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Originally published In Press as doi:10.1074/jbc.M301506200 on July 10, 2003

J. Biol. Chem., Vol. 278, Issue 38, 35940-35949, September 19, 2003
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Ectopic B-Raf Expression Enhances Extracellular Signal-regulated Kinase (ERK) Signaling in T Cells and Prevents Antigen-presenting Cell-induced Anergy*

Tara J. Dillon {ddagger}, Vladamir Karpitski §, Scott A. Wetzel ¶, David C. Parker ¶, Andréy S. Shaw § and Philip J. S. Stork {ddagger} ||

From the {ddagger}Vollum Institute and the Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97239 and the §Department of Pathology and Center for Immunology, Washington University School of Medicine, St Louis, Missouri 63110

Received for publication, February 12, 2003 , and in revised form, June 15, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
T cells that receive stimulation through the T cell receptor (TCR) in the absence of costimulation become anergic and are refractory to subsequent costimulation. This unresponsiveness is associated with the constitutive activation of the small G protein, Rap1, and the lack of Ras-dependent activation of ERK. Recent studies suggest that Rap1 can activate the MAP kinase kinase kinase B-Raf that is either endogenously or ectopically expressed. Peripheral T cells generally do not express B-Raf; therefore, to test the hypothesis that ectopic expression of B-Raf could permit Rap1 to activate ERK signaling, we generated transgenic mice expressing B-Raf within peripheral T cells. This converted Rap1 into an activator of ERK, to enhance ERK activation and proliferation following TCR engagement in the absence of costimulation. When T cells were incubated with engineered APCs presenting antigen on I-Ek and expressing low levels of B7, they became anergic, displayed constitutive activation of Rap1, and were deficient in Ras and ERK activation. However, when incubated with the same APCs, T cells expressing the B-Raf transgene proliferated upon restimulation and displayed elevated ERK activation. Thus B-Raf expression and enhanced ERK activation is sufficient to prevent anergy in a model of APC-induced T cell anergy. However, studies using anti-TCR antibody-induced anergy showed that the ability of ERKs to reverse T cell anergy is dependent on the anergic model utilized.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The MAP1 kinase cascade governs multiple cellular processes in T cell function, including proliferation and differentiation. Activation of the MAP kinase ERK (extracellular signal-regulated kinase) is required for IL-2 gene transcription and T cell proliferation via engagement of the T cell receptor (TCR) (1). In addition, the magnitude of ERK activation may regulate the response of T cells to TCR engagement (2, 3). Therefore, it is expected that both the magnitude and duration of ERK activation are tightly regulated in T cells.

One potential mechanism of ERK regulation is the modulation of Ras signaling by the small G protein, Rap1. Rap1, a member of the Ras super-family, was identified as an antagonist of Ras dependent transformation in fibroblasts (4). In many cell types, including T cells, Rap1 has been shown to inhibit ERK activation by blocking Ras-dependent activation of the MAP kinase kinase kinase, Raf-1 (5–8). Rap1 is activated upon TCR engagement in the absence of costimulation (5, 9, 10) and can limit TCR-induced signals to ERK (5, 11). Interestingly, Rap1 is inhibited by the engagement of the CD28 costimulatory receptor (10, 12), suggesting that one of the functions of Rap1 activation may be to limit ERK activation in the absence of costimulation (5). Indeed, in models of T cell anergy elicited by stimulation through the TCR alone, Rap1 is constitutively active and may limit signaling downstream of Ras (13).

Recently other functions of Rap1 have been identified, particularly the enhancement of T cell adhesion (14, 15), raising the possibility that Rap1 can regulate anergy independently of its actions on ERKs. To address this question, we selectively inhibited Rap1 antagonism of ERKs by introducing B-Raf, a non-lymphoid Rap1 effector, into T lymphocytes, and examined the consequence of this change on Rap1 signaling and the development of anergy. B-Raf, unlike Raf-1, is activated by Rap1, and when activated can activate ERK via the ERK kinase, MEK (16–18). Recent studies indicate that Rap1 activates B-Raf by recruiting B-Raf to the membrane. This recruitment of B-Raf by either Rap1 or Ras results in B-Raf activation. The requirements for Raf-1 activation are more stringent. In addition to recruitment to the membrane, additional phosphorylations are required that occur upon recruitment of Raf-1 to Ras. These phosphorylations of Raf-1 do not occur upon recruitment of Raf-1 to Rap1 (19). For this reason, Rap1 activity results in ERK activation when B-Raf is expressed, but may prevent Raf-1 activation by sequestering Raf-1 without activating it. Transfection of B-Raf into non-lymphoid cells that do not normally express this Raf isoform has been proposed to allow Rap1 to couple to ERK (17, 20, 21). Since T cells do not generally express B-Raf, we tested the hypothesis that the transgenic expression of B-Raf in peripheral T cells might have a similar action.

Recent studies examining the role of ERK inhibition in T cell anergy have utilized constitutively active or interfering mutants of upstream activators of ERKs (22) as well as the pharmacological inhibition of the ERK kinase MEK (23). These manipulations result in the activation or inhibition of ERK without mirroring physiological levels of regulation (24–26). By introducing B-Raf into T cells, we hoped to test the hypothesis that B-Raf expression can permit Rap1 to couple positively to ERKs. By selectively enhancing ERK signaling through Rap1 without altering ERK activation through Ras, ectopic B-Raf expression in T cells might provide the opportunity to examine the consequence of enhancing ERK selectively in the absence of costimulation.

We show that B-Raf expression in T cells converted Rap1 into an activator of ERKs. Additionally, B-Raf-expressing T cells proliferated in the absence of costimulation. Moreover, these cells could not be anergized by APCs expressing low levels of B7, which were deficient in providing costimulation. Wild-type and B-Raf-expressing cells stimulated with APCs expressing low levels of B7 were characterized by constitutive Rap1 activation and lack of Ras activation. The only detectable biochemical difference between B-Raf-expressing cells and wild-type cells from B-Raf negative littermates was elevated ERK activity, suggesting that elevated ERK activation could prevent anergy in this setting.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Transfections—Cells from the human T cell leukemia cell line Jurkat (clone E6–1, from A.T.C.C., Manassas, VA) were maintained in RPMI 1640 media (Invitrogen) containing 10% fetal calf serum (Hyclone, Logan, NJ) at 37 °C with 5% CO2. For transient transfections, 5 x 107 Jurkat T cells were transfected by electroporation (250 V/950 µF) with 10 µg of RapV12 (constitutively active Rap1) and 10 µg of Raf-1 or 10 µg of B-Raf plasmids. All cells were transfected with the reporter constructs, Gal4-Elk1and 5xGal4-E1b/luciferase (3 µg each) and activity was measured by luciferase assay as previously described (17). The total DNA transfected was made constant with the addition of pcDNA3 (vector). After transfection, cells recovered for 24 h in media.

Ltk- fibroblasts were obtained from ATCC and displayed a broad range of endogenous B7.1 (CD80) and no detectable B7.2 (CD86) expression. To generate B7hi and B7lo fibroblasts, Ltk- cells were stained with anti-CD80 FITC (16–10A1, BD PharMingen), sorted on a FACSCalibur (BD PharMingen) and cloned at limiting dilution. Cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum (Hyclone) and supplemented with 1 mM L-glutamine, sodium pyruvate (100 mg/ml), 5 x 10-5 M {beta}-mercaptoethanol, essential and non-essential amino acids (Invitrogen), 100 units/ml penicillin G, 100 units/ml streptomycin, and 50 µg/ml gentamycin) at 37 °C with 5% CO2. Fibroblasts were transfected with a plasmid encoding an MCC:I-Ek chimera (27). The MCC:I-Ek chimera used for the B7lo line included the GyrB domains attached to the cytoplasmic tail, while that used for the B7hi line included green fluorescent protein. These cytoplasmic domains do not affect presentation of the peptide complex on the surface of the APCs to T cells (27) and data not shown. Transfectants were selected with 500 µg/ml G418 active drug, and drugresistant cells were screened for I-Ek surface expression by flow cytometry and cloned by limiting dilution.

