| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Departments of Medical Biophysics and Immunology, Ontario Cancer Institute, Toronto, Ontario, Canada; and
Ontario Cancer Institute and Amgen Institute, Toronto, Ontario, Canada; and
Department of Immunology, University of Toronto, Toronto, Ontario, Canada
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Many studies have addressed the role of various mitogen-activated
protein (MAP) kinase pathways during thymocyte develop-ment.
Studies have shown that mice overexpressing dominant neg-ative
components of the Ras (MAP) kinase signaling pathway (Ras/raf-1/MAP
kinase kinase (MEK)) exhibit partially blocked positive
selection (5, 6, 7, 8) and extracellular signal-regulated kinase
(ERK) 1-deficient thymocytes show impaired positive selection
(9). A selective role for ERK in positive selection was
also shown in mice expressing a mutation in the TCR
-chain-connecting peptide (CPM). This mutation results in
abrogation of positive selection but not negative selection and
prevents CD3
from being recruited to the TCR-CD3 complex
(10). Accordingly, positive selection is also attenuated
in mice deficient in CD3 and biochemical analysis revealed that ERK
activation is severely impaired in these mice (11). Recent
evidence has also shown that a novel guanine nucleotide exchange factor
for Ras, RasGRP, is involved in thymocyte-positive selection
(12). These studies along with studies using
pharmacological inhibitors of the ERK pathway (13, 14, 15)
clearly support the role for ERK during positive selection.
The role for ERK in negative selection remains controversial. Studies
using transgenic mice expressing dominant negative effectors in the
Ras/raf/MEK pathway suggest that Ras/raf/MEK do not play a role in
negative selection (5, 6, 7). However, experiments have shown
that modifying the ERK pathway by using pharmacological inhibitors can
shift negative selection to positive selection (14, 15).
In addition, studies have also shown that the absence of CD3 not
only impairs positive selection and ERK activation (11),
but also impairs negative selection (16).
Other studies have suggested that p38 and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase MAP kinases are important in mediating negative selection (17, 18, 19). Based upon the existing data, the currently favored model predicts that positive and negative selection results from the stimulation of unique signals involving discrete molecules. The induction of ERK leads to positive selection, while the induction of p38 and JNK results in negative selection. However, it is not clear how a strong signal that is associated with negative selection could induce p38 and JNK, and not ERK. Accordingly, recent studies suggest that ERK is activated during negative selection (10, 14, 15).
To gain further insights into TCR-mediated signals that determine cell fate, we developed a culture system in which a population of naive DP thymocytes were triggered with a defined set of ligands that induced either positive or negative selection. Biochemical analysis revealed that while positively selecting ligands triggered sustained low-level ERK activation, negatively selecting ligands induced strong but transient ERK activation. The ability to induce sustained vs transient ERK activation correlated with surface TCR levels. Furthermore, the transient ERK activation induced by negatively selecting ligands triggered important downstream events, since inhibition of such initial ERK activation abrogated clonal deletion. Together, our data suggest that the extent and duration of ERK activation could influence both positive and negative thymocyte selection.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Generation of P14 TCR recombination-activating gene (RAG)
2-deficient H-2d/d mice has been previously
described (15). C57BL/6 mice,
2-microglobulin
(2m)-deficient mice, and RAG-1-deficient mice
were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were
bred and maintained according to institutional guidelines.
Reagents
The MEK1 inhibitor PD98059 was purchased from Calbiochem (La Jolla, CA). U0126 was kindly provided by M. Favata (DuPont Pharmaceuticals, Wilmington, DE). The peptides p33 (KAVYNFATM), S7A (KAVYNFSTM), L6F (KAVYNLATM), and AV (SGPSNTPPEI) have been characterized before (20).
In vitro culture of thymocytes
C57BL/6 mice or 2m-deficient mice were
used for harvesting day 4 thioglycolate-activated peritoneal
macrophages as APCs. Macrophages were plated at 3 x
105 cells/well in 24-well plates in IMDM
supplemented with 10% FCS, 2 mM glutamine, 5 x
10-5 M 2-ME, penicillin, and streptomycin. After
overnight incubation, nonadherent cells were gently washed away. The
APCs were then pulsed with appropriate concentrations of peptides for
2 h. Thymocytes were harvested from P14
RAG2-/- H-2d/d mice and
kept on ice for 2 h as a single-cell suspension. For peptide/APC
stimulation, thymocytes (2 x 106
cells/well) were then complexed with the peptide-pulsed macrophages by
quick-spinning the cells at 1300 rpm for 30 s. The cells were
placed in the 37°C incubator for appropriate periods of time.
