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. 2010 Aug;24(8):2818–2828. doi: 10.1096/fj.09-144295

The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling

Sandra Pastorino *,1, Hemamalini Renganathan *,1, Maisel J Caliva *, Erin L Filbert †, John Opoku-Ansah *, Florian J Sulzmaier *, Joanna E Gawecka *, Guy Werlen ‡, Andrey S Shaw †,§, Joe W Ramos *,2
PMCID: PMC2909277  PMID: 20354143

Abstract

PEA-15 is a death effector domain-containing phosphoprotein that binds ERK and restricts it to the cytoplasm. PEA-15 also binds to FADD and thereby blocks apoptosis induced by death receptors. Abnormal expression of PEA-15 is associated with type II diabetes and some cancers; however, its physiological function remains unclear. To determine the function of PEA-15 in vivo, we used C57BL/6 mice in which the PEA-15 coding region was deleted. We thereby found that PEA-15 regulates T-cell proliferation. PEA-15-null mice did not have altered thymic or splenic lymphocyte cellularity or differentiation. However, PEA-15 deficient T cells had increased CD3/CD28-induced nuclear translocation of ERK and increased activation of IL-2 transcription and secretion in comparison to control wild-type littermates. Indeed, activation of the T-cell receptor in wild-type mice caused PEA-15 release of ERK. In contrast, overexpression of PEA-15 in Jurkat T cells blocked nuclear translocation of ERK and IL-2 transcription. Finally, PEA-15-null T cells showed increased IL-2 dependent proliferation on stimulation. No differences in T cell susceptibility to apoptosis were found. Thus, PEA-15 is a novel player in T-cell homeostasis. As such this work may have far reaching implications in understanding how the immune response is controlled.—Pastorino, S., Renganathan, H., Caliva, M. J., Filbert, E. L., Opoku-Ansah, J., Sulzmaier, F. J., Gawecka, J. E., Werlen, G., Shaw, A. S., Ramos, J. W. The death effector domain protein PEA-15 negatively regulates T-cell receptor signaling.

Keywords: ERK MAP kinase, IL-2, proliferation, Jurkat T cells

PEA-15 (phosphoprotein enriched in astrocytes, 15 kDa) is a small death effector domain (DED)-containing protein expressed in a variety of human and mouse tissues. It can bind several proteins including ERK (1) and FADD (2). Which of these proteins it binds is determined by phosphorylation at two serine residues, S104 and S116 (2). PEA-15 has at least two distinct roles within the cell: that of blocking apoptosis by interfering with the assembly of the death-induced signaling complex (3) and that of regulating ERK1/2 localization by sequestering them in the cytoplasm (1, 4), thereby preventing ERK-mediated transcription (1). To date, its physiological function is less clear.

The activation of T lymphocytes plays a central role in the regulation of immune responses. An optimal T-cell response is orchestrated by exact modulation of a series of signaling events that start with the engagement of the T-cell receptors and coreceptors. On TCR ligation, the Src-family protein kinases Lck and Fyn phosphorylate the ITAM motifs within the TCR-associated CD3-complex, leading to the recruitment and phosphorylation of ZAP70 (5, 6). Activated ZAP70 then phosphorylates several proteins, including LAT, Gads, and SLP-76 (7, 8), that induce the formation of a membrane-associated signaling platform. This signalosome triggers multiple events, including recruitment and activation of PLC-γ1 (7), PKC, and RasGRP, and generation of inositol 1,4,5-trisphosphate (IP3), followed by IP3-mediated Ca2+ release. Downstream signaling events include the sequential activation of the kinases Raf-1, MEK1/2, and ERK1/2 (9).

ERK1/2 activation and its differential kinetics after receptor engagement lead to specific functional outcomes, including differentiation, proliferation, survival, or apoptosis (9,10,11). ERK1/2 has been implicated in pre-TCR-mediated proliferation of immature double negative (DN) thymocytes (10). Similarly, the fate of double positive (DP) thymocytes is determined by differential modulation of ERK1/2 activity (12, 13), and the duration of ERK signaling influences CD4+CD8+ lineage commitment (14, 15). In mature T cells, ERK1/2 plays a critical role in the formation and activation of the AP-1 transcription factor. AP-1, along with other transcription factors, binds to the interleukin-2 (IL-2) promoter and results in IL-2 production (16, 17). Recently, a positive feedback loop in which ERK phosphorylates Lck in a regulatory manner to prevent SHP-1 deactivation of Lck was shown to sustain TCR signaling (18). A downstream positive feedback loop between ERK-mediated immediate early gene expression (10) and ERK has also been described (19). Interestingly, the different kinetics of ERK1/2 activation correlate with subcellular localization of this kinase (20, 21), leading to distinctive outcomes. The dynamic integration of these multiple regulatory mechanisms both upstream and downstream of ERK1/2 may yield a strong dependence of T-cell responses to ERK activity and localization.

Here, we analyzed the phenotype of PEA-15-deficient (PEA-15−/−) mice and found that through its ability to modify ERK1/2 localization, PEA-15 can affect T-cell activation. PEA-15−/− T cells showed increased ERK nuclear translocation and IL-2 transcription. PEA-15 deficiency did not alter the general apoptotic properties of these cells. PEA-15−/− T cells were hyperproliferative in response to CD3/CD28 and produced higher levels of IL-2 after in vivo immune challenge. Moreover, PEA-15 in wild-type T cells released ERK1/2 on CD3/CD28 ligation and the use of anti-IL-2 antibodies or an ERK inhibitor reversed the hyperproliferative phenotype of PEA-15−/− T cells. These data suggest that PEA-15 restriction of ERK localization is responsible for the defects observed in the immune system of PEA-15−/− animals. These findings indicate that PEA-15 is a critical adaptor protein that regulates T-cell activation and the immune response. This finding is the first evidence for a physiological function of PEA-15 in regulating proliferation in the immune system.