Transgenic Mice—The cDNA encoding B-Raf was inserted into a unique XhoI site downstream of the CD4 promoter under control of a T cell-specific enhancer element with the cis-acting silencer element deleted (28) (gift from Paul Allen, Washington University). This construct was used to generate transgenic mouse lines in C57BL6/J mice by standard procedures. Potential founders were genotyped using both tail and ear DNA by Southern blot using random-primed probes derived from the full-length cDNA and by PCR analysis using vector specific primers. The founders that were identified by these methods were mated with wild-type C57BL6/J mice and F1 litters genotyped by PCR. Three lines were established and shown to have the same phenotype. One B-Raf-expressing line was chosen to cross to the AD10 TCR transgenic line. Experiments were performed on adult mice, except for thymocyte staining which was performed on thymocytes from 3-week-old mice. For anergy experiments B-Raf transgenic animals were crossed with the TCR transgenic mouse line, AD10 (29). T cells from the AD10 TCR transgenic mice express a recombined TCR, which recognizes pigeon cytochrome c (PCC) peptide (amino acids 88–104) or moth cytochrome c (MCC) peptide (amino acids 88–103) presented by MHC class II I-Ek. B-Raf transgenic mice were also crossed with the TCR transgenic mouse line, AND, which also recognizes the PCC and MCC peptides presented by I-Ek (29). In all animal studies, experiments were performed in compliance with the relevant laws and institutional guidelines and were approved by the IACUC.

Flow Cytometry—The surface expression of CD8 and CD4 molecules on thymocytes was analyzed by direct staining with FITC-labeled anti-CD8 mAb (53–6.7, BD PharMingen) and Cy-Chrome-labeled anti-CD4 mAb (RM4-5, BD PharMingen). CD69 expression was analyzed on CD4+, V{alpha}11+, V{beta}3+T cells. Cells were stained with Cy-Chrome-labeled anti-CD4 mAb, PE-labeled anti-V{alpha}11 mAb (RR8-1, BD PharMingen), FITC-labeled anti-V{beta}3 mAb (KJ25, BD PharMingen) and biotinylated anti-CD69 mAb (H1.2F3, BD PharMingen) followed by streptavidin APC (BD PharMingen). Intracellular staining for B-Raf expression was performed following fixation and permeabilization with anti-B-Raf antibody (c-19, Santa Cruz Biotechnology Inc., Santa Cruz, CA) or rabbit IgG (Southern Biotechnology Inc.) followed by Cy5-labeled anti-rabbit antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA).

Surface expression of B7.1, B7.2, and I-Ek was determined by surface staining of fibroblasts with FITC-labeled anti-CD80, PE-labeled anti-CD86 (GL1, BD PharMingen) and D-4 mAb, which recognizes I-Ek:MCC (30), followed by PE-labeled anti-mouse IgG1 (Southern Biotechnology Inc.). The stained cells were analyzed by flow cytometry using a FACSCalibur. Viable cells were gated using forward and side scatter.

Primary T Cell Isolation and Stimulation—CD4+ T cells from the spleen or lymph nodes were purified by negative selection with monoclonal antibodies using SpinSep (17031, Stemcell Technologies, Vancouver, BC) according to the manufacturer's instructions. Ten million purified T cells were incubated with 0.5 µg/ml anti-CD3 antibody (clone 145-2C11, BD PharMingen) and/or 10 µg/ml anti-CD28 antibody (clone 37.51, BD PharMingen) at 4 °C for 30 min, washed, then stimulated with the addition of 10 µg/ml of goat anti-hamster immunoglobulin (Southern Biotechnology Assoc. Inc.) for 5 min at 37 °C.

T Cell Anergy—The two protocols for the induction of anergy used were modified from the methods of Paul Allen and co-workers (31) and Ronald Schwartz and co-workers (32). T cell blasts were generated from AD10 TCR transgenic and AD10xB-Raf mice by culturing splenocytes for 4–6 days with 2.5 µM PCC peptide in T cell media (RPMI 1640) containing 10% fetal calf serum and supplemented with 1 mM L-glutamine, sodium pyruvate (100 mg/ml), 5 x 10-5 M {beta}-mercaptoethanol, essential and non-essential amino acids, 100 units/ml penicillin G, 100 units/ml streptomycin, and 50 µg/ml gentamycin).

To initiate anergy via exposure to B7 low APCs, viable cells were recovered over a density gradient (Lympholyte M, Cedarlane, Hornby, Ontario) and 5 x 106 cells/ml were incubated with equal numbers of B7hi or B7lo expressing fibroblasts for 24 h. T cells were separated from fibroblasts by density gradient centrifugation and subsequent culture for 1 h on plates to remove any remaining adherent cells. T cells were then recovered and rested for 4–6 days prior to experimental use.

To initiate anergy via antibody stimulation, viable cells were recovered over a density gradient and 5 x 106cells/ml were incubated overnight with plate bound anti-TCR{beta} antibody (10 µg/ml, H57-957, BD PharMingen) or isotype control. T cells were removed from the plates and rested for 24 h prior to experimental use. For experiments using PD98059 (Sigma), the drug was applied at 20 µM during the initial 24-h incubation with fibroblast APCs.

Raf-1 and B-Raf Kinase Assays—T cell blasts were generated from wild-type and B-Raf transgenic mice by culturing splenocytes for 4–6 days with 0.25 µg/ml anti-CD3 antibody (clone 145-2C11) in T cell media (as described above). Clones were then rested 24 h prior to use in kinase assays. B-Raf and Raf-1 proteins were immunoprecipitated from 1000 µg of cell lysate with anti Raf-1 (E10, Santa Cruz Biotechnology Inc.) or anti-B-Raf antibody (C19, Santa Cruz Biotechnology Inc.) coupled to GammaBind Plus Sepharose (Amersham Biosciences) at 4 °C overnight. Kinase assays were performed as previously described (19).

In Vivo Rap1 and Ras Activation Assays—This assay was performed as previously described (5). Isolated T cells were stimulated as described above and lysed in ice-cold Rap1 lysis buffer with inhibitors, and the protein content of lysates determined by BioRad assay. 300 µg of total cell lysate was incubated with 40 µg of glutathione S-transferase (GST)-Ral-GDS-Ras binding domain (RBD) fusion protein (gift of J. L. Bos, Utrecht University, Utrecht, The Netherlands) coupled to glutathione-agarose beads for 1 h at 4 °C to recover activated Rap1 (Rap-GTP). Beads were pelleted and washed and the protein eluted from the beads by boiling in Laemmli buffer. Activated Ras (Ras-GTP) was isolated from cell lysates using agarose-coupled GST fused to the Ras-binding domain (RBD) of Raf-1 (GST-Raf-RBD) provided in a Ras activation kit (Upstate Biotechnology, Inc., Lake Placid, NY) according to the manufacturer's instructions. Proteins were resolved by SDS-PAGE and detected by Western blot (see below).

Preparation of Nuclear Extracts—Control and anti-TCR-treated T cells (2.5 x 106/ml) were rested for 48 h, then restimulated at 37 °C for 3 h in 6-well plates coated with 2 µg/ml anti-CD3 and 10 µg/ml anti-CD28 mAbs. Cells were then collected and washed with ice-cold phosphate-buffered saline and lysed in cytosolic buffer (10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.2% Nonidet P-40) containing 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 5 µg/ml leupeptin for 5 min on ice. Nuclei were removed by centrifugation, washed once in cytosolic buffer, then lysed in hypertonic buffer (50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol) containing 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, and 5 µg/ml leupeptin for 30 min on ice and then centrifuged for 15 min at 14,000 rpm at 4 °C to remove the insoluble fraction. Proteins were resolved by SDS-PAGE and detected by Western blot (see below).

