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J. Biol. Chem., Vol. 277, Issue 24, 21529-21536, June 14, 2002
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From the Goldyne Savad Institute of Gene Therapy, Hadassah
University Hospital, Jerusalem 91120, Israel
Received for publication, February 17, 2002, and in revised form, April 7, 2002
Triggering of the T cell receptor (TCR) leads to
the production of intracellular intermediates with half-life of a few
minutes. Signaling kinetics of events originating from serial TCR
triggering and its relation to antigen dose was investigated. In this
study we documented incremental accumulation of short-lived
intermediates of the extracellular signal-regulated kinase (ERK)
family, produced during successive TCR triggering. The rate and extent
of the intermediate accumulation are essentially determined by the
level of TCR engagement and are augmented by costimulation. ERK-1 and
ERK-2 exhibit different rates of accumulation following serial receptor
triggering. The data indicate that the quantitative kinetic differences
in downstream signaling pathways induce qualitatively distinct
biological outcomes. Although CD69, interleukin-2, and interferon- A single peptide antigen-major histocompatibility complex
(MHC)1 complex on the surface
of an antigen-presenting cell (APC) can serially engage and trigger up
to ~200 T cell receptors (TCRs) on a responding T cell (1). As a
consequence of this "antigenic efficiency," relatively small
numbers of peptide-MHC complexes on APC surfaces can trigger the
threshold numbers of TCRs that are required for T cell activation (2).
This intriguing feature of the T cell activation process makes
physiological sense, in face of the antigenic complexity, as well as
the consequent low frequency of any particular antigen, on the surface
of any given APC in vivo.
Any explanation of how serially receptor triggering leads to T cell
activation must reconcile two opposing kinetic features. On the one
hand, the signal emanating from each triggered TCR is short-lived and,
in the absence of a continuous TCR triggering, the signal will stop.
This point has been established by the demonstration that interference
with continuous TCR engagement by peptide-MHC complexes can terminate
signaling within minutes (3, 4). Furthermore, productive TCR engagement
results in rapid TCR internalization and degradation (reviewed in Ref.
5). However, on the other hand, prolonged intracellular signaling over
several hours is required for T cell activation (6).
The intracellular signaling events underlying the serial triggering
phenomenon are of considerable interest. The binding of TCR to a
specific peptide-MHC complex initiates intracellular signaling cascade
in the T cell (reviewed in Ref. 7). The earliest known step in this
cascade is the induction of protein-tyrosine kinase activity, followed
by downstream events, which include, among others, activation of
members of the mitogen-activated protein kinase (MAPK) families
(reviewed in Ref. 8) and the increase in the level of intracellular
calcium. Experiments that followed calcium response in individual cells
using calcium imaging have revealed that there is a delay between the
TCR triggering (T cell-APC or antibody-coated bead contact) and the
onset of the calcium signal. The duration of this delay decreased with
increasing anti-CD3 mAb density on beads or with increasing stimuli
strength (i.e. different combinations of agonist and
antagonist ligands) (9, 10). These findings have led to the suggestion
that the delay in calcium and other T cell responses are a consequence
of the requirement for the accumulation of a threshold level of
intracellular signaling complexes or events with a half-life of a few
minutes (9). Thus, a pivotal aspect of T cell activation by serially triggered receptors is the accumulation of transient signaling events
over the time required for activation.
Studies on the mechanisms of serial TCR triggering and sustained
signaling have for the most part been performed on T cells stimulated
with high, saturating stimuli doses. Although informative, such studies
failed to follow the level of signaling intermediates at the initial
steps of T cell activation. In the present study, we have explored the
kinetics of TCR-induced signaling events and their relationships to
functional responses using human T cells stimulated by a wide range of
stimuli doses. We primarily followed the activation of the MAPK
cascade, which consists of the extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2), and demonstrated that TCR-evoked signaling
events are gradually accumulating rather than being sustained over the
period of time following T cell-APC interaction and T cell activation. This incremental accumulation of ERK-1/2, which depended on the nature
of the antigen and its dose, is especially evident in low (physiological) stimuli concentrations. Interestingly, despite their
similar activation threshold (11), we found that ERK-1 and ERK-2 differ
in both the extent and rate of accumulation upon serial receptor
triggering. In addition, in view of the distinct role played by ERK in
the activation of CD69, IFN- In aggregate, the interpretation of the results suggest that signaling
events originating from serially triggered TCRs are not simply
sustained for the duration of time required for activation, but are
gradually accumulated up to a threshold required for activation.
Cells--
Peripheral blood mononuclear cells (PBMCs) were
purified from the venous blood of healthy donors by density gradient
centrifugation as described (13). As a source of APCs, monocytes were
isolated from mononuclear cell populations by adherence to plastic.