MATERIALS AND METHODS

Mice

PEA-15-null mice were generated by homologous recombination, as described previously (22). Mice were bred in our facility and backcrossed to a C57BL/6J background for 7 generations. Animals were maintained under specific pathogen-free conditions and handled in accordance with National Institutes of Health (NIH) guidelines for the care and use of animals. Mice were used at 3 to 6 mo of age, and all experiments were conducted with either backcrossed animals or sex-matched littermates from PEA-15+/− intercrosses. All experiments were approved by the Institutional Review Board of the University of Hawaii.

Flow cytometry and antibodies

Cell suspensions of spleen or thymus were prepared by standard protocols. Cell were preincubated with anti-CD16/CD32 antibodies to block Fc receptors, then stained for analysis by flow cytometry using PBS containing 2% FCS and 2 mM EDTA. The following antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-CD3e (clone 17A2), allophycocyanin (APC)-conjugated anti-CD4 (clone GK1.5), peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5)- or FITC-conjugated anti-CD4 (clone RM4-5), phycoerytrin (PE)- or PE-Cy5-conjugated anti-CD8 (clone 53-6.2), APC-conjugated anti-CD25 (clone PC61.5), APC-conjugated anti-B220 (clone RA3-6B2), FITC-conjugated anti-CD11b (M1/70–15), APC-conjugated anti-CD49b (clone DX5), APC-conjugated anti-CD44 (clone IM7), and PE-conjugated anti-CD122 (clone 5H4). For intracellular staining, cells were fixed, permeabilized, and stained with PE-conjugated anti-FOXP3 (clone FJK-16s) following the manufacturer’s recommendations (all antibodies from eBioscience, San Diego, CA, USA). At least 30,000 viable cells were live-gated on a BD FACScalibur using Cell Quest Pro or on a BD FACSAria using Cell Quest Diva software (BD Biosciences, San Jose, CA, USA) and analyzed by FlowJo software (Tree Star, Ashland, OR, USA).

T-cell activation, proliferation, and apoptosis

T cells were isolated from total splenocytes by negative selection using the Pan T-cell Isolation Kit with routinely >95% purity (Miltenyi Biotech, Bergisch Gladbach, Germany). A total of 1 × 106 T cells/ml were seeded in complete RPMI on 96-well plates coated with anti-CD3e (clone 145–2C11) at the indicated concentrations. T cells were cultured for 64 h in the presence of CD3 and CD28 (clone 37.51) at the indicated concentrations, then pulsed with 1 μCi/well of [3H]thymidine (Perkin Elmer, Wellesley, MA, USA) for 8 h. Cells were harvested using a Skatron cell harvester (Skatron Instruments, Lier, Norway), and [3H]thymidine incorporation was measured with a Tri Carb 2900TR scintillation counter (Packard Instruments, Meriden, CT, USA). For measurement of apoptosis, splenocytes were treated for 18 h with anti-Fas (JO 95; BD Pharmingen, San Diego, CA, USA) plus protein G sepharose, anti-CD3ε only (2 μg/ml plate bound), anti-CD3ε (coated at 10 μg/ml) plus anti-CD28 (2 μg/ml soluble), or phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) plus ionomycin (100 ng/ml) and stained with Annexin V-PE and 7-amino-actinomycin D (7-AAD) according to manufacturer’s protocol (Annexin V-PE Apoptosis Detection Kit; BD Pharmingen). The extent of apoptosis was measured by flow cytometry. For measurement of activation-induced cell death in vitro, T cells were stimulated for 48 h with plate-bound anti-CD3e (10 μg/ml) and anti-CD28 (2 μg/ml) to induce proliferation. Cells were washed and rested for 3 d with IL-2 (10 U/ml). Cells were then restimulated for 48 h with plate-bound anti-CD3e and CD28. Cell viability was measured by staining with annexin V and 7-AAD.

Measurement of T-cell division

Cycling of cells was measured as dilution of the carboxylfluorescein diacetate succinimidyl diester (CFSE) dye. CFSE labeling was performed with a Vybrant CFSE-SE Cell Tracer kit (Molecular Probes, Eugene, OR, USA) following the manufacturer’s directions. Labeled cells were stimulated as described above and CFSE staining was analyzed by flow cytometry.

Real-time quantitative PCR analysis

Total RNA was extracted from 2 to 6 × 106 activated T cells using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s protocol. RNAs (0.5 μg) were digested with DNase I Amplification Grade (Invitrogen) and subsequently transcribed into cDNA using Invitrogen’s SuperScript II First Strand Synthesis System. The levels of IL-2 mRNA expression were determined by real-time PCR (ABI Prism 9700 HT Sequence Detector System, Applied Biosystems, Foster City, CA, USA) using QuantiTec SYBR Green kit (Qiagen, Valencia, CA, USA). The following primers for detection of mouse 18S and HPRT were used to normalize input cDNA: HPRT-FW 5′-TCCTCCTCAGACCGCTTTT-3′, HPRT-RV 5′-CCTGGTTCATCATCGCTAATC-3′; 18S-FW 5′-TGCCAGAGTCTCGTTCGTTAT-3′, 18S-RV 5′-CGACCAGAGCGAAAGCATTTA-3′. All real-time PCR samples were run in triplicate. The Biogazelle qBase Plus 3.3 program (Biogazelle, Ghent, Belgium) was used to analyze the data and quantitate mRNA expression. We used qBase to normalize the data against two references (HPRT and 18S) and correct for efficiency.

