Isopentenyl Pyrophosphate, a Mycobacterial Non-peptidic Antigen, Triggers Delayed and Highly Sustained Signaling in Human gamma delta T Lymphocytes without Inducing Down-modulation of T Cell Antigen Receptor*

Virginie LafontDagger, Janny Liautard, Magali Sablé-Teychené, Yannis Sainte-Marie, and Jean Favero§

From INSERM U431, Microbiologie et Pathologie Cellulaire Infectieuse, Université Montpellier 2, Place Eugène Bataillon, cc 100, Montpellier 34095, cedex 5, France

Received for publication, September 22, 2000, and in revised form, December 13, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Vgamma 9Vdelta 2 T cell subset, which represents up to 90% of the circulating gamma delta T cells in humans, was shown to be activated, via the T cell receptor (TcR), by non-peptidic phosphorylated small organic molecules. These phosphoantigens, which are not presented by professional antigen-presenting cells, induce production of high amounts of interferon-gamma and tumor necrosis factor (TNF-alpha ). To date, the specific signals triggered by these antigens have not been characterized. Here we analyze proximal and later intracellular signals triggered by isopentenyl pyrophosphate (IPP), a mycobacterial antigen that specifically stimulates Vgamma 9Vdelta 2 T cells, and compare these to signals induced by the non-physiological model using an anti-CD3 antibody. During antigenic stimulation we noticed that, except for the proximal p56lck signal, which is triggered early, the signals appear to be delayed and highly sustained. This delay, which likely accounts for the delay observed in TNF-alpha production, is discussed in terms of the ability of the antigen to cross-link and recruit transducing molecules mostly anchored to lipid rafts. Moreover, we demonstrate that, in contrast to anti-CD3 antibody, IPP does not induce down-modulation of the TcR·CD3 complex, which likely results in the highly sustained signaling and release of high levels of TNF-alpha .


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

T cells expressing the gamma delta T cell receptor (TcR)1 represent in humans a relatively low T lymphocyte population and, particularly in peripheral blood, these cells account for only 1-5% of the circulating T cells (see Ref. 1 for review). In an adult the majority of these circulating T cells are classified as Vgamma 9Vdelta 2 subset (up to 90%). It has been shown that this gamma delta T cell subset dramatically increases during infection by intracellular pathogens of bacterial, viral, or parasitic origin (2-12). One of the particularity of the Vgamma 9Vdelta 2 T cells is to be activated by components identified as non-peptidic, phosphorylated small organic molecules (13-18). Some of these components have been purified from bacteria or parasites and are thought to be responsible for the in vivo expansion of Vgamma 9Vdelta 2 T cells during the acute phase of the infection process. There is so far no formal proof that these small molecules do bind to the Vgamma 9Vdelta 2 T cell receptor, however, transfer experiments of the Vgamma 9Vdelta 2 TcR in TcR-negative Jurkat cell mutants have provided strong evidence to suggest that the recognition of the phosphoantigens is mediated by TcR (17).

Stimulation of Vgamma 9Vdelta 2 T cells by phosphoantigens results mainly in the production of high amounts of interferon-gamma (19-22) and tumor necrosis factor-alpha (TNF-alpha ) (19, 23). However, to date, the specific signals that are triggered in Vgamma 9Vdelta 2 T cells upon stimulation with phosphoantigens have not been characterized. This aspect is of great importance for the possible pharmacological control of TNF-alpha release (24, 25) by these cells, because an overproduction of this cytokine could result in immunopathology (26).

In alpha beta T cells, it is well established that activation occurs as a result of multimolecular interactions between T cells and antigen-presenting cells (27-29). These interactions include the recognition of the peptide/major histocompatibility (MHC) complex by the T cell receptor and the binding of CD4 coreceptor to non-polymorphic regions on the MHC class II molecules. The cytoplasmic tail of CD4 associates with the Src family tyrosine kinase p56lck, which plays a key role in the early events of T cell activation. However, in the case of Vgamma 9Vdelta 2 T cell activation, the non-peptidic antigens do not need to be presented in the context of MHC molecules (30, 31). Moreover, it has been shown that Vgamma 9Vdelta 2 T cells do not express CD4 and poorly express CD8 (32, 33), which normally interact with MHC class II or class I molecules, respectively. Therefore, there is not, as with alpha beta T cells, recruitment of these coreceptors, which stabilize TcR·ligand interaction and are essential for the formation of the "immunological synapse," which determines the extent and qualitative nature of the transduced signal (27, 34, 35).

