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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The V T cells expressing the Stimulation of V In Recently we studied TNF- 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 V Cell Culture--
Peripheral blood mononuclear cells (PBMC) were
isolated from healthy donors. Human Preparation of Supernatants for Measurement of TNF- 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
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 [ 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 V Production of TNF- Study of ERK Activation--
Purified V
Previous studies have shown that IPP induces cell proliferation and
cytokine release in
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 V 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
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 Study of PKB Phosphorylation--
Several papers have shown that
TNF- 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 The present paper studies signals triggered in V p56lck has been described in 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 9V
2 T cell subset, which represents up
to 90% of the circulating
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-
and tumor necrosis
factor (TNF-
). 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 V
9V
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-
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-
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 V
9V
2 subset (up to 90%). It has been
shown that this
T cell subset dramatically increases during
infection by intracellular pathogens of bacterial, viral, or parasitic
origin (2-12). One of the particularity of the V
9V
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 V
9V
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 V
9V
2 T cell receptor, however, transfer experiments of the
V
9V
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).
9V
2 T cells by phosphoantigens results mainly in
the production of high amounts of interferon-
(19-22) and tumor
necrosis factor-
(TNF-
) (19, 23). However, to date, the specific
signals that are triggered in V
9V
2 T cells upon stimulation with
phosphoantigens have not been characterized. This aspect is of great
importance for the possible pharmacological control of TNF-
release
(24, 25) by these cells, because an overproduction of this cytokine
could result in immunopathology (26).
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 V
9V
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 V
9V
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
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).
release by V
9V
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-
production does
not involve, as is the case in
T cells, CD28 costimulation. Moreover, we noticed that the cytokine production in V
9V
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
V
9V
2 T cells upon stimulation with isopentenyl pyrophosphate, a
physiological non-peptidic mycobacterial phosphoantigen known to be
specifically mitogenic for V
9V
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.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 mAb, anti-TcR V
9 mAb, anti-TcR V
1, and
anti-TcR V
1, anti-
chain mAb-conjugated or not, were purchased
from Immunotech (Marseille, France).
9
2 T lymphocytes were
purified from PBMC, by positive immunoselection using anti-TcR V
2
mAb and magnetic beads coated with anti-mouse IgG (Dynal, Compiegne,
France). After spontaneous detachment,
9
2 T cells were
specifically activated in the presence of syngeneic monocytes, IPP (50 µM), and rIL2 (20 ng/ml). Human
1
1 T lymphocytes
were purified from PBMC, by positive immunoselection using anti-TcR
V
1 mAb and magnetic beads coated with anti-mouse IgG. After
spontaneous detachment,
1
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
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.
Production--
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-
using a human
TNF-
ELISA kit (OptEIA set: human TNF-
, PharMingen, San Diego,
CA) according to the manufacturer's instructions.
chain) were lysed in 1 ml of lysis buffer. After cell lysis,
p56lck, ZAP-70, or
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-
chain. Immune complexes were collected using protein
A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), washed three
times with lysis buffer for
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.
-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 [
-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 [
-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).
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
by Anti-CD3 mAb- or IPP-stimulated V
9V
2
T Cells--
We previously established that high amounts of TNF-
are produced by V
9V
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-
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-
production by V
9V
2 T cells stimulated either with
anti-CD3 mAb or with IPP (36).
View larger version (12K):
[in a new window]
Fig. 1.
TNF- production
by
T cells. Human
peripheral blood-derived V
9V
2 T cells were stimulated by IPP (50 µM) or UCHT1 (2 µg/ml). After different times of
stimulation as indicated, TNF-
production was measured in the
culture supernatants using an ELISA kit. Each experiment is
representative of at least four experiments.
9V
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.
View larger version (29K):
[in a new window]
Fig. 2.
Kinetics of ERK activation in human
peripheral blood-derived T
cells.
9
2 T cells (A) or
1
1 T cells
(B) were stimulated for the indicated times by UCHT1 (10 µg/ml) or by IPP (100 µM). When indicated
9
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.
9
2 T cells but not in other subsets of
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
T cells, which do not express the
9
2 TcR
complex, it could trigger intracellular signals. To investigate
this, we studied ERK2 activity in
1
1 T cells (which are another
important subset of
T cells in human blood). As shown in Fig.