IL-2 luciferase reporter assay

Jurkat T cells were electroporated with the indicated amounts of GFP-PEA-15 plasmid and 10 μg of IL-2 luciferase reporter plasmid. Jurkat and Daudi B cells were seeded in a 96-well plate at 105/well with or without staphylococcal enterotoxin E (Toxin Technology, Sarasota, FL, USA). Cells were incubated at 37°C for 8 h, lysed, and assayed according to the Promega Luciferase Assay System protocol (Promega, Madison, WI, USA) using a luminometer.

Detection of IL-2 secretion

Levels of IL-2 in culture supernatants or in mouse serum was determined by enzyme-linked immunosorbent assay (ELISA; eBioscience, San Diego, CA, USA), according to the manufacturer’s instructions. Levels were normalized for cell number.

KLH immune challenge

Mice were immunized subcutaneously with 100 μg 2,4,6-trinitrophenol (TNP)-KLH (Biosearch Technologies, Inc., Novato, CA, USA) in complete Freund adjuvant (CFA; Sigma, St. Louis, MO, USA), and boosted intraperitoneally with TNP-KLH in IFA (Sigma) at 21 d. For antigen-specific T-cell proliferation, T cells were isolated from the draining lymph nodes at d 7 and 15 and stimulated in vitro with TNP-KLH at the indicated concentrations. T cells were stimulated in parallel with anti-CD3 and anti-CD28. Proliferation was then assessed as described above. For antibody production, serum samples were obtained from jaw bleeds on d 28 after immunization. Hapten-specific immunoglobulin levels were quantified in microtiter wells, coated with TNP-OVA. Serial dilution of sera were added to wells, followed by HRP-conjugated goat anti-mouse Igs.

Subcellular fractionation, immunoprecipitation and Western blotting

For total cell lysates, cells were lysed in ice-cold M2 buffer (0.5% Nonidet P-40; 20 mM Tris, pH 7.6; 250 mM NaCl; 5 mM EDTA; 3 mM ethylene glycol-bis-(b-aminoethyl ester)-N,N,N9,N9-tetraacetic acid; 20 mM sodium phosphate; 20 mM sodium pyrophosphate; 3 mM b-glycerophosphate; 1 mM sodium orthovanadate; 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF; and 10 mg/ml each of leupeptin and aprotinin). For subcellular fractionation, the Nuclear Extract Kit from Active Motif (Carlsbad, CA, USA) was used. For immunoprecipitation, T cells were stimulated as described and then lysed in Nonidet P-40 buffer (20 mM Tris, pH 8; 137 mM NaCl; 10% glycerol; and 1% Nonidet P-40) supplemented with protease inhibitors and sodium orthovanadate. Lysates were incubated with 0.4 μg of PEA-15 antibody (rabbit polyclonal). The immune complexes were precipitated with 50 μl of protein-A sepharose for 1 h at 4°C. The precipitates were washed with Nonidet P-40 lysis buffer supplemented with 1 M NaCl. Immune complexes were resuspended in 50 μl of sample buffer and immunoblotted. Lysates were resolved by 10% SDS-PAGE gel and transferred to PVDF membrane. Blots were hybridized with the following antibodies: anti-phospho ERK1/2, anti-ERK-2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho IκBα, (Cell Signaling, Boston, MA, USA), anti-IκBα (Cell Signaling), anti-actin (Santa Cruz) and anti-lamin B (Santa Cruz). PEA-15 phosphoepitope antibodies (pS104 and pS116) were developed in collaboration with BioSource (Camarillo, CA, USA) and were previously described (2).

RESULTS

PEA-15 affects T-cell proliferation but not apoptosis

To determine the physiological function of PEA-15, we made use of a PEA-15-null mouse on the C57BL/6 background. The derivation of the PEA-15−/− mice has been described previously (22). Homozygote PEA-15−/− mice were born at the expected Mendelian ratio and were healthy and fertile. The animals were monitored for >18 mo for weight, fertility, and behavior and were periodically sacrificed for histological evaluation. We noted that mice in facilities with pathogen problems had markedly enlarged spleen and lymph nodes compared to wild-type littermates. In pathogen-free facilities, no consistent difference in spleen size was found.

PEA-15 can regulate both cell proliferation and apoptosis (1, 22,23,24) and is expressed in thymocytes and splenocytes. We therefore tested whether PEA-15 deficiency would affect these processes in T cells. PEA-15−/− purified splenic T cells were hyperproliferative as assessed by measuring thymidine incorporation after stimulation with plate-bound anti-CD3 antibody and soluble anti-CD28 antibodies or with PMA plus ionomycin (Fig. 1A). The difference in proliferation between PEA-15−/− and wild-type T cells was most apparent with suboptimal stimulation conditions, indicating that PEA-15 may modulate the threshold for T-cell activation (data not shown). In complementary experiments, we labeled T cells with CFSE before stimulation and analyzed them by flow cytometry to assess cell division. The results confirmed that PEA-15−/− T cells proliferate more than their wild-type counterparts (Fig. 1B). Again, the differences in numbers of cell divisions were more notable at lower doses of anti-CD3/anti-CD28, indicating that PEA-15 might normally serve to dampen TCR-mediated signaling, necessitating a more potent stimulus for a productive response (data not shown). The increase in proliferation in the PEA-15−/− T cells could be due to differences in intrinsic signaling within the cell or to differences in the T-cell populations (e.g., fewer Tregs). We therefore isolated a purified population of naive splenic CD4+ T cells (CD4+CD62L+) in which Tregs and γ/δ T cells had been removed. Stimulation of these naive CD4+ T cells resulted in a similar hyperproliferation in the PEA-15−/− T cells compared to wild-type cells (Fig. 1C). These data suggest that PEA-15 is an intrinsic negative regulator of T-cell proliferation. As PMA activates PKC and Ras, which are downstream on the TCR signaling cascade, our results suggest that PEA-15 acts downstream of PKC and Ras functions during T-cell activation.