Thymocytes were then harvested by gentle pipetting. Usually thymocytes
from two to three wells were pooled to obtain a sufficient number of
cells to perform biochemistry. Residual macrophages were removed by
incubating these thymocytes with anti-F4/80 Abs conjugated with
anti-rat-IgG Dynal magnetic beads for 30 min at 4°C and
subjecting them to a magnetic field. For studies using PD98059,
thymocytes were preincubated with the inhibitor for 1 h at 37°C
before stimulation.
Fetal thymic organ cultures (FTOC)
Day 15 fetal thymic lobes from C57BL/6 mice were cultured for 5 days in 1.35 mM deoxyguanosine (Sigma, St. Louis, MO). The lobes were washed three times in medium. Briefly, 2 x 105 thymocytes that had been stimulated for 16 h in the monolayer signaling cultures were placed in Terasaki wells along with deoxyguanosine-treated lobes. These were cultured overnight as hanging drops in a humidified chamber in the 37°C incubator. Reconstituted lobes were then placed on 0.8-µm polycarbonate filters (Costar, Cambridge, MA), and further incubated for 40 h in medium containing 12.5% FCS. After this incubation, the thymic lobes were teased apart, the cells were enumerated by trypan blue exclusion and stained for flow cytometric analysis with anti-CD4-PE, CD8-FITC, and H-2Kd-biotin.
Flow cytometry
Thymocytes were stained with PE-conjugated anti-CD4,
FITC-conjugated anti-CD8, and biotin-conjugated anti-CD69,
anti-CD5, anti-V2, or H-2Kd. All Abs
were purchased from BD PharMingen (San Diego, CA). Cell viability was
determined by enumerating trypan blue-negative cells or by flow
cytometry, staining thymocytes with FITC-conjugated annexin V and
propidium iodide (BioSource International, Camarillo, CA) or vital
chromogenic dye 7-amino actinomycin D.
Western blot analysis
Peptide-APC-stimulated thymocytes were then washed once in PBS and pelleted. Whole cell lysates were prepared by addition of lysis buffer containing 50 mM Tris (pH 7.5), 20 mM EDTA, and 1% Triton X-100 supplemented with 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 0.2 mM PMSF on ice for 20 min. The lysates were cleared by centrifugation. Protein concentrations were determined using a commercially available kit (Bio-Rad, Hercules, CA). Cell lysates with equivalent protein content were electrophoresed in a 12% SDS-polyacrylamide gel, transferred to a nylon membrane, and detected for "activated" ERK1/2 using a PhosphoPlus p44/42 MAP kinase (Thr202/Tyr204) Ab kit (NEB, Beverly, MA). Signals were detected using the ECL system (Amersham, Arlington Heights, IL). Densities of phospho-ERK bands were quantitated using ImageQuant software. To account for loading variability, values were normalized based on the density of total ERK bands. Values are expressed as fold increase over baseline stimulation conditions. For studies using PD98059 or U0126, thymocytes were preincubated with the inhibitor for 2 h at 37°C before stimulation and washed.
Ca2+ flux analysis
Thymocytes were loaded with Indo-1 (10 µM) for 1 h at 37°C in IMDM supplemented with 2% FCS. Indo-1+ cells exhibiting a large forward scatter corresponding to thymocyte-APC duplexes were analyzed with FACSVantage (BD Biosciences, Mountain View, CA) and CellQuest software as previously described (20). For stimulation of thymocytes, appropriate macrophages were pulsed with various peptides for 2 h. Macrophages (2 x 106 cells) were mixed with thymocytes (1 x 106 cells), centrifuged, and warmed to 37°C for 3 min. Cells were gently resuspended and immediately analyzed. The basal level of Ca2+ observed in thymocytes in the presence of unpulsed APCs was calibrated at 200, as arbitrary value. The Ca2+ flux induced by nonstimulatory AV peptide was superimposable on this basal Ca2+ flux and hence should be read as the baseline response.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 x
106 thymocytes are required for Western blot
analysis. Another limitation is that thymocytes should synchronously
receive a defined TCR signal that will induce positive or negative
selection. To follow the induction of the ERK pathway during thymocyte
selection, we established an in vitro system for thymocyte selection.
P14-transgenic mice that express an
H-2b-restricted lymphocytic choriomeningitis
virus-glycoprotein-specific TCR was bred onto a nonselecting
H-2d/d RAG2-deficient background (referred to as
P14 RAG2-/- H-2d/d).