PBMCs (2 × 106 cells/ml) were incubated in serum-free
medium for one h at 37 °C. The adherent cells were washed
extensively with medium to remove any residual nonadherent cells, and
fresh complete medium was added. Adherent monocytes were removed by
gentle scraping with a plastic cell scraper. Cell viability was >80%
as determined by trypan blue exclusion. Cells were washed with medium
and resuspended at 10 × 106 cells/ml and then loaded
with the staphylococcal enterotoxins (SE) SEA or SEB at the indicated
concentrations for 2 h at 4 °C. The loaded cells were then
washed three times with medium and diluted to 1 × 106
cells/ml and incubated in culture medium at 37 °C for 20 min before
adding them to cultures. CD4+ T cells were isolated from
the PBMC pool by first depleting monocytes by adherence to tissue
culture flasks, as described above, and then further purifying the
nonadherent cells by positive selection of CD4+ T cells
using magnetic cell isolation system (Miltenyi Biotec, Bergisch
Gladbach, Germany). The cells were maintained in RPMI medium
(Biological Industries, Beit-Haemek, Israel) supplemented with 10%
heat-inactivated fetal calf serum (Biological Industries), 2 mM glutamine, and penicillin/streptomycin.
The Jurkat cell line was obtained from the American Type Cell Culture
Collection (Manassas, VA) and was maintained in the above medium.
Cell Stimulation and Assessment of Total Tyrosine and MAPK
Phosphorylation--
To stimulate cells with anti-CD3 mAbs (OKT3),
cells (0.25 × 106 cells) were incubated for varying
times with either soluble anti-CD3 mAb or were plated into wells (50 µl/well) of a 96-well plate, which were pre-coated with various
concentrations of OKT3 at 4 °C for 16 h, and then washed. In
other experiments, monocytes were preloaded with various concentrations
of superantigens. These monocytes were combined with CD4+ T
cells, centrifuged, and incubated at 37 °C for varying times. Cells
were quickly sedimented and lysed for 30 min on ice in Nonidet P-40
lysis buffer with protease and phosphatase inhibitors, as previously
described (14). Western immunoblotting was performed as described (13)
using the anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology) or
anti-MAPK dually phosphorylated mAb (Sigma). T cells and monocytes
incubated separately or T cells stimulated with unloaded monocytes were
used as controls.
Analysis of Receptor Down-modulation--
For CD3
down-modulation, cells were stimulated with various concentrations of
plate-bound OKT3. Following stimulation the cells were collected,
washed, and incubated on ice with OKT3 (1 µg/ml). The primary
antibody was detected by Alexa 488 F(ab)2 fragment goat
anti-mouse IgG (Molecular Probes, Eugene, OR). For quantitative
analysis of TCR expression, CD4+ T cells, which were
stimulated by either SEA- or SEB-pulsed monocytes, were stained
directly with either fluorescein isothiocyanate-conjugated anti-V Cytokine Production--
Cultures containing 106
peripheral blood CD4+ T cells and 106
pre-activated monocytes loaded with the various concentrations of
superantigens were plated in 1 ml of RPMI containing 10% fetal bovine
serum in individual wells of a 24-well culture plate. Cells were
stimulated for the indicated times, and conditioned media were
collected. IL-2, IL-5, and IFN- Progressive Stimulation of MAPK Phosphorylation by Immobilized
Anti-CD3, but Not by Soluble Anti-CD3 mAb--
To fully activate T
cells, antigen-stimulated TCR signaling needs to be sustained for up to
several hours (6). As the signaling evoked by individual TCR is
short-lived, sustained signaling results from ongoing TCR triggering.
Surface-attached antibodies were shown to be more effective than
soluble Ab in sustaining signaling (15, 16). For example, for sustained
tyrosine phosphorylation and MAPK activation, an immobilized stimulus
is required, whereas stimulation with soluble cross-linked anti-CD3
results in only a transient response (16).
We first compared the kinetics of TCR down-modulation induced by the
two-stimulation method. Toward this end, Jurkat cells were
stimulated either with soluble or with immobilized OKT3 for varying
times and were then assessed for receptor down-modulation by indirect
CD3 immunofluorescence and flow cytometry analysis. Stimulation of
Jurkat cells with immobilized Ab induced a prolonged TCR-down-modulation that lasted up to 1 h (Fig.
1A, upper
panel). The extent and frequency of TCR down-modulation
correlated with the concentration of the immobilized anti-CD3 used.
Stimulation with soluble anti-CD3 mAbs, on the other hand, resulted in
TCR down-modulation that lasted only 15 min following stimulation, with
no further receptor down-modulation at later times (Fig. 1A,
lower panel).
