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J Biol Chem, Vol. 274, Issue 36, 25743-25748, September 3, 1999

Evidence for a p21^ras/Raf-1/MEK-1/ERK-2-independent Pathway in Stimulation of IL-2 Gene Transcription in Human Primary T Lymphocytes^*

Virginie Lafont^§, Florence Ottones^¶, Janny Liautard^¶, and Jean Favero^¶

From the  Lymphocyte Activation Laboratory, Imperial Cancer Research Fund, London, United Kingdom WC2A 3PX and ^¶ INSERM U431, Microbiologie et Pathologie Cellulaire Infectieuse, Université de Montpellier II, Place Eugène Bataillon, cc 100, 34095 Montpellier cedex 05, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

T cell stimulation leads to triggering of signals transmitted from the cell membrane to the nucleus through TCR/CD3 proteins. Characterization of these signals largely results from the use of cell lines stimulated with anti-CD3 monoclonal antibodies. These studies have established that activation caused a rapid increase in the formation of GTP-bound Ras, which stimulates the mitogen-activated protein kinase pathway involving the extracellular-regulated kinase-2 (ERK-2) and activates the nuclear factor of activated T cells (NF-AT) that regulates interleukin-2 (IL-2) gene transcription. In the present study, we used human primary T cells, and we investigated the intracellular signals triggered by two different anti-CD3 monoclonal antibodies (UCHT1 and X-35), which both strongly induce cell proliferation. We found that, in contrast to the commonly used UCHT1, X-35 activated IL-2 gene transcription without stimulation of the Raf-1/mitogen-activated ERK kinase-1 (MEK-1)/ERK-2 phosphorylation cascade; we also showed that X-35 stimulation, which triggers an ERK-2-independent pathway, does not involve activation of p21^ras. In addition to demonstrating that activation of p21^ras and of its Raf-1/MEK-1/ERK-2 effector pathway is not an event obligatorily triggered upon TCR/CD3 ligation, these results provide the first evidence of the existence of a p21^ras/ERK-2-independent pathway for IL-2 gene transcription in human primary T lymphocytes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Binding of monoclonal antibodies to the CD3 complex has been used as model system that mimics antigen recognition to characterize the biochemical events leading to interleukin-2 production and T cell proliferation. Several studies have brought evidence that the intracellular signals that mediate activation of transcription factors regulating IL-2¹ gene transcription in human T cells involve p21^ras-mediated signaling pathways (1-5). These studies obtained with T cell lines collectively suggest that the Raf-1/MEK-1/ERK-2 phosphorylation cascade is the necessary (6-8) (if not sufficient (9)) p21^ras effector pathway for nuclear factor of activated T cell (NF-AT) induction in human T cells. These conclusions, which mainly result from the expression of dominant negative or constitutively active p21^ras (4-6), Raf-1 (10), or MEK-1 (4, 9, 11) mutants in Jurkat cells, have created a paradigm that p21^ras/ERK-2 pathway is the major route for activation of IL-2 gene transcription in TCR/CD3 induced activation of T lymphocytes. However, it cannot be excluded that an ERK-2-independent pathway might be used in primary T cells. Indeed a result obtained with splenocytes from transgenic mice expressing an inactive form of MEK-1 (12) suggested the possibility of the existence of a TCR/CD3-induced MEK-1/ERK-2-independent pathway even though one can question whether these cells, which developed in the absence of positive selection, are representative of a normal T lymphocyte population. Therefore, a clear physiological involvement of the MEK/ERK cascade in T cell activation is still a matter of debate, in part due to the fact that molecular genetic approaches are limited to cell lines or transgenic animals. Our aim was to study whether the stimulatory signals from the TCR/CD3 complex that promote IL-2 gene transcription obligatorily involve the p21^ras/Raf-1/MEK-1/ERK-2 pathway in primary T cells. We used highly purified CD4⁺ human lymphocytes that we stimulated with UCHT1 or X-35, two mitogenic anti-CD3 mAb (13, 14) recognizing the  chain of the CD3 complex (15) but presenting a pan thymocyte reactivity and a specific medullary thymocyte reactivity, respectively (14). We analyzed the effect of these antibodies on the Raf-1/MEK-1/ERK-2 phosphorylation cascade. We found that, in contrast to what happens with UCHT1, activation of IL-2 gene transcription triggered upon X-35 ligation occurred without activation of the Raf-1/MEK-1/ERK-2 pathway; moreover, we showed that this ERK-2-independent pathway does not involve activation of p21^ras. Altogether, the results we present herein demonstrate that activation of p21^ras/Raf-1/MEK-1/ERK-2 phosphorylation cascade is not an obligatory event triggered upon TCR/CD3 ligation; moreover, they bring evidence that activation of this cascade is not essential for IL-2 gene transcription in human T lymphocytes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chemicals and Reagents