2B, anti-CD3 stimulation induces a strong and rapid ERK2
activation in
1
1 T cells; however, IPP is not able to trigger any
ERK2 activation in these cells following either short or prolonged stimulation.
9V
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
1
1 T cells
(Fig. 3B) and cycloheximide pretreatment does not modify
IPP-induced p38 activation in
9
2 T cells (Fig.
3C).
View larger version (32K):
[in a new window]
Fig. 3.
Kinetics of p38 MAPK activation in human
peripheral blood derived T
cells.
9
2 T cells (A) or
1
1 T cells
(B) were stimulated for the indicated times by UCHT1 (10 µg/ml) or by IPP (100 µM). When indicated,
9
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.
T
cell stimulation. Fig. 4A
shows that the immunoprecipitated p56lck, in the presence of
[
-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
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
T cells.
View larger version (23K):
[in a new window]
Fig. 4.
Autophosphorylation and kinase activity of
p56lck in human peripheral blood derived
T cells. Human peripheral
blood-derived
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
[
-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.
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
chain and ZAP-70 are also rapidly activated. First, we
immunoprecipitated the
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
9
2 T cells. However, we showed that
in the immunocomplex, ZAP-70, is coprecipitated with the
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
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.
View larger version (23K):
[in a new window]
Fig. 5.
Co-precipitation of ZAP-70 with
chain and kinase activity of ZAP-70 in human
peripheral blood-derived
T
cells. Human peripheral blood-derived
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,
chain was immunoprecipitated from the clarified supernatants
with an anti-
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-
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
[
-32P]ATP as described under "Experimental
Procedures." The phosphorylation of LAT was quantified by
PhosphorImager analysis. This experiment was repeated twice.
production in several cell types, including T cells, is
dependent on phosphoinositide 3-kinase (PI3K) activation (45,
46). Moreover, in
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 V
9V
2 T cells, TNF-
is also dependent on PI3K
activation. As shown in Fig.
6A, TNF-
production induced
with either IPP or anti-CD3 mAb is inhibited by LY294002, an inhibitor
of PI3K, suggesting that TNF-
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).
View larger version (27K):
[in a new window]
Fig. 6.
Effect of LY294002 inhibitor on
TNF- production and kinetics of PKB activation
in Human peripheral blood derived
T cells. A, human peripheral blood-derived
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-
production
was then measured in the culture supernatants using an ELISA kit. This
experiment is representative out of four. B, human
peripheral blood-derived
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.
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-V
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).
View larger version (14K):
[in a new window]
Fig. 7.
Analysis of TcR·CD3 down-modulation in
human peripheral blood derived
T cells. Human peripheral blood-derived
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-V
9 mAb and analyzed by flow cytometry. Each
analysis has been repeated at least three times.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
9V
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
T cell activation by physiological antigen
(51), those of V
9V
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-
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
9
2 T cells. Moreover, its enzyme activity can be
detected through its autophosphorylation and by phosphorylation of
enolase used as exogenous substrate. In
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
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
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
9
2 T cells. In
V
9V
2 T cells, similarly to what happens in
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
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 V
9V
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 V
9V
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
T
cells recently put forward by Bürk and co-workers (59).
T cells to phosphorylate
ITAM on the CD3
chain and TcR
(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
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-
release. Of course, the results we present
herein are in vitro results obtained with purified
V
9V
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-
production by V
9V
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
TcR. We did not notice any difference either in
the kinetics or in the amounts of TNF-
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-
production by V
9V
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
9
2 T cells (62).
TNF-
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-
production was identical (data not shown).
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
V
9V
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-
detected in activated V
9V
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.