Actin cytoskeleton was shown to be the major motor for sustained
signaling that results from prolonged TCR triggering (3). Actin
cytoskeleton disruption by the drug cytochalasin E inhibits signaling
induced by immobilized Ab but not by cross-linked soluble anti-CD3
stimulus (16). In our hands, 4 µM cytochalasin E
abolished TCR down-modulation induced by immobilized antibodies (data
not shown).
These observations are consistent with the idea that, upon incubation
with soluble Ab, there is a uniform distribution of Ab on the cell
surface with most receptors engaged at once, and this therefore does
not enable continuous receptor triggering and sustained signaling. On
the other hand, stimulation with immobilized Ab provides conditions
that favor receptor redistribution and serial association of TCRs with
the surface-bound antibody, which leads to prolonged TCR engagements
and sustained signaling.
To study the kinetics of early signaling intermediates, Jurkat cells
were stimulated with various concentrations of soluble anti-CD3 mAbs,
and whole cell lysates were separated on SDS-PAGE and immunoblotted
with anti-phosphotyrosine and with anti-dually phosphorylated MAPK
mAbs. Following stimulation with soluble Ab (1000 ng/ml), a transient
phosphotyrosine response was evident between 1 and 15 min, which
gradually declined thereafter (Fig. 1B, upper
panel). Comparable kinetics of tyrosine phosphorylation was
observed when cells were treated with 100 and 10 ng/ml anti-CD3 mAb
(data not shown). The same membranes were re-probed with a specific Ab
for the dually phosphorylated active form of the MAPK family members,
ERK-1 and ERK-2 (Fig. 1B, lower
panel). Interestingly, although MAPK phosphorylation is a
relatively distal event in the TCR signaling cascade, its activation
followed a time course similar to that for total tyrosine
phosphorylation (Fig. 1B, lower panel). This is in agreement with the results previously
shown by Berg et al. (16)). As can be seen in Fig.
1B (lower panel), increasing the
soluble anti-CD3 concentration from 10 to 1000 ng/ml produced higher
levels of phosphorylated MAPK. However, even a 100-fold increase in
anti-CD3 concentration did not significantly change the time course of
the signal, which peaked at the same time interval at all
concentrations and declined thereafter.
In parallel experiments, plate-bound antibodies were used as
stimulators (Fig. 1C). As was seen with soluble Ab
stimulation (Fig. 1B), the maximal level of MAPK
phosphorylation increased with increasing the dose of anti-CD3, and
this was most pronounced when antibody dose was raised from 100 to 1000 ng/ml. However, in contrast to soluble Ab, alterations in antibody
concentrations resulted in marked differences in the signaling kinetic.
More specifically, MAPK phosphorylation levels rapidly increased during the initial period of time following stimulation with high anti-CD3 dose (1000 ng/ml), reaching its maximum by 7.5-15 min, and was sustained for ~30 min. The decrease seen at a later time point (60 min) is probably the result of inactivation and degradation of kinases,
such as Lck, following extensive and persistent TCR triggering
(17, 18). Significantly, the level of MAPK phosphorylation gradually
increased during the 60 min tested when 10-100 ng/ml anti-CD3 were
used, and the response latency was inversely correlated to the anti-CD3
mAb dose. MAPK activation was hardly detected at all time points in
cells stimulated with 1 ng/ml plate-bound anti-CD3 (data not shown).
To test whether the results shown above present a general phenomenon,
subsequent experiments employed peripheral blood CD4+ T
cells in place of Jurkat cells as responders. Stimulation of CD4+ T cells with soluble anti-CD3 resulted in a rapid
accumulation of MAPK activation within 15 min, which declines
thereafter (Fig. 1D, upper panel). On
the other hand, when CD4+ T cells were stimulated with
plate-bound anti-CD3 mAbs, gradual accumulation of signaling events was
observed (Fig. 1D, lower panel). The
detection of MAPK activation in CD4+ T cells stimulated by
soluble or plate bound anti-CD3 required significantly higher
concentrations of Ab as compared with Jurkat cells.
The results demonstrate that cells triggered by immobilized antibodies
gradually accumulate signaling events over time in an antibody
dose-dependent manner and in correlation with the frequency
of TCR occupancy. These findings are compatible with the notion that a
minimum rate of TCR triggering is required for the accumulation of
sufficient concentrations of signaling events.