Recently we studied TNF-alpha release by Vgamma 9Vdelta 2 T cells when stimulated either with a monoclonal antibody directed against the CD3 complex or with the mycobacterial phosphoantigen isopentenyl pyrophosphate (IPP) (36). We demonstrated that TNF-alpha production does not involve, as is the case in alpha beta T cells, CD28 costimulation. Moreover, we noticed that the cytokine production in Vgamma 9Vdelta 2 T cells was highly delayed (~10-h difference) when the cells were activated by a physiological phosphoantigen ligand (IPP) instead of anti-CD3 monoclonal antibody (mAb). This delayed cytokine production could be the result of a delayed triggered signaling or of the recruitment of different signaling molecules according to the stimulating agent used. In the present paper, we therefore studied the signals triggered in Vgamma 9Vdelta 2 T cells upon stimulation with isopentenyl pyrophosphate, a physiological non-peptidic mycobacterial phosphoantigen known to be specifically mitogenic for Vgamma 9Vdelta 2 T cells (14, 15), and compared these to the signals induced by anti-CD3 mAb. We show that the kinetics of the signals triggered upon IPP stimulation is quite different from that of the signals induced upon anti-CD3 mAb stimulation; the signals are largely delayed when the cells are stimulated with the non-peptidic antigen compared with those induced upon anti-CD3 mAb activation except for p56lck. But this delay cannot be assigned to the synthesis of de novo proteins. Moreover, we show that the majority of the phosphoantigen-induced signals, in contrast to the anti-CD3-triggered ones, are highly sustained and last for several hours. This long-lasting cell signaling observed with IPP stimulation is possibly related to the lack of induction of TcR·CD3 down-modulation that we demonstrate herein.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Chemicals and Reagents-- Recombinant IL2 (rIL2) was purchased from Chiron (Emeryville, CA), isopentenyl pyrophosphate (IPP) and enolase from Sigma Chemical Co. (St. Louis, MO), and LY 294002 from Calbiochem Corp. (Nottingham, UK). Anti-phospho-p42/44 MAPK antibody (Ab), anti-phospho-p38 MAPK Ab, anti-p38 MAPK Ab, anti-phospho(Ser-473)-PKB Ab, and anti-PKB Ab were all purchased from New England BioLabs (Beverly, MA). Anti-ERK2 Ab was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-ZAP-70 Ab, anti-p56lck Ab, and recombinant LAT protein were from Upstate Biotechnology Inc. (Lake Placid, NY). Horseradish peroxidase-conjugated anti-mouse Ab and anti-rabbit Ab were from Amersham Pharmacia Biotech (Paris, France). UCHT1 (anti-CD3 monoclonal antibody (mAb)), anti-TcR Vdelta 2 mAb, anti-TcR Vgamma 9 mAb, anti-TcR Vdelta 1, and anti-TcR Vgamma 1, anti-zeta chain mAb-conjugated or not, were purchased from Immunotech (Marseille, France).

Cell Culture-- Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors. Human gamma 9delta 2 T lymphocytes were purified from PBMC, by positive immunoselection using anti-TcR Vdelta 2 mAb and magnetic beads coated with anti-mouse IgG (Dynal, Compiegne, France). After spontaneous detachment, gamma 9delta 2 T cells were specifically activated in the presence of syngeneic monocytes, IPP (50 µM), and rIL2 (20 ng/ml). Human gamma 1delta 1 T lymphocytes were purified from PBMC, by positive immunoselection using anti-TcR Vdelta 1 mAb and magnetic beads coated with anti-mouse IgG. After spontaneous detachment, gamma 1delta 1 T cells were specifically activated in the presence of syngeneic monocytes, PHA (1 µg/ml) and rIL2 (20 ng/ml). Human peripheral blood-derived gamma delta T lymphoblasts were generated as described above and maintained in RPMI 1640 supplemented with 5% fetal calf serum (FCS), 5% human AB serum, 2 mM glutamine, and rIL2 (20 ng/ml) at 37 °C in 5% CO2 humidified atmosphere for 4 or 5 weeks.

Preparation of Supernatants for Measurement of TNF-alpha Production-- gamma delta T cells (2 × 106 cells/ml) were cultured in 24-well tissue culture plates in RPMI 1640 supplemented with 5% FCS + 5% human AB serum in a total volume of 0.5 ml per well. When mentioned cells were pretreated with inhibitors (LY 294002, 5 µM) for 30 min at 37 °C, then stimulated with IPP (50 µM) or UCHT1 (2 µg). At different times, supernatants were harvested and assayed for TNF-alpha using a human TNF-alpha ELISA kit (OptEIA set: human TNF-alpha , PharMingen, San Diego, CA) according to the manufacturer's instructions.