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-, tumor necrosis factor
;
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Haas, W., Pereira, P., and Tonegawa, S. (1993) Annu. Rev. Immunol. 11, 637-685[CrossRef][Medline] [Order article via Infotrieve] |
2. | Barnes, P. F., Grisso, C. L., Abrams, J. S., Band, H., Rea, T. H., and Modlin, R. L. (1992) J. Infect. Dis. 165, 506-512[Medline] [Order article via Infotrieve] |
3. | De Paoli, P., Gennari, D., Martelli, P., Cavarzenari, V., Comoretto, R, and Santini, G. (1990) J. Infect. Dis. 161, 1013-1016[Medline] [Order article via Infotrieve] |
4. | De Paoli, P., Gennari, D., Martelli, P., Basaglia, G., Crovatto, M., Battistin, S., and Santini, G. (1991) Clin. Exp. Immunol. 83, 187-191[Medline] [Order article via Infotrieve] |
5. | Hara, T., Mizuno, Y., Takaki, K., Takada, H., Akeda, H., Aoki, T., Nagata, N., Ueda, K., Matzuzaki, G., Yoshikai, Y., and Nomoto, K. (1992) J. Clin. Invest. 90, 204-210[Medline] [Order article via Infotrieve] |
6. | Ho, M., Webster, H. K., Tongtawe, P., Pattanapanyasat, K., and Weidanz, W. P. (1990) Immunol. Lett. 25, 139-141[CrossRef][Medline] [Order article via Infotrieve] |
7. | Jouen-Beades, F., Paris, E., Dieulois, C., Lemeland, J.-F., Barre-Dezelus, V., Marret, S., Humbert, G., Leroy, J., and Tron, F. (1997) Infect. Immun. 65, 4267-4272[Abstract] |
8. | Perrera, M. K., Carter, R., Goonewardene, R., and Mendis, K. N. (1994) J. Exp. Med. 179, 311-315[Abstract] |
9. |
Poquet, Y.,
Kroca, M.,
Halary, F.,
Stenmark, S.,
Peyrat, M.-A.,
Bonneville, M.,
Fournié, J.-J.,
and Sjöstedt, A.
(1998)
Infect. Immun.
66,
2107-2114 |
10. | Bertotto, A., Gerli, R., Spinozzi, F., Muscat, C., Scalize, F., Castellucci, G., Sposito, M, candio, F., and Vaccaro, R. (1993) Eur. J. Immunol. 23, 1177-1180[Medline] [Order article via Infotrieve] |
11. | Raziuddin, S., Telmasani, A. W., El-Hag El-Awad, M., Al-Amari, O., and Al-Janadi, M. (1992) Eur. J. Immunol. 22, 1143-1148[Medline] [Order article via Infotrieve] |
12. | Scalize, F., Gerli, R., Castellucci, F., Spinozzi, F., Fabietti, G. M., Crupi, S., Sensi, L., Britta, R., Vaccaro, R., and Bertotto, A. (1992) Immunology 76, 668-670[Medline] [Order article via Infotrieve] |
13. | Constant, P., Davodeau, F., Peyrat, M.-A., Poquet, Y., Puzo, G., Bonneville, M., and Fournié, J.-J. (1994) Science 264, 267-270[Medline] [Order article via Infotrieve] |
14. | Tanaka, Y., Sano, S., Nieves, E., De Libero, G., Rosa, D., Modlin, R. L., Brenner, M. B., Bloom, B. R., and Morita, C. T. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8175-8179[Abstract] |
15. | Tanaka, Y., Morita, C. T., Tanaka, Y., Nieves, E., Brenner, M. B., and Bloom, B. R. (1995) Nature 375, 155-158[CrossRef][Medline] [Order article via Infotrieve] |
16. | Bürk, M. R., Mori, L., and De Libero, G. (1995) Eur. J. Immunol. 25, 2052-2058[Medline] [Order article via Infotrieve] |
17. |
Bukowski, J. F.,
Morita, C. T.,
Tanaka, Y.,
Bloom, B. R.,
Brenner, M. B.,
and Band, H.
(1995)
J. Immunol.
154,
998-1006 |
18. | Bukowski, J. F., Morita, C. T., and Brenner, M. B. (1999) Immunity 11, 57-65[Medline] [Order article via Infotrieve] |
19. | Battistini, L., Borsellino, G., Sawicki, G., Poccia, F., Salvetti, M., Ristori, G., and Brosnan, C. F. (1997) J. Immunol. 159, 3723-3730[Abstract] |
20. | Follows, G. A., Munk, M. E., Gatrill, A. J., Conradt, P., and Kaufmann, S. H. E. (1992) Infect. Immun. 60, 1229-1231[Abstract] |
21. | Garcia, V. E., Sieling, P. A., Gong, J. H., Barnes, P. F., Uyemura, K., Tanaka, Y., Bloom, B. R., Morita, C. T., and Modlin, R. L. (1997) J. Immunol. 159, 1328-1335[Abstract] |
22. | Goodier, M. R., Lundquist, C., Hammarström, M. L., Troye-Blomberg, M., and Langhorne, J. (1995) Parasite Immunol. 17, 413-423[Medline] [Order article via Infotrieve] |
23. |
Lang, F.,
Peyrat, M.-A.,
Constant, P.,
Davodeau, F.,
David-Ameline, J.,
Poquet, Y.,
Vié, H.,
Fournié, J.-J.,
and Bonneville, M.