Western Blot—Cells were lysed in ice-cold Rap1 lysis buffer with inhibitors and the protein content of lysates determined by BioRad assay as described above (5). Proteins were separated in a 10–12% gel, followed by transfer to a polyvinylidine difluoride membrane. Membranes were blocked in 5% bovine serum albumin and probed with either anti-Rap1 polyclonal antibody (Rap1 (121), Santa Cruz Biotechnology Inc.) or anti-Ras mAb (clone 10, Upstate Biotechnology, Inc.) and then with a horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Proteins were detected by enhanced chemiluminesence. Phosphorylation of ERK1, ERK2, JNK1, and JNK2 were detected from 30 µg of total cell lysate by immunoblotting with phosophospecific MAP kinase polyclonal antibodies (Cell Signaling Technology, Beverly, MA) and total ERKs and JNKs were detected by immunoblotting with anti-ERK and anti-JNK polyclonal antibodies (Santa Cruz Biotechnology). Levels of c-Fos were detected from 4 µg of total nuclear proteins by Western blot using anti-c-Fos antibody (H-125, Santa Cruz Biotechnology).

CD69 Expression—Anergic and non-anergic AD10 and AD10xB-Raf T cells (106 cells/ml) were stimulated with 2 µg/ml anti-CD3 and 10 µg/ml anti-CD28 plate bound Abs (BD PharMingen) for 18 h at 37 °C. Cells were harvested on ice and stained for CD69 expression with biotinylated anti-CD69 mAb (H1.2F3, BD PharMingen) followed by streptavidin APC (see "Flow Cytometry").

T Cell Proliferation Assay—Naïve splenocytes at a concentration of 106 cells/ml were added to 96-well plates (100 µl) in the presence of a range of concentrations of anti-CD3 antibody. Alternatively, purified naïve T cells at a concentration of 5 x 104 cells/ml were added to 96-well plates with varying concentrations of B7hi or B7lo fibroblasts. Purified AD10 and AD10xB-Raf T cells that were rested following anergy induction (5 x 105 cells/ml) were added to 96-well plates with 2 x 106 cells/ml irradiated B10.BR splenocytes in the presence of varying concentrations of PCC peptide. Assays were incubated for 72 h at 37 °C and pulsed with 1 µCi of [3H]thymidine/well for the final 16–18 h of culture. The plates were harvested using a Packard 96-well Filtermate Harvester and counted on a Packard Top Count Scintillation counter.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of B-Raf but Not Raf-1 Allows Rap1 to Activate Elk-1—It has been proposed that Rap1 is a potent activator of ERKs in B-Raf-expressing cells (8, 17). To test whether a constitutively active mutant of Rap1 (RapV12), could augment ERK signaling in T cells-transfected with B-Raf, we used a transcription-coupled assay monitoring the ERK-dependent phosphorylation of the transcription factor Elk-1 (17, 33). The data show that neither B-Raf nor RapV12 activated Elk-1 when transfected alone. However, when transfected together, B-Raf and RapV12 co-operated to activate Elk-1-dependent transcription. This action of B-Raf was specific for this isoform of the Raf family kinases, as co-transfection of Raf-1 with RapV12 did not enhance Elk-1 activity (Fig. 1A).



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  		FIG. 1.
Expression of the B-Raf transgene in murine T lymphocytes allows Rap1 to activate Elk-1. A, expression of B-Raf, but not Raf-1, allows Rap1 to activate Elk-1. Jurkat cells were transfected with both Gal4-Elk1and 5xGal4-E1b/luciferase as the reporter constructs and co-transfected with constitutively active Rap1 (RapV12) or empty vector (pcDNA3), and B-Raf or Raf-1. Elk-1 activation was measured as luciferase activity + S.E. B, expression of B-Raf in the thymus and lymph node of transgenic animals. Western blot for B-Raf expression in lymphocytes isolated from the thymus and lymph nodes taken from wild-type mice (wt) and B-Raf transgenic (tr) mice. Lysates were separated by SDS-PAGE and the proteins visualized by Western blot. T cells from both the lymph node and thymus of transgenic mice show expression of B-Raf protein at 95 kDa. Endogenous expression of B-Raf in PC12 cells was used as a positive control (PC12). C, B-Raf expression in thymocyte subsets of transgenic animals. B-Raf expression in thymocytes isolated from wild-type (gray line) and B-Raf transgenic (black line) mice was compared with wild-type and B-Raf thymocytes stained with a control antibody (gray shading). The staining is representative of two experiments. D, normal thymocyte development in B-Raf transgenic animals. CD4 and CD8 expression on thymocytes isolated from AD10, AD10xB-Raf, AND, and ANDxB-Raf animals.

 

The B-Raf Transgene Is Expressed in Transgenic but Not Wild-type Murine T Cells—To examine the role of ERKs in regulating T cell proliferative responses, we generated transgenic mice expressing the 95-kDa isoform of B-Raf under a modified CD4 promoter (34). In the thymus and lymph nodes of B-Raf transgenic mice, a 95-kDa band, representing the expressed B-Raf protein, was seen in Western blots, in contrast to wild-type animals, which showed no expression of B-Raf (Fig. 1B). B-Raf was also expressed within splenic T cells (data not shown). Intracellular B-Raf staining was identified in CD4/CD8 double negative (DN), double positive (DP), CD4+/CD8-, and CD4-/CD8+ T cells from the B-Raf-transgenic, but not in the wild-type mice (Fig. 1C).

To examine if the introduction of B-Raf had an effect on T cell development, we examined thymocyte development in AD10 TCR transgenic and AD10xB-Raf animals by examining CD4 and CD8 expression. The AD10 TCR is specific for pigeon or moth cytochrome c (PCC or MCC) presented on I-Ek. No differences were seen in positive selection of T cells into the CD4 compartment (Fig. 1D). Similarly, there were no differences in thymocyte development when B-Raf-expressing mice were crossed with AND TCR transgenic mice (Fig. 1D), whose TCR shares specificity with the AD10 TCR.

Transgenic Expression of B-Raf Enhances the Proliferation and ERK Activation of T Cells in a Costimulation-independent Manner—To evaluate the functional consequences of B-Raf expression in T cells, we tested the proliferative capacity of splenocytes following CD3 stimulation by cross-linking antibodies. B-Raf-expressing T cells showed an increased proliferation rate following CD3 stimulation compared with wild-type cells (Fig. 2A).