Figure 1.

Figure 1

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Proliferation and survival of wild-type and PEA-15−/− T cells. A) Proliferation of purified splenic T cells stimulated for 72 h with anti-CD3 and anti-CD28 at 1 mg/ml and 0.1 mg/ml, respectively, or with PMA and ionomycin. Analysis was performed by [3H]thymidine incorporation. Data shown are means ± sd. Inset: immunoblot showing that PEA-15−/− splenocytes lack PEA-15 and that purified CD3+ splenic T cells express PEA-15. B) Flow cytometry of CFSE-labeled splenocytes on CD3/CD28 stimulation. Proliferation was analyzed for cells gated on the lymphocyte population. Numbers indicate percentage of cells in each division. C) Proliferation of purified naive (CD3+CD4+CD62L+) splenic T cells stimulated for 72 h with anti-CD3+anti-CD28 (TCR stimulation) at 1 mg/ml and 0.1 mg/ml, respectively. Cells were purified using the Miltenyi Biotec CD4+CD62L+ T-cell isolation kit II. In this purification, regulatory T cells and TCRγ/δ+ T cells are depleted. Analysis was performed by [3H]thymidine incorporation. D) Apoptosis of purified splenic T cells treated with the indicated stimuli. E) Activation-induced cell death of purified splenic T cells pretreated for 48 h with anti-CD3 and anti-CD28, then incubated for 3 d with IL-2 and restimulated for 48 h with anti-CD3 and anti-CD28. F) Jurkat T cells were electroporated with 20 μg of expression vectors encoding either PEA-15, MC159, or vector alone. After 2 d in culture, cells were treated with the indicated amounts of anti-Fas antibody for 3 h. Cells were lysed and analyzed by Western blotting for PEA-15 expression (right panel). Percentage of apoptotic cells was measured by AnnexinV/7AAD staining (left panel). Data are representative of 3 independent experiments.

We further investigated whether the apparent hyperproliferation of PEA15−/− T cells was due to increased cell survival. On treatment with anti-CD3e, anti-CD3e plus CD28, dexamethasone, or PMA plus ionomycin, apoptotic rates were similar in wild-type and PEA-15−/− T cells (Fig. 1D). Also, no difference was found in apoptosis when preactivated T cells were restimulated to mimic the activation-induced cell death program (Fig. 1E). These results suggest that PEA-15 affects T-cell proliferation and not T-cell survival. PEA-15 is a known inhibitor of Fas-induced apoptosis in several cell types. We, therefore, investigated whether PEA-15−/− T cells were more susceptible to Fas-induced apoptosis than those from wild-type mice. Treatment with anti-Fas antibody activated apoptosis to similar levels in both PEA-15−/− and wild-type T cells (Fig. 1D). Similarly, Jurkat T cells overexpressing PEA-15 remained sensitive to Fas-activated apoptosis, whereas control cells expressing the antiapoptotic viral FLIP protein (MC159) did not (Fig. 1F). Thus, PEA-15 does not act to prevent apoptosis in mouse T cells in the contexts tested.

Normal lymphoid development in PEA-15−/− mice

One possible explanation for the differences in proliferation for the PEA-15−/− T cells is that differences occur in the development of T cells or the number of Tregs or memory T cells in the null animals. PEA-15 protein is expressed in the spleen, and deletion in the PEA-15−/− mice was verified by immunoblotting (Fig. 2A) and PCR genotyping. No major defects were observed in the architecture or morphology of the spleen or other organs and tissues, and the animals were of normal weight and size (data not shown). Wild-type and PEA-15−/− mice had similar cellularity of peripheral blood (Table 1) and lymphoid organs (Fig. 2B). Wild-type and PEA-15−/− mice had similar numbers of thymic CD4+ and CD8+ T cells (Fig. 2C) and of peripheral CD4+, CD8+, Treg (CD4+CD25+FOXP3+), and memory T cells (CD4+CD44+CD122+ and CD8+CD44+CD122+) (Fig. 2D). The splenic B, NK, and macrophage populations were also largely unaffected by PEA-15 deficiency (Fig. 2E and data not shown). These data suggest that PEA-15 is not essential for the development of the lymphoid or the myeloid compartments but may affect subpopulations of cells within these compartments. We, therefore, focused on intrinsic differences in T-cell activation.

Figure 2.

Figure 2

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Lymphoid development in PEA-15−/− mice. A) Spleen total lysates were prepared from WT and KO mice. Top panel: detection of PEA-15 protein by immunoblot. Bottom panel: photomicrographs of explanted spleens. Left images: WT; right images: PEA-15−/−. A modest splenomegaly was observed with aging. B) Absolute cell numbers in spleen and thymus of individual sex- and age-matched mice. At least 10 animals of each genotype were analyzed. Error bars = sd. C) Percentage of CD8+ and CD4+ T cells in thymus of wild-type and PEA-15−/− mice. D) Percentage of CD4+, CD8+, Treg, and memory cells in spleen of wild-type and PEA-15−/− mice. Treg cells were identified by FOXP3 and CD4+ markers; for memory cells expression of the surface markers CD44 and CD122 was analyzed on CD3+CD4+ and CD3+CD8+ populations. E) Percentage of T cells (CD3+), B cells (CD19+), NK cells (CD49b), and macrophages (CD11b) in the spleen of wild-type and PEA-15−/− mice. Also shown is the percentage of IgM- and IgD-containing B cells. Cells in top left quadrant are IgM+IgD− (immature B cells); top right are IgM+IgD+ (mature B cells). Numbers indicate cell percentage in gated areas of dot plots and histograms. All data are representative of ≥3 independent experiments.

