The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, D.
Right arrow Articles by McMahon, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, D.
Right arrow Articles by McMahon, M.
The Journal of Immunology, 1999, 163: 5796-5805.
Copyright © 1999 by The American Association of Immunologists

Sustained Activation of the Raf-MEK-ERK Pathway Elicits Cytokine Unresponsiveness in T Cells1

Dan Chen2,*, Victoria Heath{dagger}, Anne O’Garra{dagger}, James Johnston* and Martin McMahon3,*

Departments of * Cell Signaling and {dagger} Immunobiology, DNAX Research Institute, Palo Alto, CA 94304


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Activation of T cells via the TCR and other costimulatory receptors triggers a number of signaling cascades. Among them, the Ras-activated Raf-mitogen-activated protein/extracellular signal-related kinase (ERK) kinase (MEK)-ERK signaling cascade has been demonstrated to be crucial for both T cell development and activation. It has previously been demonstrated that high doses of Ag or anti-CD3 mAb are able to induce in T cells a nonresponsive state to subsequent treatment with cytokines such as IL-2. The precise biochemical mechanisms underlying this effect are not fully characterized. In this study, we demonstrate that cytokine nonresponsiveness is accompanied by the induction of the cyclin-dependent kinase inhibitor p21Cip1 that is mediated, at least in part, by the activation of the Raf-MEK-ERK pathway. Furthermore, we demonstrate that selective activation of the Raf-MEK-ERK signaling pathway in T cells is sufficient to induce cytokine nonresponsiveness in both a T cell clone and naive primary T cells. In this case, nonresponsiveness is accompanied by the induction of p21Cip1 and the prevention of p27Kip1 down-regulation, leading to inhibition of cyclin E/cyclin-dependent kinase 2 activity. These data suggest that anti-CD3 mAb-induced cytokine nonresponsiveness may be a consequence of hyperactivation of the Raf-MEK-ERK pathway, leading to alterations in the expression of key cell cycle regulators. These observations may provide a novel insight into the mechanisms of induction of peripheral tolerance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lymphocyte development and activation involve continuous integration of signals delivered through the T or B cell Ag receptor and a variety of coreceptors (1, 2, 3, 4). In addition to the intrinsic properties determined by developmental or activation stages, subtle changes in environmental stimuli may result in significantly different cellular responses (5, 6, 7, 8, 9). In general, positive signaling that confers immune reactivity is triggered by the interplay and concerted action of different signaling pathways delivered through the Ag receptor and costimulatory molecules (10, 11, 12, 13, 14). On the other hand, negative signals that cause peripheral tolerance or anergy are thought to be delivered through the Ag receptor in the absence of appropriate costimulatory signals (10, 15, 16, 17, 18, 19, 20). Cytokine unresponsiveness accompanied by a failure of appropriate mitogenic responses in lymphocytes has been reported in certain models of anergy, but appears not to be due to either the lack of IL-2 or IL-2R expression. For instance, in vitro unresponsiveness in T cell clones and hybridomas can be achieved by treating cells with a high dose Ag or anti-CD3 mAb or in B lymphoma cell lines with high dose anti-IgM (21, 22, 23, 24, 25, 26). In transgenic models, persistent or chronic triggering of T or B cells through autoreactive Ag receptors, in the absence of appropriate costimulatory signals, has also been shown to induce anergy characterized by poor proliferative responses to IL-2 and IL-4 as well as to stimulation through the Ag receptors (13, 14, 27). Notably, removal of such chronic stimuli or disengagement of the Ag receptor reverses the anergic state, restoring T or B cells to full responsiveness to subsequent stimulation (28, 29). The signal transduction pathways that confer these effects on lymphocytes are not fully understood.

Engagement of the TCR leads to the activation of membrane-associated Src family protein tyrosine kinases. The subsequent phosphorylation of immunoreceptor tyrosine-based activation motifs on various accessory chains of the TCR leads to the recruitment and activation of the ZAP-70 protein tyrosine kinase. Coordinate activation of ZAP-70 and other protein tyrosine kinases leads to the tyrosine phosphorylation of a number of adaptor proteins, leading to the activation of downstream signaling pathways that serve to transmit the signal throughout the cell. Such downstream effectors include phospholipase C{gamma}1, Ras, and phosphatidylinositol 3 (PI3)4-kinase. Phospholipase C{gamma}1 elicits the production of diacylglycerol and inositol 1,4,5-triphosphate, which in turn participate in the activation of protein kinase C and the release of intracellular calcium stores, respectively. Ras, on the other hand, participates in the regulation of the c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) signaling pathways. Finally, PI3-kinase elicits the production of phosphatidylinositides, among the effects of which include activation of the protein kinase Akt/PKB. The coordinate activation of these pathways serves to regulate cytosolic and nuclear events involved in immune cell effector functions (30, 31, 32, 33). Among the different MAP kinase cascades, the Ras-activated Raf-MEK-ERK pathway has been shown to be important in the differentiation of T cells, but this pathway has also been reported to be activated in anergic B cells (29, 34, 35, 36, 37).

Using conditionally active forms of the protein kinase Raf-1 ({Delta}Raf-1:ER), we and others have previously shown that sustained activation of the ERK pathway can induce nonresponsiveness to cytokines and growth factors, which is accompanied by cell cycle arrest, in a number of primary, established, and tumor cell types (38, 39, 40, 41, 42, 43, 44). In mouse NIH 3T3 and in human IMR-90 fibroblasts, the level of MAP kinase activation determines whether cells will proliferate or be arrested in the G1 phase of the cell cycle. In general, low levels of MAP kinase activation promote cell proliferation, whereas higher levels promote cell cycle arrest that appears to be mediated by the induced expression of CDK inhibitors (CKI) such as p21Cip1 and p16INK4a (38, 39). We therefore decided to test whether nonresponsiveness in T cells involves activation of the Raf-MEK-ERK pathway and whether this could be mimicked by selective activation of this pathway in T cells.

In this work, we demonstrate that the cytokine nonresponsiveness induced in T cells in response to high doses of anti-CD3 mAb is inhibited, in part, by a specific and selective inhibitor of MEK, PD098059. This observation is further supported by the fact that conditional activation of the ERK pathway induced a cytokine unresponsive state that was similar to that induced by treatment with high dose anti-CD3 mAb. Biochemical analyses revealed that the nonresponsive state correlates with the robust induction of the CKI p21Cip1 and inhibition of cyclin E/CDK2 activity. Moreover, anti-CD3 mAb-induced expression of p21Cip1 was MEK1 dependent. These data suggest that engagement of the TCR can elicit an inhibitory signal that is most likely transmitted through the Raf-MEK-ERK pathway. Such mechanisms may participate in the generation of negative signals that lead to the induction of peripheral tolerance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture

D10G4.1 cells were cultured in the presence of IL-2 (10 ng/ml) and stimulated with conalbumin and irradiated splenocytes from CBA/J mice (The Jackson Laboratory, Bar Harbor, ME) every 2 wk (45). Primary naive T cells were isolated from DO11.10 transgenic mice that express TCR specific for the chicken OVA323–339 peptide in the context of I-Ad (46). Isolation and maintenance of the naive T cells were performed as described elsewhere (47). Briefly, splenocytes were enriched for CD4+ cells by negative selection using magnetic bead cell sorting with a mixture of Abs to B220, Mac1, and CD8 (all Abs from PharMingen (San Diego, CA) and BioMag beads from PerSeptive Biosystems, Cambridge, MA). CD4+ Mel-14high naive T cells were further purified by fluorescence-activated cell sorting using a FACStarPlus flow cytometer (Becton Dickinson, Palo Alto, CA). Sorted naive T cells were then cultured in RPMI with 10% FCS, 50 µM 2-ME (Sigma, St. Louis, MO), 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 1 mM sodium pyruvate (JRH Biosciences, Lenexa, KS), 10 mM HEPES (Life Technologies), 100 U/ml penicillin, 100 µg/ml streptomycin (Life Technologies), and 10 ng/ml IL-2. Cells were stimulated with irradiated BALB/c splenocytes and 0.6 µM of OVA323–339 peptide (Biosynthesis, Lewisville, TX) every 7 to 10 days. The retroviral packaging cell line, Phoenix E, from Dr. Gary Nolan (Stanford University, Stanford, CA), was maintained in DMEM with 10% FCS and supplements. For T cell activation analysis, 96-well flat-bottom plates (Falcon) or 150-mm tissue culture dishes (Falcon) were coated with different concentrations of anti-CD3 mAb (145-2C11; PharMingen) overnight at 4°C and were washed three times with PBS before adding cells. The specific and selective MEK inhibitor PD098058 was a generous gift from Dr. John Lyons (Onyx Pharmaceuticals, Richmond, CA). PMA (Upstate Biotechnology, Lake Placid, NY) and ionomycin (Calbiochem, La Jolla, CA) were used at final concentrations of 20 ng/ml and 1 µM, respectively. Cells were treated with different concentrations of either 4-hydroxytamoxifen (4-HT) or ICI 182,780 (ICI), which activate the {Delta}Raf:ER fusion proteins to the same extent and with similar kinetics (data not shown).