Cell Extract Preparation and Western Blot Analysis-- 20 × 106 cells were stimulated at 37 °C by UCHT1 (10 µg/ml) or IPP (100 µM) for the indicated times. After stimulation, cells were lysed in 1 ml of lysis buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 10 mM NaF, 10 mM iodoacetamide, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na2VO3, and 1 µg/ml of each protease inhibitor (leupeptin, aprotinin, chymostatin). Proteins were concentrated by precipitation with 1.5 volumes of acetone. Proteins from 5 × 106 cells were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and detected with the indicated antibodies: anti-phospho-p38 MAPK Ab (1:1000), anti-p38 MAPK Ab (1:1000), anti-phospho-p42/44 MAPK Ab (1:1000), anti-ERK2 Ab (1:5000), anti-phospho-(Ser-473) PKB Ab (1:1000), and anti-PKB Ab (1:1000). Immunoreactive bands were visualized with the chemiluminescence Western blotting system (Amersham Pharmacia Biotech).

Immunoprecipitation-- Following stimulation, 20 × 106 cells (for p56lck immunoprecipitation) or 50 × 106 cells (for ZAP-70 immunoprecipitation and zeta  chain) were lysed in 1 ml of lysis buffer. After cell lysis, p56lck, ZAP-70, or zeta  chain were immunoprecipitated from clarified supernatants, respectively, with 4 µg of anti-p56lck Ab, 5 µg of anti-ZAP-70 Ab, or 10 µg of anti-zeta chain. Immune complexes were collected using protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), washed three times with lysis buffer for zeta  chain before loading on 15% SDS-PAGE and revealed by Western blotting, or washed twice with washing buffer containing 20 mM Tris, pH 7.4, 140 mM NaCl, 1% Nonidet P-40, 500 µM Na3VO4, 1 mM PMSF, 1% aprotinin, and once with specific kinase assay buffer before performing kinase assay.

In Vitro Kinase Assay-- For p56lck kinase assay, complexes were resuspended in 50 µl of specific kinase buffer (50 mM Pipes, pH 7.5, 10 mM MgCl2, 10 mM MnCl2, 1 mM PMSF, 100 µM NA3VO4), and autophosphorylation of p56lck was determined in the presence of 5 µCi of [gamma -32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences) and incubated for 10 min at 37 °C. p56lck activity was measured by phosphorylation of the exogenous substrate enolase. Complexes were incubated in 50 µl of kinase assay buffer in the presence of 10 µg of acid-denatured enolase, 10 µCi of [gamma -32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences), and 4.5 µM unlabeled ATP. For ZAP-70 kinase assay, complexes were incubated in 25 µl of kinase buffer (100 mM Tris, pH 7.5, 125 mM MnCl2, 25 mM MnCl2, 2 mM EGTA, 250 µM Na3VO4, 2 mM dithiothreitol) in the presence of 4 µg of recombinant LAT protein, 10 µCi of [gamma -32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences), and 10 µM unlabeled ATP for 10 min at 37 °C. The reactions were stopped by addition of 2-mercaptoethanol-containing sample buffer and boiling. Radiolabeled proteins were then resolved on 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and then detected by autoradiography. Quantification of the phosphorylated bands reported in the results has been performed using a PhosphorImager Storm system driven by ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA).

Flow Cytometry-- Cells were stimulated by UCHT1 or IPP at different times, fixed in 1% paraformaldehyde for 15 min, washed in phosphate-buffered saline, and then stained with 1 µg of fluorescein isothiocyanate (FITC)-labeled anti-TcR Vgamma 9 mAb in phosphate-buffered saline supplemented with 2% fetal calf serum, 0.02% NaN3, on ice in a total volume of 50 µl. After 30 min, the cells were washed once, fixed in 1% paraformaldehyde, and analyzed by flow cytometry on a FACSCalibur (Becton Dickinson) with Cell Quest software.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production of TNF-alpha by Anti-CD3 mAb- or IPP-stimulated Vgamma 9Vdelta 2 T Cells-- We previously established that high amounts of TNF-alpha are produced by Vgamma 9Vdelta 2 T cells when stimulated with either anti-CD3 mAb or with the physiological antigen IPP (36). As shown in Fig. 1, upon anti-CD3 mAb stimulation, TNF-alpha is produced very early with its maximum reached after 3 h, whereas maximum production induced by IPP only occurs after 16 h. This delay between the two stimulation processes could reflect either a recruitment of different signals or a difference in the kinetics of the triggered signals. To test these hypotheses, we analyzed the kinetics of the extracellular regulated kinase (ERK) and p38, two mitogen-activated protein kinase (MAPK) pathways directly involved in TNF-alpha production by Vgamma 9Vdelta 2 T cells stimulated either with anti-CD3 mAb or with IPP (36).