Kinetics of MAPK Phosphorylation in T Cells Stimulated with
Superantigens--
The high affinity binding between Ab and the
TCR·CD3 complex makes antibodies less efficient for serial TCR
triggering compared with the peptide-MHC complex, which binds the TCR
with low affinity, thus allowing dissociation following triggering. On
the other hand, TCR recognition by superantigens mimics more closely
that of peptide-MHC complex, as evident by a similar kinetic pattern of
activation (19, 20). This is most likely because of their activity
being dependent on MHC binding and to the rapid dissociation of the
superantigen-TCR complex, which leads to high frequency of serial TCR
triggering. We therefore followed the kinetics of MAPK activation in T
cells stimulated with superantigens, which were presented by autologous
monocytes, as accessory cells. It is known that interaction of ligands
on the accessory cell with other T cell receptors such as the B7-CD28
interactions contribute to T cell activation. Furthermore, it was
previously shown that freshly isolated monocytes have a low level of
cell surface B7 expression and hence very little costimulatory
activity, whereas monocytes incubated for 24 h at 37 °C provide
good costimulation, which correlates with higher levels of B7 and
ICAM-1 expression on their surface (Fig.
2A and Refs. 13 and 21). Most
importantly, these incubation conditions have little effect on MHC
class II expression, in contrast to the commonly used IFN-
Various SEs such as SEB and SEA, through interaction with different T
cell receptor V
To establish the extent of TCR occupancy achieved by the two
superantigens, T cells were incubated with APCs bearing various doses
of either SEA or SEB and then stained for surface TCR-V
Thus, the level of MAPK phosphorylation induced by serially triggered
TCRs increased over a relatively long period of time, and the rate of
this increase correlated with the extent of TCR engagement as measured
by receptor down-modulation. Both the extent of TCR engagement and the
corresponding signaling depended on the nature of the antigen and its
dose. Furthermore, costimulation amplified receptor signaling and
facilitated its accumulation in the face of a fixed antigen dose.
Differential Effects of Antigen Dose on CD69, IL-2, IFN-
In view of ERK's unique role in regulating distinct T cell responses,
we sought to correlate the rate of accumulation of activated ERK,
resulting from increasing doses of the SEB and SEA stimulators, with
the level of production of individual cytokines. T cells were combined
with monocytes that were preloaded with various concentrations of
either SEB or SEA. After 48 h of culture, the level of CD69
expression on the cell surface was measured by flow cytometric
analysis, and the level of cytokines secreted to the medium was
determined by ELISA.
The results revealed that CD69 expression by individual cells as well
as IFN-
In aggregate, there is a correlation between the extent of ERK
activation (seen in Fig. 2) and the induction of
ERK-dependent T cell responses (i.e. CD69,
IFN- Antigen Dose Dictates the Stimulation Time Required for IFN- Commitment of T cells to cytokine production and proliferation
requires sustained TCR signaling for up to several hours, which is
achieved through serial receptor engagement. In the present study, we
characterized the kinetics of early signaling events during the course
of serial TCR triggering. The principal experimental finding in this
study is that signaling intermediates produced by serially triggered
TCRs are not simply sustained but are incrementally building up.
Increased signaling levels during T cell stimulation were also observed
by Muller et al. (28), at the level of phosphotyrosine staining in single T cells.
The data demonstrated that the onset of ERK phosphorylation and the
rate of its accumulation were dependent on the antigen dose. A previous
report has established that the number of TCR triggered per time
correlated with stimulus dose as well as with antigen-TCR dissociation
rate (29). Thus, the extent of accumulation of signaling events
reflected the level of antigenic stimulation, which was determined by
antigen dose and the dissociation rate of the TCR-ligand complex, with
higher dissociation rate resulting in increased rate of
intermediate accumulation. Differences in dissociation rate and
consequently the frequency of TCR engagement can explain the
differences between SEA and SEB in regard to their respective ability
to induce ERK phosphorylation and TCR down-modulation.
Hudrisier et al. (30) have shown that the magnitude of the
functional cytotoxic T lymphocyte response is directly related to the frequency of serial TCR engagement, which, in turn, is determined by the rate of ligand-TCR complex dissociation. Our data
support these findings and provide an explanation for how the priming
conditions such as the nature of the antigen and its dose are
integrated in a corresponding response. Accordingly, the amplitude of
the biological response is determined by the level of accumulation of
signaling intermediates within the cell, which in turn are determined
by the level of TCR occupancy.
Furthermore, antigen dose and the frequency of TCR engagement dictate
not only the amplitude of the response, but also the rate of
accumulation of signaling intermediates and as a result the duration of
signaling required to reach threshold level. In accord with the above
proposition, we demonstrated that the stimulation time required for
IFN- Besides the level of TCR triggering, we demonstrated that costimulation
also plays an important role in determining the extent and rate of
intermediate accumulation. In this regard, it was shown that the role
of costimulation is not to increase the number of triggered TCRs, but
rather to contribute to TCR signaling by stabilizing tyrosine
phosphorylation (18); in this way, costimulation reduces the number of
triggered TCR required to reach activation threshold. Taken together,
costimulation is thus predicted to shorten signaling time leading to a
biological response. Indeed, it was previously shown that the time
required for commitment of T cell to proliferation can be shortened by
providing costimulation and by increasing antigen dose (31).