Retroviral constructs and retroviral infection

The retroviral vector used in the experiments was a modified version of a pMX-based vector (48). The internal ribosomal entry site from encephalomyocarditis virus and the sequences encoding the enhanced green fluorescent protein (eGFP, kindly provided by Dr. Hergen Spits, the Netherlands Cancer Institute) (49) were introduced into the vector (generated and provided by Alice Mui, DNAX Research Institute, Palo Alto, CA). A kinase-active form of the {Delta}Raf-1:ER (39, 50, 51) and a similarly constructed kinase-inactive version of the protein, {Delta}Raf301:ER (39), were introduced into the vector by standard subcloning techniques (Fig. 2GoA). The human ER domain encoded in the original Raf:ER constructs was replaced by a mutated form of the mouse ER domain (ER*) that is sensitive to 4-HT and the ICI series of compounds, but is insensitive to 17-ß-estradiol and Phenol Red in the cell culture medium (52). The {Delta}Raf-1:ER* and {Delta}Raf301:ER*-encoding retrovirus constructs were used for infection of T cell clones and primary T cells.



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 2. Response of D10G4.1 cells to selective Raf activation. A, Diagram of the retrovirus constructs used to express conditionally active {Delta}Raf:ER proteins in T cells. B, D10 cells expressing {Delta}Raf-1:ER were treated with ICI or PMA for different times, and the kinetics of ERK activation was compared using a protein kinase assay, as described in Materials and Methods. C, D10 cells expressing {Delta}Raf-1:ER were treated with different concentrations of ICI or anti-CD3 mAb, and ERK activity was measured as in B. D, D10 cells expressing either {Delta}Raf-1:ER or kinase-inactive {Delta}Raf301:ER were either untreated (no tx) or treated with ethanol (ETOH), or with 300 nM ICI (ICI) or with immobilized anti-CD3 mAb ({alpha}CD3) for 16 h and stained with a PE-conjugated anti-CD69 Ab. E, {Delta}Raf-1:ER-expressing D10 cells were treated with ethanol, ICI, ICI + ionomycin (ICI + Iono), PMA, ionomycin (Iono), or PMA + ionomycin (PMA + Iono) for 4 h, as indicated. RNA samples were prepared from these cells and probed using an RNase protection assay with a panel of cytokine-specific antisense riboprobes, as indicated. As positive and negative controls, RNA prepared from PMA-treated EL4 cells and yeast tRNA, respectively, were subjected to similar analysis side by side.

 
Phoenix E cells were transfected by the lipofectamine-based method, and viral supernatants were harvested as described elsewhere (53). Retroviral infection of primary T cells was performed as described by Ferber et al. (54). Briefly, 2 days after OVA323–339/APC stimulation, primary T cells were infected with viral supernatants in the presence of 0.5 µg/ml polybrene plus IL-2. eGFP-positive cells were isolated by fluorescence-activated cell sorting 4 days after infection. Alternatively, transfected virus producing Phoenix E cells were irradiated and cocultured with D10G4.1 T cells for 1 to 2 days before expanding the cells with fresh medium. Infected T cells were selected by sorting for cells double positive for eGFP and CD4 with a FACStarPlus flow cytometer (Becton Dickinson).

RNase protection assay

D10 cells were harvested 4 h after various treatments, and RNA was extracted with Qiagen RNeasy columns. A total of 5 µg of RNA per condition was used for each assay. Multicytokine templates (PharMingen) were used to generate 32P-labeled riboprobes. Probe labeling, hybridization, RNase digestion, and final denaturing polyacrylamide gel electrophoresis were performed, as described elsewhere (55).

DNA synthesis assays

DNA synthesis was measured by plating cells on 96-well plates at 1–2 x 104 cells/well. Cells were preincubated with ICI 182,780, a gift of Dr. Alan Wakeling (Zeneca Pharmaceuticals, Macclesfield, Cheshire, U.K.) or 4-HT (Research Biochemicals) in the absence of IL-2 for 8–12 h. Cells were then treated with IL-2 (100 U/ml; DNAX) or left untreated for an additional 12 or 36 h. Methyl-[3H]thymidine was added to a final concentration of 1.5 µCi/ml for the final 4 h of culture. Incorporation of methyl-[3H]thymidine was assayed using a Skatron cell harvester and a Betaplate scintillation machine, as described previously (39).

Flow cytometry

Cell viability was measured in 96-well microtiter dishes exactly in parallel to the assays set up to measure DNA synthesis, as described above. An equal volume of PBS containing 20 µg/ml of propidium iodide was added to each well and analyzed using a FACScan II (Becton Dickinson) with CellQuest software. Three thousand individual cells were assessed for each sample. The percentage of viable cells was calculated by quantifying the propidium iodide-negative cells. Each measurement was performed in triplicate.

For cell cycle analysis, cells were fixed in solution containing 0.1% sodium citrate, 0.1% Nonidet P-40, and 50 µg/ml of propidium iodide for 30 min on ice and assayed by a FACScan II. Alternatively, cells cultured in IL-2 were labeled with 0.1 mg/ml 5-bromo-2'-deoxyuridine (BrdU; Sigma) for 30 min, washed with warm PBS, and then cultured under different conditions. At different time points, cells were collected, fixed, and stained with a FITC-coupled anti-BrdU Ab and propidium iodide, as described in the manufacturer’s protocol (Boehringer Mannheim, Indianapolis, IN). Samples were analyzed using a FACScan II.

Preparation of cell lysates and Western blotting

Cells were harvested and lysed in ELB buffer (50 mM HEPES, pH 7, 150 mM NaCl, and 0.1% (v/v) Nonidet P-40 with standard protease and phosphatase inhibitors (50)). Abs for immunoprecipitation and Western blotting were as follows: anti-ERK1, anti-ERK2, anti-cyclin E, anti-cyclin A, and anti-CDK2 (all from Santa Cruz Biotechnology, Santa Cruz, CA); anti-p21Cip1 (PharMingen); anti-p27Kip1 (Transduction Laboratories, Lexington, KY); anti-cyclin D2 was a gift of Drs. David Parry and Emma Lees (DNAX). HRP-conjugated anti-mouse and anti-rabbit Igs (Amersham, Arlington Heights, IL) were used at 1/5,000 to 1/10,000 dilutions. Western blots were visualized using the enhanced chemiluminescence technique (Amersham).

Protein kinase assays

Antisera were used to immunoprecipitate ERK1 and ERK2 from 100–200 µg of cell lysate, and a protein kinase assay was performed using myelin basic protein as a substrate according to published protocols (50). The kinase activity of ERK1 and ERK2 was quantitated using a Molecular Dynamics Storm PhosphorImager (Sunnyvale, CA).