Since thymocytes from these mice were arrested at the DP cell stage,
they were cultured in conditions that promoted either positive or
negative selection. For most experiments, macrophages were used as
APCs; however, similar results were obtained with thymic stromal cell
lines (data not shown). Previous studies using different models have
shown that other cell types, including macrophages, could promote
positive selection (21, 22, 23, 24).
To define conditions that promoted negative selection, P14
RAG2-/- H-2d/d thymocytes
were cultured on H-2b macrophages prepulsed with
either the nominal peptide p33 (KAVYNFATM, 10-6
M) or a strong agonist variant S7A (KAVYNFSTM,
10-6 M). These peptides were previously shown to
induce effective negative selection in FTOC (20, 25, 26).
After various times, thymocytes were harvested and stained with Abs
specific for CD4, CD8, CD69, CD5, and V2. In addition, cell
viability was assessed using propidium iodide and annexin V. When
thymocytes were cultured with p33 or S7A on H-2b
APCs for 20 h, CD4 and CD8 coreceptor down-regulation was observed
in conjunction with an increase in apoptotic cells (Fig. 1
, A and B). These
events have been correlated with negative selection (27).
Under these conditions, thymocytes also rapidly up-regulated the
activation markers CD69 and CD5 (Fig. 1, C and
D), reflecting the intensity of TCR triggering.
|
2m-deficient H-2b
macrophages prepulsed with either the strong agonist peptide p33
(10-7 M) or the weak agonist peptide variant L6F
(KAVYNLATM, 10-6 M). We postulated that
decreasing the level of available peptide-MHC complexes using
2m-deficient macrophages would favor positive
selection by reducing the avidity of the thymocyte-APC interactions.
Both p33 and L6F have been shown to mediate positive selection of
functional thymocytes in FTOC (15, 20). A nonstimulatory
adenovirus peptide, AV (10-6 M), was used as a
control peptide. Upon culture of thymocytes with p33 or L6F on
2m-/- APCs, initial
events associated with positive selection were detected, such as
up-regulation of CD69 (28) and CD5 (29) (Fig. 1
, C and D). Moreover, interactions that favored
positive selection did not dramatically affect cell viability (Fig. 1, A and B) and TCR levels remained high (Fig. 1E). This analysis demonstrates that these culture
conditions are able to induce early events associated with either
positive or negative selection.
It should be noted that in defining the conditions that were most
efficient in promoting either positive or negative selection, a full
titration of all of the peptides (between micromolar to picomolar
concentrations) was done on both
2m+/+ and
2m-/- APCs. A clear
correlation was observed where maximal TCR down-regulation only
correlated with the induction of negative selection, while minimal
down-regulation directly correlated with positive selection.
We next examined whether the thymocytes that had received positively
selecting stimuli could further differentiate into
CD8+ single-positive T cells. P14
RAG2-/- H-2d/d thymocytes
that had received different signals in the monolayer culture system for
16 h were transferred to culture wells containing
deoxyguanosine-treated C57BL/6 (H-2b/b) fetal
thymic lobes. After 60 h of culture, flow cytometric analysis was
performed on thymocytes that repopulated the fetal thymic lobes.
Thymocytes that received positively selecting stimuli in the initial in
vitro culture system (10-7 M
p33/2m-/- or
10-6 M
L6F/2m-/-) were able
to mature into CD8+ T cells, compared with
thymocytes that received "null stimulation" with AV peptide (Fig. 2). On the other hand, thymocytes that
initially received strong negatively selecting stimuli
(10-6 M
p33/2m+/+ or
10-6 M
S7A/2m+/+) were unable
to survive and differentiate in the fetal thymic lobes. This was
demonstrated by the 8- to 10-fold reduction in thymic cellularity and
the presence of only the double-negative thymocyte subset. This
analysis demonstrates that this in vitro system is appropriate for
examining early selection events that promote positive and negative
selection.
|
To investigate the activation of the Ras/Raf/MEK/ERK pathway
during selection events, Western blot analysis was done using an Ab
specific for the phosphorylated activated form of ERK. As shown in Fig. 3A, P14
RAG2-/- H-2d/d thymocytes
cocultured in positively selecting conditions induced sustained ERK
activation. During positive selection with either p33 or L6F, activated
ERK was still detectable after 16 h compared with
"null-selecting" AV peptide (Fig. 3, A and
B). Notably, positive selection with low-avidity
interactions (10-7 M
p33/2m-/- APCs) or
low-affinity peptides (10-6 M
L6F/2m-/- APCs) were
similar, consistent with the affinity/avidity model.
