Expression of the transcription factor ELF4 is regulated by the mTOR pathway in CD8⁺ T cells

Abstract

T cell receptor (TCR) activation inhibits expression of the ELF4 and KLF4 genes to release naïve CD8⁺ T cells from their quiescent state. Here we show that ELF4 controls the ERK-mediated proliferative response by maintaining normal levels of DUSP1 and DUSP5 in CD8⁺ T cells. In activated CD8⁺ T cells, the mTOR pathway inhibits ELF4 and KLF4 expression downstream of ERK and PI3K signaling. Our findings demonstrate that rapamycin could be used to modulate expression of this transcriptional network involved in cell cycle regulation.

Introduction

The quiescence of naïve T cells is actively regulated by cell-intrinsic factors (i.e. Foxo1, Tob, NFATc2, KLF2 and ELF4/KLF4) to avoid spontaneous proliferation in response to weak signals (1-5). We recently reported that ELF4 inhibits T cell proliferation by activating expression of the cell cycle inhibitor KLF4 downstream of T cell receptor (TCR) signaling (4). Consequently, loss of ELF4 leads to enhanced homeostatic proliferation of naïve CD8⁺ T cells and to increased frequency of memory CD8⁺ T cells after vaccination with peptide-pulsed dendritic cells (4). The identification of signals that suppress this restraint of proliferation is important to improve long-lasting immunological protection induced by vaccination and to better understand the pathogenesis of T-cell leukemias (6, 7).

The transcription factor ELF4 is a member of the Ets family of proteins that negatively regulates quiescence of hematopoietic stem cells (8). In contrast to hematopoietic stem cells, ELF4 expression is downregulated after TCR activation to induce proliferation in CD8⁺ T cells (4). Despite the importance of ELF4 for the maintenance of T cell quiescence and frequency of memory T cells, the control of ELF4 suppression by TCR signaling remains to be elucidated. Thus, the goals of this study were to investigate how ELF4 modulates TCR signaling and how TCR activation represses ELF4 expression.

The mammalian target of rapamycin (mTOR) is a protein kinase that regulates mRNA translation and energy sensing signals associated with cell growth and proliferation (9). In addition to the well-known immunosuppressive function of rapamycin, recent data indicate that inhibition of the mTOR pathway increases the number of memory CD8⁺ T cells when administered at low doses during the expansion of antigen-specific T cells (10). In addition, the mTOR pathway is involved in the metabolic switch of effector cells in their transition to resting memory T cells (11). However, the genes regulated by mTOR that control proliferation and development of memory have yet to be identified yet.

Here we have demonstrated that ELF4 is involved in T cell activation at two levels. First, the mTOR pathway inhibits ELF4 expression in activated CD8⁺ T cells, releasing them from their quiescent state. Second, ELF4 restricts ERK-mediated activity upon activation by maintaining the pool of the dual-specificity phosphatases DUSP1 and DUSP5 at steady state. Taken together, these data suggest that rapamycin could be used to restore ELF4 gene expression in CD8⁺ T cells to modulate immune response.

Materials and Methods

Mice

Elf4^−/− mice were obtained from S. Nimer. C57BL/6 (B6) and OT-1 TCR transgenic mice were purchased from Jackson Laboratories. All mice were bred and maintained under specific pathogen-free conditions at Baylor College of Medicine. All experiments were performed with the approval of the Institutional Animal Care and Usage Committee of Baylor College of Medicine.

T cell proliferation

CD8⁺ T cells were purified from spleens using the BD-IMag magnetic-bead separation system (BD Biosciences). Purified CD8⁺ T cells were labeled with 4 μM of CFSE (Invitrogen) in PBS with 0.1% BSA at 37 °C for 10 min. For in vitro stimulation, CD8⁺ T cells were cultured in anti-CD3-coated 96-well plates (Bio-Coat, BD Biosciences) at a density of 1 × 10⁵ cells/well in X-VIVO medium (Lonza) containing 5% T-Stim (BD Biosciences) and 2 μg/ml of anti-CD28 (BD Biosciences). CFSE dilution was analyzed by flow cytometry 3 or 4 days later using the FACSCanto instrument (BD Biosciences) and FlowJo software (Tree Star). The proliferation index was calculated using FlowJo or ModFit software (Verity).

Zap70 phosphorylation assay

Purified CD8⁺ T cells (1 × 10⁷ cells/ml in PBS) were incubated at 4 °C for 20 min with different doses of anti-CD3 (BD Biosciences). For TCR crosslinking, cells were washed and incubated at 4 °C for 20 min with 20 μg/ml of anti-hamster IgG (eBioscience) in PBS followed by incubation at 37 °C. Activated cells were fixed with Cytofix/Cytoperm solution (BD Biosciences) and intracellularly stained with phycoerythrin-anti-phospho-Zap70 (BD Biosciences). The mean fluorescence intensity (MFI) was calculated by FlowJo software.