Cyclin E-associated kinase assays were performed as described elsewhere (39). Briefly, 500 µg of cell lysate per sample was subject to immunoprecipitation with an anti-cyclin E polyclonal Ab (Santa Cruz Biotechnology). Immune complexes, collected with protein A-Sepharose 4B (Sigma), were washed three times with lysis buffer and once with kinase reaction buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 1 mM DTT). The kinase reaction mixtures were incubated in 20 µl of the same buffer containing 10 µCi of [{gamma}-32P]ATP, 12.5 µM ATP, and 2 µg histone H1 (Boehringer Mannheim) as substrates for 30 min at 30°C. The kinase reactions were denatured in SDS sample buffer and analyzed by SDS-PAGE and transferred onto an Immobilon P membrane (Millipore, Bedford, MA). Histone H1 phosphorylation was quantitated using a Molecular Dynamics Storm PhosphorImager. Proteins in the immune complexes were detected by probing the membrane with appropriate Abs, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The Raf-MEK-ERK pathway is involved in anti-CD3 mAb-mediated IL-2 unresponsiveness

For this study, primary naive CD4+ T cells as well as various T cell clones were utilized. However, we focused primarily on a Th2 clone D10G4.1 (D10) as a model cell line in which to dissect the biochemical mechanism of anti-CD3 mAb and Raf-induced cytokine unresponsiveness. Treatment of D10 cells with increasing concentrations of anti-CD3 mAb induced a bell-shaped mitogenic response with approximately a 50% reduction in DNA synthesis when more than 1 µg/ml of immobilized anti-CD3 mAb was utilized (Fig. 1Go, top panels). Furthermore, the inhibition of cell proliferation was not due to a lack of cytokine expression, because exogenously added IL-2 did not abrogate the inhibition of DNA synthesis observed at high concentrations of anti-CD3 mAb. Furthermore, increased doses of anti-CD3 mAb had no obvious effect on cell survival, as cell viability remained greater than 65% even at the highest dose of anti-CD3 mAb (Fig. 1Go, bottom panels). Therefore, the decreased thymidine incorporation observed at high doses of anti-CD3 mAb was not due to cell death. Similar inhibition of DNA synthesis was observed in primary naive CD4+ T cells stimulated with varying doses of anti-CD3 mAb (A. O’Garra, data not shown).



View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. MEK activity is required for the inhibition of DNA synthesis by high dose anti-CD3 mAb. In the top panels, D10G4.1 cells were cultured in the presence of immobilized anti-CD3 mAb in either the presence of DMSO (solvent control, {square}) or 10 µM PD098059 (PD) ({blacksquare}) for 24 h. IL-2 was added for the final 12 h of the experiment (right panel), and [3H]thymidine was added for the final 4 h of the experiment before harvesting and measuring [3H]thymidine incorporation. In the bottom panels, cell viability was assessed in duplicate assays by propidium iodide exclusion and quantitated using a FACStarPlus. Data are representative of two independent experiments.

 
Based on previous observations that activation of the MAP kinase pathway is able to induce nonresponsiveness to cytokines and growth factors in nonlymphoid cells (38, 39, 40, 41, 42, 43, 44), we decided to test whether the ERK pathway might be involved in the cell cycle inhibition elicited by high dose anti-CD3 mAb treatment of T cells (D10, Th2 cells). To investigate this possibility, we utilized a selective and specific MEK inhibitor, PD098059 (56). The addition of 10 µM PD098059 to the D10 cell culture partially abrogated the inhibition of thymidine incorporation observed when D10 cells were treated with high concentrations of anti-CD3 mAb (Fig. 1Go). Similar results were obtained in two independent experiments using D10 cells. The inability to fully reconstitute the levels of DNA synthesis with higher doses of PD098059 as shown in Fig. 1Go was most likely a reflection of the fact that in addition to its ability to induce cell cycle arrest, the Raf-MEK-ERK pathway is also required for cell proliferation. Indeed, the use of higher concentrations of PD098059 significantly inhibited the cell proliferation induced by low concentrations of anti-CD3 mAb (data not shown). These data are consistent with previous observations in both human and mouse fibroblasts, in which it has been shown that the Raf-MEK-ERK pathway is required for normal cell proliferation, but is also capable of eliciting cell cycle arrest and senescence when activated in a sustained fashion (38, 39, 43). These data suggest that in T cells, the Raf-MEK-ERK pathway is required for normal cell proliferation as well as in the induction of cytokine nonresponsiveness by anti-CD3 mAb.

Activation of Raf elicits cytokine nonresponsiveness and cell cycle arrest in T cells

To determine whether activation of the Raf-MEK-ERK pathway might elicit IL-2 unresponsiveness in T cells, we expressed a conditionally active form of Raf ({Delta}Raf-1:ER*) in two T cell clones (D10 and HDK1) using retroviral infection. Retroviral constructs encoding either a catalytically active form of the kinase domain of human Raf-1 ({Delta}Raf-1) or its kinase-inactive counterpart ({Delta}Raf301) were fused to a modified form of the hormone-binding domain of mouse estrogen receptor that is insensitive to 17-ß-estradiol, but retains sensitivity to 4-HT and the ICI series of compounds, as described in Materials and Methods. {Delta}Raf:ER expression constructs were introduced into T cells by retrovirus infection. To select for infected cells, the retroviral construct was also engineered to encode an enhanced form of GFP. Cells infected with these viruses express {Delta}Raf:ER and eGFP proteins from a single bi-cistronic mRNA with the translation of the 5' coding region for eGFP promoted by the presence of the internal ribosomal entry site (IRES) from encephalomyocarditis virus. Virus-infected cells were FACS sorted according to the expression of eGFP, which allowed the isolation of cells also expressing the appropriate {Delta}Raf:ER proteins.

To maximize the efficiency of virus infection, D10 T cells were cocultured with the virus-producing cell line, as described in Materials and Methods, and infected T cells were isolated by FACS sorting cells that were positive for the expression of eGFP and the T cell marker CD4. We have previously demonstrated that the level of activation of {Delta}Raf-1:ER is dose dependent on the amount of hormone added to the cell culture media (50). To characterize these cells, we first compared the kinetics of ERK activation upon the activation of {Delta}Raf-1:ER by ICI 182,780 (ICI) or by the addition of the phorbol ester PMA. As shown in Fig. 2GoB, ERK activity was induced upon either the activation of {Delta}Raf-1:ER or PMA treatment, and the maximal levels observed were comparable between the two stimuli. However, {Delta}Raf-1:ER-induced ERK activity was maintained at elevated levels, whereas that induced by PMA showed transient kinetics of induction returning to low levels 24 h after PMA addition. In addition, the level of induced ERK activity could be titrated by varying the ICI and anti-CD3 mAb concentrations, which provided a convenient way to correlate biological responses with the levels of ERK activity (Fig. 2GoC). As expected, D10 cells expressing equivalent amounts of the kinase-inactive {Delta}Raf301:ER displayed no ERK activation in response to ICI treatment (data not shown), which served as a negative control for all subsequent experiments. In addition, the kinase-inactive {Delta}Raf301:ER did not display any evidence of a dominant-negative effect either in the absence or presence of 4-HT (data not shown).

To determine the biological consequences of Raf-MEK-ERK activation in these cells, we determined whether any T cell activation markers were induced upon {Delta}Raf-1:ER activation. Initially, we examined the expression of CD69, the induction of which has been reported to occur in a Ras-dependent manner (57). Similar to the controls, untreated D10 cells expressing either {Delta}Raf-1:ER or {Delta}Raf301:ER expressed little or no CD69 in the resting state (Fig. 2GoD and data not shown). Upon addition of ICI, the expression of CD69 was significantly induced on the surface of {Delta}Raf-1:ER-expressing cells, whereas no change in CD69 expression was detected on the surface of cells expressing the kinase-inactive {Delta}Raf301:ER (Fig. 2GoD). Both populations showed similar induction of CD69 expression in response to anti-CD3 mAb stimulation; hence, neither of the forms of {Delta}Raf:ER expressed in these cells is capable of eliciting a dominant-negative effect on CD69 expression, an observation consistent with previous results. These data indicate that {Delta}Raf-1:ER is able to activate the ERK pathway, leading to the expression of at least one T cell activation marker, namely CD69.