|
2m+/+ APCs. At
various time points, cell lysates were prepared and Western blot
analysis was done using the Ab specific for the phosphorylated forms of
ERK. The activation of ERK reached maximum at 3–4 h and then declined
5–6 h after stimulation (Fig. 4A). A similar transient
kinetics was observed for negative selection that was induced by the
strong agonist ligand S7A (Fig. 4B). Therefore, negative
selection is associated with a strong transient activation of ERK,
while conditions that induce positive selection generate sustained ERK
activation.
|
2m-/- and
2m+/+ APCs that were
prepulsed with positive (10-7 M
p33/2m-/- or
10-6 M
L6F/2m-/-) or negative
(10-6 M
p33/2m+/+ or
10-6 M
S7A/2m+/+) selecting
ligands. Cultures were prepared in parallel and harvested after 3
h. Fig. 4C shows that negatively selecting
conditions induced strong ERK phosphorylation in contrast to weak
phosphorylation triggered by the positively selecting interactions. It
should be noted that in all cases a background level of ERK is seen
(Figs. 3 and 4). Our data suggest that this level is not sufficient for
the induction of positive or negative selection under these defined
culture conditions. Activation of ERK above a certain threshold may
help discriminate between death by neglect and positive vs negative
selection. Negative selection leads to attenuation of TCR signals
In these culture conditions, negative selection has been
correlated with peptides that were able to induce rapid TCR
internalization (Fig. 1E) (20). To examine
whether any remaining or re-expressed TCR were able to trigger other
downstream pathways, we examined the ability of the thymocytes to
mobilize calcium. P14 RAG2-/-
H-2d/d thymocytes were complexed with negatively
or positively selecting ligand/APCs. After 3 min, calcium mobilization
was monitored. Initially, negatively selecting interactions induced a
strong Ca2+ flux, whereas positively selecting
interactions induced a weaker Ca2+ flux (Fig. 5A). After 5 h of
coculture, thymocytes were transferred to plates with fresh APCs pulsed
with high concentration of p33 (10-6 M).
Thymocytes that were cocultured with negatively selecting ligands for
5 h were refractive to further Ca2+ flux on
re-exposure to class I+ APC pulsed with the
strong agonist p33 (Fig. 5B). On the contrary, thymocytes
that were incubated with peptide/MHC ligands that induced positive
selection (10-7 M
p33/2m-/- or
10-6 M
L6F/2m-/-) were able
to generate a Ca2+ flux in response to positively
selecting stimuli or a strong antigenic stimulus (Fig. 5B;
data not shown). These data provide direct evidence that negatively
selecting TCR-peptide-MHC interactions decrease TCR expression to the
extent where the remaining TCRs are unable to trigger other downstream
signal transduction pathways.
|
The next question we wanted to address was whether the transient
ERK activation transmits downstream signals, leading to negative
selection. To address this issue, experiments were done to block the
activation of ERK using the pharmacological inhibitor PD98059. P14
RAG2-/- H-2d/d thymocytes
were preincubated with 25 µM PD98059, a selective compound that has
been shown to inhibit the activation of MEK1/2 by upstream activators
of the MAP kinase cascade (30, 31). These thymocytes were
cultured for 1 h with
2m+/+ APCs that have
been prepulsed with 10-6 M p33. The data show
that 25 µM PD98059 markedly diminishes ERK activity in p33-induced
thymocytes (Fig. 6A).
|
2m+/+ APCs pulsed with
either the nonstimulatory AV (10-6 M) peptide or
the antigenic p33 (10-6 M) peptide. After
16 h of incubation, DP thymocytes cultured under conditions that
promoted negative selection showed a substantial down-regulation of CD4
and CD8 coreceptors, along with enhanced apoptosis (Fig. 6, B and C). In contrast, PD98059 was able to
inhibit clonal deletion by negatively selecting stimuli. Consistent
with the block in ERK activation, flow cytometric analysis showed that
up-regulation of Ras-dependent CD69 expression was also abrogated (Fig. 6D). Examining the P14-transgenic TCR levels confirmed that
the thymocytes received the p33 peptide/MHC signal and internalized the
P14 TCR (Fig. 6E). In addition to PD98059, we also used
U0126, a second, noncompetitive inhibitor of MEK (32). As
shown in Fig. 7, U0126 inhibition of MEK
had the same consequence as inhibition of PD98059 in that deletion of
DP thymocytes was significantly reduced. Together these data
demonstrate that inhibition of MEK activity could inhibit p33-induced
clonal deletion. These data suggest that the MEK/ERK pathway does
generate downstream signaling events necessary to induce negative
selection.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
). We have also shown that the
early signals received during the initial 16-h culture, either
negatively selecting signals or positively signals, were sufficient to
lead to death or promote CD8+ selection after
maturation in deoxyguanosine-treated thymic lobes, respectively (Fig. 2). Although several attempts were made to promote maturation in vitro
in the initial monolayer APC culture system, successful maturation was
only achieved in the thymic microenvironment. This is consistent with
previous studies suggesting that this environment is essential for the
maturation of thymocytes (24, 33).