Significantly, in this report, T cells stimulated with high or low
doses of anti-CD3 in combination with anti-CD28 required short and long
stimulation time for commitment, respectively, but ultimately reached
the same proliferative response. Thus, both the antigenic dose and the
presence of costimulation determine the duration of signaling required
for T cell activation.
Together, these observations are consistent with the model proposed by
Wulfing et al. (9), to explain the correlation between the
duration of delay in the onset of calcium response and the strength of
the stimulus. Accordingly, the accumulation of intracellular intermediates with a short half-life is the rate-limiting step in
triggering the calcium response. Using different experimental systems,
Gunzer et al. (32) have arrived to a similar conclusions. Having shown that a single T cell can sequentially encounter one or
more APCs in multiple short-lived interactions, which eventually trigger T cell activation, the authors have suggested a cumulative signaling threshold based on both the frequency of interactions and
their respective strength (32). The data in the present study are the
first to directly demonstrate the accumulation of short-lived
intracellular intermediates that are produced by successive receptor
triggering, most likely, up to a threshold level required for
activation of the biological response.
It was previously suggested that the level of TCR occupancy could
control the amplitude of the T cell response as well as its type (1).
In accord with this proposal, we documented differences in the
antigenic dose required for the activation of individual responses,
which correlated with the rate and extent of ERK phosphorylation. These
findings are in agreement with a previous report, which has shown that
distinct activation pathways contribute differently to the production
of various cytokines (12). Accordingly, the ERK signaling pathway is
essential for both IFN- A recent report has revealed distinct activation thresholds for the
three MAPK families: ERK, c-Jun N-terminal kinase, and p38 (11). Hence,
differences in signaling strength induce selective activation of the
different MAPK families. Although activation of other MAPK families was
not examined in the present study (the diphosphorylated c-Jun
N-terminal kinase was not detected, probably reflecting its higher
activation threshold), the data in hand (Fig. 2) demonstrate higher
level of ERK-2 (p42) phosphorylation and a faster onset of appearance
than ERK-1 (p44). These differences in the activation levels of ERK-1
and ERK-2 are most apparent at the lower superantigen doses (Fig. 2,
A and B), and are less prominent in cells
stimulated with immobilized anti-CD3 mAbs (see Fig. 1C). One
possible explanation for the asymmetries seen in the Western blot data
for ERK-1 and ERK-2 lies in the characteristics of the antibodies used
in the assay. It is possible that the antibodies are more sensitive
detectors of the ERK-2 than the ERK-1. However, the observation that
the proportion and kinetics of ERK-1 and ERK-2 activation were similar
in T cells stimulated with soluble anti-CD3 mAbs (Fig. 1B,
lower panel) suggests that this is not the case.
Moreover, this latter observation indicates that the observed
differences between ERK-1 and ERK-2 activation are attributable to
distinct accumulation rates of the two ERKs that are apparent when
serial receptor triggering is taking place. These accumulation rates
are not dependent on their activation thresholds, as both ERKs share
similar activation thresholds (Fig. 1B and Ref. 11). We
therefore suggest that there are distinct time thresholds for the
accumulation of ERK-1 and ERK-2 and possibly for other signaling pathways, which are distinct from activation thresholds. These differences in signal strength can induce quantitatively and
qualitatively different signaling pathways. In this regard, Pages
et al. (36) have demonstrated, using ERK-1-deficient mice,
that although for most part ERK-1 was dispensable, and ERK-2 may have
compensated for its loss, thymocytes stimulated by anti-TCR mAbs had
severely reduced proliferative response, and thymocyte maturation was
also defective in those mice. Thus, ERK-1 and ERK-2 apparently have distinct roles in thymocyte development. Furthermore, it was suggested that the two ERK isoforms compete with each other for the upstream MAPK
kinase activator (36). This latter point provides a possible mechanism
for our observation regarding different activation rates for the two isoforms.
Thus, the data demonstrate distinct accumulation rates of intermediates
of various signaling pathways according to their intrinsic properties.
In turn, the degree of activation of the various signaling pathways,
which have qualitatively distinct roles in regulating T cell response,
determines T cell fate and the quantitative and qualitative aspects of
the biological outcome. This mechanistic insight into the kinetics of
signaling events during TCR serial triggering provides a useful
framework for further exploring the intracellular pathways influenced
by this process and its impact on T cell fate.
We thank E. Galun and H. Giladi for helpful
discussion and critical reading of the manuscript.
*
This work was supported by the Greensboro Community
Foundation.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.