UCHT1 (IgG1) and X-35 (IgG2a) anti-CD3 mAb were from Immunotech (Marseille, France), mouse anti-phosphotyrosine mAb (4G10) was from Upstate Biotechnology Inc. (Lake Placid, New York); rabbit anti-ERK-2 Ab, rabbit anti-Raf-1 Ab, and rabbit anti-ZAP-70 Ab were from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated rabbit anti-mouse and donkey anti-rabbit were from Amersham Pharmacia Biotech. Rabbit anti-phosphoserine 473 PKB and PD098059 were from New England Biolabs Inc. (Beverly, MA), and PMA and myelin basic protein (MBP) were from Sigma. GST-Elk-1 was obtained from Dr. A. Nordheim (Tubingen University, Germany).

Cell Preparation and Culture

Peripheral blood monocyte cells were isolated from peripheral blood from healthy donors. Monocytes were removed by plastic adherence, and CD4⁺ T cells were purified (>99% pure) by positive immunoselection using magnetic beads coated with anti-CD4 mAb (Dynal International, Oslo, Norway) according to the manufacturer's instructions. Before being used, CD4⁺-purified T cells were left 15 to 18 h in RPMI 1640 supplemented with 10% fetal calf serum and gentamycin at 37 °C in a 5% CO₂-humidified atmosphere. CD4⁺ cells were stimulated (72 h) either with soluble anti-CD3 mAb or with anti-CD3 coated on anti-IgG-conjugated beads. Proliferation was estimated by a 4-h [³H]thymidine incorporation.

Analysis of IL-2 mRNA Expression by Reverse Transcription-Polymerase Chain Reaction

CD4⁺ T cells were stimulated for 6 h in the presence of 1 µM ionomycin and anti-CD3 mAb (1 µg/ml). The cells were treated with PD098059 (30 µM) as described. Total RNA isolation, reverse transcription reaction, and polymerase chain reaction were performed as already described (16).

Analysis of MAP Kinase Activation

Analysis of MAP Kinase Phosphorylation-- This analysis was performed as described (16). Briefly, CD4⁺ cells (5 × 10⁶/ml) were stimulated with anti-CD3 mAb (10 µg/ml) or phorbol esters (PMA or phorbol dibutyrate (PDBu)). The supernatants were resolved in a 12.5% SDS-PAGE, and the gel was transferred onto a PVDF membrane (polyscreen, NEN Life Science Products). After blocking of nonspecific binding, the membrane was probed with anti-ERK-2 Ab (0.2 µg/ml) and revealed with horseradish peroxidase-conjugated anti-rabbit antibody (1:20,000) followed by enhanced chemiluminescence detection system (NEN Life Science Products). Reprobing of the same blots with the anti-phosphotyrosine mAb 4G10 (1 µg/ml) was performed after stripping of bound Ab. The membrane was revealed with 1:10,000 solution of horseradish peroxidase-conjugated-anti-mouse Ab and the chemiluminescence detection system. Raf-1 was similarly revealed on PVDF membranes electroblotted from 8% SDS-PAGE (anti-Raf-1 antibody was used as 0.5 µg/ml).

Analysis of MAP Kinase Activity Using MBP as Substrate-- This experiment was done as described (16). Stimulated cells were lysed, and ERK-2 was immunoprecipitated with 1 µg of anti-ERK antibody and protein A-Sepharose. After washing, in vitro phosphorylation was carried out for 20 min at 30 °C in kinase buffer with 10 µg of MBP, 50 µM ATP, 1 µCi of [-³²P]ATP. Proteins were resolved in 10% SDS-PAGE, electroblotted and visualized by autoradiography, and quantitatively analyzed with a PhosphorImager (Molecular Dynamics, Inc.).