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  		FIG. 2.
Transgenic expression of B-Raf increases T cell proliferation and ERK activation in the absence of costimulation. A, B-Raf transgenic splenocytes proliferate more than wild-type splenocytes. Equal numbers of splenocytes from wild-type (open square) and B-Raf transgenic mice (closed circle) were incubated in the presence of increasing concentrations of soluble anti-CD3 antibody (anti-CD3). Data represent [3H]thymidine incorporation in counts per minute (c.p.m.) + S.E., from a representative experiment (n = 3). B, B-Raf transgenic T cells show greater ERK activation than wild-type T cells in the absence of costimulation. T cells were isolated from the spleens of wild-type and B-Raf transgenic mice. T cells were stimulated with anti-CD3 antibody (CD3), anti-CD28 antibody (CD28), or both (CD3+CD28) and cross-linking secondary antibody for 5 min or left-untreated (U) as indicated. Lysates were prepared and the proteins separated by SDS-PAGE followed by Western blot. The levels of pERK1 and pERK2 are indicated, in a representative Western blot (n = 2). C, B-Raf transgenic T cells show a decrease in B-Raf kinase activity and an increase in Raf-1 kinase activity in the presence of costimulation. Rested T cell blasts from wild-type and B-Raf transgenic mice were stimulated with anti-CD3 antibody (CD3), anti-CD28 antibody (CD28), or both (CD3+CD28) and cross-linking secondary antibody for 5 min or left-untreated (U) as indicated. B-Raf and Raf-1 were immunoprecipitated from lysates and kinase assays performed using GST-MEK as the substrate. The proteins were separated by SDS-PAGE followed by Western blot. The levels of phosphorylated GST-MEK1 (pGst-MEK) are indicated; in a representative Western blot (n = 2). D, model of signaling through Ras and Rap1 in T cells. 1) In wild-type T cells, the engagement of the TCR/CD3 complex via MHC/peptide activates both Ras and Rap1. Because Rap1 limits Ras activation of Raf-1 and ERKs, stimulation of the TCR in the absence of costimulation results in modest ERK activation. However, engagement of CD28 by B7 inhibits Rap1 and enhances ERK activation by removing the Rap1 block. 2) In B-Raf-expressing T cells, the expression of B-Raf alters the consequence of both TCR and CD28 signaling. Instead of limiting ERK activation, in the presence of B-Raf, Rap1 activation by TCR augments ERK signaling. As in AD10 T cells, CD28 activation inhibits Rap1. However, because TCR activation of Rap1/B-Raf-activated ERKs, this inhibition by CD28 blocks the Rap1/B-Raf component of ERK activation by the TCR. E, CD28 inhibits proliferation in B-Raf transgenic T cells. Splenocytes from wild-type (Wt) and B-Raf transgenic (B-Raf) animals were incubated in the presence of 0.25 µg/ml soluble anti-CD3 antibody (3) with 10 µg/ml or without anti-CD28 antibody (28). Data represent [3H]thymidine incorporation in counts per minute (c.p.m.) + S.E., (n = 3).

 

Rap1 is activated by TCR stimulation (5, 9, 10). To test if the expression of B-Raf in T cells allows activated Rap1 to couple positively to ERKs, we compared the activation of ERKs in wild-type and B-Raf transgenic T cells by Western blot (Fig. 2B). In isolated B-Raf-expressing splenic T cells, CD3 ligation alone caused a larger increase in the activity of ERK1 and ERK2 compared with wild-type cells.

We also examined the effect of B-Raf on the ability of costimulation via CD28 ligation to augment ERK activation. In wild-type cells, costimulation enhanced the magnitude of ERK activation (Fig. 2B). Interestingly, in the B-Raf-expressing cells, co-ligation of CD3 and CD28 did not lead to enhanced ERK activation. Rather, it inhibited ERK activation to below the levels seen by CD3 ligation alone (Fig. 2B), suggesting that the increased ERK activation seen in CD3-stimulated B-Raf-expressing cells could be inhibited by CD28 costimulation. The pattern of ERK activation in wild-type and B-Raf-expressing T cells reflects the activity of the Raf kinase isoforms. Endogenous Raf-1, which is expressed to similar levels in both wild-type and transgenic T cells, showed increased activation under conditions of costimulation in both cell types (Fig. 2C). In contrast, the activity of B-Raf (expressed only in transgenic T cells) was greater in the absence of costimulation (Fig. 2C). Under conditions of costimulation, Raf-1 activity was increased while B-Raf activity was decreased. The opposing regulation of Raf-1 and B-Raf following costimulation rules out a primary role of Ras in these actions, as Ras is thought to regulate Raf-1 and B-Raf similarly, and points to a role for molecules like Rap1, whose actions on Raf-1 and B-Raf are opposite. This is consistent with the ability of CD28 to negatively regulate Rap1 activity as shown previously in wild-type cells. The model predicts that inhibition of Rap1 by CD28 in B-Raf-expressing cells would inhibit rather than activate ERKs. It has been shown that CD28 stimulation selectively inhibits Rap1 but not Ras (5). Therefore, we suggest that the augmentation of CD3-induced ERK activation in the B-Raf-expressing T cells was likely mediated through Rap1, not Ras. This model is depicted in Fig. 2D.

To further compare the actions of CD28 in wild-type and B-Raf-expressing T cells, proliferation assays were performed using splenocytes isolated from either wild-type or B-Raf-expressing animals stimulated with anti-CD3 antibody in the presence or absence of anti-CD28 antibody (Fig. 2E). As expected, wild-type T cell proliferation increased in the presence of anti-CD28 antibody. Interestingly, proliferation of B-Raf-expressing T cells in the presence of anti-CD28 antibody was reduced from the level seen following CD3 stimulation alone to a level below that seen in wild-type T cells costimulated with anti-CD3 and anti-CD28. Proliferation of T cells was inhibited by the pharmacological inhibition of ERKs (data not shown). These data are consistent with CD28 inhibition of Rap1 resulting in decreased ERK activation and proliferation in B-Raf-expressing T cells.

B-Raf-expressing T Cells Proliferate in Response to B7loAPCs—Although antibody stimulation of the TCR has been widely used to recreate TCR signaling, the use of APCs expressing either high or low levels of B7 provides a more relevant model to examine the role of costimulatory molecules in antigen-dependent activation (35–37). We tested the ability of AD10 TCR transgenic and AD10xB-Raf T cells to proliferate in response to fibroblast APCs that expressed either high levels of B7 (B7hi APCs) or low levels of B7 (B7lo APCs). B7hi and B7lo fibroblasts expressed similar levels of I-Ek with an antigenic peptide covalently attached to the {beta}-chain. B7lo fibroblasts expressed less than 5% of the level of surface B7.1 than B7hi fibroblasts. B7.2 was undetectable in both lines (Fig. 3A). Following incubation with B7hi APCs, both AD10 and AD10xB-Raf T cells proliferated to the same degree, demonstrating that B7hi APCs provide functional costimulation (Fig. 3B). Following incubation with B7lo APCs, AD10 T cells were unable to proliferate, indicating that these APCs were not capable of providing functional costimulation (Fig. 3B). In contrast, B-Raf-expressing T cells proliferated better in response to B7lo APCs than in response to B7hi APCs, although their basal proliferation was the same. These data suggest that B-Raf-expressing T cells are able to be stimulated to proliferate in the absence of functional costimulation (Fig. 3B).



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  		FIG. 3.
B-Raf-expressing T cells proliferate following exposure to B7lo APCs. A, expression of MHC:peptide and B7 molecules on fibroblast APCs. Cell surface expression of B7.1, B7.2, and MCC:I-Ek on B7hi (thick black line) and B7lo (thin black line) fibroblasts was compared with the background staining obtained with an isotype control antibody (gray shading). B, AD10xB-Raf T cells show enhanced proliferative responses to costimulatory deficient (B7lo) APCs. Equal numbers of CD3+ T cells from AD10 TCR transgenic mice were incubated with B7hi APCs (AD10/B7hi, open triangle) or B7lo APCs (AD10/B7lo, closed triangle) expressing APCs. AD10xB-Raf T cells were incubated with B7hi APCs (AD10xB-Raf/B7hi, closed circle) or B7lo APCs (AD10xB-Raf/B7lo, open circle). Data represent [3H]thymidine incorporation in counts per minute (c.p.m.) + S.E., from a representative experiment (n = 2). C, AD10xB-Raf T cells do not become anergic after culture with B7lo APCs. AD10 T cells previously co-cultured with B7hi APCs (AD10/B7hi, open triangle) or B7lo APCs (AD10/B7lo, closed triangle) and AD10xB-Raf T cells previously co-cultured with B7hi APCs (AD10xB-Raf/B7hi, closed circle) or B7lo APCs (AD10xB-Raf/B7lo, open circle), were rested then incubated with irradiated B10.BR splenocytes in the presence of increasing concentrations of MCC peptide. Data represent [3H]thymidine incorporation in counts per minute (c.p.m.) + S.E., from a representative experiment (n = 3).