Published, JBC Papers in Press, April 8, 2002, DOI 10.1074/jbc.M201613200
The abbreviations used are:
MHC, major
histocompatibility complex;
APC, antigen-presenting cell;
TCR, T cell
receptor;
MAPK, mitogen-activated protein kinase;
ERK, extracellular
signal-regulated kinase;
PBMC, peripheral blood mononuclear cell;
SE, staphylococcal enterotoxin;
ELISA, enzyme-linked immunosorbent assay;
Ab, antibody;
mAb, monoclonal antibody;
IL, interleukin;
IFN-
Serial Triggering of T Cell Receptors Results in Incremental
Accumulation of Signaling Intermediates*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(IFN-) were primarily produced by high antigen doses that supported
high MAPK phosphorylation, maximal interleukin-5 expression is induced
by low and intermediate stimulus doses that do not support significant
accumulation of activated ERK. We further demonstrated that the rate of
phosphorylated ERK accumulation correlates with the duration of delay
between T cell stimulation and the onset of IFN- response, with
stronger stimuli giving a more rapid IFN- response. This delay might
reflect the time required for the accumulation of signaling
intermediates up to a threshold level that is necessary for activation.
Thus, the data suggest that signaling events originating from
serially triggered TCR are not simply sustained but are gradually
accumulated and are integrated in a corresponding response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and IL-2 as opposed to IL-4 and IL-5
(12), we correlated the degree of ERK accumulation with the level of
production of individual cytokines, and ERK's accumulation rate with
the onset of IFN- response.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5
or phycoerythrin-conjugated anti-V17 (Jackson Immunoresearch, West
Grove, PA). Cells (104) were analyzed using FACScalibur and
Cell Quest Analysis software (Becton Dickinson, San Jose, CA). The data
were calculated using the mean fluorescence values of the cell.
levels in the conditioned media were
assayed by ELISA (R&D Systems, Minneapolis, MN).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Time and antibody density dependence of TCR
down-modulation, total tyrosine, and MAPK phosphorylation following
soluble and immobilized anti-CD3 stimulation. A, Jurkat
cells (2.5 × 105) were stimulated with immobilized
anti-CD3 mAb (OKT3) at the indicated concentrations, at 37 °C for
the indicated times (upper panel). The extent of
TCR-down modulation by soluble anti-CD3 (0.1 µg/ml; open
symbols) was compared with that achieved by similar
concentration of immobilized anti-CD3 (closed
symbols; lower panel). The expression
of CD3 is shown as percentage of CD3 down-modulation as compared with
the level of CD3 expression on unstimulated cells. B, Jurkat
cells were stimulated with soluble anti-CD3 mAb (OKT3: 10 and 1000 ng/ml) and incubated at 37 °C for the indicated times. Cell lysates
were subjected to SDS-PAGE and anti-phosphotyrosine immunoblotting
(upper panel) and anti-dually phosphorylated MAPK
(lower panel; exposure times of 3 min and 30 s are shown for 10 and 1000 ng/ml SEB, respectively). C,
Jurkat cells were stimulated with immobilized anti-CD3 mAbs as in
A, for the indicated times. Cell lysates were subjected to
SDS-PAGE and anti-dually phosphorylated MAPK immunoblotting
(upper panel in each anti-CD3 concentration).
D, CD4+ T cells (1 × 106) were
stimulated with 10 µg/ml anti-CD3 Abs that were either soluble
(upper panel) or immobilized (lower
panel) for the indicated times. Immunoblotting for Vav
confirmed comparable loading of total protein in each lane
(lower panel in each anti-CD3 concentration).
Similar results were obtained in two (A) and three
additional (B-D) experiments. U, unstimulated T
cells.
treatment
for accessory cell stimulation (21). To study the kinetics of
TCR-induced intracellular signaling, and the contribution of accessory
receptors to this process, we compared MAPK activation in T cells
stimulated with various concentrations of the superantigen SEB
presented by either freshly isolated or pre-incubated monocytes (Fig.
2, B and C). T cells stimulated with
pre-incubated monocytes (Fig. 2C) had considerably higher
levels of MAPK phosphorylation compared with T cells stimulated with
freshly isolated monocytes loaded with the same concentrations of SEB
(Fig. 2B). Significantly, both the latency of the response
and the time required to reach a maximal signal in each SEB
concentration were greatly reduced in T cells stimulated with the
pre-incubated monocytes. T cells incubated separately or with unloaded
monocytes were used as controls and demonstrated insignificant levels
of MAPK phosphorylation (Fig. 2E).
View larger version (39K):
[in a new window]
Fig. 2.
Kinetics of MAPK phosphorylation and TCR
down-modulation following superantigen stimulation. A,
expression of cell surface antigens on resting and activated monocytes.