(1995)
J. Immunol.
154,
5986-5994 |
24. | Badger, A. M., Bradbeer, J. N., Votta, B., Lee, J. C., Adams, J. L., and Griswold, D. E. (1996) J. Pharmacol. Exp. Ther. 279, 1453-1461[Abstract] |
25. |
Wadsworth, S. A.,
Cavender, D. E.,
Beers, S. A.,
Lalan, P.,
Schafer, P. H.,
Malloy, E. A.,
Wu, W.,
Fahmy, B.,
Olini, G. C.,
Davis, J. E.,
Pellegrino-Gensey, J. L.,
Wachter, M. P.,
and Siekierka, J. J.
(1999)
J. Pharmacol. Exp. Ther.
291,
680-687 |
26. | Kabelitz, D., Wesch, D., and Hinz, T. (1999) Spring. Semin. Immunopathol. 21, 55-75[CrossRef] |
27. | Lanzavecchia, A., and Sallusto, F. (2000) Curr. Opin. Immunol. 12, 92-98[CrossRef][Medline] [Order article via Infotrieve] |
28. | Acuto, O., and Cantrell, D. (2000) Ann. Rev. Immunol. 18, 165-184[CrossRef][Medline] [Order article via Infotrieve] |
29. | Kane, L. P., Lin, J., and Weiss, A. (2000) Curr. Opin. Immunol. 12, 242-249[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Bigby, M.,
Markowitz, J. S.,
Bleicher, P. A.,
Grusby, M. J.,
Simha, S.,
Siebrecht, M.,
Wagner, M.,
Nagler-Anderson, C.,
and Glimcher, L. H.
(1993)
J. Immunol.
151,
4465-4475 |
31. | Morita, C. T., Beckman, E. M., Bukowski, J. F., Tanaka, Y., Band, H., Bloom, B. R., Golan, D. E., and Brenner, M. B. (1995) Immunity 3, 495-507[Medline] [Order article via Infotrieve] |
32. | Groh, V., Porcelli, S., Fabbi, M., Lanier, L. L., Picker, L. J., Anderson, T., Warnke, R. A., Bham, A. K., Strominger, J. L., and Brenner, M. B. (1989) J. Exp. Med. 169, 1277-1294[Abstract] |
33. | Parker, C. M., Groh, V., Band, S. A., Porcelli, C., Morita, M., Fabbi, D., Glass, D., Strominger, J. L., and Brenner, M. B. (1990) J. Exp. Med. 171, 1597-1612[Abstract] |
34. |
Grakoui, A.,
Bromley, S. K.,
Sumen, C.,
Davis, M. M.,
Shaw, A. S.,
Allen, P. M.,
and Dustin, M. L.
(1999)
Science
285,
221-227 |
35. | Dustin, M. L., and Cooper, J. A. (2000) Nature Immunol. 1, 23-29[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Lafont, V.,
Liautard, J.,
Gross, A.,
Liautard, J.-P.,
and Favero, J.
(2000)
J. Biol. Chem.
275,
19282-19287 |
37. | Winkler, D. G., Park, I., Kim, T., Payne, N. S., Walsh, C. T., Strominger, J. L., and Shin, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5176-5180[Abstract] |
38. |
Joung, I.,
Kim, T.,
Stolz, L. A.,
Payne, G.,
Winkler, D. G.,
Walsh, C. T.,
Strominger, J. L.,
and Shin, J.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
5778-5782 |
39. |
Soula, M.,
Rothhut, B.,
Camoin, L.,
Guillaume, J. L.,
Strosberg, D.,
Vorherr, T.,
Burn, P.,
Meggio, F.,
Fisher, S.,
and Fagard, R.