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Fig. 1.   TNF-alpha production by gamma delta T cells. Human peripheral blood-derived Vgamma 9Vdelta 2 T cells were stimulated by IPP (50 µM) or UCHT1 (2 µg/ml). After different times of stimulation as indicated, TNF-alpha production was measured in the culture supernatants using an ELISA kit. Each experiment is representative of at least four experiments.

Study of ERK Activation-- Purified Vgamma 9Vdelta 2 T cells were stimulated with either anti-CD3 mAb or with an optimal dose of IPP over a broad time range, and activation of ERK1/ERK2 was studied. As shown in Fig. 2A, phosphorylation of ERK1/ERK2 upon anti-CD3 mAb activation is very rapid (maximum reached at 5-min stimulation) and decreases but lasts at a high degree for around 30 min. After 30-min activation, the intensity is reduced to a very low phosphorylation level even though it still remains detectable after 2 h. In contrast, when the cells are stimulated with IPP, the phosphorylation/activation of ERK1/ERK2 begins to be faintly detectable after 1 h and reaches a plateau after 3 h, which lasts for at least one more hour. After 6 h, even though the phosphorylation signal begins to decrease, it still remains high.


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Fig. 2.   Kinetics of ERK activation in human peripheral blood-derived gamma delta T cells. gamma 9delta 2 T cells (A) or gamma 1delta 1 T cells (B) were stimulated for the indicated times by UCHT1 (10 µg/ml) or by IPP (100 µM). When indicated gamma 9delta 2 T cells were pretreated 30 min with cycloheximide (10 µg/ml) before performing stimulation (C). Total cellular proteins were separated on 10% SDS-PAGE and revealed by Western blot analysis using an anti-phospho-p42/44 MAPK Ab (which recognizes the phosphorylated and active forms of ERK-1 and ERK-2), and reprobed with anti-ERK2 Ab, after Ab stripping. This experiment is representative of three experiments.

Previous studies have shown that IPP induces cell proliferation and cytokine release in gamma 9delta 2 T cells but not in other subsets of gamma delta T cells (13-15, 17, 23). However, we could not rule out the possibility that, even if IPP was not able to trigger biological responses in gamma delta T cells, which do not express the gamma 9delta 2 TcR complex, it could trigger intracellular signals. To investigate this, we studied ERK2 activity in gamma 1delta 1 T cells (which are another important subset of gamma delta T cells in human blood). As shown in Fig. 2B, anti-CD3 stimulation induces a strong and rapid ERK2 activation in gamma 1delta 1 T cells; however, IPP is not able to trigger any ERK2 activation in these cells following either short or prolonged stimulation.

We also wondered if the observed delay in IPP-induced ERK2 activation could be assigned to a necessity to synthesize de novo proteins. As shown in Fig. 2C, pretreatment with cycloheximide, a protein synthesis inhibitor, does not modify the activation of ERK2 induced by IPP. In addition we confirmed that cycloheximide efficiently blocks protein synthesis at the concentration used in these experiments (10 µg/ml; data not shown).

Study of p38 MAPK Activation-- We similarly studied activation of p38 kinase in Vgamma 9Vdelta 2 T cells that have been stimulated either with anti-CD3 mAb or with IPP. Fig. 3A shows that, as for ERK activation, p38 MAPK phosphorylation appears to be delayed in IPP stimulation compared with anti-CD3 stimulation. The kinetics are very similar to that of ERK1/ERK2 with the maximum activation in IPP stimulation occurring 2 h after triggering and lasting as a plateau for at least 4 more hours. In anti-CD3 mAb stimulation, the maximum is already reached within 5 min and then decreases, but the activated form remains elevated for at least 2 h. As we have shown for ERK2 MAPK, IPP does not trigger p38 activation in gamma 1delta 1 T cells (Fig. 3B) and cycloheximide pretreatment does not modify IPP-induced p38 activation in gamma 9delta 2 T cells (Fig. 3C).


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Fig. 3.   Kinetics of p38 MAPK activation in human peripheral blood derived gamma delta T cells. gamma 9delta 2 T cells (A) or gamma 1delta 1 T cells (B) were stimulated for the indicated times by UCHT1 (10 µg/ml) or by IPP (100 µM). When indicated, gamma 9delta 2 T cells were pretreated 30 min with cycloheximide (10 µg/ml) before performing stimulation (C). Total cellular proteins were separated on 10% SDS-PAGE and revealed by Western blot analysis using an anti-phospho-p38 MAPK Ab (which specifically reveals the phosphorylated and active form of p38) and reprobed with an anti-p38 MAPK Ab after Ab stripping. This experiment is representative of three experiments.