Analysis of MAP Kinase Activity Using GST-Elk-1 as Substrate-- GST-Elk-1 phosphorylation was performed as already reported (17). Briefly, activated cells were lysed, and the supernatants were mixed with GST fusion protein kinase substrate and glutathione-agarose (Sigma) and incubated overnight at 4 °C. The substrate-agarose complexes were washed, and in vitro phosphorylation was carried out for 20 min at 30 °C in kinase assay buffer. Proteins were fractionated by 10% SDS-PAGE, electrotransferred, and revealed by autoradiography.

Analysis of p21^ras Activation

Purified CD4⁺ cells (20.10⁶/ml) were incubated with anti-CD3 mAb (10 µg/ml) or phorbol esters (PDBu, 50 ng/ml) and then lysed for 30 min in cold lysis buffer (50 mM Hepes, pH 7.4, 10 mM NaF, 10 mM iodoacetamide, 75 mM NaCl, 1% Nonidet P-40, 10 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₂VO₃, and pepstatin A, chymostatin, and leupeptin (each at 1 mg/ml). The supernatant were mixed with GST-Ras binding domain of Raf (RDB) fusion protein (10 µg/ml) (a generous gift from Dr. Julian Downward, Imperial Cancer Research Fund, London) and glutathione-agarose (Sigma) for 2 h at 4 C. GST-RBD contains the Ras binding domain of Raf and binds only the active form of Ras (19). Protein-agarose complexes were washed and solubilized in electrophoresis buffer. Proteins were resolved in 15% SDS-PAGE and electroblotted. The membrane was revealed with an anti-Ras mAb (Calbiochem).

Analysis of p56^lck Autophosphorylation

These experiments were done as in Lafont et al. (18). Briefly, 10⁷ CD4⁺ T cells stimulated with PDBu or with anti-CD3 mAb were lysed, and p56^lck was immunoprecipitated with rabbit anti-p56^lck polyclonal Ab (a generous gift from Dr. S. Fisher, Hôpital Cochin, Paris, France) and protein A-Sepharose. Complexes were resuspended in kinase buffer, and autophosphorylation of p56^lck was determined in the presence of 5 µCi of [-³²P]ATP (6000 Ci/mmol, NEN Life Science Products). Radiolabeled protein were then resolved on 8% SDS-PAGE, blotted onto PVDF membrane. and detected by autoradiography.

Analysis of ZAP-70 Tyrosine Phosphorylation

5 ×10⁷ unstimulated CD4⁺ T cells as well as X-35-or UCHT1-stimulated cells (5 min at 37 °C) were lysed in lysis buffer, and ZAP-70 was immunoprecipitated with rabbit anti-ZAP-70 polyclonal antibody and protein A-Sepharose. Immunoprecipitate complexes were recovered and solubilized in electrophoresis buffer. After SDS-PAGE and electroblotting, the membrane was revealed with antiphosphotyrosine 4G10. The blot was then reprobed after stripping of bound Ab with anti-ZAP-70 antibody.

Analysis of PKB/Akt Phosphorylation

Purified CD4⁺ cells (5.10⁶/ml) were incubated with anti-CD3 mAb (10 µg/ml) for the indicated times and lysed in a lysis buffer comprised of 50 mM Hepes, pH 7.9, 1% Nonidet P-40, 150 mM NaCl, 0.1 mM EDTA, 10 mM NaF, 1 mM Na₃VO₃, 1 mM phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml chymostatin. The supernatants were resolved in a 7.5% SDS-PAGE, and the gel was transferred onto a PVDF membrane. After blocking of nonspecific binding, the membrane was probed with a rabbit antiphosphoserine 473 PKB Ab (1:1000) and revealed with horseradish peroxidase-conjugated anti-rabbit antibody (1:2000) followed by enhanced chemiluminescence detection. Reprobing of the same blots with the anti-PKB Ab (1:1000) was performed after stripping of bound Ab. The membrane was revealed with 1:2000 solution of horseradish peroxidase-conjugated-anti-rabbit Ab and the chemiluminescence detection system. Raf-1 was similarly revealed on PVDF membranes electroblotted from 8% SDS-PAGE (anti-Raf-1 antibody was used as 0.5 µg/ml).