Purified resting monocytes or monocytes that were "pre-activated"
by incubation in medium at 37 °C were stained with anti-CD86,
CTLA-4Ig, anti-CD54, and anti-MHC class II (solid
lines). The dashed lines represent
staining with control reagents. CD4+ T cells (2.5 × 105) were conjugated with either freshly isolated
(B) or pre-activated (C-F) autologous monocytes
(2.5 × 105) that were pulsed with the indicated
concentrations of the superantigens SEB (B and C)
or SEA (D). The conjugates were then incubated at 37 °C
for the indicated times. E, negative controls: T cells
incubated for 120 min in the absence of monocytes (lane 1),
monocytes loaded with 1000 ng/ml SEB incubated for 120 min in the
absence of T cells (lane 2), T cells that were conjugated
with unpulsed monocytes and incubated for 120 min (lane 3),
and T cells and monocytes loaded with 1000 ng/ml SEB at time 0 (lane 4). Dually phosphorylated MAPK was detected in
detergent lysates of these cells after Western blotting as described in
Fig. 1. Immunoblotting for Vav confirmed comparable loading of protein
in each lane as in Fig. 1C. F, CD4+ T
cells and either SEA- or SEB-pulsed monocytes were allowed to form
conjugates as described above and were then incubated at 37 °C for
16 h. The level of TCRV 5 or TCRV17 expression in SEA and SEB
stimulated cells, respectively, was determined using the appropriate
anti-TCR antibodies and fluorescence-activated cell sorting analysis.
The data are presented as percentage of TCR down-modulation. 100%
expression is the level of TCR expression on T cells cultured with
unpulsed APCs. The data are from one experiment; similar results were
obtained in two other experiments.
chain, can activate distinct populations of T cells.
We compared the ability of superantigens SEB and SEA to activate MAPK,
using Western blotting analysis as described above. We found that both
SEA and SEB induced MAPK phosphorylation, which gradually accumulated
over time, reaching approximately the same maximal levels (Fig. 2,
C and D). However, SEA induced significant levels
of MAPK phosphorylation at earlier time points, compared with
stimulation with SEB, and required considerably lower concentrations.
These differences in the effective stimulatory concentrations of SEA
and SEB might reflect their respective affinities for the MHC class II
molecules (22). However, in the present study, the superantigens were
loaded onto the monocytes at 4 °C; at this temperature, no
significant differences in the binding affinities of the two
superantigens to class II were observed (22). It is possible,
therefore, that other factors contribute to the observed differences
between SEA and SEB activity. One such factor could be differences in
the affinities of the two SEs to the TCR, which lead to different
frequencies of receptor engagement. In fact, several studies have
indicated a relationship between the overall extent of TCR
down-modulation and ligand potency (1, 23-25).
5 or -V17,
respectively. The maximal level of TCR down-modulation was similar in
both SEA- and SEB-stimulated cells. However, in agreement with
expectations, SEA was much better in inducing TCR down-modulation as
compared with SEB, at the lower antigen dosage (Fig. 2F).
Although no V17 down-modulation was induced by 0.1-1 ng/ml SEB, a
total of 10-20% of V5 TCR are down-modulated when cells are
stimulated with the same concentrations of SEA.
, and
IL-5 Production--
In light of the results presented above, we
hypothesized that the quantitative differences in strength and duration
of downstream signaling pathways can induce qualitatively distinct
biological outcomes. Dumont et al. (26, 27) have previously
demonstrated that distinct signaling pathways contribute differently to
the regulation of cytokine response in T cells. The authors have
disrupted ERK-1/2 activation by using PD98059, a selective inhibitor of MAPK kinase 1 (26, 27). Treatment of stimulated T cells with PD98059
resulted in marked inhibition of CD69, IL-2, and IFN- expression.
However, even at high concentrations, PD98059 not only failed to
suppress IL-4 and IL-5, but it significantly augmented their production
(12).
and IL-2 secretion to the culture media increased with
increasing SEB concentrations. IL-5 secretion, on the other hand,
peaked at 100-fold lower SEB concentration (10 ng/ml) and decreased at
higher doses (Fig. 3). Giving the
differential role ERK activation plays in the expression of these
proteins, their levels of expression correlated with the extent of
phosphorylated ERK accumulation at each antigen dose. Likewise, SEA
with its capacity to induce rapid ERK-1/2 activation at lower stimuli
doses, as compared with SEB (i.e. 0.1-10 ng/ml), induced
significant levels of CD69 and IFN- at these doses, whereas only
marginal levels of these proteins were detected in SEB-stimulated cells (Fig. 3). The levels of IL-5 were relatively low when SEA was used as
stimulator at all doses except for a peak at 0.1 ng/ml, consistent with
its ability to induce significant ERK phosphorylation even at the
intermediate dosage range. Of note, the levels of IL-4 detected in the
culture supernatants under the conditions used in these experiments
were too low to permit reliable measurements.
View larger version (18K):
[in a new window]
Fig. 3.