Immunoblot analysis of activated T cells

Activated CD8⁺ T cells were lysed directly with LDS Sample Buffer (Invitrogen) and loaded onto NuPAGE Bis-Tris gels (Invitrogen). Immunoblots were incubated with anti-ERK1/2 (Cell Signaling Technology), anti-phospho-ERK1/2 (Cell Signaling Technology), anti-DUSP1 (Abcam), anti-DUSP5 (Sigma-Aldrich), anti-p38 (Cell Signaling Technology), anti-phospho-p38 (Cell Signaling Technology), anti-JNK1 (Santa Cruz), anti-phospho-JNK1/2 (Cell Signaling Technology), anti-IKKα/β (Santa Cruz), anti-phospho-IKKα/β (Cell Signaling Technology), anti-β-actin (Sigma-Aldrich), or rabbit anti-ELF4 serum. Anti-rabbit IgG or anti-mouse IgG conjugated with peroxidase (GE Healthcare) was used as the secondary antibody. Band intensities were analyzed using the FluorChemHD2 chemi-imager (Alpha Innotech).

Inhibition of T cell activation and quantitative real-time PCR

CD8⁺ or CD4⁺CD25⁻ T cells were purified from B6 mice and cultured in anti-CD3-coated 96-well plates (Bio-Coat, BD Biosciences) at 1 × 10⁵ cells/well in X-VIVO medium in the presence of Cyclosporin A (Calbiochem), PD98059 (Calbiochem), LY294002 (Sigma-Aldrich) or rapamycin (Sigma-Aldrich). After 24 h of in vitro culture, total RNA was isolated using the RNeasy Mini kit (Qiagen). Then, cDNA was synthesized from 100-500 ng RNA using random hexamer primers and a SuperScript III kit (Invitrogen). Quantitative real-time PCR was performed using LightCycler FastStart DNA Master SYBR Green I (Roche) as specified by the manufacturer. Primer sequences for PCR were as follows: β-actin forward, 5′-GTGGGCCGCTCTAGGCACCA-3′ and reverse, 5′-CGGTTGGCCTTAGGGTTCAGGGG-3′; ELF4 forward, 5′-CGGAAGTGCTTTCAGACTCC-3′ and reverse, 5′-GGTCAGTGACAGGTGAGGTA-3′; and KLF4 forward, 5′-CTGAACAGCAGGGACTGTCA-3′ and reverse, 5′-GTGTGGGTGGCTGTTCTTTT-3′. Reactions were run on a Smart Cycler (Cepheid) or Mx3005P (Stratagene). Relative expression was calculated by normalization to β-actin.

Results and Discussion

ELF4 expression is regulated via the mTOR pathway

We recently showed that Elf4^−/− CD8⁺ T cells proliferate more than Elf4^+/+ CD8⁺ T cells in response to homeostatic and antigen-driven stimuli (4). This increased proliferation correlated with downregulation of ELF4 and KLF4 gene expression following TCR activation (Fig. 1A and D). However, it is not clear how ELF4 expression is suppressed by TCR signaling and whether ELF4 controls proliferation in naïve CD8⁺ T cells solely by activating KLF4. Thus, we examined several signaling pathways downstream of TCR to evaluate their involvement in the inhibition of ELF4 transcription.

FIGURE 1.

The mTOR pathway represses ELF4 gene expression. (A) ELF4 mRNA expression was measured by qPCR in Elf4^+/+ (+/+) and Elf4^−/− (−/−) CD8⁺ T cells activated with anti-CD3 and anti-CD28 (α-CD3/CD28). PI, proliferation index; ND, not detectable. (B) ELF4 mRNA expression was measured 24 hours after polyclonal TCR activation of CD8⁺ T cells with plate-bound α-CD3 in the presence of different concentrations of rapamycin (Rapa). (C) Immunoblot analysis of ELF4 expression in activated CD8⁺ T cells in the presence of Rapa. β-actin, loading control. (D) ELF4 and KLF4 mRNA expression in CD8⁺ T cells activated with α-CD3 in the presence of Rapa. (E) ELF4 mRNA expression in CD8⁺ T cells activated with α-CD3, α-CD28 or α-CD3/CD28 in the presence of Rapa. (F) ELF4 and KLF4 mRNA expression in CD4⁺ CD25⁻ T cells activated in the presence of Rapa. Two-tailed Student's t-test (*, P < 0.05; **, P < 0.01; n = 3). Data are representative of three independent experiments.