To further examine the response of T cells to {Delta}Raf-1:ER activation, we analyzed the effects of Raf activation on the expression of cytokine genes as monitored by an RNase protection assay (Fig. 2GoE). Upon PMA treatment or Raf activation, D10 cells were induced to express IL-5, IL-6, IL-10, and IL-13 mRNAs. Elevation of intracellular calcium with ionomycin led to induced expression of IL-4, IL-10, and IL-13 mRNAs, and synergized with {Delta}Raf-1:ER activation or PMA to superinduce the expression of the IL-5, IL-6, IL-10, and IL-13 mRNAs. As expected, we observed no induction of IL-2 or IFN-{gamma} mRNAs, because D10 cells are prototypical Th2 cells that do not express such Th1 cytokines. In these experiments, it was apparent that Raf activation had almost identical effects on cytokine gene expression as PMA treatment of cells. Indeed, the level of cytokine mRNA expression elicited either by Raf alone or in conjunction with ionomycin was very similar to that elicited by PMA. Hence, sustained Raf-MEK-ERK activation is not leading to supraphysiological induction of cytokine mRNAs at least when compared with treatment of cells with PMA.

Because D10 cells do not produce IL-2 and because {Delta}Raf-1:ER activation was insufficient to induce IL-4 expression in these cells, we added exogenous IL-2 to determine whether activation of {Delta}Raf-1:ER could render D10 cells unresponsive to IL-2. In the absence of exogenous IL-2, D10 cells displayed a low basal level of DNA synthesis. Activation of {Delta}Raf-1:ER was sufficient to induce a modest increase in thymidine incorporation (Fig. 3GoA). As seen previously in NIH 3T3 cells (39), D10 cells displayed a bell-shaped mitogenic response to activation of {Delta}Raf-1:ER, suggesting that low levels of Raf-MEK-ERK activation induced proliferation, whereas higher levels did not. The addition of IL-2 to resting D10 cells stimulated high levels of thymidine incorporation (Fig. 3GoA). Under these conditions, activation of {Delta}Raf-1:ER inhibited IL-2-induced DNA synthesis. The addition of ICI had no effect on either the proliferation or viability of parental D10 cells or cells expressing the kinase-inactive {Delta}Raf301:ER (Fig. 3GoA), indicating that Raf kinase activity is required for both {Delta}Raf-1:ER-induced cell proliferation and arrest. The lower total level of [3H]thymidine incorporation in the {Delta}Raf301:ER-expressing cells in this experiment was not reproducible in other iterations of this experiment and most likely reflects a smaller number of cells in the proliferation assay. The surface expression of the IL-2R ({alpha}-, ß-, and {gamma}-chains) was not changed upon activation of {Delta}Raf-1:ER (data not shown). Thus, the lack of mitogenic response to IL-2 was not due to the down-regulation of the IL-2R. Because D10G4.1 are Th2 cells, they proliferate in response to IL-4. Consequently, we examined the proliferative response of these cells to IL-4. We found that, similar to the response to IL-2, {Delta}Raf-1:ER activation inhibited IL-4-induced cell proliferation (data not shown). Importantly, cell viability was not significantly altered by {Delta}Raf-1:ER activation (Fig. 3GoA and data not shown), suggesting that the ability of Raf to inhibit IL-2- and IL-4-induced proliferation did not result from increased apoptosis. On the contrary, in the absence of cytokine, Raf activation appeared to provide a survival signal, as demonstrated by a moderate increase in cell viability (Fig. 3GoA). Similar retroviral infection and proliferation assays were also performed in an IL-2-dependent Th1 clone, a cytokine-independent T cell line, and an IL-3-dependent pro-B cell line. In all cases, we observed that activation of {Delta}Raf-1:ER led to inhibition of cell cycle progression (data not shown).



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 3. Inhibition of IL-2-induced DNA synthesis by selective activation of Raf. A, D10 cells expressing {Delta}Raf-1:ER were treated with increasing concentrations of ICI for 12 h. Cells were either untreated or treated with IL-2 for an additional 12 h, at which time [3H]thymidine was added for an additional 4 h before harvesting. Each point was measured in triplicate or quadruplicate. B, D10 cells expressing {Delta}Raf-1:ER were treated as in A, except that at the end of the culture the cells were fixed and stained with propidium iodide for cell cycle analysis. C, D10 cells were cultured for 12 h in either noncoated plates or plates coated with 10 µg/ml anti-CD3 mAb and were then either treated with IL-2 or left untreated for an additional 12 h. Cells were then fixed and stained with propidium iodide, as described above.

 
Raf activation induces G1 arrest in T cells

High levels of MAP kinase activation have been shown to induce cell cycle arrest in nonlymphoid cells (38, 39, 40, 42, 43, 44). To examine whether {Delta}Raf-1:ER activation could elicit cell cycle arrest in T cells, D10 cells expressing {Delta}Raf-1:ER were cultured in the presence or absence of ICI and/or IL-2, and the effects of {Delta}Raf-1:ER activation were compared. Cell cycle status was assessed by staining cell nuclei with propidium iodide (Fig. 3GoB).

In the absence of stimulation, ~70% of cells displayed a 2N DNA content indicative of cells in the G0/G1 phase of the cell cycle. The remaining 30% of cells were either in the S or G2/M phases of the cell cycle. Following 12 h of IL-2 treatment, the percentage of cells in G0/G1 was decreased to 50%, with a corresponding increase in the numbers of cells in S and G2/M phase, consistent with the expected mitogenic effects of IL-2. However, activation of {Delta}Raf-1:ER significantly inhibited the mitogenic response of D10 cells to IL-2 treatment, as under these conditions the percentage of cells in G0/G1 phase was maintained at 77%, and the percentage of cells in S and G2/M phase was reduced from 50% to 23%. Importantly, cells expressing {Delta}Raf301:ER, the kinase-inactive form of the protein, displayed no such inhibition of IL-2-induced mitogenesis, indicating that Raf activity is required for the observed inhibition of cell cycle progression (Fig. 3GoB).

In a separate experiment, we examined the inhibition of IL-2-induced mitogenesis by treatment of parental D10 cells with high doses of anti-CD3 mAb. In this experiment, 22% of untreated cells were in the S or G2/M phases of the cell cycle before IL-2 treatment, and this was increased to 59% after IL-2 addition. Prior treatment of these cells with high dose anti-CD3 mAb reduced the percentage of cells in S and/or G2/M from 58% to 38% (Fig. 3GoC). Hence, high dose anti-CD3 mAb also induces a G0/G1 arrest in these cells.

Additionally, we performed a pulse-chase experiment, in which D10 cells were pulse labeled with BrdU for a short period of time. Following the removal of BrdU, the cells were cultured in the presence of IL-2, and their transit time through the cell cycle into the next S phase was assessed. In the absence of {Delta}Raf-1:ER activation, cells reentered S phase ~12 h following the initial BrdU labeling. However, when {Delta}Raf-1:ER was activated, cells had failed to reenter S phase even up to 14 h after the initial BrdU labeling (data not shown). These data further support the hypothesis that {Delta}Raf-1:ER elicits a G1 cell cycle arrest in T cells.

Raf activation inhibited IL-2-dependent cell cycle progression in naive primary T cells

We wished to address whether the observations made in D10 cells also held true for primary T cells cultured in vitro. To this end, we isolated a population of naive CD4+Mel-14high T cells from DO11.10 TCR transgenic mice and cultured them in the presence of the OVA323–339 antigenic peptide and irradiated APC (46, 47, 58). T cells were then infected with retroviruses encoding either kinase-active {Delta}Raf-1:ER or kinase-inactive {Delta}Raf301:ER. Infected cells were selected for eGFP expression by fluorescence-activated cell sorting and then treated with different concentrations of 4-HT to activate the MAP kinase pathway in the presence or absence of IL-2.