 

The absence of costimulation induces T cell anergy characterized by a lack of proliferation, loss of ERK activation, and the absence of IL-2 production in response to subsequent exposure to antigen with costimulation (38, 39). To examine whether B-Raf expression could overcome anergy, we measured the ability of AD10 and AD10xB-Raf T cells to proliferate in response to restimulation following exposure to B7hi or B7lo APCs. Both AD10 and AD10xB-Raf T cells stimulated with B7hi APCs were able to proliferate upon restimulation (Fig. 3C). AD10 T cells previously stimulated with B7lo APCs were anergic and unable to proliferate upon restimulation (Fig. 3C). This block in proliferation could be overcome by the addition of exogenous IL-2 (data not shown). In contrast, AD10xB-Raf cells previously stimulated with B7lo APCs were able to proliferate upon restimulation, suggesting that B-Raf activation of ERKs was sufficient to prevent anergy induction under conditions of low B7 (Fig. 3C).

Rap1 Is Constitutively Active in T Cells Stimulated with B7lo Fibroblasts and Correlates with Increased ERK Activation in B-Raf-expressing but Not Wild-type T Cells—We examined Rap1 and ERK activation in AD10 T cells 4–6 days after incubation with B7hi or B7lo APCs. When AD10 T cells previously incubated with B7hi fibroblasts were re-stimulated with anti-CD3 antibody, both Rap1 and Ras were activated, and there was a corresponding moderate activation of ERKs. Under conditions of costimulation, Rap1 activation was reduced and ERK activation was enhanced, while Ras activation remained the same (Fig. 4A, B7hi). Under the anergizing conditions (B7lo), Rap1 was constitutively activated (Fig. 4A, B7lo), and Ras and ERKs show no activity under all conditions of restimulation in these cells (Fig. 4A, B7lo).



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  		FIG. 4.
Rap1and ERK are activated in B-Raf-expressing T cells co-cultured with B7lo fibroblasts. A and B, ERKs are constitutively active in AD10xB-Raf T cells following culture with B7lo APCs. T cells from AD10 (A) and AD10xB-Raf (B) TCR transgenic mice previously co-cultured with B7hi APCs (B7hi) or B7lo APCs (B7lo), were restimulated with anti-CD3 antibody (3), anti-CD28 antibody (28), or both (3 + 28) and cross-linking secondary antibody for 5 min or left untreated (UT) as indicated. Panel 1, the level of Rap1 activation (Rap1-GTP) is shown. Panel 2, the level of Ras activation (Ras-GTP) is shown. Panel 3, The level of ERK activation (pERK1/2) is indicated. The levels of total ERK1 and ERK2 are indicated in panel 4. Each panel is a representative Western blot (n = 2).

 

We also examined Rap1 and ERK activation in AD10xB-Raf T cells. The expression of B-Raf did not alter Ras or Rap1 regulation in AD10xB-Raf cells, but dramatically altered the ERK response. Following incubation with B7hi APCs, ERK activation in B-Raf-expressing T cells followed the levels of Rap1 activation and was highest following CD3 stimulation and was reduced by co-stimulation (Fig. 4B, B7hi).

Following incubation of AD10xB-Raf T cells with B7lo APCs, Rap1, and ERKs were constitutively activated, whereas Ras showed no activity (Fig. 4B, low B7). These data provide strong evidence that Rap1 is signaling through B-Raf to activate ERKs. Like ERKs, the stress-activated protein kinases [SAP kinases, or c-Jun N-terminal kinases (JNKs)] are activated upon costimulation (40–42) and are inhibited during anergy (43, 44) (Fig. 5). Importantly, B-Raf expression did not affect JNK signaling (Fig. 5), demonstrating that the absence of JNK activation is not sufficient to induce anergy. Indeed, B-Raf has only one known action, the activation of MEK and ERKs. Therefore, these data suggest that the activation of ERKs is sufficient to prevent these T cells from becoming anergic.



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  		FIG. 5.
JNK activation is deficient in T cells co-cultured with B7lo APCs. A, AD10 T cells previously co-cultured with B7hi APCs (B7hi) or B7lo APCs (B7lo) were re-stimulated with anti-CD3 antibody (3), anti-CD8 antibody (28), or both (3 + 28), and cross-linking secondary antibody for 5 min or left untreated (UT) as indicated. B, AD10xB-Raf T cells were treated as in A. The levels of JNK activation (pJNK1/2, upper panel) and total JNK1/2 (lower panel) are indicated. Each panel is a representative Western blot (n = 2).

 

The Expression of B-Raf Is Not Sufficient to Rescue Anti-TCR Antibody-induced Anergy—To examine whether the expression of B-Raf could overcome antibody-induced anergy, we measured proliferation in AD10 and AD10xB-Raf T cells rested for 24 h following overnight incubation with anti-TCR-antibody (anti-TCR) or isotype control antibody (control). Control AD10 and AD10xB-Raf T proliferated in response to antigenic signals (Fig. 6A) and as expected, AD10 T cells stimulated with anti-TCR antibody were anergic and did not proliferate. Surprisingly, anti-TCR stimulation of AD10xB-Raf T cells also induced anergy, as these cells did not proliferate (Fig. 6A). The addition of exogenous IL-2 rescued the proliferation defect of both the AD10 and AD10xB-Raf anergic cells (data not shown), suggesting that these cells were anergic secondary to a deficiency in IL-2 production.



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  		FIG. 6.
Anergy in antibody-treated cells is not prevented by B-Raf, despite enhanced ERK activity. A, AD10xB-Raf T cells become anergic following stimulation with anti-TCR{beta}. Control and anti-TCR-treated T cells from AD10 and AD10xB-Raf animals were incubated with irradiated B10.BR splenocytes in the presence of increasing concentrations of PCC peptide. Control AD10 cells (open circle), anti-TCR treated AD10 cells (closed circle), control AD10xB-Raf cells (open square), and anti-TCR treated AD10xB-Raf cells (closed square). Data represent [3H]thymidine incorporation in counts per minute (c.p.m) + S.E. (B–E) Constitutive ERK activation in AD10xB-Raf T cells following stimulation with anti-TCR{beta}. Control AD10 (B) and anti-TCR-treated AD10 T cells (C), control AD10xB-Raf (D), and anti-TCR treated AD10xB-Raf T cells (E) were stimulated with anti-CD3 antibody (3), anti-CD28 antibody (28) or both (3 + 28), and cross-linking secondary antibody for 5 min or left untreated (UT) as indicated. Panel 1, the levels of Rap1 activation (Rap1-GTP) is shown. Panel 2, the levels of Ras activation (Ras-GTP) is shown. Panel 3, the levels of ERK activation (pERK1/2) are indicated. The levels of total ERK1 and ERK2 are indicated in panel 4. Each panel is a representative Western blot (n = 3).

 

Rap 1 and ERKs Are Constitutively Active in B-Raf-expressing T Cells following Antibody-induced Anergy—The biochemical effects seen following anergy induction by plate-bound anti-TCR-antibody (Fig. 6, B–E) mimicked those seen following incubation with APCs (Fig. 4). Control AD10 T cells responded to subsequent CD3 stimulation by activating ERKs to a modest degree. Costimulation (3 + 28) inhibited Rap1 activation and potentiated ERK activation, without further increasing Ras activation (Fig. 6B). Anti-TCR stimulated cells showed biochemical hallmarks of anergy: constitutive activation of Rap1, the absence of Ras activation, and absence of ERK activation under all conditions of restimulation (Fig. 6C).