(1993)
J. Biol. Chem.
268,
27420-27427 |
40. | Veillette, A., Horak, I. D., Horak, E. M., Bookman, M. A., and Bolen, J. B. (1988) Mol. Cell. Biol. 8, 4353-4361[Medline] [Order article via Infotrieve] |
41. | Danielian, S., Fagard, R., Alcover, A., Acuto, O., and Fisher, S. (1989) Eur. J. Immunol. 19, 2183-2189[Medline] [Order article via Infotrieve] |
42. |
Marth, J. D.,
Lewis, D. B.,
Cooke, M. P.,
Mellins, E. D.,
Gearn, M. E.,
Samelson, L. E.,
Wilson, C. B.,
Miller, A. D.,
and Perlmutter, R. M.
(1989)
J. Immunol.
142,
2430-2437 |
43. | Veillette, A., Horak, J. D., and Bolen, J. B. (1988) Oncogene Res. 2, 385-401[Medline] [Order article via Infotrieve] |
44. | Cantrell, D. (1998) Trends Cell Biol. 8, 180-182[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Hayes, A. L.,
Smith, C.,
Foxwell, B. M.,
and Brennan, F. M.
(1999)
J. Biol. Chem.
274,
33455-33461 |
46. |
Ramirez, M.,
Fernandez-Troy, N.,
Buxade, M.,
Casaroli-Marano, R. P.,
Benitez, D.,
Perez-Maldonado, C.,
and Espel, E.
(1999)
Int. Immunol.
11,
1479-1489 |
47. |
Cuevas, B.,
Lu, Y.,
Watt, S.,
Kumar, R.,
Zhang, J.,
Siminovitch, K. A.,
and Mills, G. B.
(1999)
J. Biol. Chem.
274,
27583-27589 |
48. |
August, A.,
Sadra, A.,
Dupont, B.,
and Hanafusa, H.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11227-11232 |
49. | Boudewijn, M., Burgering, T. H., and Koffer, P. J. (1995) Nature 376, 599-602[CrossRef][Medline] [Order article via Infotrieve] |
50. | Brown, V. I., and Green, M. I. (1991) DNA Cell Biol. 10, 399-409[Medline] [Order article via Infotrieve] |
51. | Valitutti, S., Dessing, M., Aktories, K., Gallati, H., and Lanzavecchia, A. (1995) J. Exp. Med. 181, 577-584[Abstract] |
52. |
Lanzavecchia, A.
(1997)
J. Exp. Med.
185,
1717-1719 |
53. |
Itoh, Y.,
Hemmer, B.,
Martin, R.,
and Germain, R. N.
(1999)
J. Immunol.
162,
2073-2080 |
54. | Straus, D. B., and Weiss, A. (1992) Cell 70, 585-593[Medline] [Order article via Infotrieve] |
55. | Danielian, S, Alcover, A., Polissard, L., Stefanescu, M., Acuto, O., Fisher, S., and Fagard, R. (1992) Eur. J. Immunol. 22, 2915-2921[Medline] [Order article via Infotrieve] |
56. | Hivroz, C., Mazerolles, F., Soula, M., Fagard, R., Graton, S., Meloche, S., Sekaly, R. P., and Fisher, A. (1993) Eur. J. Immunol. 23, 600-607[Medline] [Order article via Infotrieve] |
57. | Gauen, L. K. T., Kong, A.-N. T., Samelson, L. E., and Shaw, A. S. (1992) Mol. Cell. Biol. 12, 5438-5446[Abstract] |
58. | Ley, S. C., Marsh, M., Bebbington, C. R., Proudfoot, K., and Jordan, P. (1994) J. Cell Biol. 125, 639-649[Abstract] |
59. |
Bürk, M. R.,
Carena, I.,
Donda, A.,
Mariani, F.,
Mori, L.,
and De Libero, G.
(1997)
J. Exp. Med.
185,
91-97 |
60. | Barber, E. K., Dasdupta, J. D., Schlossman, S. F., Trevillyan, J. M., and Rudd, C. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 3277-3281[Abstract] |
61. |
Janes, P. W.,
Ley, S. C.,
and Magee, A. I.
(1999)
J. Cell Biol.
147,
447-461 |
62. | Ottones, F., Liautard, J., Gross, A., Rabenoelina, F., Liautard, J. P., and Favero, J. (2000) Immunology 100, 252-258[CrossRef][Medline] [Order article via Infotrieve] |