Unlike the situation in D10 cells, activation of {Delta}Raf-1:ER alone in primary T cells did not induce DNA synthesis, whereas treatment with IL-2 induced DNA synthesis by more than 10-fold (Fig. 4GoA). Activation of {Delta}Raf-1:ER for 24 h in the presence of IL-2 led to a pronounced (~65%) inhibition of the mitogenic response to IL-2. No inhibition of the IL-2-proliferative response was observed in primary T cells expressing kinase-inactive {Delta}Raf301:ER. Once again, the effects of {Delta}Raf-1:ER were on cell cycle progression and not on cell viability, as the percentage of viable cells did not change following {Delta}Raf-1:ER activation (Fig. 4GoA). Similar results were obtained when cells were analyzed over the course of 48 h (data not shown). These observations were confirmed by analysis of cellular DNA content using propidium iodide. Treatment of the {Delta}Raf-1:ER-expressing populations of DO11.10 T cells with IL-2 led to a 10-fold increase in the population of cells with a greater than 2N DNA content that were presumably in the S/G2/M phase of the cell cycle. However, {Delta}Raf-1:ER activation caused a 50% inhibition of the response to IL-2, an effect that was not observed with the kinase-inactive {Delta}Raf301:ER (Fig. 4GoB). Taken together, these data strongly suggest that, similar to high dose exposure to Ag, {Delta}Raf-1:ER activation is capable of eliciting cell cycle arrest in naive primary T cells.



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 4. Raf activation elicits cell cycle arrest in naive T cells. A, Naive T cells from DO11.10 mice were infected with retroviruses encoding {Delta}Raf-1:ER or {Delta}Raf301:ER. Infected cells expressing eGFP were selected by FACS. eGFP+ cells expressing either {Delta}Raf-1:ER or {Delta}Raf301:ER were cultured in the absence or presence of 4-HT for 6–8 h, and were then either untreated or treated with IL-2 for an additional 16 h, as indicated. [3H]thymidine was added for last 4 h of the experiment before harvesting the cells and measuring [3H]thymidine incorporation. Parallel cell viability assays were performed by propidium iodide exclusion using a FACScan. Data are representative of three independent experiments. B, Cells from the above experiment were cultured for an additional 24 h and were collected, fixed, and stained with propidium iodide. In this experiment, 4-HT was used at a concentration of 300 nM.

 
Effects of Raf activation on cell cycle regulatory proteins

The experiments described above suggested that {Delta}Raf-1:ER activation could elicit G1 arrest in T cells that could not be overcome by exogenous IL-2. We have previously described a similar situation in mouse NIH 3T3 fibroblasts in which Raf activation induced a potent cell cycle arrest mediated, in large part, by the induced expression of the CKI, p21Cip1 (39). To determine whether either {Delta}Raf-1:ER or anti-CD3 mAb-induced G1 arrest in T cells is mediated by alterations in the expression or activity of key components of the cell cycle machinery, we examined the expression and activity of some of the proteins that are known to control the G1 to S phase transition in mammalian cells.

D10 cells expressing {Delta}Raf-1:ER were treated with two different concentrations of ICI (30 or 300 nM) to activate {Delta}Raf-1:ER or with a high dose of anti-CD3 mAb in the presence or absence of IL-2. Different concentrations of ICI were used in an effort to activate the MAP kinase pathway to different extents, as indicated in Fig. 1GoC. However, similar results were obtained with both concentrations of ICI used. After 12 h, cell extracts were prepared and the expression of cell cycle regulators and the activity of cyclin E/CDK2 were assessed.

Treatment of D10 cells with IL-2 alone led to increased expression of both cyclin E and cyclin A, a modest increase in the expression of p21Cip1, and decreased expression of p27Kip1 (Fig. 5GoA). Cyclin E/CDK2 activity was measured in the same extracts and, as expected, was shown to be strongly induced by IL-2 treatment. Consistent with this was the observation that cyclin E/CDK2 complexes from IL-2-treated cells contained little or none of the CKI, p21Cip1, and p27Kip1 as compared with untreated cells (Fig. 5GoB).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition of IL-2-induced cyclin E/CDK2 activity following Raf activation. A, D10 cells expressing {Delta}Raf-1:ER were pretreated with ethanol, ICI (30 or 300 nM), or 10 µg/ml immobilized anti-CD3 mAb for 24 h. One-half of each cell sample was then treated with IL-2 for the last 12 h before harvesting. Cell lysates were prepared, and the expression of cyclin E, cyclin A, cyclin D2, p21Cip1, and p27Kip1 was assessed by Western blotting with the appropriate antisera. Equal loading of cell extract was confirmed using an Ab that recognizes ERK1 and ERK2, which serves as a convenient loading control. B, Lysates from the same experiment described above were immunoprecipitated with anti-cyclin E antiserum, and associated CDK2 kinase activity was measured using histone H1 (HH1) as a substrate, as described in Materials and Methods. The presence of cyclin E, p21Cip1, and p27Kip1 in the immunoprecipitates was confirmed by Western blotting with the appropriate antiserum. C, D10G4.1 cells were stimulated with 10 µg/ml of immobilized anti-CD3 mAb in the absence or presence of PD098059 for 4 or 7 h, and lysates were prepared for Western blot analysis with an antisera that recognizes p21Cip1. D, The presence of CDK2 in immunoprecipitates of cyclin D2 was detected by Western blot analysis of cell lysates prepared as described in A or from cells treated with 0.3 or 15 µg/ml of immobilized anti-CD3 mAb for 24 h that were immunoprecipitated with an antiserum that recognizes cyclin D2 and probed with an antiserum that recognizes CDK2 or cyclin D2. IP, antiserum for immunoprecipitation; WB, antiserum for Western blotting.

 
Activation of {Delta}Raf-1:ER alone or treatment of cells with a high dose of anti-CD3 mAb led to induction of cyclin D2 and cyclin E, but not cyclin A (Fig. 5GoA). Moreover, activation or {Delta}Raf-1:ER or treatment with anti-CD3 mAb led to highly elevated expression of p21Cip1. In addition, these treatments largely prevented the reduced expression of p27Kip1 that normally occurs following IL-2 treatment. The apparent consequence of these events was that the activation of cyclin E/CDK2 complexes by IL-2 was significantly inhibited (Fig. 5GoB). Consistent with this was the fact that we could readily detect the presence of both p21Cip1 and p27Kip1 in cyclin E/CDK2 immune complexes (Fig. 5GoB). These data suggest that the observed effects of {Delta}Raf-1:ER and high dose anti-CD3 mAb on p21Cip1 and p27Kip1 expression may be responsible for the cell cycle arrest observed in response to these agents.

To determine whether the induction of p21Cip1 by high dose anti-CD3 mAb was mediated by the Ras-activated Raf-MEK-ERK pathway, D10 cells were treated with anti-CD3 mAb for 4 or 7 h in the absence or presence of 20 or 40 µM of the MEK-specific inhibitor PD098059 (Fig. 5GoC). Consistent with the partial ability of PD098059 to reverse the antiproliferative effects of high dose anti-CD3 mAb, the induced expression of p21Cip1 was inhibited, although not entirely abrogated, by the presence of the MEK inhibitor. Equal loading of the gel lanes in Fig. 5Go, A and C, was confirmed by probing the Western blots with an antisera that recognizes ERK1 and ERK2, which, due to the low resolving power of these polyacrylamide gels, are not resolved according to their phosphorylation status. These data support a model in which high dose anti-CD3 mAb induces cell cycle arrest in T cells by activating the Raf-MEK-ERK pathway leading to induced expression of p21Cip1 and a failure to down-regulate the expression of p27Kip1.