Analysis of Tyrosine Phosphorylation

Cells were stimulated and lysed as described for MAP kinase phosphorylation studies. The proteins were resolved on 10% SDS-PAGE, and the gel was transferred onto a PVDF membrane. After blocking of nonspecific binding, the membrane was probed with the anti-phosphotyrosine 4G10 antibody (1 µg/ml) and revealed with a 1:10,000 solution of horseradish peroxidase-conjugated anti-mouse Ab followed by enhanced chemiluminescence detection system.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Analysis of MAP Kinase Pathway Activation in CD3-stimulated Cells-- We first showed that highly purified CD4⁺ T cells that do not respond to soluble anti-CD3 mAb are activated by and proliferate in response to both X-35 and UCHT1 when coated on beads (Table I).

We then analyzed phosphorylation (appearance on electroblot of a slow migrating band) (20, 21) and activation of ERK-2. Fig. 1A shows that a shifted band, not present after 1-min stimulation, is clearly detected by anti-ERK-2 Ab after a 5-min activation with UCHT1, then diminishes after 15 min and is no more detectable after 20 min. Conversely, no shifted band can be detected in X-35 stimulation at any time of the analysis. These experiments were performed using 10 µg/ml anti-CD3 mAb. We checked that the difference between the two mAb in term of ERK-2 activation is also observed in a large concentration range (1 to 20 µg/ml) (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Study of the activation of ERK-2 upon X-35 and UCHT1 stimulation in CD4+-purified T cells. CD4+-purified T cells were stimulated with X-35 or with UCHT1 for 1 or 20 min (a 5-min PMA stimulation was used as control). A, ERK-2 corresponding bands were detected on blotted membranes using anti-ERK-2 antibody. The result shows the presence of a shifted form in PMA and UCHT1 activation and not of X-35 stimulation. B, parallel detection of ERK-2 with anti-ERK-2 Ab and tyrosine phosphorylation of ERK-2 with anti-phosphotyrosine Ab after a 5-min stimulation confirm that phosphorylation of ERK-2 occurs in PMA- and UCHT1- but not in X-35-activated cells. C, kinase activity of immunoprecipitated ERK-2 protein was estimated using MBP as an exogenous substrate. The percent intensity estimated on the PhosphorImager was as follow: nonstimulated (NS) 100%, PMA 272%, X-35 (5) 104%, UCHT1 (5) 161%, X-35 (15) 106%, UCHT1 (15) 100%. D, kinase activity of ERK-2 from stimulated cell lysates was estimated using GST-Elk-1 as substrate. The two latter results demonstrate that ERK-2 enzyme activity can only be detected in PMA- and UCHT1-stimulated cells and not in X-35-treated lymphocytes. Each experiment is representative of three.

A parallel study analyzing tyrosine phosphorylation after a 5-min stimulation similarly showed (Fig. 1B) that ERK-2 was tyrosine-phosphorylated in UCHT1 but not in X-35-stimulated cells. We also established that only ERK-2 immunoprecipitated from UCHT1 (or PMA)-treated cells was able to phosphorylate (maximum after a 5-min stimulation) over basal level MBP when used as exogenous substrate (Fig. 1C). Enzymatic activity was confirmed using GST-Elk-1, the GST fusion protein of ERK-2 physiological substrate (22), which was highly phosphorylated by lysates from cells pretreated with PMA or UCHT1 but not from cells treated with X-35 (Fig. 1D). It is noteworthy that we did not detect phosphorylation of ERK1 in neither X-35- nor UCHT1-stimulated cells (not shown).

