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*
Department of Immunology, University of Toronto, Toronto, Ontario, Canada; and
Howard Hughes Medical Institute and Rockefeller University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IL-4 secretion whether the costimulatory signal was
delivered via 4-1BB or CD28. In contrast, following differentiation
into Th1 or Th2 cells, p38 inhibition blocked IL-2 and IFN- without
affecting IL-4 secretion. Nevertheless, IL-4 secretion by Th2 cells
remained costimulation-dependent. Thus, critical T cell signaling
events diverge following Th1 vs Th2
differentiation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4-1BB is expressed on activated CD4+ and CD8+ T cells (5). 4-1BBL is expressed on activated APC including B cells, macrophages and dendritic cells (6, 7). Several studies have demonstrated a role for 4-1BB in T cell activation using either transfected ligand, anti-4-1BB Abs, or blocking studies with a soluble form of 4-1BB (5). The extracellular domain of 4-1BBL (soluble 4-1BBL; s4-1BBL), when immobilized together with anti-CD3, is a potent activator of resting T cells from both CD28+ and CD28- mice, resulting in proliferation and IL-2 secretion (8). Studies using anti-4-1BB Abs have demonstrated higher levels of proliferation and rescue from cell death of CD8+ compared with CD4+ T cells (9, 10). Moreover, ligation of 4-1BB by anti-4-1BB or by 4-1BBL promotes the development of CTL activity and anti-tumor immunity (9, 11, 12, 13, 14). In addition to its effects on CD8+ T cells, 4-1BB has also been shown to augment CD4+ T cell responses (15, 16, 17, 18) and to play a role in sustaining Th1 responses after down-modulation of CD28 (17).
A role for 4-1BB/4-1BBL in the immune response has been substantiated using 4-1BBL-/- mice (14, 19, 20). 4-1BBL-/- mice show an impaired CTL response to influenza virus (14) but can clear lymphocytic choriomeningitis virus as effectively as wild-type mice (14, 19). However, 4-1BBL can play a role in the development of a lymphocytic choriomeningitis virus-specific CD8 T cell response under suboptimal conditions of antigenic stimulation (14, 20). Both CD28-/- and 4-1BBL-/- mice reject skin allografts as effectively as wild-type mice, whereas doubly deficient CD28-/-4-1BBL-/- mice show a delay in skin allograft rejection (14). Thus, the accumulating evidence suggests a role for 4-1BB/4-1BBL in augmenting and sustaining suboptimal immune responses.
The molecular mechanisms by which 4-1BB can provide costimulatory
signals in the absence of CD28 are beginning to emerge. Members of the
TNFR family signal via TNFR-associated factors (TRAFs) that act as
adapters to downstream signaling events (21).
Costimulatory members of the TNFR family have in common the ability to
activate stress-activated protein kinases (SAPK) otherwise known as
c-Jun N-terminal kinase (JNK) and NF-
B (4). CD28
costimulation also involves JNK/SAPK and NF-B activation
(22, 23, 24, 25, 26). TRAF2 deficient mice
(TRAF2-/-) and mice expressing a
dominant-negative form of TRAF2 (TRAF2DN) revealed that TRAF2 is
necessary for JNK/SAPK but not for NF-B activation following TNF-
or CD40 aggregation (27, 28). 4-1BB aggregation induces
TRAF1 and 2 recruitment (8, 29, 30) resulting in its
interaction with and activation of apoptosis-signal-regulating kinase 1
(ASK1; Ref. 31). ASK1 can activate the JNK/SAPK cascade,
whereas a dominant-negative ASK1 interferes with 4-1BB mediated
costimulation and IL-2 production. Thus, ASK1 completes the link
between TNFR family members, TNFR and 4-1BB, with TRAF2 and the
JNK/SAPK pathway (31, 32, 33, 34).