Study of p56lck Activation-- We questioned whether the delay observed for ERK and p38 kinase activation, which are later signals in the transduction cascade, could be due to a difference in the triggering of a signal directly related to TcR·CD3 ligation. For that purpose, we studied activation of the Src family kinase p56lck, which represents one of the earliest events in alpha beta T cell stimulation. Fig. 4A shows that the immunoprecipitated p56lck, in the presence of [gamma -32P]ATP, displays a 60-kDa-shifted band in anti-CD3 stimulation, which corresponds to the conversion of p56lck to an Lck form phosphorylated on Ser-59 (37, 38). This hyperphosphorylation was already observed in alpha beta T cells stimulated with anti-CD3 mAb or phorbol 12-myristate 13-acetate (39) and was shown not to be concomitant with an increase of the kinase activity but rather to be accompanied by a decrease in the kinase activity (40-43). Similarly, in our experiments, we could not detect, through phosphorylation of enolase used as exogenous substrate, any activity of p56lck in anti-CD3 stimulated gamma delta T cells.


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Fig. 4.   Autophosphorylation and kinase activity of p56lck in human peripheral blood derived gamma delta T cells. Human peripheral blood-derived gamma delta T cells were stimulated for the indicated times by UCHT1 (10 µg/ml) or by IPP (100 µM). Immunoprecipitations were performed using a total lysates from 2 × 107 cells with anti-Lck Ab. Activation of p56lck was estimated by its autophosphorylation (A) and by phosphorylation of enolase used as exogenous substrate (B); these experiments were carried out in the presence of [gamma -32P]ATP as described under "Experimental Procedures." The amount of p56lck in each lane was evaluated by Western blot using mouse anti-p56lck Ab (data not shown). The phosphorylation of enolase was quantified by PhosphorImager analysis. This experiment was repeated twice.

In IPP stimulation (Fig. 4B) conversion of p56lck to a slower migrating form also exists but occurred along with an increased intensity of the p56 band (as a control we checked that the amounts of immunoprecipitated p56lck loaded on the gel were similar in each sample; data not shown). This increased intensity of the p56lck band reflects autophosphorylation of the kinase and its activation. Indeed, kinase activity was detectable through phosphorylation of enolase. It has to be noted that activation of p56lck in IPP-stimulated cells is rapid (detectable at 5 min) and peaked at 30-45 min. Therefore, because p56lck activation in IPP stimulation is high and rapid, it can hardly be accountable for the delay observed in the MAPK late signals.

Study of ZAP-70 Kinase Activity-- It is generally accepted that immunoreceptor tyrosine-based activation motifs (ITAM) of the signal-transducing subunits of CD3, as well as the zeta  chain, are phosphorylated by Lck, making them competent to associate with zeta-associated protein (ZAP)-70 (reviewed in Ref. 28). Once recruited, ZAP-70 is activated through its phosphorylation by Lck and then is able to recruit and phosphorylate its own substrates, SLP76 and a linker for activation of T cells (LAT) (28, 29, 44). Because we observed a rapid activation of p56lck upon IPP stimulation, we questioned whether the zeta  chain and ZAP-70 are also rapidly activated. First, we immunoprecipitated the zeta  chain proteins and studied their phosphorylation in samples from unstimulated or stimulated cells. We were unable to detect by Western blot, using an anti-phosphotyrosine Ab, phosphorylation of this protein in either unstimulated or stimulated samples (data not shown). This is probably due to the low rate of expression and phosphorylation of this protein in non-transformed cells such as gamma 9delta 2 T cells. However, we showed that in the immunocomplex, ZAP-70, is coprecipitated with the zeta  chain and the amount of coprecipitated ZAP-70 is higher in the immunocomplex from anti-CD3- or IPP-stimulated cells than from unstimulated-cells. This indicates that the zeta  chain may also be more phosphorylated in these samples (Fig. 5A). Following stimulation with IPP, the maximum amount of coprecipitated ZAP-70 protein is observed after 30-min IPP stimulation. Moreover, we studied ZAP-70-induced phosphorylation using recombinant LAT as an exogenous substrate. As shown in Fig. 5B, recombinant LAT is phosphorylated by immunoprecipitated (IP)-ZAP-70 from cells stimulated by IPP. However, as for ERK and p38, phosphorylation of LAT is largely delayed compared to activation of p56lck, indeed phosphorylation is detectable only with IP-ZAP-70 from 30-min-activated cells and lasts as a plateau with IP-ZAP-70 from at least 2-h-activated cells. As a control, LAT appears to be highly phosphorylated with IP-ZAP-70 from cells activated for 5 min with anti-CD3 mAb. The delayed phosphorylation of recombinant LAT thus demonstrates that ZAP-70 is activated tardily in IPP stimulation.