The mammalian target of rapamycin complex 1 (mTORC1) has a dual function as an immunosuppressor and an immunomodulator in a dose- and time-dependent manner. We showed that suppression of ELF4 gene expression in CD8⁺ T cells was blocked with the mTOR inhibitor rapamycin at both the transcript and the protein level (Fig. 1B and C). KLF4 mRNA expression was measured after TCR activation in the presence of rapamycin to confirm that KLF4 induces cell cycle arrest downstream of ELF4 as previously described (4). Indeed, the same dose of rapamycin released the inhibition of both ELF4 and KLF4 gene expression in activated CD8⁺ T cells (Fig. 1D). However, rapamycin was unable to block repression of ELF4 gene expression when CD8⁺ T cells were activated by TCR crosslinking and anti-CD28 (Fig. 1E). This finding indicates that TCR-mediated activation of CD8⁺ T cells leads to a weaker suppression of the ELF4/KLF4-mediated proliferation brake. Co-stimulation elicited additional signals, independent of mTOR, which inhibited ELF4 expression and allowed full activation and proliferation. We also examined ELF4 expression in OT-1 CD8⁺ T cells stimulated with splenocytes pulsed with OVA peptide and observed that ELF4 downregulation was not inhibited by rapamycin (even at higher doses) or LY294002 (supplemental Fig. 1). However, a combination of rapamycin and LY294002 was required to repress ELF4 downregulation. A combination of TCR and CD28 signals is needed for PI3K and Akt activation to release resting naïve CD8⁺ T cells from quiescence (G0 to G1 transition) (9). A proper balance of signals ultimately drives activation of T cells, whereas the lack of co-stimulation could lead to anergy. Our data show that both receptors trigger additive signals to fully repress the proliferation brake mediated by the transcription factors ELF4 and KLF4, thereby ensuring differentiation to effector T cells. The PI3K and mTOR pathways are also involved in the trafficking of activated lymphocytes by repressing KLF2 (12). Thus, signaling derived from TCR and CD28 can release naïve T cells from their quiescent state and modulate their mobilization to peripheral tissues (downregulating CD62L and CCR7) by repressing the transcription factors ELF4, KLF4 and KLF2. Finally, we investigated whether mTOR mediates a similar mechanism in CD4⁺ T cells. In contrast to CD8⁺ T cells, ELF4 repression, but not KLF4, was prevented only by increasing the dose of rapamycin in CD4⁺ CD25⁻ T cells (Fig. 1F). This finding suggests that the inhibitory pathway mediated by ELF4 and KLF4 is not active in CD4⁺ T cells. The functional role of ELF4 and KLF4 in the proliferation and differentiation of CD4⁺ T cells is currently being investigated.

ELF4 regulates ERK-mediated activity in CD8⁺ T cells

Loss of ELF4 results in increased expression of cyclins D1 and D3 and reduced levels of p21 (4). In addition, T cell activation with different concentrations of cognate antigen suggests a lower threshold of activation (4). To further address this issue, we measured tyrosine phosphorylation of Zap70, which is an early event following TCR ligation (13). We found no significant differences in the kinetics or the threshold of phosphorylation (Fig. 2A). We next examined activation of protein kinases downstream of Zap70 phosphorylation by immunoblots. Both Elf4^+/+ and Elf4^−/− CD8⁺ T cells showed a peak of phosphorylation of IKKα/β, JNK1/2, P38 and ERK at 5 min after TCR crosslinking (Fig. 2B). The activation kinetics of IKKα/β, JNK1/2 and p38 did not show any significant difference between Elf4^+/+ and Elf4^−/− CD8⁺ T cells (Fig. 2B). Conversely, Elf4^−/− CD8⁺ T cells displayed increased levels of phospho-ERK2 compared to Elf4^+/+ CD8⁺ T cells after 10 min of activation (Fig. 2B). A closer examination of the kinetics of ERK activation revealed a significant delay in the dephosphorylation of ERK in Elf4^−/− CD8⁺ T cells (Fig. 2C and D). In a global gene expression analysis, we previously identified a significant reduction of the dual-specificity phosphatases, DUSP1 and DUSP5, in Elf4^−/− CD8⁺ T cells (5.9- and 7.1-fold reduction, respectively) (4). Consistent with this observation, immunoblot analysis showed that levels of DUSP1 and DUSP5 were significantly reduced in Elf4^−/− CD8⁺ T cells (Fig. 2E), suggesting that a sustained ERK activation may contribute to the deregulated control of proliferation observed in Elf4^−/− CD8⁺ T cells. Among a large family of dual-specificity phosphatases, DUSP1, DUSP2, DUSP5 and DUSP10 play important roles in the immune system, particularly in T cell development and function (14-18). Even though transcription of DUSPs increases upon T cell activation (19), in our experiment, the protein levels remained low within 10 min after activation of CD8⁺ T cells (Fig. 2E).