Analysis of Raf-1, the upstream kinase in the MAP kinase cascade, shows that this MAP kinase kinase kinase is also phosphorylated upon UCHT1 and PMA stimulation but not upon X-35 activation (Fig. 2).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Study of the activation of Raf-1. CD4+-purified T cells were stimulated with X-35 or with UCHT1 for 5 and 15 min (PMA stimulation was used as control). Raf-1 was detected using anti-Raf-1 antibody; the result shows the presence of a shifted form of Raf-1 upon UCHT1 stimulation and not upon X-35 treatment. Each experiment is representative of at least three. NS, nonstimulated.

Analysis of Interleukin-2 mRNA Expression in CD3-stimulated Cells-- IL-2 gene transcription, a key event in T cell activation and proliferation, is regulated by the coordinate action of multiple nuclear factors including NF-AT. Previous results have brought evidence that NF-AT activation is directly dependent on stimulation of Raf-1/MEK-1/ERK-2 phosphorylation cascade. Since our preceding results suggested that this pathway is not activated in X-35 stimulation, we questioned whether IL-2 gene transcription could occur when the ERK-2 pathway is blocked with PD098059, a specific inhibitor of MEK-1 (23). Fig. 3A confirms that ERK-2 activation, which only occurs in UCHT1 and PMA stimulation (as assessed by the appearance of a slower migrating band and the phosphorylation of GST-Elk-1), is indeed prevented by PD098059. In parallel, Fig. 3B shows that, in the absence of inhibitor, IL-2 mRNA expression is induced by both mAbs, whereas in the presence of inhibitor, IL-2 mRNA expression is blocked in UCHT1-stimulated cells and is not in X-35-activated cells. This result demonstrates that IL-2 gene transcription triggered upon X-35 ligation does not involve activation of Raf-1/MEK-1/ERK-2 pathway.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   Parallel analysis of IL-2 mRNA expression and ERK-2 activation. Effect of PD098059. A, activation of ERK-2 from X-35- or UCHT1-stimulated T cells was evaluated according to its phosphorylation state (using anti-ERK-2 Ab) and kinase activity (using GST-Elk-1 as substrate). PMA stimulation was used as the control. This experiment was performed in the absence or presence of PD098059. This representative set of experiments was performed twice. B, IL-2 mRNA from purified CD4+ cells stimulated with X-35 or UCHT1 in the presence of ionomycin was detected by reverse transcription-polymerase chain reaction. These experiments, performed in the absence or in the presence of PD098059, a specific inhibitor of MEK-1 which blocks activation of ERK-2, show that inhibition of ERK-2 has no effect on IL-2 mRNA expression induced upon X-35 stimulation. 2-Microglobulin mRNA expression is used as an internal standard. This is a representative experiment of three. NS, nonstimulated.

Effect of X-35 and UCHT1 Treatment on p21^ras Activation-- Raf-1/MEK-1/ERK-2 has been described as a p21^ras effector pathway for NF-AT induction in Jurkat T cells. Rac-1, along with other possible pathways, has also been shown in Jurkat cells to participate in this stimulation as downstream effectors of p21^ras (9), confirming that this small G protein played a pivotal role in lymphocyte stimulation. We therefore questioned whether the ERK-2-independent pathway, which is triggered in human primary T lymphocytes upon X-35 stimulation, involves activation of p21^ras. Fig. 4 shows that p21^ras activation can be detected in UCHT1 (the most intense band appearing at 5 min) as well as in phorbol ester-treated cells (used as a control) but not in X-35-stimulated lymphocytes. This results strongly suggest that X-35 binding to CD3 on purified T cells triggers IL-2 gene transcription through a stimulation pathway independent of p21^ras activation.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   p21ras activation in anti-CD3 stimulation. Purified CD4+ T cells were stimulated (5 and 15 min) with X-35 or UCHT1 or with PDBu (5 min) as a control. After cell lysing, the active form of Ras was pulled down using GST-RDB fusion protein. After electrophoresis and electroblotting, the membrane was revealed with anti-Ras mAb. NS, nonstimulated.