It has previously been suggested that the formation of complexes between D-type cyclins and CDK2 may be involved in the transmission of an antiproliferative signal in mammalian cells (59). Because {Delta}Raf-1:ER and anti-CD3 mAb, but not IL-2, induced cyclin D2 expression, we assessed whether cyclin D2 formed a complex with CDK2 in cells arrested as a consequence of {Delta}Raf-1:ER activation or treatment with anti-CD3 mAb. Interestingly, we were readily able to detect CDK2 in immunoprecipitates of cyclin D2 from cells that were arrested as a consequence of {Delta}Raf-1:ER activation or high dose anti-CD3 mAb treatment (Fig. 5GoD). Although not definitive proof of the hypothesis, these data are consistent with the suggestions of others that the formation of cyclin D2/CDK2 complexes may play a role in the inhibition of cell cycle progression.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The mitogenic response of resting T cells is mediated initially by interactions of Ag bound to MHC with the TCR, which elicits a plethora of early signaling events (60, 61). This initial signaling interaction leads to the expression of a series of activation markers as well as the expression of cytokines such as IL-2 and its cognate high affinity receptor. The autocrine effects of secreted cytokines lead to the induction of D- and E-type G1 cyclins and their cognate CDKs and the down-regulation of the CKI p27Kip1, allowing cells to transit from G1 into S phase (60, 61).

Previous observations indicated that high intensity signaling through the Raf-MEK-ERK pathway could render cells resistant to the effects of growth factors and cytokines (38, 39, 40, 41, 42, 43, 44). Such observations prompted us to explore a possible role for this pathway in the cytokine nonresponsive phenotype that is elicited in T cells by either a high dose of Ag or treatment with anti-TCR mAb. In this work, we demonstrate that activation of the ERK pathway either through the TCR or by specific activation of the Raf-MEK-ERK pathway leads to a cytokine unresponsive state. This inability of T cells to respond to IL-2 was not associated with cell death or a lack of IL-2R expression, but appeared to be due to cell cycle arrest. We found that both anti-TCR mAb and Raf-induced cell cycle arrest were associated with enhanced expression of the CKI p21Cip1 and that the level of induced p21Cip1 expression correlated with the signal strength elicited by TCR or Raf activation. Importantly, the level of induction of p21Cip1 elicited by Raf and by anti-TCR mAb treatment was similar, suggesting that the effects of Raf on T cell cycle were not a consequence of supraphysiological induction of p21Cip1 expression. Second, the induction of p21Cip1 elicited by anti-TCR mAb treatment was mediated, at least in part, by the activation of the Raf-MEK-MAP pathway, which is consistent with the ability of Raf to induce p21Cip1 and thereby induce cell cycle arrest. The induced association of p21Cip1 with the cyclin E/CDK2 complexes led to decreased activity of this complex consistent with the observed G1 cell cycle arrest, as has been demonstrated previously (39). In addition to observing the induced expression of p21Cip1, both Raf and anti-CD3 mAb also prevented the normal down-regulation of p27Kip1 expression from cyclin E/CDK2 complexes that occurs in response to IL-2 treatment. Typically, down-regulation of p27Kip1 expression is associated with the initial activation of CDK2 that leads to phosphorylation of p27Kip1, thereby targeting it for degradation. Because the consequence of induced p21Cip1 expression is the inhibition of cyclin E/CDK2 complexes, it is possible that the failure to down-regulate p27Kip1 expression is secondary to the induction of p21Cip1. Nonetheless, the induced expression of p21Cip1, the sustained expression of p27Kip1, or most likely a combination of both may be important in the induction of the cytokine nonresponsive state in T cells. This stands in contrast to the situation in NIH 3T3 and primary mouse embryo fibroblasts in which the induction of p21Cip1 appears to be most important to the induction and maintenance of a growth-factor nonresponsive state. Indeed, in this situation, NIH 3T3 cells arrest with high levels of p21Cip1, but low levels of p27Kip1 expression. Furthermore, in p21Cip1-/- mouse embryonic fibroblasts, activation of {Delta}Raf-1:ER failed to cause cell cycle arrest, even though the cells continue to express p27Kip1 (39). Similarly, p21Cip1, but not p27Kip1, was found to be associated with cyclin E/CDK2 in a B lymphoma cell line that was arrested in the cell cycle by treatment with anti-IgM (26). Therefore, we believe that the induction of p21Cip1 by the Raf-MEK-ERK pathway may be the trigger for cell cycle arrest in a number of circumstances.

It has been suggested that the assembly of complexes containing cyclin D2 and CDK2 might be involved in the induction of cell cycle arrest in fibroblasts and in a T cell lymphoma (59, 62). It is interesting, therefore, that in T cells arrested either by high dose anti-CD3 mAb or by {Delta}Raf-1:ER activation, there was a robust induction of cyclin D2 as well as the formation of cyclin D2/CDK2 complexes. These complexes were not seen upon cytokine withdrawal or in response to mitogenic stimulation. It is not clear how such complexes are assembled, but the recent demonstration that p21Cip1 serves as an assembly factor for D-type cyclins and their cognate CDKs suggests that the induced expression of this CKI may play a role. Even less clear is the potential role of the cyclin D2/CDK2 complex in the induction and maintenance of the nonresponsive state, although the availability of cyclin D2 knockout mice makes such analyses feasible in the future.

It has been reported that Ag-induced IL-2 unresponsiveness can be attributed to activation-induced cell death both in vivo and in vitro (24, 63). When we examined the D10 cells and primary T cells from DO11.10 mice following either anti-CD3 mAb treatment or {Delta}Raf-1:ER activation, there was no obvious induction of apoptosis within the first 24 h of treatment. Indeed, activation of {Delta}Raf-1:ER appeared to protect cells from the apoptotic cell death that normally occurs after the withdrawal of cytokines. However, at later times following Raf activation, we observed increased cell death in these cultures. It is possible that the initial response to Raf activation is cell cycle arrest that is followed at later times by apoptosis. Our results are consistent with the observation that in Jurkat T cells and PBL, activation-induced apoptosis occurs following late G1 arrest (64).

The availability of a system for the conditional activation of the Raf-MEK-ERK pathway in T cells should provide a useful tool to study how costimulatory signals such as the Ca2+-calcineurin-NF-AT pathway, and other MAPK pathways such as the c-Jun N-terminal kinase pathway interact to synergistically regulate nuclear events that control immune cell function. Indeed, we and others have previously demonstrated the feasibility of such studies by demonstrating that the activation of c-Fos and MAP kinase phosphatase-1 expression in fibroblasts requires the synergistic action of the MAP kinase pathway and a calcium signal (65, 66). The availability of conditional systems for the regulation of MEK kinase, PI3-kinase, and Akt will further facilitate such studies (67, 68, 69). Moreover, it will be interesting to determine whether the activation of costimulatory receptors such as CD28 or other signaling molecules might modify the cell cycle arrest elicited by the activation of the Raf-MEK-ERK pathway. Such an approach is all the more interesting in view of the recent observation that pathways regulated by the Rho GTPase can antagonize the induction of p21Cip1 and thereby prevent the cell cycle arrest induced by activated Ras in Swiss 3T3 cells (70). Indeed, the Rho GTPase has previously been shown to be important for thymic development (71, 72).

In this study, we have demonstrated that the constitutive activation of the Raf-MEK-ERK signaling pathway was sufficient to render T cells insensitive to subsequent mitogenic stimulation. This finding may provide a partial explanation for the induction of anergy in lymphoid cells as a consequence of high dose Ag or anti-TCR stimulation. It is possible that the sustained activation of ERKs in the absence of costimulatory signals results in the sustained expression of CKI such as p21Cip1 and p27Kip1. It is interesting to note, therefore, that memory T cells from aged mice with autoimmune disease exhibit a predominant G1 arrest and are refractory to anti-CD3 mAb-induced proliferation and apoptosis. It has been suggested that the unresponsive state of T cells in these mice may be a consequence of cumulative chronic self Ag stimulation, and so it is interesting to note that the phenotype correlates with increased expression of p21Cip1 and p27Kip1. (73). Furthermore, it has been demonstrated that anergic B cells from mice engineered to express anti-hen egg lysozyme IgM and IgD and soluble hen egg lysozyme display low level constitutive activation of the ERK pathway (29). However, it is unclear whether the cell cycle arrest elicited under these conditions is mediated by any of the CKI implicated in this study.