TABLE 1.

Hemograms from wild-type and PEA-15−/− mice

Parameter WT/WT KO/KO
WBC count (×103) 13.28 ± 3.53 16.83 ± 7.17 (NS)
RBC count (×106) 9.47 ± 0.78 9.24 ± 1.48 (NS)
HGB (g/dl) 15.25 ± 0.98 14.78 ± 2.59 (NS)
HCT (%) 46.42 ± 3.09 44.7 ± 7.07 (NS)
MCV (fl) 49.12 ± 4.44 48.5 ± 3.16 (NS)
MCH (pg) 16.05 ± 1.26 15.98 ± 0.75 (NS)
MCHC (g/dl) 32.75 ± 1.23 33.06 ± 2.09 (NS)
Neutrophil segs (%) 12 ± 1.36 11 ± 3.50 (NS)
Neutrophil bands (%) 19.75 ± 33.88 10.5 ± 29.69 (NS)
Lymphocytes (%) 64.5 ± 32.84 73.6 ± 28.29 (NS)
Monocytes (%) 4.75 ± 2.80 5.5 ± 2.44 (NS)
Eosinophils (%) 0.25 ± 0.81 0.625 ± 1.18 (NS)
Abs neutrophil segs (ml−1) 1573 ± 499.73 1724.5 ± 546.14 (NS)
Abs neutrophil bands (ml−1) 2440.6 ± 4776.7 1921.6 ± 5435.17 (NS)
Abs lymphocytes (ml−1) 8698.5 ± 6322. 12295 ± 7482.31 (NS)
Abs monocytes (ml−1) 643.37 ± 461.67 932.5 ± 658.72 (NS)
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Peripheral blood was collected from the maxillary facial vein of mice 12–15 wk old. Cell counts were performed using an automated cell counter with veterinary parameters and reagents. Differential counts were performed manually on Wright-Giemsa-stained smears. WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCN, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; Abs, absolute; NS, nonsignificant. Values are means ± sd; n = 16/group. P values were calculated using the nonparametric Mann- Whitney U test (comparison with WT). Values of P < 0.05 were significant.

PEA-15 regulates T-cell activation in vivo

To investigate whether the enhanced in vitro proliferation of PEA-15−/− cells correlated with in vivo T-cell activation, we immunized age- and sex-matched wild-type and PEA-15−/− mice with keyhole limpet hemocyanin (KLH) in the presence of CFA, which induces a mixed T-helper 1 and T-helper 2 response. After KLH restimulation, PEA-15−/− T cells showed higher proliferation levels than their wild-type counterparts. Similar results were obtained when in vivo challenged PEA15−/− cells were restimulated with plate-bound CD3 and soluble CD28, or CD3 alone (Fig. 3A–C). Next, to determine whether increased T-cell proliferation in PEA-15−/− mice leads to an altered T-cell-dependent humoral immune responses, we measured antigen-specific antibody production in immunized mice. We found that PEA-15−/− mice produced higher amounts of immunoglobulins after both primary and secondary immunization. (Fig. 3D). These results indicate that PEA-15 has a role in modulating T-cell-dependent immune response in vivo.

Figure 3.

Figure 3

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Increased T-cell activation in PEA-15−/− mice. A–C) Wild-type and PEA-15−/− mice were immunized subcutaneously with T-cell-dependent antigen (TNP-KLH), and T-cell proliferation was assessed 15 d after immunization. T cells were in vitro restimulated with different concentrations of TNP-KLH (A), anti-CD3 plus anti-CD28 (B), or anti-CD3 alone (C). Proliferation was measured as [3H]thymidine incorporation. D) Anti-TNP Ig levels from preimmune serum and serum collected weekly after first and second immunization were determined by ELISA with TNP-OVA. Data are means ± se of 3–4 mice. *P= 0.046 vs. control; unpaired 2-tailed t test.

Increased IL-2 secretion and transcription in PEA-15-null T cells

IL-2 has a major role in T-cell activation and proliferation. To dissect the mechanism by which PEA-15 alters these processes, we first determined the effect of PEA-15 overexpression on IL-2 production by Jurkat T cells using a luciferase reporter assay. Overexpression of PEA-15 inhibited IL-2 production in a dose-dependent manner (Fig. 4A). We confirmed these findings by measuring the production of IL-2 following CD3/CD28 stimulation in wild-type and PEA-15−/− mice. Indeed, PEA-15−/− T cells showed higher secretion of IL-2 in vitro on CD3 and CD28 stimulation (Fig. 4B). Increased IL-2 secretion in PEA-15−/− T cells was preceded by an increase in the IL-2 mRNA levels, which suggests that PEA-15 might be required to control transcription of IL-2 (Fig. 4C). The increase in IL-2 secretion was also present in KLH-challenged mice with a similar increase in interferon-γ (Fig. 4D). No significant differences were seen in TNF-α, IL-4, or IL-5 levels (Fig. 4D, E). This finding suggests that Th1 cells may be the predominant CD4 cell type affected. To assess whether the increase in IL-2 transcription and secretion could account for the hyperproliferation observed in PEA-15−/− T cells, we performed CD3/CD28 stimulation in the presence of either an antibody blocking IL-2 or in the presence of excess of IL-2. Blocking IL-2 decreased proliferation of wild-type and PEA-15−/− T cells to the same levels. In contrast, excess IL-2 treatment increased proliferation to the same extent (Fig. 4F, G). Finally, IL-2 receptor levels are equivalent in both wild-type and PEA-15−/− T cells (Fig. 4H). These results support the idea that PEA-15 deficiency leads to hyperproliferation of the T-cell compartment through increased IL-2 transcription and secretion.