Effect of UCHT1 and X-35 on Early Signals Linked to TCR Activation-- Upstream of the p21^ras/MAP kinase phosphorylation cascade, the events that are directly induced upon engagement of the TCR are tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAM) of  and CD3 chains by Src family tyrosine kinases Fyn and Lck (2). These phosphorylated motifs provide docking sites for the protein-tyrosine kinases ZAP-70 and Syk, that are phosphorylated and activated (2, 24). Activation of these protein-tyrosine kinases has been shown to be necessary for propagating downstream signaling. We therefore studied phosphorylation of p56^lck, p59^fyn, and of ZAP-70 in primary T cells stimulated either with UCHT1 or X-35; as shown in Fig. 5A, a shifted band can be observed in p56^lck only after a 15-min stimulation with UCHT1 and not with X-35. No activation of p59^fyn could be detected either with UCHT1 or X-35 stimulation (not shown). Concerning ZAP-70, the presence of which is detected by anti-ZAP-70 Ab, it appears to be phosphorylated in cells stimulated by both anti-CD3 mAb (Fig. 5B).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Study of p56lck, ZAP-70, and PKB/Akt phosphorylation in anti-CD3-stimulated T cells. A, CD4+ T cells were unstimulated or stimulated (5 and 15 min) with X-35 or UCHT1. A 5-min stimulation with PdBu was used as a control. After cell lysis, p56lck was immunoprecipitated. Activation of p56lck was estimated by its autophosphorylation in the presence of [-32P]ATP. This experiment has been repeated twice. NS, nonstimulated. B, ZAP-70 was immunoprecipitated from unstimulated cells or from cells previously treated for 5 min with X-35 or UCHT1. Tyrosine phosphorylation of ZAP-70 was analyzed using 4G10 anti-phosphotyrosine mAb. This is a representative experiment of three. C, CD4+ T cells were unstimulated or stimulated (5 and 15 min) with X-35 or UCHT1. After cell lysis, total proteins were run on 7.5% SDS-PAGE. Phosphorylation of PKB/Akt was analyzed using a phosphoserine 473 PKB antibody. This is a representative experiment of two.

Recently, it was shown that the two serine/threonine kinases PKB/Akt (29) and the Tec kinase ITK (30, 31) are phosphorylated and activated during T cell activation via TCR/CD3. PKB/Akt as well as ITK have a pleckstrin homology domain (32, 33) that interacts with membrane-phosphorylated inositol lipids generated by activated phosphatidylinositol 3-kinase; after recruitment to the cell membrane, PKB/Akt and ITK are able to be phosphorylated and activated by PDK1/PDK2 kinases and Src kinases, respectively. PKB/Akt and ITK appear as downstream targets for phosphatidylinositol 3-kinase (33, 34), the activity of which has been detected in immunoprecipitates of the  and  chains of the TCR following cell surface engagement of the TCR (35, 36). We therefore studied activated phosphatidylinositol 3-kinase-dependent phosphorylation of PKB/Akt and ITK. Most of the studies on ITK activation have been performed using the Jurkat T cell line. Using highly purified primary T cells we actually failed to bring evidence of any phosphorylation of ITK immunoprecipitated from cells activated with one or the other anti-CD3 mAb. In contrast, we showed (Fig. 5C) that the serine/threonine PKB/Akt, which is immunoprecipitated in equal quantity from one or the other anti-CD3 mAb (visualized by a pan-PKB antibody), was phosphorylated only in cells stimulated with UCHT1 but not in cells activated by X-35 as revealed by an anti-phospho-PKB antibody. This result suggests that X-35 does not trigger activation of TCR-related phosphatidylinositol 3-kinase-dependent pathway.

It appears that, except for ZAP-70, which is phosphorylated in both cases, none of the signals we studied that are commonly described as activation signals in T cell stimulation via TcR/CD3, are triggered upon X-35 stimulation. This is in line with what can be observed on phosphorylation electrophoretic profiles obtained with lysates from UCHT1- or X-35-stimulated cells (Fig. 6). Indeed, in UCHT1, several bands appear phosphorylated, whereas in X-35, the profile is very similar to that from unstimulated cells except for one single band at 58 kDa, which is present in X-35- and not in UCHT1-activated cells. This 58-kDa band, which we have not yet characterized, could represent an important signaling intermediate in the Ras/MEK/ERK-independent pathway triggered by X-35 anti-CD3 mAb.