Control AD10xB-Raf cells responded to subsequent CD3 stimulation by activating Rap1, Ras, and ERKs robustly. Costimulation (3 + 28) inhibited Rap1 activation, and reduced ERK activation, with no effect on Ras (Fig. 6D). Anti-TCR stimulated cells showed constitutive activation of Rap1, absence of Ras, and elevated ERK activity, under all conditions of restimulation (Fig. 6E). These data provide strong evidence that B-Raf expression converted Rap1 into an activator of ERKs. Moreover, these data demonstrate that both models of anergy induction (APC- and antibody-induced) induced similar changes in Rap, Ras, and ERKs and that the introduction of B-Raf selectively augmented ERKs in both models. However, in antibody-induced anergy, the constitutive activation of ERKs in AD10xB-Raf cells was not sufficient to block anergy.

ERK Activation in Anergic T cells Can Activate Downstream Effectors—To see if this constitutive ERK activation could couple to downstream targets, we examined the regulation of two T cell gene products whose expression is ERK-dependent; the T cell activation marker CD69 (45) and the transcription factor c-Fos (41, 46). CD69 expression in control AD10 and AD10xB-Raf T cells was enhanced to the same degree following restimulation. In contrast, the expression of CD69 in anti-TCR treated AD10xB-Raf T cells was significantly higher than that seen in anti-TCR treated AD10 T cells (Fig. 7A). Protein levels of c-Fos were increased following costimulation of control AD10 and AD10xB-Raf T cells, and this increase was lost in anti-TCR-treated AD10 T cells. In contrast, in anti-TCR-treated AD10xB-Raf T cells levels of c-Fos protein expression were much higher (Fig. 7B). Taken together the data demonstrate that increased ERK activity detected in anti-TCR-treated AD10xB-Raf cells were coupled to downstream effectors. Therefore, during antibody-induced anergy, ERK activation was not sufficient to rescue the proliferative defect of anergic T cells.



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  		FIG. 7.
ERK-dependent expression of CD69 and c-Fos are enhanced during antibody-induced anergy in AD10xB-Raf T cells. A, up-regulation of CD69 on AD10xB-Raf T cells following stimulation with anti-TCR{beta}. Control and anti-TCR treated AD10 and AD10xB-Raf T cells were stimulated for 18h with plate-bound anti-CD3 and anti-CD28 antibodies. Cell surface expression of CD69 on unstimulated (gray shading) and stimulated (black line) control and anti-TCR-treated T cells was compared with the background staining obtained with an isotype control APC-labeled mouse IgG (gray line). The staining was representative of three experiments. B, increased c-Fos expression in AD10xB-Raf T cells following stimulation with anti-TCR{beta}. Control and anti-TCR-treated AD10 and AD10xB-Raf T cells were re-stimulated (st) for 3 h with plate-bound anti-CD3 and anti-CD28 antibodies or left unstimulated (un). Nuclear extracts were prepared and c-Fos expression was measured by Western blot. The position of c-Fos is shown in a representative Western blot (n = 3).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Rap1 is constitutively activated when the TCR is engaged in the absence of costimulation, and may mediate anergy by inhibition of Ras-dependent ERK activation (13). It has been proposed that Rap1 activation during anergy limits signals from Ras to ERKs (13). This is consistent with the view that Rap1 can antagonize Ras signaling at the level of Raf-1 (47), although other mechanisms by which Rap1 can antagonize Ras have been proposed (48).

The distinct specificities of Ras and Rap1 for Raf-1 are probably not due to differences in affinity for Raf-1, but rather may be related to the subcellular localization of Ras and Rap1. Ras recruits Raf-1 to specific membrane locales where Raf-1 can be further activated, by phosphorylation, whereas Rap1 fails to do so (19). The inability of Rap1 to activate Raf-1 can be overcome by activating mutations at these sites of phosphorylation. These activating mutants encode residues that are present in B-Raf and provide an explanation for the specificity of Rap1 for B-Raf versus Raf-1.

We took advantage of this pattern of Rap1 activation to engineer T cells that expressed B-Raf, which allows Rap1 to activate ERKs. Exposure to APCs expressing high or low levels of B7 provided the control and anergizing conditions, respectively. B7lo APCs induced anergy in AD10 T cells but not in AD10xB-Raf T cells. Both kinds of T cells expressed the biochemical hallmarks of anergy (low Ras, and elevated Rap1 activity), but ERK activation was restored in the B-Raf-expressing T cells. Therefore, the absence of Ras activation in anergic T cells is not sufficient to maintain anergy in the presence of elevated ERK activity. Moreover, Rap1 activation correlated well with ERK activation in B-Raf-expressing cells, suggesting that Rap1 was capable of coupling to ERKs via B-Raf. We propose that elevated ERK activity during anergy induction and challenge is sufficient to prevent or reverse anergy in this model.

B-Raf Enhances ERK Signaling and Proliferation in the Absence of Costimulation—In wild-type cells, anti-CD3 stimulation in the absence of costimulation was a poor activator of Raf-1 and ERKs, and a potent activator of Rap1. In contrast, in B-Raf-expressing cells, anti-CD3 stimulation in the absence of costimulation was a potent activator of B-Raf and ERKs, yet remained a poor activator of Raf-1, suggesting that B-Raf activation by CD3 engagement was selectively enhanced. Since Ras can activate B-Raf and Raf-1 equally, we propose that the selective activation of B-Raf was not dependent on Ras. The introduction of B-Raf into T cells did not enhance basal or Ras-stimulated ERK activity, but only augmented Rap1-dependent ERK activation. This rules out any function of basal B-Raf activity, or Ras-dependent activation of B-Raf in ERK activation, and is consistent with the ability of B-Raf to activate ERKs in response to Rap1 activation. Interestingly, the effects of enhancing ERK signaling on T cell proliferation was only seen using unfractionated splenocytes containing splenic APCs or with purified T cells incubated with fibroblast APCs. In the absence of any APCs, purified wild-type and B-Raf-expressing T cells activated by plate-bound anti-CD3 antibody show no differences in proliferation (data not shown). This suggests that Rap1/B-Raf-dependent ERK activation by itself was not sufficient to enhance T cell proliferation unless additional signals were provided by APCs.

CD28 enhanced ERK activation without enhancing Ras activity, which suggested that this action of CD28 was mediated by the inhibition of Rap1 (5). Two previous results in Jurkat cells are consistent with this model. First, expression of a constitutively active mutant of Rap1 in Jurkat cells blocked the enhancement of ERKs by CD28. Second, expression of Rap1GAP, a selective inhibitor of Rap1, was able to augment ERK activity but prevented any further enhancement of ERKs by CD28 in these cells (5). Here, in both wild-type primary T cells and wild-type T cell blasts, CD28 inhibited Rap1 and enhanced Raf-1 and ERK activity, without any effect on Ras. In B-Raf transgenic primary T cells and T cell blasts, CD28 inhibited Rap1 and inhibited B-Raf and ERK activity, suggesting that the Rap1-B-Raf-ERK pathway may be predominant in these transgenic T cells. Taken together, these data strongly suggest that Rap1 inhibition was a target of CD28 action. The effect of CD28 on ERK activation was also reflected in T cell proliferation. In B-Raf-expressing cells, CD28 costimulation reduced proliferation, whereas in wild-type cells CD28 enhanced T cell proliferation. Similarly, B7hi APCs induced a higher level of proliferation in AD10 T cells compared with B7lo APCs. However, B7hi APCs reduced the level of proliferation of AD10xB-Raf T cells compared with B7lo APCs. These data suggest that CD28 regulation of ERKs can influence T cell proliferation in the presence of APCs.