Although it is possible that maturation of CD8+ T
cells may require extended signals from the class I molecules
encountered in the fetal thymic lobes, the initial culture conditions
definitely provided the appropriate signals to direct positive
selection of these cells. We observed that the nonstimulatory peptide
AV (10-6
M/2m-/-) induced
minimal maturation of CD8+ T cells (6%), whereas
the weak agonist ligand (10-6 M
L6F/2m-/-) or low
concentrations of strong agonist ligand (10-7 M
p33/2m-/-) triggered
maturation of a significant proportion of CD8+ T
cells (27 and 30%, respectively). The ability of weak agonists or low
concentrations of strong ligands to promote positive selection is fully
consistent with an affinity/avidity model for selection. The
inefficient maturation of nonstimulatory peptide AV-induced thymocytes
in C57BL/6 FTOC may appear peculiar at first, since P14 TCR-transgenic
T cells are effectively selected in this genetic background. However, a
60-h culture in the FTOC may not be sufficient for maturation via
interactions with natural ligands. Cumulative nuclear signals may be
required for positive selection and, as such, thymocytes that were
induced with positive-selecting ligands in the initial monolayer
culture would have acquired a temporal advantage. Accordingly, our data
demonstrate that thymocytes cultured in the positively selecting
conditions acquire active ERK molecules over a sustained period
compared with thymocytes cultured in null-selecting conditions.
This study demonstrates that exposure to either strong agonist peptide,
p33 or S7A, induces intracellular events that trigger negative
selection. Evidence for apoptosis was detected after 16–20 h in the
initial culture, and further culture in the thymic microenvironment
demonstrated that no detectable DP or single-positive cells repopulated
the thymic lobes (Fig. 2). Interactions with relatively higher
concentrations of strong agonist ligands led to extensive TCR
internalization (Fig. 1E), to a degree where remaining TCRs
were unable to transmit further signals, as measured by the
mobilization of intracellular calcium (Fig. 5). Biochemical analysis
revealed that under conditions that induced negative selection, ERK
phosphorylation was much stronger compared with positively selecting
conditions (Fig. 4C). In addition, the strong ERK activation
is transient as it declines after 5 h (Fig. 4A).
Does this strong ERK activity observed during the initial TCR
triggering under negatively selecting conditions contribute to the
induction of clonal deletion? This contention is supported by studies
that have shown that reducing the intensity of ERK signals may shift
negative selection to positive selection (14, 15). In
addition, several studies have now shown that negative selection is
correlated with a transient ERK signal (10, 13). However,
the latter studies do not explicitly address whether the transient ERK
activation has any biological significance for clonal deletion. Using
our novel in vitro culture system, we investigated the requirement of
ERK activation during negative selection by abrogating the initial ERK
activity in thymocytes by preincubating them with either of two
independent MEK inhibitors, PD98059 or U0126. These two MEK inhibitors
used in this study do not block JNK or p38 activation
(14). In addition, this protocol selectively inhibits MEK
only in thymocytes and obviates the concerns surrounding the effects of
the drugs on APC function during negative selection. Our studies
demonstrate that abrogating the ERK activity in thymocytes blocked the
rapid clonal deletion in response to strong negatively selecting
stimuli (Figs. 6 and 7).
Previously, using the P14 TCR
2m-/- transgenic
system in FTOC, we have shown that peptide-mediated negatively
selecting stimuli could be converted into positively selecting signals
by diminishing MEK-mediated ERK activity with the use of PD98059
(15). In these FTOC assays, the switch from negative to
positive selection was observed at p33 peptide concentrations closer to
the negative/positive selection thresholds. In this previous model, we
showed that the MEK inhibitor could reduce, but not completely block,
ERK activation. This is in contrast to this report where ERK activation
is ablated. PD98059 may not effectively reduce ERK activity and block
negative selection in FTOC for a variety of reasons. In FTOC, the lobes
are not completely submersed in media. Therefore, the ability of the
drug to permeabilize each cell at the same concentration is very
unlikely. It is likely that a gradient is achieved where the levels of
active ERK is reduced and not completely eliminated. Also in FTOC, it
is unlikely that all thymocytes are synchronously receiving the
negative selection signal. As the activity of PD98059 declines in
culture, some thymocytes may receive a slightly reduced ERK signal and
become positively selected. Nonetheless, these studies clearly show a
role for ERK during negative selection and support a model where
negative selection signals are stronger than positive selection
signals.