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Fig. 5.   Co-precipitation of ZAP-70 with zeta  chain and kinase activity of ZAP-70 in human peripheral blood-derived gamma delta T cells. Human peripheral blood-derived gamma delta T cells (5 × 107 cells) were stimulated for the indicated times by UCHT1 (10 µg/ml) or by IPP (100 µM). A, after cell lysis, zeta  chain was immunoprecipitated from the clarified supernatants with an anti-zeta chain Ab, and the amount of co-precipitated ZAP-70 was evaluated by Western blot using an anti-ZAP Ab and reprobed with an anti-zeta chain Ab after Ab stripping. B, after cell lysis, ZAP-70 was immunoprecipitated from the clarified supernatants with an anti-ZAP Ab. The amount of ZAP-70 was evaluated on Western blot using an anti-ZAP-70 Ab. Activation of ZAP-70 was estimated by phosphorylation of recombinant LAT proteins used as exogenous substrate; these experiments were carried out in the presence of [gamma -32P]ATP as described under "Experimental Procedures." The phosphorylation of LAT was quantified by PhosphorImager analysis. This experiment was repeated twice.

Study of PKB Phosphorylation-- Several papers have shown that TNF-alpha production in several cell types, including T cells, is dependent on phosphoinositide 3-kinase (PI3K) activation (45, 46). Moreover, in alpha beta T cells, it was demonstrated that TcR engagement results in rapid phosphorylation of Tyr-685 in the p85 subunit of PI3K (47), and that this phosphorylation and the consequent activation of PI3K have been attributed to Lck. We therefore studied whether, in Vgamma 9Vdelta 2 T cells, TNF-alpha is also dependent on PI3K activation. As shown in Fig. 6A, TNF-alpha production induced with either IPP or anti-CD3 mAb is inhibited by LY294002, an inhibitor of PI3K, suggesting that TNF-alpha release is dependent on activation of this kinase. We also studied activation of PI3K through phosphorylation of protein kinase B (PKB), one of its secondary substrates (48, 49), upon stimulation with anti-CD3 mAb or IPP. Even though activation of PI3K is directly dependent on Lck activation, its response is largely delayed in IPP activation (maximum after 2-h stimulation) compared to anti-CD3 mAb activation (maximum after 5 min stimulation) (Fig. 6B).


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Fig. 6.   Effect of LY294002 inhibitor on TNF-alpha production and kinetics of PKB activation in Human peripheral blood derived gamma delta T cells. A, human peripheral blood-derived gamma delta T cells were preincubated or not 30 min with LY 294002 inhibitor (5 µM) and then stimulated with UCHT1 (2 µg/ml) for 3 h or with IPP (50 µM) for 16 h. TNF-alpha production was then measured in the culture supernatants using an ELISA kit. This experiment is representative out of four. B, human peripheral blood-derived gamma delta T cells were stimulated for the indicated times by UCHT1 (10 µg/ml) or IPP (100 µM). Total cellular proteins were separated on 10% SDS-PAGE and revealed by Western blot analysis using an anti-phospho-(Ser-473) PKB Ab (which specifically reveals the phosphorylated form of PKB) and reprobed with an anti-PKB Ab after Ab striping. This experiment is representative of three experiments.

Study of TcR·CD3 Down-modulation upon IPP or Anti-CD3 mAb Stimulation-- One of the most striking characteristics of the signals triggered by IPP compared with those induced by anti-CD3 mAb, aside from the fact that they are delayed, is that they last for a long period of time, i.e. several hours. Receptor internalization following ligand binding is generally considered to be an important mechanism that limits both the quantity of signals received by the cell and the duration of the triggered signals (50). Concerning TcR, several papers have shown that sustained signaling results from prolonged T cell receptor occupancy (51-53). We therefore questioned whether the long-lasting signaling triggered in IPP stimulation compared with that induced during anti-CD3 stimulation could be parallel to a difference in the rate of TcR down-modulation. We therefore stimulated gamma delta T cells with either anti-CD3 mAb or with IPP. The cells were paraformaldehyde-fixed at different times after stimulation and analyzed for TcR expression using FITC-conjugated anti-Vgamma 9 mAb. As shown in Fig. 7, TcR is down-modulated in a time-dependent manner upon anti-CD3 stimulation whereas, with IPP, it remains unmodified. In parallel, as a control of stimulation efficacy, we analyzed ERK phosphorylation, which showed the same kinetics as that presented above (data not shown).