View larger version (62K): [in this window] [in a new window]   Fig. 6.   Tyrosine phosphorylation of cellular proteins upon X-35 and UCHT1 stimulation in CD4+-purified T cells. CD4+ purified T cells were not stimulated (NS) or stimulated with anti-CD3 (X-35 and UCHT1, 10 µg/ml) or with PMA (50 ng/ml) for 5 min. After cell lysis, total proteins were run on 10% SDS-PAGE. Tyrosine-phosphorylated proteins were analyzed using an anti-phosphotyrosine mAb 4G10. This is a representative experiment of three.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The results we present herein provide evidence that activation of the p21^ras/Raf-1/MEK-1/ERK-2 phosphorylation cascade is not an event obligatorily triggered upon stimulation of purified T lymphocytes through the TCR/CD3 complex. Moreover they support the related conclusion that, in primary T cells, IL-2 gene transcription may occur independently of the activation of the MAP kinase pathway. Indeed we have shown that, in contrast to the commonly used UCHT1, which triggers MAP kinase activation, the anti-CD3 mAb X-35 triggers lymphocyte stimulation leading to IL-2 gene transcription and cell proliferation without activating ERK-2; moreover, using the MEK-1 inhibitor PD098059, we demonstrated that the blockade of ERK-2 phosphorylation has no effect on IL-2 mRNA expression induced by X-35. These results demonstrate that the Ras/Raf-1/MEK-1/ERK-2 phosphorylation cascade is not an exclusive and necessary pathway in TCR/CD3-induced T cell activation. The possibility of the existence of a MAP kinase-independent stimulation of T cells has been suggested using splenocytes from transgenic mice expressing an inactive form of MEK-1 (12); however, as pointed out by others (11), it is unclear whether the splenic T cells in these transgenic mice, which developed in the absence of positive selection, are representative of a normal T lymphocyte population.

It has been demonstrated that Rac-1 participates in the stimulation process in parallel and in addition to ERK-2 pathway but still as an effector of p21^ras (9); however, a hypothesis of an involvement of Rac-1 as an effector of Ras seems unlikely since we showed that p21^ras is not activated upon X-35 binding. However, the possibility remains that in X-35 stimulation, Rac-1 could act instead of p21^ras. Recently Rac-1 and/or CDC-42 were shown to be involved in NF-AT activation through activation of the serine threonine kinase Pak-1 (26); however, evidence has been provided that Pak-1 acts upstream of Ras and participates in a signaling event required for TCR-mediated Ras activation (26). We analyzed Jun N-terminal kinase (25) stimulation in both UCHT1 and X-35 activation (data not shown); this kinase appeared very faintly but similarly stimulated in both cases and, therefore, is probably not involved in this phenomenon.

The difference between the two antibodies in their differential capacity to stimulate ERK-2 cannot be attributed to the fact that they are of different isotype; indeed we studied MAP kinase activation using purified CD4⁺ T cells totally depleted from Fc receptor-expressing cells. Moreover, we found that OKT3, an anti-CD3 mAb of the same isotype than X-35, behaves as UCHT1, i.e. it stimulates ERK-2.

As already described, the two antibodies immunoprecipitate the same proteins, and their different abilities would therefore be more likely related to different epitopes recognized by each anti-CD3 mAbs, likely on the CD3  chain as demonstrated by Tunnacliffe et al. (15). Recognition of different functional epitopes by the two antibodies was confirmed in several other studies (37, 38). It is noteworthy that, on tissue section of human thymus, UCHT1 has been shown to present a reactivity to medullary and cortical thymocytes, whereas X-35 reacted only with medullary thymocytes (14).

Our results on proliferative response of purified T cells using immobilized anti-CD3 or of peripheral blood monocyte cells using soluble mAb (not shown) bring evidence that the response to X-35 is higher than that obtained with UCHT1. Such a difference in the response level between the two antibodies was already observed by others (14). A higher proliferative response induced by X-35 can be explained by the fact that higher amounts of IL-2 are produced by X-35 than by UCHT1 (not shown). This higher production of the protein correlates with a higher expression of IL-2 mRNA in X-35 stimulation. Therefore it seems that stimulation of the p21^ras/ERK-2-independent pathway triggered by X-35 could be more efficient for IL-2 gene transcription and IL-2 production. One could then question whether inhibition of the MAP kinase pathway could be a potentiating factor in activation of primary T cells. This appears unlikely since we showed that MEK/ERK inhibition by PD098059 results in the inhibition of IL-2 mRNA production in UCHT1-stimulated cells. A recent study (39) has also shown on primary T cells stimulated with a mouse mAb to CD3 (IgE isotype) that the blockade of the MEK/ERK pathway inhibited IL-2 production but differentially modulated the production of other cytokines.

