-IRDye 680RD (LiCor). The membranes were imaged on the Odyssey CLx (LiCor) and analyzed using the Image Studio software (LiCor).
Prism software was used to perform two-way analysis of variance for data with multiple timepoints and unpaired t-tests for data from a single timepoint. Error bars show the standard error of the mean. P values less than 0.05 were considered significant.
To study the role of REDD1 in normal T cell function, we first determined the level of REDD1 expression in primary human CD4 T cells in response to activation signals. REDD1 mRNA was significantly upregulated by PHA and beads coated with anti-CD3 and anti-CD28 antibodies (
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
The pronounced upregulation of REDD1 mRNA and protein upon stimulation led us to explore its potential role in T cell activation. To determine the role of REDD1 in T cell proliferation, we took advantage of REDD1 knockout mice [
Wildtype (WT) and knockout (KO) mouse lymph node cells were labeled with CFSE and stimulated with 1.5 μg/ml PHA for 72 hours.
To determine the role of REDD1 in other aspects of T cell activation, we compared the upregulation of activation markers CD69 and CD25 by wildtype and REDD1 knockout T cells in response to PHA stimulation. CD4 and CD8 T cells from REDD1 knockout mice and wildtype controls displayed closely matched patterns of CD69 and CD25 expression during the course of a 72 hour stimulation (
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.
Our lab first became interested in the role of REDD1 in T cells with our report of REDD1 upregulation in HIV-infected human T cells that are resistant to apoptosis [
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.
To confirm these results, we repeated the same set of experiments with PI/Annexin V staining as another measure of cell viability. Live cells are PI-/Annexin V-. As cells undergo apoptosis, they expose phosphatidylserine (PS) on the outside of their plasma membrane, allowing Annexin V to bind, and become Annexin V+. At later timepoints, apoptotic cells lose membrane integrity and become PI+/Annexin V+. Necrotic cells, however, directly become PI+/Annexin V+ [
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.
REDD1 is highly conserved, and is known to be upregulated following many stresses, including DNA damage, hypoxia, and dexamethasone treatment [
In order to study the role of REDD1 in mature T cells, we took advantage of a previously developed REDD1 knockout mouse [
REDD1 is a stress response gene that has been shown to function as an inhibitor of mTOR in several cell types, including thymocytes stimulated with the stress hormone dexamethasone [
To determine if REDD1 knockout T cells were capable of being normally activated, we looked at the effect of PHA stimulation on the upregulation of activation markers on wildtype and knockout T cells. Neither CD69 nor CD25 expression after PHA stimulation was affected by the lack of REDD1 over the same time period as the defect in proliferation was observed (
We next looked at REDD1’s effect on T cell survival. While REDD1 expression has been shown to have either a pro- or anti-survival effect in different cell types [
In order to better understand what type of cell death REDD1 is involved in regulating, we used PI/Annexin V staining to measure apoptosis of REDD1 knockout cells. With PI/Annexin V staining, early apoptotic cells are Annexin V+, while late apoptotic and necrotic cells are PI+/Annexin V+. Because this method is unable to discriminate between late apoptotic and necrotic cells, we refer to them simply as dead cells. In an effort to catch any effect of REDD1 on early apoptosis, we took measurements at several timepoints following stimulation. The PI/Annexin V staining shows a decrease in live unstimulated REDD1 knockout CD4 and CD8 T cells and a corresponding increase in dead cells (
It has recently become clear that a type of regulated necrosis linked to autophagy called necroptosis, is separate from, and cross-inhibitory to, apoptosis. Caspase 8 deficient T cells stimulated with anti-CD3 and CD28 antibodies have been shown to die by necroptosis [
In summary, we show for the first time that REDD1 is upregulated during T cell activation. We also show that the absence of REDD1 causes a decrease in the proliferation of PHA stimulated T cells and a decrease in survival of stimulated and unstimulated T cells, but does not appear to significantly affect T cell activation. Further study is needed to elucidate the biochemical mechanisms of these effects of REDD1 on T cell homeostasis.
We thank Dr. Elena Feinstein and Quark Pharmaceuticals for very kindly providing the REDD1 knockout mice.