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Fig. 7.   Analysis of TcR·CD3 down-modulation in human peripheral blood derived gamma delta T cells. Human peripheral blood-derived gamma delta T cells were stimulated for the indicated times by UCHT1 (10 µg/ml) (left panel) or by IPP (100 µM) (right panel). After stimulation, cells were paraformaldehyde-fixed and stained with FITC-conjugated anti-Vgamma 9 mAb and analyzed by flow cytometry. Each analysis has been repeated at least three times.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

The present paper studies signals triggered in Vgamma 9Vdelta 2 T cells by IPP, a physiological antigen specific for this T cell subpopulation. It has first to be noted that, in contrast to in vitro studies of alpha beta T cell activation by physiological antigen (51), those of Vgamma 9Vdelta 2 T cell stimulation do not require that the antigen be presented by antigen-presenting cells. The IPP-induced signals were compared with those triggered by the non-physiological model, using an anti-CD3 mAb. It appears that signals triggered by IPP leading to TNF-alpha release were delayed compared with those induced by anti-CD3 mAb, and this delay cannot be assigned to the synthesis of de novo proteins as shown in the experiments in the presence of cycloheximide. In contrast, the Src family kinase p56lck, the activation of which represents one of the earliest events in T cell stimulation (28, 54), appears to be triggered very early in IPP-stimulated gamma 9delta 2 T cells. Moreover, its enzyme activity can be detected through its autophosphorylation and by phosphorylation of enolase used as exogenous substrate. In alpha beta T cells, p56lck enzyme activation was shown to occur upon TcR·CD3 ligation, but this was demonstrated in cell lines (mostly Jurkat cells) (41, 55). In primary alpha beta T cells, engagement of the TcR·CD3 complex by anti-CD3 mAb leads to hyperphosphorylation (on Ser-59) of p56lck, observed in SDS-PAGE as a slower migrating band (60 kDa) (37, 38), with no increase but even a decrease in kinase activity (40-43). Autophosphorylation activation of the kinase in primary alpha beta T cells is, however, detectable when co-receptors CD4 or CD8 are engaged by interacting components such as anti-CD4 mAb or human immunodeficiency virus external glycoprotein gp120 in CD4+ cells (56). In this case, p56lck activation is detectable early (5 min after activation), as is the case in IPP stimulation of gamma 9delta 2 T cells. In Vgamma 9Vdelta 2 T cells, similarly to what happens in alpha beta T cells, anti-CD3 stimulation leads to the appearance of a 60-kDa band, but there is no visible increase in enzymatic activity. Signal triggering in anti-CD3 stimulation, leading to activation of downstream signaling pathways, can involve, as has been postulated in alpha beta T cells, another Src family kinase, p59fyn, which has been shown to be directly associated with the T cell receptor complex (57, 58). The fact that Vgamma 9Vdelta 2 T cells do not express CD4 and express CD8 poorly (the cells we used in the present study were CD4-,CD8-; data not shown) and that IPP stimulation leads to p56lck activation could suggest that this antigen, in addition to engaging the TcR·CD3 complex, could also engage another cell surface molecule as a co-receptor. Such an hypothesis that responsiveness of Vgamma 9Vdelta 2 T cells is modulated by the expression of a (unknown) molecule with a co-receptor-like function is similar to that described for CD4 and CD8 co-receptors in alpha beta T cells recently put forward by Bürk and co-workers (59).