How does ERK signaling contribute to thymocyte-negative selection? One possible downstream target gene that is regulated by ERK signaling is Id3. Id gene products lack DNA-binding domains, but exert their transcriptional influence by dimerizing with E proteins and thereby, disrupting E protein activity (34). In this manner, Id gene products enhance TCR-mediated responses by attenuating E protein activity. Recent data from Bain et al. (35) have positioned ERK MAP kinase signaling and Id3 gene regulation in a common pathway . They also show that PD98059 inhibits Id3 gene induction in a dose-dependent manner. Interestingly, previous studies have shown that Id3 deficiency can perturb both positive and negative selection (36). In addition to Id3, up-regulation of Nur77 has also been shown to be sensitive to PD98059-mediated ERK inhibition (13). Nur77 is an orphan member of the steroid nuclear superfamily and is rapidly induced by TCR-mediated signaling in immature thymocytes (37, 38). Transgenic mice that overexpress dominant-negative forms of Nur77 show protection from thymocyte apoptosis (39, 40), whereas overexpression of the wild-type Nur77 facilitated apoptosis (39, 41). These studies suggest that the ERK-MAP kinase module regulates genes that are involved in both thymocyte-positive and -negative selection. In this instance, the outcome of the thymocyte fate may be determined by the extent and/or timing of activation of ERK-sensitive nuclear genes.
Therefore, our data favor a model in which a strong but transient ERK
activation does play a role in negative selection (Fig. 8). This ERK signal along with signals
from other pathways may lead to the induction or inactivation of a
subset of genes that lead to cell death. Conversely, an alternative
scenario exists where the lack of sustained ERK activation during
negative selection may not afford sufficient protection for thymocytes
from JNK or p38-mediated apoptosis. Several studies have shown that JNK
and p38 are involved in negative selection (16, 17, 18, 19), and
there is evidence in other biological models where it has been shown
that the dynamic balance between growth-activated ERK and
stress-activated JNK-p38 pathways is important in determining whether a
cell survives or undergoes apoptosis (42). On the other
hand, positively selecting low-affinity/avidity ligands may achieve
sustained signaling by their inability to induce maximal TCR
internalization and the TCR complexes that remain on the surface are
able to relay continuous signals for survival and differentiation. This
is consistent with observations from other models that show that
multiple interactions are required to promote thymocyte differentiation
(43, 44). The current literature supports the role of ERK
in positive selection (5, 6, 7, 8, 9, 11, 12) and other reports
show that sustained ERK signals are observed during positive selection
(10, 13).
|
).
On the contrary, epidermal growth factor (EGF)-driven proliferation of
PC12 cells is coupled with rapid receptor internalization and transient
ERK activation. Similarly, thymocytes undergoing negative selection
rapidly internalize their TCRs and become refractive to further
stimulation. Intracellular signals such as ERK and calcium flux are
transient (Figs. 1, 4, and 5). It should be noted that other mechanisms
may also blunt any sustained ERK or Ca2+
activities in response to strong, negatively selecting stimulation in
DP thymocytes. In addition, negatively selecting ligands can stimulate
a robust proliferation in mature T cells in analogy with EGF-driven
proliferation in the PC12 model (20). Interestingly, if
PC12 cells are engineered to overexpress EGF receptors, there is
prolonged ERK and, as a result, cells differentiate but do not
proliferate in response to EGF stimulation (46).
Sustained ERK activation also promotes differentiation in other models.
Protein kinase C-mediated sustained ERK activation has been
shown to induce megakaryocyte differentiation in K562 cells. In this
system,12-O-tetradecanoylphorbol-13-acetate (TPA) and
bryostatin are known to activate protein kinase C but paradoxically
have opposing effects on megakaryocyte differentiation. TPA, a
differentiation inducer, caused sustained ERK activation (>24 h),
whereas bryostatin, a differentiation blocker, only transiently
activated ERK (6 h) and attenuated the activation of ERK by TPA
(47, 48).