Effect of antigen dose on cell surface
expression of CD69 and the production of IL-2,
IFN- , and IL-5. CD4+ T cells
were conjugated with SEB- or SEA-pulsed autologous monocytes as in Fig.
2, and then cultured at 37 °C for 48 h. The expression
of CD69 was detected by immunofluorescence and flow cytometry. The data
are calculated from the mean fluorescence intensity above unstimulated
cells. IL-2, IFN-, and IL-5 secretion was determined in the
conditioned medium by ELISA. The data represent the mean of triplicate
samples. Comparable results were obtained in three experiments.
, and IL-2) by each antigen and its respective dose. In
contrast, maximal IL-5 expression is induced by stimulus doses that do
not support significant accumulation of activated ERK. A similar
correlation among antigen dose, the extent of TCR
down-modulation, and the induction of effector function was described
previously (24). It is especially interesting to note, in this regard,
that CD69 up-regulation was elicited at low antigen concentrations
whereas up to 1000-fold more antigen was required for IFN- secretion
and proliferation.
Production--
The data above demonstrated that the magnitude of
ERK-dependent T cell responses, as measured by IFN-,
IL-2, and CD69 expression, correlates with the antigen dose. These
findings are in agreement with the notion that higher stimulus dose
gives stronger T cell response. However, the data also demonstrated
that antigen dose determines the time lag between T cell stimulation
and the onset of ERK activation, and the rate of its accumulation.
Therefore, it seems reasonable to assume, based on the present data,
that an increased stimuli dose, which reduces the accumulation time required to reach a threshold level of signaling intermediates, will
consequently shorten the time required for the cytokine response. To
test this assumption, T cells were stimulated by monocytes loaded with
various doses of SEB and conditioned medium was collected after
different times of incubation and tested for the presence of IFN-
(Fig. 4). Interestingly, the time
required for the onset of the IFN- response correlated well with SEB
concentration. IFN- production by T cells stimulated with high SEB
doses (100 and 1000 ng/ml) was rapid and took place within 5 h of
stimulation. A longer period of 8 and 20 h was required for
IFN- production by cells stimulated with 10 and 1 ng/ml,
respectively. Remarkably, despite the differences in the antigenic
doses used, similar initial levels of IFN- were detected in culture
media of cells stimulated with the various concentrations, although
detected at different time points following stimulation, suggesting a
threshold effect for IFN- production. This latter observation is not
a consequence of the IFN- detection sensitivity, as these
concentrations (~100 pg/ml) are significantly higher than the lower
limit of detection by the ELISA used (5 pg/ml). Thus, stimulus dose
augments T cell activation, at least in part, by reducing the time
required for IFN- secretion.
View larger version (25K):
[in a new window]
Fig. 4.
The time of signaling required for
IFN- production is dependent on the dose of
antigenic stimulation. CD4+ T cells were conjugated
with SEB-pulsed autologous monocytes as in Fig. 2, and then
cultured at 37 °C for the indicated times. IFN- secretion was
determined in the conditioned medium by ELISA. The data represent the
mean of triplicate samples. A representative experiment of three is
shown.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
production inversely correlated with antigenic dose, with
higher doses giving a more rapid response. The results also
demonstrated that, although they emanated after different times
following stimulation, the initial levels of IFN- induced by low and
high antigen concentrations were similar, suggesting a threshold
effect. We suggest that the frequency of TCR engagement, which depends
on antigen nature and dose, dictates the accumulation rate of triggered
events and, hence, the time required to reach threshold levels and activation.
and IL-2 production, which are associated
with Th1 response, and inhibition of this pathway augments
Th2-associated cytokines. Our findings, as well as previous
reports (33, 34), imply that, whereas intermediate dose of stimuli
induced T cell response that is skewed toward Th2-like
cytokine (IL-5), priming with higher doses of stimuli favored increased
production of Th1-like cytokines (IFN-, IL-2), correlating with the respective abilities of the various stimuli doses
to induce ERK-phosphorylation. Fully polarized response requires
prolonged TCR stimulation (31) and the presence of certain cytokines,
which was not tested here. However, these results propose that the
extent of antigenic stimulation biases the response toward certain
Th-like phenotypes and contributes to the subsequent acquisition of
distinct type of response. Furthermore, the data provide a possible
mechanistic insight into this process. Thus, although
Th1/Th2 development is primarily driven by
cytokines (such as IL-12 and IL-4), priming conditions such as the
strength of antigenic stimulation play an important role in the
decision (33-35).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Goldyne Savad Inst. of
Gene Therapy, Hadassah University Hospital, P. O. Box 12000, Jerusalem 91120, Israel. Tel.: 972-2-6777848; Fax:
972-2-6430982; E-mail: rjacob@hadassah.org.il.
ABBREVIATIONS
, interferon-.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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