It appears, however, that the proximal activation induced by both mAbs after their ligation on CD3 involves phosphorylation of ZAP-70, suggesting that the respective pathways induced by UCHT1 or X-35 diverge downstream in this protein-tyrosine kinase. Concerning p56^lck or p59^fyn, their autophosphorylation is difficult to detect in primary T cells, and the phosphorylated p56^lck band that appears only in UCHT1-activated cells after a relatively long time activation (15 min) is probably not due to its direct autophosphorylation but is likely due to phosphorylation induced by activated ERK-2 as described previously (27, 28). This result is in line with the fact that UCHT1 triggers ERK-2 activation, whereas X-35 does not.

We also considered two other TCR-related signals, i.e. activation of the two protein kinases PKB/Akt and ITK; activation of these kinases is dependent on activation of phosphatidylinositol 3-kinase normally triggered following engagement of the TcR/CD3 complex. We failed to detect phosphorylation of ITK in both cases, but our results show that PKB/Akt is phosphorylated upon UCHT1 treatment and not upon X-35 stimulation, suggesting that the latter anti-CD3 mAb does not induce activation of the TCR-related phosphatidylinositol 3-kinase-dependent pathway.

Studying the overall tyrosine phosphorylation of total lysates from UCHT1- or X-35-stimulated cells, it appears that a single band around 58 kDa is present in X-35 and not in UCHT1 activation. This band unlikely represents phosphorylated p56^lck or p59^fyn, since we showed that phosphorylation of these protein-tyrosine kinases are difficult to observe in primary T cells even using [-³²P]ATP. However, this band, which is not yet characterized, could represent an important signaling molecule involved in the Ras/MEK/ERK-independent pathway triggered by X-35.

Previous studies to explore the role of MAP kinases in TCR function have looked at regulation of the transcription factor NF-AT in the Jurkat cell line. In these cells, experiments with inhibitory mutants of the MAP kinase pathway have suggested that NF-AT activation is dependent on the Ras/Raf/MEK/ERK signaling cascade. These data now show that in peripheral blood T cells ERK-2 activation is not an obligatory signal for IL-2 gene transcription. This illustrates that Jurkat cells, although a good model for the initial receptor proximal biochemical processes associated with T cell activation, may not be an appropriate model for cytokine gene regulation as it relates to primary human T cells. Interestingly, many of the signaling pathways worked out in Jurkat cells, particularly in the context of TCR/Ras/MEK/ERK-2 pathways, have been proven to be important as predicted in TCR function in the thymus.

	ACKNOWLEDGEMENTS

We thank Dr. D. Cantrell (Imperial Cancer Research Fund, London) for helpful discussion of the results and for critical reading of the manuscript and Veronica Athié-Morales for her skillful help in performing Ras experiments.

	FOOTNOTES

^* This work was financially supported by the Fondation Singer Polignac (France).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of a grant from the federation of European Biochemical Societies.

To whom correspondence should be addressed. Tel.: 33 467 14 42 44; Fax: 33 467 14 33 38; E-mail: favero@univ-montp2.fr.

	ABBREVIATIONS

The abbreviations used are: IL-2, interleukin-2; MAP, mitogen-activated protein; ERK-2, extracellular-regulated kinase-2; MEK-1, mitogen-activated ERK kinase; NF-AT, nuclear factor of activated T cells; mAb, monoclonal antibody; PKB, protein kinase B; PMA, phorbol 12-myristate 13-acetate; MBP, myelin basic protein; GST, glutathione S-transferase; PDBu, phorbol dibutyrate; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; RDB, Ras binding domain of Raf.

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