Figure 4.

Figure 4

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PEA-15 controls optimal IL-2 expression, and IL-2 is responsible for the hyperproliferative phenotype of PEA-15−/− mice. A) Jurkat T cells were cotransfected with 10 μg IL-2 luciferase reporter construct and the indicated amounts of GFP-PEA-15 24 h following transfection, cells were seeded in a 1:1 ratio with uncoated or SEE-coated Daudi B cells (APC), lysed 8 h later, and assayed for luciferase activity. Data are derived from 2 independent experiments each done in triplicate. B) IL-2 protein secretion in splenic purified T cells from wild-type and PEA-15−/− mice stimulated with anti-CD3 and anti-CD28. Cells were stimulated in vitro, and protein levels were analyzed by ELISA. C) IL-2 mRNA expression in CD3/CD28-stimulated wild-type and PEA-15−/− T cells. IL-2 mRNA levels were measured by real-time PCR and normalized using expression of HPRT and 18S control mRNAs. Data are means ± sd of triplicate wells and are representative of 2 independent experiments (A–C). D, E) Wild-type and PEA-15−/− mice were immunized subcutaneously with T-cell-dependent antigen (TNP-KLH), and levels of IL-2, IFN-γ, and TNF-α (D) or IL-4 and IL-5 (E) were determined using a cytokine bead array assayed by flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). F, G) Proliferation of wild-type and PEA-15−/− T cells on CD3/CD28 stimulation (F) in the presence of anti-IL-2 antibodies (2 mg) (F) or in the presence of excess of recombinant IL-2 (20 ng) (G). Proliferation was measured by [3H]thymidine incorporation. Data are means ± se of 2 mice from 2 independent experiments (D–G). H) Levels of IL-2 receptor α were determined by flow cytometry using an antibody to mouse IL-2 receptor α. Numbers indicate percentage of IL-2α+ cells in the gate.

PEA-15 regulates distal CD3/CD28 signaling

The observation that PEA-15−/− T cells underwent similar hyperproliferation in response to stimulation with either CD3/CD28 or PMA and ionomycin suggested that PEA-15 regulates T-cell proliferation downstream of the activation of the T-cell receptor complex. PEA-15 is a known modulator of ERK function. We, therefore, investigated whether alteration of the MAPK/ERK pathway occurs in the PEA-15−/− T cells on CD3/CD28 stimulation. We found that absence of PEA-15 does not alter the phosphorylation of ERK in response to CD3/CD28 stimulation (Fig. 5A). We previously reported that PEA-15 can restrict ERK localization to the cytoplasm, we therefore examined whether PEA-15−/− T cells had altered localization of ERK. We observed a consistent increase in the amount of nuclear ERK in the PEA-15−/− T cells at 2 and 4 h after TCR stimulation (Fig. 5B, C). This result is consistent with reports that PEA-15 prevents ERK translocation to the nucleus and supports the possibility that PEA-15 regulates IL-2 transcription by affecting ERK nuclear translocation. Interestingly, ERK is present in the nucleus at increased amounts prior to in vitro stimulation (Fig. 5B, C, E). Notably, the levels of PEA-15 did not change after activation in the wild-type T cells, and NFκB activation was largely unaffected (Fig. 5D). If PEA-15 controls ERK translocation, then it would be expected that CD3/CD28 stimulation would cause the release of ERK from PEA-15 in wild-type T cells. We immunoprecipitated PEA-15 from purified wild-type T cells or Jurkat T cells that had been CD3/CD28-stimulated. We then detected coprecipitating ERK by immunoblot. PEA-15 was found associated with ERK in unstimulated cells and released ERK in response to CD3/CD28 ligation (Fig. 5F). Moreover, PEA-15 was increasingly phosphorylated at serine 104 in a manner corresponding to its release of ERK (Fig. 5F). Phosphorylation at serine 116 decreased during this same time. Overall, these data suggest a previously unknown mechanism by which PEA-15 negatively regulates TCR activation of IL-2 transcription by controlling ERK localization in the T cell.

Figure 5.

Figure 5

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PEA-15 regulates CD3/CD28 activation via modulation of ERK localization. A) Purified T cells from wild-type and knockout mice were left untreated (0) or treated with anti-CD3ε and anti-CD28 antibodies for 0.5, 1, or 4 h. ERK phosphorylation was determined by immunoblot. B) Detection of ERK protein localization by immunoblot analysis of nuclear and cytoplasmic fractions from PMA-stimulated T cells. Anti-Lamin B1 antibody was used to determine purity of the fractions. C) Splenocytes from wild-type and knockout mice were left untreated (0) or treated with anti-CD3ε and anti-CD28 antibodies for 2 and 4 h. Nuclear fractions were resolved by SDS-PAGE. Amount of ERK in the nuclear fraction was visualized by immunoblotting. Densitometry was performed on 3 independent nuclear ERK1/2 immunoblots using ImageJ and correcting for extracted nuclear protein levels by normalizing to lamin expression (shown as relative ERK intensity). Data are means ± se. *P = 0.038 vs. wild type; Student’s t test. D) WT and PEA-15−/− splenocytes were activated as in A, and PEA-15, phosphorylated IkBα, NFkB, ERK, and actin levels were determined by Western blotting. E) WT and KO splenocytes were activated as in A, and ERK localization was determined by immunofluorescence (×100, Zeiss Axiovert). F) PEA-15 was immunoprecipitated from wild-type (WT) purified T cells or Jurkat T cells activated by CD3 and CD28 as in A. As a control, PEA-15−/− T cells were identically treated. Precipitated PEA-15 and coprecipitated ERK were visualized by immunoblotting; 5% of input ERK2 is shown. In Jurkat cells, phosphorylation of immunoprecipitated PEA-15 at serines 104 and 116 was visualized using phosphoepitope antibodies.