B-Raf-dependent ERK Activation Prevents APC-induced Anergy—The absence of functional costimulation induces anergy in T cells, defined by the inability to proliferate upon restimulation. Here, AD10 T cells became anergic following TCR engagement with B7lo APCs. AD10 T cells did not activate ERKs upon subsequent costimulation, and Rap1 was constitutively active in these cells, consistent with previous findings in murine and human T cell clones (13, 44, 49). However, AD10xB-Raf T cells did not become anergic following exposure to B7lo APCs and were able to proliferate in response to subsequent costimulation. In these cells Rap1 was constitutively active and Ras activation was inhibited, similar to that seen in anergic AD10 T cells. The only difference between these cells was a consequence of B-Raf expression, which, in the face of constitutive Rap1 activation, resulted in constitutive ERK activation. Therefore, we propose that Rap1/B-Raf/ERK signaling was able to prevent anergy in these cells by providing an ERK signal in the absence of Ras activation. B-Raf may also act during the induction of anergy by providing an adequate ERK signal in the absence of costimulation. We found that prevention of anergy was blocked by application of the MEK inhibitor PD98059 during the initial exposure of AD10xB-Raf T cells with B7lo APCs (data not shown). Therefore, we propose that B-Raf expression also prevented anergy by promoting ERK-dependent signaling during the initiation of the anergic protocol. Indeed, the lack of proliferation during initial stimulation of T cells has been associated with anergy (50, 51). However, we cannot rule out other actions of ERKs. Although ERK activation is sufficient to prevent anergy it may not be necessary if other costimulatory pathways are intact (23).

Antibody-induced Anergy Cannot be Prevented by B-Raf Expression—B-Raf-expressing T cells became anergic following stimulation with plate-bound anti-TCR{beta}. This is despite the ability of B-Raf to augment ERK activation and the ERK effector pathways including c-Fos and CD69 expression. These data suggest ERK activation is not sufficient to block antibody-induced anergy. This suggests that antibody-mediated anergy may be qualitatively different than anergy induced in response to B7lo APCs, despite inducing similar biochemical alterations in Ras, Rap1, and ERKs. Additional negative regulation of the IL-2 gene at the promoter level independent of upstream signaling has been shown (52–54). Such additional blocks in anergic cells were invoked by Crespi et al. (22) who showed that ERK activation via expression of a constitutively active mutant of Ras was not sufficient to prevent anti-CD3 antibody-induced anergy. Non-physiological activation of the TCR complex by plate-bound antibodies may also account for the inability of Ras-ERK pathways to overcome antibody-induced anergy. Alternatively, given the low but detectable levels of B7.1 expressed on B7lo APCs, it is also possible that CTLA-4 is involved.

We confirm previous results that constitutive activation of Rap1 is a consistent feature of anergic cells (13). However, no mechanism of Rap1 action in anergy has been established. Much recent attention has focused on actions of Rap1 that are independent of ERK (15, 55). However, the studies presented here demonstrate that constitutive activation of Rap1 by itself is not sufficient to induce anergy, if ERKs are also activated. This is consistent with the classical role of Rap1 as an antagonist of Ras signaling but does not rule out other actions of Rap1. Rap1 has been shown to have a positive role in the activation of T cells by enhancing integrin-dependent T cell/APC interactions (12, 15). It is possible that while Rap1 initially potentiates T cell activation by promoting integrin-dependent interactions, it may interfere with TCR signaling to ERKs at later time points. It is possible that this explains the necessity to inhibit Rap1 signaling after initial T cell adhesion through engagement of CD28 (5, 10, 12, 15). Interestingly, CD28 inhibition of Rap1 was absent in anergic T cells. Perhaps this loss of inhibitory function of CD28 contributes to the inability of anergic T cells to respond to costimulation.

In summary, our results demonstrate that reconstituting ERK activation in anergic T cells via the expression of B-Raf is sufficient to prevent APC-induced anergy. This is the first example of enhanced ERK signaling blocking the induction of anergy in T cells. However, anergy induction by cross-linking antibodies in the absence of APCs could not be overcome by ERK activation, suggesting that additional pathways are required to maintain T cell responsiveness. Triggering anergy by APCs rather than cross-linking antibodies may better reflect anergy induction seen in physiological and pathophysiological settings. Moreover, these studies suggest that inducing B-Raf expression in T cells may selectively enhance T cell function in anergic cells.


   FOOTNOTES
 
* This work was supported by the National Institutes of Health (to D. C. P., A. S. S., and P. J. S. S.; RO1 AI47337) and the Washington-Monsanto Biomedical Research Agreement (to A. S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Vollum Institute, L-474, Oregon Health Sciences University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3098. Tel.: 503-494-5494; Fax: 503-494-4976; E-mail: stork{at}ohsu.edu.

1 The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; IL, interleukin; mAb, monoclonal antibody; TCR, T cell receptor; FITC, fluorescein isothiocyanate; PCC, pigeon cytochrome c; MCC, moth cytochrome c; APC, antigen presenting cell; GST, glutathione S-transferase; RBD, Ras-binding domain. Back