In a previous study, we demonstrated that the activation of the Raf-MEK-ERK pathway in primary human fibroblasts elicits an irreversible cell cycle arrest that is accompanied by the expression of a variety of cellular senescence markers. The induction of irreversible arrest in this context was associated with the induced expression of p16Ink4a that sequesters CDK4 and CDK6 into an inactive complex (38, 74). In contrast, the cell cycle arrest induced by {Delta}Raf-1:ER in D10 T cells was fully reversed by the removal of the activating agent, indicating that Raf is most likely not inducing a senescence-like response in these cells. It is not clear whether this observation is a peculiarity of lymphoid cells or reflects a fundamental difference between mouse and human cells, but this phenomenon warrants further investigation in both primary and established mouse and human T cells. As described above, the availability of conditional forms of various signaling molecules and efficient systems for gene transfer into lymphoid populations will significantly facilitate such analyses.

In conclusion, we show in this study that selective activation of the Raf-MEK-ERK pathway in T cells can mimic the antiproliferative signals transduced by the TCR. In both cases, the induced expression of p21Cip1 is implicated in the cell cycle arrest that ensues following these treatments. Further study will be necessary to elucidate the involvement of other mechanisms in TCR-induced nonresponsiveness and how such mechanisms relate to peripheral tolerance.


   Acknowledgments
 
We thank the members of the McMahon and Lees labs for reagents, discussions, and suggestions; Alice Mui for providing the retroviral vector; Hyun-Jun Lee and Iris Ferber for technical advice on retrovirus vector construction and retrovirus infection; and Steven Hurst for critical review of the manuscript. We also thank the DNAX FACS facility for technical assistance in cell sorting.


   Footnotes
 
1 DNAX Research Institute is supported by Schering Plough Corporation. Back

2 Current address: SyStemix Inc., 3155 Porter Drive, Palo Alto, CA 94304. Back

3 Address correspondence and reprint requests to Dr. Martin McMahon at the current address: Cancer Research Institute, University of California San Francisco/Mt. Zion Cancer Center, 2340 Sutter Street, Box 0128, S-329, San Francisco, CA 94143-0128. E-mail address: Back

4 Abbreviations used in this paper: PI3, phosphatidylinositol 3; 4-HT, 4-hydroxytamoxifen; BrdU, 5-bromo-2'-deoxyuridine; CDK, cyclin-dependent kinase; CKI, CDK inhibitor; eGFP, enhanced green fluorescent protein; ERK, extracellular signal-related kinase; MAP, mitogen-activated protein; MEK, MAP/ERK kinase; {Delta}Raf-1:ER, fusion protein of the kinase domain of Raf-1 and the hormone-binding domain of estrogen receptor; {Delta}Raf301:ER, kinase-inactive fusion protein. Back