To further test the hypothesis that the defect observed in PEA15−/− T cells is related to ERK signaling, we treated wild-type and PEA-15−/− T cells with a MEK inhibitor (U0126) before TCR stimulation. U0126 inhibited proliferation in wild-type and PEA-15−/− T cells, indicating that ERK is responsible in part for the hyperproliferative phenotype of the PEA-15−/− T cells (Fig. 6). Again, addition of excess IL-2 overrode the effect of the inhibitor (Fig. 6), corroborating the hypothesis that, by keeping ERK out of the nucleus, PEA-15 limits IL-2 transcription and production. Overall these data suggest a previously unknown mechanism by which PEA-15 negatively regulates TCR activation of IL-2 transcription by controlling ERK translocation.

Figure 6.

Figure 6

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PEA-15 effects on CD3/CD28 activation are dependent on ERK activation. Top panel: proliferation of CD3/CD28-stimulated T cells in the presence of excess of IL-2 and the ERK1/2 inhibitor U0126. Purified T cells from wild-type and knockout mice were left untreated (CTRL) or treated with anti-CD3ε and anti-CD28 antibodies for 4 h (CD3/CD28). U0126 (10 μM) or IL-2 (20 ng/ml) was added as indicated. Proliferation was measured by [3H]-thymidine incorporation. Data are means ± se of 3 mice. Bottom panel: phospho-ERK2 immunoblot indicates the effectiveness of the U0126 inhibitor in these experiments.

DISCUSSION

Our results have revealed that the DED protein PEA-15 is an important regulator of TCR signaling. In PEA-15−/− mice, T cells underwent increased proliferation and cytokine production compared to wild-type T cells after CD3/CD28 stimulation. On CD3/CD28 activation, PEA-15−/− T cells transcribed and produced excess IL-2. Moreover, enforced expression of PEA-15 in Jurkat T cells suppresses IL-2 transcription. It is well documented that ERK activity influences T-cell activation in part through activation of IL-2 transcription. Our results indicate that PEA-15 regulates T-cell activation by modulating ERK translocation to the nucleus (Fig. 7). These data provide the first evidence for a physiological function of PEA-15 in the immune system.

Figure 7.

Figure 7

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PEA-15 negatively regulates T-cell proliferation by limiting ERK nuclear translocation. PEA-15 limits T-cell proliferation in part by acting downstream of T-cell receptor activation to control ERK translocation to the nucleus and thereby IL-2 transcription. PEA-15 binds directly to ERK and prevents its translocation. On TCR engagement, PEA-15 releases ERK. Release of ERK may be activated by phosphorylation of PEA-15 at serine 104 by PKC, while serine 116 is concomitantly dephosphorylated.

The observation that a hyperproliferative phenotype could be triggered by CD3/CD28 or CD3 alone suggests that the defect in PEA-15−/− T cells is intrinsic. Our data further indicate that PEA-15 modulates CD3 activation of T cells via ERK signaling. We demonstrated previously that PEA-15 interacts with ERK1/2, restricting it to the cytoplasm where it is targeted to specific substrates (1, 25). Consistent with our previous results, here we show that ablation of PEA-15 led to accumulation of ERK in the nucleus in PEA-15−/− T cells. Other investigators have reported PEA-15 interaction with ERK1/2 and its relevance in the modulation of ERK1/2 functions (26,27,28); however, these studies relied mainly on in vitro expression systems. This is the first report that indicates the importance of this interaction in a physiologically significant context in the immune system.

ERK1/2 signaling is involved in different stages of T-cell development: while ERK2 knockout is embryonic lethal, ERK1−/− mice have a reduction in the number of mature single-positive thymocytes (29). Moreover, ERK1−/− mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune encephalomyelitis (30). In our study, we did not observe major developmental defects in the lymphoid organs of the PEA-15−/− mice; however, a deregulated TCR response was evident. Perhaps PEA-15 has different functions in thymocytes and in peripheral T cells, as TCR signaling may be regulated differently among these populations (31,32,33). We propose that PEA-15 might play a more critical role in mature T cells. It is, however, possible that subtler defects in T-cell development exist. It is notable that the PEA-15−/− mice do not have spontaneous adenopathy, given that they show increased T-cell proliferation and no difference in cell death. This is likely further evidence that deletion of PEA-15 predominantly causes changes only on stimulation and clonal expansion, as our mice are kept in pathogen-free facilities. Homeostasis appears to be unaffected and what minimal proliferation occurs in lymph nodes and the spleens does not cause the massive lymphocyte expansion associated with adenopathy.