   ACKNOWLEDGMENTS
 
We thank M. White of the Immunology Microinjection Facility (Washington University) for help with generating the transgenic mice. Dr. D. Littman for providing the CD4-promoter construct and M. Findlay for technical assistance. We appreciate Drs. K. Carey, A. Baird, Q. Low, and P. Allen for scientific discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Whitehurst, C. E., and Geppert, T. D. (1996) J. Immunol. 156, 1020-1029[Abstract]
  2. van den Brink, M. R., Kapeller, R., Pratt, J. C., Chang, J. H., and Burakoff, S. J. (1999) J. Biol. Chem. 274, 11178-11185[Abstract/Free Full Text]
  3. Chen, T., Cho, R. W., Stork, P. J., and Weber, M. J. (1999) Cancer Res. 59, 213-218[Abstract/Free Full Text]
  4. Kitayama, H., Sugimoto, Y., Matsuzaki, T., Ikawa, Y., and Noda, M. (1989) Cell 56, 77-84[CrossRef][Medline] [Order article via Infotrieve]
  5. Carey, K. D., Dillon, T. J., Schmitt, J. M., Baird, A. M., Holdorf, A. D., Straus, D. B., Shaw, A. S., and Stork, P. J. S. (2000) Mol. Cell. Biol. 20, 8409-8419[Abstract/Free Full Text]
  6. Hu, C. D., Kariya, K., Kotani, G., Shirouzu, M., Yokoyama, S., and Kataoka, T. (1997) J. Biol. Chem. 272, 11702-11705[Abstract/Free Full Text]
  7. Okada, S., Matsuda, M., Anafi, M., Pawson, T., and Pessin, J. E. (1998) EMBO J. 17, 2554-2565[CrossRef][Medline] [Order article via Infotrieve]
  8. Schmidt, M., Goebeler, M., Posern, G., Feller, S. M., Seitz, C. S., Brocker, E. B., Rapp, U. R., and Ludwig, S. (2000) J. Biol. Chem. 275, 41011-41017[Abstract/Free Full Text]
  9. Amsen, D., Kruisbeek, A., Bos, J. L., and Reedquist, K. (2000) Eur. J. Immunol. 30, 2832-2841[CrossRef][Medline] [Order article via Infotrieve]
  10. Reedquist, K. A., and Bos, J. L. (1998) J. Biol. Chem. 273, 4944-4949[Abstract/Free Full Text]
  11. Czyzyk, J., Leitenberg, D., Taylor, T., and Bottomly, K. (2000) Mol. Cell. Biol. 20, 8740-8747[Abstract/Free Full Text]
  12. Katagiri, K., Hattori, M., Minato, N., and Kinashi, T. (2002) Mol. Cell. Biol. 22, 1001-1015[Abstract/Free Full Text]
  13. Boussiotis, V. A., Freeman, G. J., Berezovskaya, A., Barber, D. L., and Nadler, L. M. (1997) Science 278, 124-128[Abstract/Free Full Text]
  14. de Bruyn, K. M., Rangarajan, S., Reedquist, K. A., Figdor, C. G., and Bos, J. L. (2002) J. Biol. Chem. 277, 29468-29476[Abstract/Free Full Text]
  15. Sebzda, E., Bracke, M., Tugal, T., Hogg, N., and Cantrell, D. A. (2002) Nat. Immunol. 3, 251-258[CrossRef][Medline] [Order article via Infotrieve]
  16. Ohtsuka, T., Shimizu, K., Yamamori, B., Kuroda, S., and Takai, Y. (1996) J. Biol. Chem. 271, 1258-1261[Abstract/Free Full Text]
  17. Vossler, M., Yao, H., York, R., Rim, C., Pan, M.-G., and Stork, P. J. S. (1997) Cell 89, 73-82[CrossRef][Medline] [Order article via Infotrieve]
  18. York, R. D., Yao, H., Dillon, T., Ellig, C. L., Eckert, S. P., McCleskey, E. W., and Stork, P. J. S. (1998) Nature 392, 622-625[CrossRef][Medline] [Order article via Infotrieve]
  19. Carey, K. D., Watson, D. E., Pessin, J. E., and Stork, P. J. S. (2003) J. Biol. Chem. 278, 3185-3196[Abstract/Free Full Text]
  20. Barberis, L., Wary, K. K., Fiucci, G., Liu, F., Hirsch, E., Brancaccio, M., Altruda, F., Tarone, G., and Giancotti, F. G. (2000) J. Biol. Chem. 275, 36532-36540[Abstract/Free Full Text]
  21. Dugan, L. L., Kim, J. S., Zhang, Y., Bart, R. D., Sun, Y., Holtzman, D. M., and Gutmann, D. H. (1999) J. Biol. Chem. 274, 25842-25848[Abstract/Free Full Text]
  22. Crespi, D., Massa, S., Basso, V., Colombetti, S., Mueller, D. L., and Mondino, A. (2002) Eur. J. Immunol. 32, 2500-2509[CrossRef][Medline] [Order article via Infotrieve]
  23. DeSilva, D. R., Jones, E. A., Favata, M. F., Jaffee, B. D., Magolda, R. L., Trzaskos, J. M., and Scherle, P. A. (1998) J. Immunol. 160, 4175-4181[Abstract/Free Full Text]
  24. Faris, M., Kokot, N., Lee, L., and Nel, A. E. (1996) J. Biol. Chem. 271, 27366-27373[Abstract/Free Full Text]
  25. Izquierdo, M., Bowden, S., and Cantrell, D. (1994) J. Exp. Med. 180, 401-406[Abstract/Free Full Text]
  26. Rayter, S. I., Woodrow, M., Lucas, S. C., Cantrell, D. A., and Downward, J. (1992) EMBO J. 11, 4549-4556[Medline] [Order article via Infotrieve]
  27. Wetzel, S. A., McKeithan, T. W., and Parker, D. C. (2002) J. Immunol. 169, 6092-6101[Abstract/Free Full Text]
  28. Sawada, S., Scarborough, J. D., Killeen, N., and Lipmann, D. R. (1994) Cell 77, 917-929[CrossRef][Medline] [Order article via Infotrieve]
  29. Kaye, J., Vasquez, N. J., and Hedrick, S. M. (1992) J. Immunol. 148, 3342-3353[Abstract]
  30. Reay, P. A., Matsui, K., Haase, K., Wulfing, C., Chien, Y. H., and Davis, M. M. (2000) J. Immunol. 164, 5626-5634[Abstract/Free Full Text]
  31. Sloan-Lancaster, J., Evavold, B. D., and Allen, P. M. (1993) Nature 363, 156-159[CrossRef][Medline] [Order article via Infotrieve]
  32. Jenkins, M. K., Chen, C. A., Jung, G., Mueller, D. L., and Schwartz, R. H. (1990) J. Immunol. 144, 16-22[Abstract]
  33. Marais, R., Wynne, J., and Treisman, R. (1993) Cell 73, 381-393[CrossRef][Medline] [Order article via Infotrieve]
  34. Sawada, S., and Littman, D. R. (1991) Mol. Cell. Biol. 11, 5506-5515[Abstract/Free Full Text]
  35. Gimmi, C. D., Freeman, G. J., Gribben, J. G., Gray, G., and Nadler, L. M. (1993) Proc. Natl. Acad. Sci. U. S. A., U. S. A. 90, 6586-6590[Abstract/Free Full Text]
  36. Ragazzo, J. L., Ozaki, M. E., Karlsson, L., Peterson, P. A., and Webb, S. R. (2001) Proc. Natl. Acad. Sci., U. S. A. 98, 241-246[Abstract/Free Full Text]
  37. Chen, C., Faherty, D. A., Gault, A., Connaughton, S. E., Powers, G. D., Godfrey, D. I., and Nabavi, N. (1994) J. Immunol. 152, 2105-2114[Abstract]
  38. Jenkins, M. K., Burrell, E., and Ashwell, J. D. (1990) J. Immunol. 144, 1585-1590[Abstract]
  39. Kang, S. M., Beverly, B., Tran, A. C., Brorson, K., Schwartz, R. H., and Lenardo, M. J. (1992) Science 257, 1134-1138[Abstract/Free Full Text]
  40. Su, B., Jacinto, E., Hibi, M., Kallunki, T., Karin, M., and Ben-Neriah, Y. (1994) Cell 77, 727-736[CrossRef][Medline] [Order article via Infotrieve]
  41. Mondino, A., Whaley, C. D., DeSilva, D. R., Li, W., Jenkins, M. K., and Mueller, D. L. (1996) J. Immunol. 157, 2048-2057[Abstract]
  42. Werlen, G., Jacinto, E., Xia, Y., and Karin, M. (1998) EMBO J. 17, 3101-3111[CrossRef][Medline] [Order article via Infotrieve]
  43. DeSilva, D. R., Feeser, W. S., Tancula, E. J., and Scherle, P. A. (1996) J. Exp. Med. 183, 2017-2023[Abstract/Free Full Text]
  44. Li, W., Whaley, C. D., Mondino, A., and Mueller, D. L. (1996) Science 271, 1272-1276[Abstract]
  45. D'Ambrosio, D., Cantrell, D. A., Frati, L., Santoni, A., and Testi, R. (1994) Eur. J. Immunol. 24, 616-620[Medline] [Order article via Infotrieve]
  46. Murphy, L. O., Smith, S., Chen, R. H., Fingar, D. C., and Blenis, J. (2002) Nat. Cell Biol. 4, 556-564[Medline] [Order article via Infotrieve]
  47. Cook, S. J., Rubinfeld, B., Albert, I., and McCormick, F. (1993) EMBO J. 12, 3475-3485[Medline] [Order article via Infotrieve]
  48. Stork, P. J. S. (2003) Trends Biochem. Sci. 28, 267-275[CrossRef][Medline] [Order article via Infotrieve]
  49. Fields, P., Fitch, F. W., and Gajewski, T. F. (1996) J. Mol. Med. 74, 673-683[CrossRef][Medline] [Order article via Infotrieve]
  50. DeSilva, D. R., Urdahl, K. B., and Jenkins, M. K. (1991) J. Immunol. 147, 3261-3267[Abstract]
  51. Wells, A. D., Walsh, M. C., Bluestone, J. A., and Turka, L. A. (2001) J. Clin. Invest. 108, 895-903[CrossRef][Medline] [Order article via Infotrieve]
  52. Becker, J. C., Brabletz, T., Kirchner, T., Conrad, C. T., Brocker, E. B., and Reisfeld, R. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2375-2378[Abstract/Free Full Text]
  53. Kitagawa-Sakakida, S., and Schwartz, R. H. (1996) J. Immunol. 157, 2328-2339[Abstract]
  54. Telander, D. G., Malvey, E. N., and Mueller, D. L. (1999) J. Immunol. 162, 1460-1465[Abstract/Free Full Text]
  55. Enserink, J. M., Christensen, A. E., De Rooij, J., Van Triest, M., Schwede, F., Genieser, H. G., Doskeland, S. O., Blank, J. L., and Bos, J. L. (2002) Nat. Cell Biol. 4, 901-906[CrossRef][Medline] [Order article via Infotrieve]

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