FIGURE 2.

ELF4 restricts ERK activity in CD8⁺ T cells. (A) Zap70 phosphorylation was measured by flow cytometry in Elf4^+/+ and Elf4^−/− CD8⁺ T cells activated with 10 μg/ml α-CD3 (left) or following 2 min activation with the indicated concentration of α-CD3 (right). MFI, mean fluorescent intensity. (B, C) Phosphorylation of IKKα/β, JNK1/2, p38 and ERK proteins in activated Elf4^+/+ and Elf4^−/− CD8⁺ T cells was monitored by immunoblots. (D) p-ERK2 expression was normalized to total ERK2 by densitometry of immunoblots. (E) Immunoblot analysis of DUSP1 and DUSP5 expression. β-actin, loading control. Data are representative of at least two independent experiments.

To correlate ERK activation with cell proliferation, we measured cell division by CFSE dilution in the presence of Cyclosporin A or PD98059, which are known to selectively inhibit calcium/calmodulin and ERK but not p38 or JNK in the MAPK pathway respectively (20). Inhibition of the ERK pathway blocked proliferation in Elf4^−/− CD8⁺ T cells compared to wild type cells, indicating that the hyperproliferation of Elf4^−/− CD8⁺ T cells was due to prolonged ERK activation (supplemental Fig. 2). Of note, complete restoration of ELF4 protein expression was not achieved in this experiment (supplemental Fig. 2). In contrast, Elf4^−/− CD8⁺ T cells proliferated more than their wild type counterparts in the presence of Cyclosporin A (supplemental Fig. 2). Addition of Cyclosporin A during activation did not prevent downregulation of ELF4 expression (Fig. 3), suggesting that ELF4 function is independent of the Ca²⁺/calcineurin pathway.

FIGURE 3.

ERK and PI3K pathways mediate repression of ELF4 gene expression. ELF4 mRNA expression was measured by qPCR in Elf4^+/+ CD8⁺ T cells activated with α-CD3 or α-CD3/CD28 in the presence of PD98059, LY294002 (LY), rapamycin (Rapa) or a combination of Rapa and LY. Data are representative of two independent experiments. mTORC1, mTOR complex 1.

Mechanism of control of T cell quiescence

Having demonstrated that ELF4 gene expression upon TCR activation was blocked by treatment with rapamycin (Fig. 1), we focused on the signaling pathways that activate mTOR. The use of ERK and PI3K inhibitors prevented downregulation of ELF4 transcripts in T cells activated by plate-bound anti-CD3 (Figure 3). Co-stimulation with anti-CD28 prevented restoration of ELF4 transcripts by treatment with rapamycin, indicating that activation of PI3K via CD28 is able to inhibit ELF4 expression independent of mTOR (Fig. 1E). Thus, treatment with rapamycin or LY294002 alone was unable to fully restore ELF4 gene expression when CD8⁺ T cells were activated with anti-CD3 and anti-CD28 (Fig. 3). Furthermore, an additive effect is evident from the reversion of the inhibitory effect by activation in the presence of both rapamycin and LY294002.

Collectively, we propose that in resting naïve T cells, ELF4 has a dual function by inducing quiescence via KLF4 and maintaining normal levels of DUSP1 and DUSP5 to modulate the extent of proliferation upon activation (Fig. 4). Thus, DUSP levels dictate the duration of activation and, as a consequence, the extent of proliferation. ELF4 can therefore prevent activation in response to weak signals by maintaining normal levels of DUSP1 and DUSP5. In activated T cells, sustained ERK activation triggers downregulation of ELF4 and KLF4 via the mTOR pathway (Fig. 4). Co-stimulation reinforces the repression of ELF4 by a mechanism independent of mTOR (Fig. 4). Distinct functions of ELF4 in resting versus activated CD8⁺ T cells suggest a temporal and spatial mechanism that modulates the immune response to infection or vaccination.

FIGURE 4.

Model of regulation of CD8⁺ T cell proliferation by the transcription factor ELF4. In resting T cells, ELF4 maintains normal levels of DUSP1 and DUSP5 to modulate the extent of proliferation upon activation. Engagement of TCR and co-stimulation led to ERK- and PI3K-mediated activation of the mTOR pathway and inhibition of ELF4 and KLF4 expression in activated T cells.

Abbreviations used in this paper

ELF4

E74-like factor 4

KLF4

Krüppel-like factor 4

mTOR

mammalian target of rapamycin

FOXO1

Forkhead box O1

Tob

Transducer of ERBB1/2