p56lck has been described in alpha beta T cells to phosphorylate ITAM on the CD3 epsilon  chain and TcR zeta  (60) chain, rendering them competent for recruitment of the Syk family kinase ZAP-70. Subsequent phosphorylation activation of this kinase triggers the downstream signaling pathways regulating the transcription of genes essential for cytokine production. In IPP stimulation, the events that are directly dependent on p56lck activation, i.e. recruitment and activation of ZAP-70 and PI3K as well as later signals like ERK and p38 kinase, appeared delayed in reference to the kinetics of p56lck activation, or in comparison to that induced in anti-CD3 stimulation. This delay is likely not to be attributable to a slow rate in IPP·TcR interaction, because p56lck is activated early upon IPP stimulation. On the other hand, it is now generally accepted that in T cells, microdomains of the plasma membrane, commonly referred to as lipid rafts, play an important role in TcR signaling (61). They are mostly involved through the recruitment of transducing molecules like Lck and LAT, which are anchored to them. Aggregation of lipid raft-associated proteins and TcR·CD3 complex can be induced in T cell stimulation through cross-linking with anti-CD3 mAb, thus taking part in the formation of the immunological synapse (35). It has recently been shown in an elegant study that TcR is naturally associated with lipid rafts even though this association is sensitive to nonionic detergent (61). However, aggregation of the TcR by anti-CD3 cross-linking causes aggregation of raft-associated proteins, which leads to triggering of tyrosine phosphorylation of zeta  chain, ZAP-70 activation, and downstream signal transduction. It thus appears that triggering of the signaling cascade must occur when the TcR·CD3 proximal signaling molecules are brought into close contact through cross-linking. A possibility therefore exists that the small non-peptidic molecule IPP, which likely does not have multivalent TcR-interacting sites and is not presented to TcR by MHC or MHC-like molecules, is probably not able to rapidly and efficiently cross-link with the TcR·CD3 complex and the lipid rafts. As a possible consequence, the rate for the physical recruitment of a sufficient number of engaged TcR·CD3 complexes to colocalize with the rafts anchored transducing molecules is slower than that occurring in anti-CD3 stimulation, leading to a delay in cell signaling (ZAP-70, PI3K, MAPKs) and TNF-alpha release. Of course, the results we present herein are in vitro results obtained with purified Vgamma 9Vdelta 2 T cells, and it cannot be totally ruled out that in vivo the antigens could be presented by other cell types through cell surface molecules not yet determined. According to such an hypothesis, the kinetics of the triggered signals could be faster. To investigate this point, we studied TNF-alpha production by Vgamma 9Vdelta 2 T cells stimulated by IPP in the presence of syngeneic paraformaldehyde-fixed PBMC. This experiment was done to allow IPP to bind to putative cell surface molecules involved in its possible presentation to gamma delta TcR. We did not notice any difference either in the kinetics or in the amounts of TNF-alpha produced upon stimulation with IPP alone or with IPP in the presence of fixed PBMC (data not shown). Moreover, we cannot totally rule out that when non-peptidic antigens are expressed on the surface of pathogens, they do not behave as a monovalent antigen and thus could engage several TcR·CD3 complexes together, in this case the kinetics of the triggered signals could be faster. To test this hypothesis, we compared the kinetics of TNF-alpha production by Vgamma 9Vdelta 2 T cells induced by a non-peptidic antigen such as IPP and by a whole pathogen. We chose to use as a pathogen a strain of Brucella, which we have shown produces a non-peptidic antigen that is able to stimulate gamma 9delta 2 T cells (62). TNF-alpha production that we measured was lower in supernatants from cells stimulated with gentamicin-killed bacteria than from those stimulated with IPP (probably due to the lower concentration of non-peptidic antigen present on the surface of the bacteria compared with the IPP concentration that we used), but the kinetics of TNF-alpha production was identical (data not shown).

One of the striking features concerning IPP-induced signals, is that they are highly sustained compared with those induced by anti-CD3 mAb or with those described in alpha beta T cells stimulated either with anti-CD3 mAb or physiological antigens presented in the context of MHC molecules. It has been shown that sustained signaling can be related to TcR occupancy (51). Here we demonstrated that, in contrast to anti-CD3 mAb, IPP does not induce down-modulation of the TcR·CD3 complex. Therefore, a possibility exists that the sustained signals observed in Vgamma 9Vdelta 2 T cells stimulated by IPP results from a long lasting interaction between the antigen and the T cell antigen receptor. This possibility could account for the high and durable production of TNF-alpha detected in activated Vgamma 9Vdelta 2 T cells and which has been shown in several cases to result in immunopathology (26).

    FOOTNOTES

* This work was supported in part by an Ecos-Anuies program (France-Mexico) grant (action number PM99S01).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.

Dagger Recipient of a grant from the Société de Secours des Amis des Sciences (France).

§ To whom correspondence should be addressed: INSERM U431, Université Montpellier 2, Place Eugène Bataillon, cc100, Montpellier 34095, cedex 5, France. Tel.: 33-0-467-14-42-44; Fax: 33-0-467-14-33-38; E-mail: favero@crit.univ-montp2.fr.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M008684200

    ABBREVIATIONS

The abbreviations used are: TcR, T cell receptor; TNF-alpha , tumor necrosis factor alpha ; IPP, isopentenyl pyrophosphate; PBMC, peripheral blood mononuclear cells; ERK, extracellular regulated kinase; MAPK, mitogen-activated protein kinase; Ab, antibody; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; rIL2, recombinant interleukin-2; ITAM, immunoreceptor tyrosine-based activation motif; ZAP-70, zeta-associated protein-70; PI3K, phosphoinositide 3-kinase; LAT, linker for activation of T cells; PKB, protein kinase B; PAGE, polyacrylamide gel electrophoresis; MHC, major histocompatibility complex; FCS, fetal calf serum; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; Pipes, 1,4-piperazinediethanesulfonic acid; IP, immunoprecipitated.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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