Intriguingly, in several models including thymocyte selection, cell fate determination is influenced by the dynamics of receptor engagement and signaling. Further experiments are necessary to elucidate the interplay of MAPK pathways and the induction or inhibition of genes that are necessary to induce positive or negative thymocyte selection.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Pamela S. Ohashi, Ontario Cancer Institute, 610 University Avenue, 8-327, Toronto, Ontario M5G 2 M9, Canada. E-mail address: pohashi{at}uhnres.utoronto.ca
3 Abbreviations used in this paper: DP, double positive; MAP, mitogen-activated protein; MEK, MAP kinase kinase; ERK, extracellular signal-regulated kinase; RAG, recombination activating gene; FTOC, fetal thymic organ culture; 2m, 2-microglobulin; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate.
Received for publication April 23, 2001. Accepted for publication August 30, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T-cell receptor controls positive selection by modulating ERK activity. Nature 406:422.[Medline]
couples T-cell receptor signalling to ERK activation and thymocyte positive selection. Nature 406:426.[Medline]
deficiency arrests development of the but not the 
T cell lineage. EMBO J. 16:1360.[Medline]
This article has been cited by other articles:
|
|
 |
|
  N. K. Y. Wong, J. C. Y. Lai, D. Birkenhead, A. S. Shaw, and P. Johnson CD45 Down-Regulates Lck-Mediated CD44 Signaling and Modulates Actin Rearrangement in T Cells J. Immunol., November 15, 2008; 181(10): 7033 - 7043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Morley, K. S. Weber, H. Kao, and P. M. Allen Protein Kinase C-{theta} Is Required for Efficient Positive Selection J. Immunol., October 1, 2008; 181(7): 4696 - 4708. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Guo, C.-K. Wan, J. H. Carpenter, T. Mousallem, R.-M. N. Boustany, C.-T. Kuan, A. W. Burks, and X.-P. Zhong Synergistic control of T cell development and tumor suppression by diacylglycerol kinase {alpha} and {zeta} PNAS, August 19, 2008; 105(33): 11909 - 11914. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Fujii, S. Matsuda, G. Takayama, and S. Koyasu ERK5 is involved in TCR-induced apoptosis through the modification of Nur77. Genes Cells, May 1, 2008; 13(5): 411 - 419. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Gong, R. Zhang, J. Zhang, L. Xu, F. Zhang, W. Xu, Y. Wang, Y. Chu, and S. Xiong {alpha}-Dystroglycan is involved in positive selection of thymocytes by participating in immunological synapse formation FASEB J, May 1, 2008; 22(5): 1426 - 1439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Baldwin and K. A. Hogquist Transcriptional Analysis of Clonal Deletion In Vivo J. Immunol., July 15, 2007; 179(2): 837 - 844. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamasaki, E. Ishikawa, M. Sakuma, O. Kanagawa, A. M. Cheng, B. Malissen, and T. Saito LAT and NTAL Mediate Immunoglobulin E-Induced Sustained Extracellular Signal-Regulated Kinase Activation Critical for Mast Cell Survival Mol. Cell. Biol., June 15, 2007; 27(12): 4406 - 4415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Y. Lee, Y. I. Choi, J. Kim, J. W. Choi, D. H. Sohn, C. Lee, S. H. Jeon, and R. H. Seong Down-Regulation of the SWI/SNF Chromatin Remodeling Activity by TCR Signaling Is Required for Proper Thymocyte Maturation J. Immunol., June 1, 2007; 178(11): 7088 - 7096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Kemp, L. Wille, C. L. Lewis, L. B. Nicholson, and D. A. Lauffenburger Quantitative Network Signal Combinations Downstream of TCR Activation Can Predict IL-2 Production Response J. Immunol., April 15, 2007; 178(8): 4984 - 4992. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. Graham, M. P. Bell, C. J. Huntoon, M. D. Griffin, X. Tai, A. Singer, and D. J. McKean CD28 Ligation Costimulates Cell Death but Not Maturation of Double-Positive Thymocytes due to Defective ERK MAPK Signaling J. Immunol., November 1, 2006; 177(9): 6098 - 6107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. J. Priatel, X. Chen, S. Dhanji, N. Abraham, and H.-S. Teh RasGRP1 Transmits Prodifferentiation TCR Signaling That Is Crucial for CD4 T Cell Development J. Immunol., August 1, 2006; 177(3): 1470 - 1480. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Clements, S. A. John, and L. A. Garrett-Sinha Impaired Generation of CD8+ Thymocytes in Ets-1-Deficient Mice J. Immunol., July 15, 2006; 177(2): 905 - 912. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. E. Keir, Y. E. Latchman, G. J. Freeman, and A. H. Sharpe Programmed Death-1 (PD-1):PD-Ligand 1 Interactions Inhibit TCR-Mediated Positive Selection of Thymocytes J. Immunol., December 1, 2005; 175(11): 7372 - 7379. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. K. McNeil, T. K. Starr, and K. A. Hogquist A requirement for sustained ERK signaling during thymocyte positive selection in vivo PNAS, September 20, 2005; 102(38): 13574 - 13579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Kortum, D. L. Costanzo, J. Haferbier, S. J. Schreiner, G. L. Razidlo, M.-H. Wu, D. J. Volle, T. Mori, H. Sakaue, N. V. Chaika, et al. The Molecular Scaffold Kinase Suppressor of Ras 1 (KSR1) Regulates Adipogenesis Mol. Cell. Biol., September 1, 2005; 25(17): 7592 - 7604. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nekrasova, C. Shive, Y. Gao, K. Kawamura, R. Guardia, G. Landreth, and T. G. Forsthuber ERK1-Deficient Mice Show Normal T Cell Effector Function and Are Highly Susceptible to Experimental Autoimmune Encephalomyelitis J. Immunol., August 15, 2005; 175(4): 2374 - 2380. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Auger, I. Motta, K. Benihoud, D. M. Ojcius, and J. M. Kanellopoulos A Role for Mitogen-activated Protein KinaseErk1/2 Activation and Non-selective Pore Formation in P2X7 Receptor-mediated Thymocyte Death J. Biol. Chem., July 29, 2005; 280(30): 28142 - 28151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Sommers, J. Lee, K. L. Steiner, J. M. Gurson, C. L. DePersis, D. El-Khoury, C. L. Fuller, E. W. Shores, P. E. Love, and L. E. Samelson Mutation of the phospholipase C-{gamma}1-binding site of LAT affects both positive and negative thymocyte selection J. Exp. Med., April 4, 2005; 201(7): 1125 - 1134. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Fisher, M. Nagarkatti, and P. S. Nagarkatti 2,3,7,8-Tetrachlorodibenzo-p-dioxin Enhances Negative Selection of T Cells in the Thymus but Allows Autoreactive T Cells to Escape Deletion and Migrate to the Periphery Mol. Pharmacol., January 1, 2005; 67(1): 327 - 335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kwon, H.-S. Jun, L.-Y. Khil, and J.-W. Yoon Role of CTLA-4 in the Activation of Single- and Double-Positive Thymocytes J. Immunol., December 1, 2004; 173(11): 6645 - 6653. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tsukamoto, A. Irie, and Y. Nishimura B-Raf Contributes to Sustained Extracellular Signal-regulated Kinase Activation Associated with Interleukin-2 Production Stimulated through the T Cell Receptor J. Biol. Chem., November 12, 2004; 279(46): 48457 - 48465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Sinnett-Smith, E. Zhukova, N. Hsieh, X. Jiang, and E. Rozengurt Protein Kinase D Potentiates DNA Synthesis Induced by Gq-coupled Receptors by Increasing the Duration of ERK Signaling in Swiss 3T3 Cells J. Biol. Chem., April 16, 2004; 279(16): 16883 - 16893. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yamasaki, E. Ishikawa, M. Kohno, and T. Saito The quantity and duration of FcR{gamma} signals determine mast cell degranulation and survival Blood, April 15, 2004; 103(8): 3093 - 3101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Hare, J. Pongracz, E. J. Jenkinson, and G. Anderson Modeling TCR Signaling Complex Formation in Positive Selection J. Immunol., September 15, 2003; 171(6): 2825 - 2831. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Ariel, N. Chiang, M. Arita, N. A. Petasis, and C. N. Serhan Aspirin-Triggered Lipoxin A4 and B4 Analogs Block Extracellular Signal-Regulated Kinase-Dependent TNF-{alpha} Secretion from Human T Cells J. Immunol., June 15, 2003; 170(12): 6266 - 6272. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Werlen, B. Hausmann, D. Naeher, and E. Palmer Signaling Life and Death in the Thymus: Timing Is Everything Science, March 21, 2003; 299(5614): 1859 - 1863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. T. Donlin, C. A. Roman, M. Adlam, A. G. Regelmann, and K. Alexandropoulos Defective Thymocyte Maturation by Transgenic Expression of a Truncated Form of the T Lymphocyte Adapter Molecule and Fyn Substrate, Sin J. Immunol., December 15, 2002; 169(12): 6900 - 6909. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-W. He Orphan nuclear receptors in T lymphocyte development J. Leukoc. Biol., September 1, 2002; 72(3): 440 - 446. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