The detailed mechanism of PEA-15 regulation of TCR signaling and immune responses is still under investigation. However, here we show that PEA-15 acts in part by altering the localization of ERK1/2, which in the absence of PEA-15 is free to translocate to the nucleus. Indeed, we find that ERK is already somewhat increased in the nucleus of T cells before in vitro stimulation. Thus, PEA-15 might be required to enforce ERK cytoplasmic localization in the unstimulated T cell. Absence of PEA-15 may thereby permit ERK to translocate in response to lesser stimuli. Alternatively, PEA-15 may be required to return ERK to the cytoplasm as reported previously (1). Thus, the resident nuclear ERK may be the result of previous in vivo ERK activation. Interestingly, the MEK inhibitor U0126 did not completely prevent the increased proliferation of the PEA-15−/− T cells. It is possible that the modest increase in IkB phosphorylation leads to activation of NFkB and the remaining proliferation. Alternatively, the nuclear ERK in the knockout T cells may be unphosphorylated yet still synergize with the actively translocated JNK to promote proliferation. We continue to test these hypotheses.

Increased nuclear ERK is likely to be responsible for the increased IL-2 transcription, secretion, and consequent increased proliferation observed in PEA-15−/− T cells. This conclusion is supported by studies showing that ERK1/2 is required for IL-2 transcription (16, 34, 35). Moreover, blocking IL-2 overcame the increased proliferation of PEA-15−/− T cells. In addition, excess IL-2 equalized proliferation of wild-type and PEA-15−/− T cells. These data indicate that ERK is required for the hyperproliferative phenotype of PEA-15−/− mice and that this phenotype is secondary to increased IL-2 secretion. These data also suggest that PEA-15 does not affect functions downstream of IL-2 receptor activation.

PEA-15 belongs to the family of DED proteins, which includes FADD, FLIP, and caspase 8. These proteins have been traditionally associated with regulation of apoptosis; however, they have critical functions in both apoptosis and proliferation of T cells (3, 36, 37). PEA-15 binds to FADD and has been proposed to exert an antiapoptotic function by preventing the formation the death initiation signaling complex (DISC), by either blocking FADD or caspase 8 recruitment (22, 23). However, other investigators found that while PEA-15 inhibits Fas-mediated apoptosis, it increases tumor necrosis factor receptor-1 (TNF-R1)-mediated apoptosis in the same cell type in a ligand-dependent manner. Moreover, no evidence showed interaction between PEA-15 and caspase-8 in these experiments (38). In the present study, we evaluated whether the hyperproliferative phenotype of PEA-15−/− T cells resulted from a defect in apoptosis. We did not see any difference in apoptotic rates between PEA-15−/− and wild-type T cells on TCR stimulation, nor with other proapoptotic stimuli or during activation-induced cell death. In addition, the analysis of proliferation performed by CFSE staining confirmed that PEA-15−/− T cells are hyperproliferative. These results do not support a general pro- or antiapoptotic function of PEA-15 in the T-cell compartment, although we cannot exclude the possibility that PEA-15 might regulate apoptosis in a different context or tissue.

DED proteins are required for the survival and proliferation of T cells (39). For example, the analysis of knockouts of DED proteins, such as cFLIP, FADD, or caspase-8, revealed proliferative defects, rather than a phenotype related to either pro- or antiapoptotic functions (40). In all cases, the ablation of the DED protein led to decreased proliferation and IL-2 secretion (36). FADD is, at the same time, proapoptotic, through recruitment and aggregation of caspases, and proproliferative, presumably through ERK activation (41); however, the mechanism for the latter function is still unknown. It is an intriguing possibility that PEA-15 may regulate proliferation and apoptosis by controlling both FADD functions. Interestingly, PEA-15 has two phosphorylation sites, and we have shown that phosphorylation of PEA-15 decreases PEA-15 binding to ERK and increases its affinity for FADD, thereby enhancing PEA-15 antiapoptotic function (2). In particular, we reported previously that in vitro phosphorylation of PEA-15 at serine 104 impairs ERK binding (2). Moreover, phosphomimetic PEA-15 constructs in which serines 104 and 116 are replaced by aspartic acid residues do not bind as well to ERK (27). Similarly, we show here that CD3/CD28 stimulation of purified splenic T cells or Jurkat T cells leads to an immediate drop in ERK binding to PEA-15 and concomitant increase in phosphorylation of PEA-15 at serine 104. Phosphorylation at serine 116 decreases during this same timeframe. Phosphorylation at serine 104 can be mediated by PKCs, while phosphorylation at serine 116 is mediated by AKT and Cam kinase II (42). Thus, on CD3/CD28 activation of T cells, phosphorylation of PEA-15 at serine 104 by PKC might release ERK from PEA-15 and allow it to translocate into the nucleus (Fig. 7). In summary, in our present study, we have identified a previously unknown regulatory function of PEA-15 in TCR signaling, which may alter the physiopathology of the immune response.

Acknowledgments

The authors thank Dr. Hervé Chneiweiss (Centre Paul Broca, Paris, France) for providing the original PEA-15−/− mice. The authors thank E. Collier for expert technical assistance and Dr. P. Hoffmann for helpful suggestions. The authors also thank Drs. Y. Shimizu and M. Matter for critically reviewing this manuscript. The authors thank the staff of the University of Hawaii at Manoa Centers of Biomedical Research Excellence (COBRE; P20RR018727) and Research Centers in Minority Institutions (RCMI; G12RR003061), National Center for Resource Resources, NIH flow cytometry core facility for excellent technical assistance. G.W. is supported by the New Jersey Commission on Cancer Research, grant 08-1089-CCR-EO. This work was supported by the National Cancer Institute of the NIH (CA03849 to J.W.R.). S.P. designed and performed experiments, analyzed the data, and wrote the manuscript; H.R., M.J.C., F.J.S., and E.L.F. designed and performed experiments and analyzed the data; J.E.G. and J.O-A. performed experiments; J.W.R., G.W., and A.S.S. designed experiments, analyzed the data and edited the manuscript. The authors declare that they have no competing financial interests.

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