Received for publication May 7, 1999. Accepted for publication September 13, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Geppert, T., L. Davis, H. Gur, M. Wacholtz, P. Lipsky. 1990. Accessory cell signals involved in T cell activation. Immunol. Rev. 117:5.[Medline]
  2. Leung, H. T., P. S. Linsley. 1994. The CD28 costimulatory pathway. Ther. Immunol. 1:217.[Medline]
  3. Weintraub, B. C., C. C. Goodnow. 1998. Immune responses: costimulatory receptors have their say. Curr. Biol. 8:575.
  4. Buhl, A. M., J. C. Cambier. 1997. Co-receptor and accessory regulation of B-cell antigen receptor signal transduction. Immunol. Rev. 160:127.[Medline]
  5. Madrenas, J., R. N. Germain. 1996. Variant TCR ligands: new insights into the molecular basis of antigen-dependent signal transduction and T cell activation. Semin. Immunol. 8:83.[Medline]
  6. Kersh, G. J., P. M. Allen. 1996. Essential flexibility in the T-cell recognition of antigen. Nature 380:495.[Medline]
  7. Chau, L. A., J. A. Bluestone, J. Madrenas. 1998. Dissociation of intracellular signaling pathways in response to partial agonist ligands of the T cell receptor. J. Exp. Med. 187:1699.[Abstract/Free Full Text]
  8. Healy, J. I., C. C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 16:645.[Medline]
  9. Schwartz, R. H.. 1994. A cell culture model for T lymphocyte clonal anergy. Science 248:1349.
  10. Mueller, D. L., M. K. Jenkins, R. H. Schwartz. 1989. Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Annu. Rev. Immunol. 7:445.[Medline]
  11. Kearney, E. R., K. A. Pape, D. Y. Loh, M. K. Jenkins. 1994. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo. Immunity 1:327.[Medline]
  12. McCormack, J. E., J. Kappler, P. Marrack. 1994. Stimulation with specific antigen can block superantigen-mediated deletion of T cells in vivo. Proc. Natl. Acad. Sci. USA 91:2086.[Abstract/Free Full Text]
  13. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, R. J. Trent, A. Basten. 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334:676.[Medline]
  14. Cooke, M. P., A. W. Heath, K. M. Shokat, Y. Zeng, F. D. Finkelman, P. S. Linskley, M. Howard, C. C. Goodnow. 1994. Immunoglobulin signal transduction guides the specificity of B cell-T cell interactions and is blocked in tolerant self-reactive B cells. J. Exp. Med. 179:425.[Abstract/Free Full Text]
  15. Jenkins, M. K., R. H. Schwartz. 1987. Antigen presentation by chemically modified splenocytes induces antigen-specific T-cell unresponsiveness in vitro and in vivo. J. Exp. Med. 165:302.[Abstract/Free Full Text]
  16. Jenkins, M. K., J. D. Ashwell, R. H. Schwartz. 1988. Allogeneic non-T spleen cells restore the responsiveness of normal T cell clones stimulated with antigen and chemically modified antigen-presenting cells. J. Immunol. 140:3324.[Abstract]
  17. Jenkins, M. K., C. A. Chen, G. Jung, D. L. Mueller, R. H. Schwartz. 1990. Inhibition of antigen-specific proliferation of type 1 murine T cell clones after stimulation with immobilized anti-CD3 monoclonal antibody. J. Immunol. 144:16.[Abstract]
  18. Harding, F. A., J. G. McArthur, J. A. Gross, D. H. Raulet, J. P. Allison. 1992. CD28-mediated signalling co-stimulates murine T-cells and prevents induction of anergy in T-cell clones. Nature 356:607.[Medline]
  19. Li, W., C. D. Whaley, A. Mondino, D. L. Meuller. 1996. Blocked signal transduction to the ERK and JNK protein kinases in anergic CD4+ T-cells. Science 271:1272.[Abstract]
  20. Fields, P. E., T. F. Gajewski, F. W. Fitch. 1996. Blocked Ras activation in anergic CD4+ T-cells. Science 271:1276.[Abstract]
  21. Nau, G. J., R. L. Moldwin, D. W. Lancki, D. K. Kim, F. W. Fitch. 1987. Inhibition of IL 2-driven proliferation of murine T lymphocyte clones by supraoptimal levels of immobilized anti-T cell receptor monoclonal antibody. J. Immunol. 139:114.[Abstract]
  22. Williams, M. E., A. H. Lichtman, A. K. Abbas. 1990. Anti-CD3 antibody induces unresponsiveness to IL-2 in Th1 clones but not in Th2 clones. J. Immunol. 144:1208.[Abstract]
  23. Ashwell, J. D., R. E. Cunningham, P. D. Noguchi, D. Hernandez. 1987. Cell growth cycle block of T cell hybridomas upon activation with antigen. J. Exp. Med. 165:173.[Abstract/Free Full Text]
  24. Lenardo, M. J.. 1991. Interleukin-2 programs mouse {alpha}ß T lymphocytes for apoptosis. Nature 353:858.[Medline]
  25. Ezhevsky, S. A., H. Toyoshima, T. Hunter, D. W. Scott. 1996. Role of cyclin A and p27 in anti-IgM-induced G1 growth arrest of murine B-cell lymphomas. Mol. Biol. Cell 7:553.[Abstract]
  26. Marches, R., R. H. Scheuermann, J. W. Uhr. 1998. Cancer dormancy: role of cyclin-dependent kinase inhibitors in induction of cell cycle arrest mediated via membrane IgM. Cancer Res. 58:691.[Abstract/Free Full Text]
  27. Maier, C. C., M. I. Greene. 1998. Biochemical features of anergic T cells. Immunol. Res. 17:133.[Medline]
  28. DeSilva, D. R., K. B. Urdahl, M. K. Jenkins. 1991. Clonal anergy is induced in vitro by T cell receptor occupancy in the absence of proliferation. J. Immunol. 147:3261.[Abstract]
  29. Healy, J. I., R. E. Dolmetsch, L. A. Timmerman, J. G. Cyster, M. L. Thomas, G. R. Crabtree, R. S. Lewis, C. C. Goodnow. 1997. Different nuclear signals are activated by the B cell receptor during positive versus negative signaling. Immunity 6:419.[Medline]
  30. Cantrell, D.. 1996. T-cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14:259.[Medline]
  31. Qian, D., A. Weiss. 1997. T cell antigen receptor signal transduction. Curr. Opin. Cell Biol. 9:205.[Medline]
  32. Carrera, A. C., L. Rodriguez-Borlado, C. Martinez-Alonso, I. Merida. 1994. T cell receptor-associated {alpha}-phosphatidylinositol 3-kinase becomes activated by T cell receptor cross-linking and requires pp56lck. J. Biol. Chem. 269:19435.[Abstract/Free Full Text]
  33. Reif, K., S. Lucas, D. Cantrell. 1997. A negative role for phosphoinositide 3-kinase in T-cell antigen receptor function. Curr. Biol. 7:285.[Medline]
  34. Alberola-lla, J., K. A. Forbush, R. Seger, E. G. Krebs, R. M. Perlmutter. 1995. Selective requirement for MAP kinase activation in thymocyte differentiation. Nature 373:620.[Medline]
  35. Alberola-lla, J., K. A. Hogquist, K. A. Swan, M. J. Bevan, R. M. Perlmutter. 1996. Positive and negative selection invoke distinct signaling pathways. J. Exp. Med. 184:9.[Abstract/Free Full Text]
  36. Crompton, T., K. C. Gilmour, M. J. Owen. 1996. The MAP kinase pathway controls differentiation from double-negative to double-positive thymocyte. Cell 86:243.[Medline]
  37. Sharp, L. L., D. A. Schwarz, C. M. Bott, C. J. Marshall, S. M. Hedrick. 1997. The influence of the MAPK pathway on T cell lineage commitment. Immunity 7:609.[Medline]
  38. Zhu, J., D. Woods, M. McMahon, J. M. Bishop. 1998. Senescence of human fibroblasts induced by oncogenic Raf. Genes Dev. 12:2997.[Abstract/Free Full Text]
  39. Woods, D., D. Parry, H. Cherwinski, E. Bosch, E. Lees, M. McMahon. 1997. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol. Cell. Biol. 17:5598.[Abstract]
  40. Tombes, R. M., K. L. Auer, R. Mikkelsen, K. Valerie, M. P. Wymann, C. J. Marshall, M. McMahon, P. Dent. 1998. The mitogen-activated protein (MAP) kinase cascade can either stimulate or inhibit DNA synthesis in primary cultures of rat hepatocytes depending upon whether its activation is acute/phasic or chronic. Biochem. J. 330:1451.
  41. Ravi, R. K., M. McMahon, Z. Yangang, J. R. Willimas, L. E. Dillehay, B. D. Nelkin, M. Mabry. 1999. Raf-1-induced cell cycle arrest in LNCaP human prostate cancer cells. J. Cell. Biochem. 15:458.
  42. Ravi, R. K., W. Weber, M. McMahon, J. R. Williams, S. Baylin, A. Ma, M. L. Harter, L. E. Dillehay, P. P. Claudio, A. Giordano, et al 1998. Activated Raf-1 causes growth arrest in human small cell lung cancer cells. J. Clin. Invest. 101:153.[Medline]
  43. Lloyd, A. C., F. Obermuller, S. Staddon, C. F. Barth, M. McMahon, H. Land. 1997. Cooperating oncogenes converge to regulate cyclin/cdk complexes. Genes Dev. 11:663.[Abstract/Free Full Text]
  44. Auer, K. L., J.-S. Park, P. Seth, R. J. Coffe, G. Darlington, A. Abo, M. McMahon, R. A. DePinho, P. B. Fisher, P. Dent. 1998. Prolonged activation of the mitogen-activated protein kinase pathway promotes DNA synthesis in primary hepatocytes from p21Cip-1/WAF1-null mice but not in hepatocytes from p16INK4a-null mice. Biochem. J. 336:551.
  45. Kaye, J., S. Procelli, J. Tite, B. Jones, Jr C. A. Janeway. 1983. Both a monoclonal antibody and antisera specific for determinants unique to individual cloned helper T cell lines can substitute for antigen and antigen-presenting cells in the activation of T cells. J. Exp. Med. 158:836.[Abstract/Free Full Text]
  46. Murphy, K. M., A. B. Heimberger, D. Y. Loh. 1990. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo. Science 250:1720.[Abstract/Free Full Text]
  47. Hsieh, C.-S., A. B. Heimberger, J. S. Gold, A. O’Garra, K. M. Murphy. 1992. Differential regulation of T helper phenotype development by interleukins 4 and 10 in an {alpha}ß T-cell-receptor transgenic system. Proc. Natl. Acad. Sci. USA 89:6065.[Abstract/Free Full Text]
  48. Onishi, M., S. Kinoshita, Y. Morikawa, A. Shibuya, J. Phillips, L. L. Lanier, D. M. Gorman, G. P. Nolan, A. Miyajima, T. Kitamura. 1996. Applications of retrovirus-mediated expression cloning. Exp. Hematol. 24:324.[Medline]
  49. Heemskerk, M. H., B. Blom, G. Nolan, A. P. Stegmann, A. Q. Bakker, K. Weijer, P. C. Res, H. Spits. 1997. Inhibition of T cell and promotion of natural killer cell development by the dominant negative helix loop helix factor Id3. J. Exp. Med. 186:1597.[Abstract/Free Full Text]
  50. Samuels, M. L., M. J. Weber, J. M. Bishop, M. McMahon. 1993. Conditional transformation of cells and rapid activation of the mitogen-activated protein kianse cascade by an estradiol-dependent human raf-1 protein kinase. Mol. Cell. Biol. 13:6241.[Abstract/Free Full Text]
  51. Bosch, E., H. Cherwinski, D. Peterson, M. McMahon. 1997. Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1. Oncogene 15:1021.[Medline]
  52. Danielian, P. S., R. While, S. A. Hoare, S. E. Fawell, M. G. Parker. 1993. Identification of residues in the estrogen receptor that confer differential sensitivity to estrogen and hydroxytamoxifen. Mol. Endocrinol. 7:232.[Abstract]
  53. Pear, W. S., G. P. Nolan, M. L. Scott, D. Baltimore. 1993. Production of high-titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90:8392.[Abstract/Free Full Text]
  54. Ferber, I. A., H.-J. Lee, F. Zonin, V. Heath, A. Mui, N. Arai, A. O’Garra. 1999. GATA-3 significantly down-regulates IFN-{gamma} production developing Th1 cells in addition to inducing IL-4 and IL-5 levels. Clin. Immunol. 91:134.-144. [Medline]
  55. McCarthy, S. A., M. L. Samuels, C. A. Pritchard, J. A. Abraham, M. McMahon. 1995. Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes. Genes Dev. 9:1953.[Abstract/Free Full Text]
  56. Alessi, D. R., A. Cuenda, P. Cohen, D. R. Dudley, A. R. Saltiel. 1995. PD098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270:27489.[Abstract/Free Full Text]
  57. D’Ambrosio, D., D. A. Cantrell, L. Frati, A. Santoni, R. Testi. 1994. Involvement of p21ras activation in T cell CD69 expression. Eur. J. Immunol. 24:616.