p38 mitogen-activated protein kinase (MAPK), like JNK/SAPK, is also activated in response to cellular stress and by members of the TNFR family (35). Several groups have demonstrated an essential role for p38 MAPK in CD28-mediated costimulation (36, 37, 38, 39, 40, 41). p38 MAPK is important for cytokine production (42) as well as in the response to cytokines (43, 44, 45). In view of the ability of 4-1BB to replace CD28 costimulatory signals for cytokine production as well as the ability of CD28 to induce p38 MAPK activation, we examined the role of p38 MAPK in 4-1BB-mediated costimulation. Here we report that p38 MAPK is essential for 4-1BB-dependent cytokine production by primary T cells. Furthermore, TRAF2 is required for 4-1BB-dependent p38 MAPK activation, and p38 MAPK activation can occur via TRAF2 recruitment and activation of ASK1. We also demonstrate that p38 MAPK activation during 4-1BB- or CD28-dependent responses is essential for the development of both Th1 and Th2 cells. However, p38 MAPK activation is not required for IL-4 production by committed Th2 effector cells, although the response of these cells remains costimulation dependent. Thus the intracellular signals required for cytokine production diverge following T cell differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD28- mice backcrossed on to the H-2b background (n = 10) were obtained from Dr. T. W. Mak (1). TRAF2DN transgenic mice have been described (28). C57BL/6 and BALB/c mice were obtained from Charles River Laboratories (St. Constant, Quebec, Canada) and used at 8–12 wk of age. All mouse studies were approved by the University of Toronto animal care committee.
The autoreactive T cell hybrid, C8.A3, obtained from Dr. L. Glimcher (Harvard Medical School, Boston, MA) responds to Ak plus an unidentified peptide expressed on B lymphomas. Although C8.A3 cells can respond to anti-CD3 alone, the response of these T cells to MHC/peptide requires costimulation (15, 46). The BALB/c B cell lymphomas M124.1 and K46J were originally described by Kim et al. (47). K46J73.35 is an Ak transfectant of K46J (48). K46J lymphomas constitutively expresse high levels of 4-1BBL and low levels of CD80 and CD86 (15). In this report, we identified a variant of the BALB/c B lymphoma M12.4.1, which when treated overnight with cAMP to up-regulate costimulatory molecules, expressed low levels of 4-1BBL but moderate levels of CD80 and high levels of CD86.
The CT.4S, IL-4-dependent cell line, described by Li-Hu et al.
(49) was provided by Dr. G. Mills (M.D. Anderson Cancer
Center, Houston, TX). The anti-CD3-producing hybridoma 145-2C11
(50) was provided by Dr. J. Bluestone (University of
Chicago, IL). The 12CA5-producing hybridoma (51) that
secretes an Ab to influenza hemaglutinin (HA) epitope tag was obtained
from Dr. B. Phillips (University of Toronto, Toronto, Ontario, Canada).
The hybridomas N418 (anti-CD11c), Y-3P
(anti-Ab), MKD6
(anti-Ad), RA3-6B2 (anti-B220), TIB-128
(anti-MAC-1), M1/69 (anti-heat stable Ag), RG7/7.6H2
(anti-rat Ig -chain), GL-1 (anti-B7-2), S4B6
(anti-IL-2), and 11B11 (anti-IL-4) and the IL-2 dependent line
CTLL were obtained from the American Type Culture Collection (Manassas,
VA). The hybridoma line YN-1, secreting an ICAM-1 specific rat IgG Ab
was kindly provided by Dr. F. Takei (University of British Columbia,
Vancouver, British Columbia, Canada). The anti-CD28-secreting
hybridoma 37.51.1 (52) was provided by Dr. J. Allison
(University of California, Berkeley, CA). The anti-B7-1 hybridoma
16.10A1 (53) was provided by Dr. H. Reiser (Dana Faber
Cancer Institute, Boston, MA). A cell line producing CTLA4.Ig was
provided by Dr. P. Lane (University of Birmingham, Birmingham, U.K.).
Cells were maintained in RPMI 1640 containing 10% FCS (Cansera,
Rexdale, Ontario, Canada), 50 µM 2-ME, MEM nonessential amino acids
(Life Technologies, Gaithersburg, MD), antibiotics, pyruvate, and
glutamine as previously described (54).
CTLA4.Ig and the Abs described above were purified from hybridoma supernatants using protein G- or protein A-Sepharose (Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. 3T3 cells secreting 4-1BB linked to alkaline phosphatase (AP) were provided by Dr. B. Kwon (Indiana University, Indianapolis, IN). 4-1BB.AP was purified on anti-AP-Sepharose as previously described (55). AP from human placenta was obtained from Sigma (St. Louis, MO). The generation and purification of the soluble 4-1BB ligand (s4-1BBL) was previously described (8).
Anti-4-1BB (1AH2) was purchased from PharMingen (San Diego, CA). Anti-p38 was purchased from New England Biolabs (Beverly, MA). Anti-JNK1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit Ig HRP, goat anti-mouse Ig HRP, sorbitol, dibutyryl cAMP, and rat Ig were purchased from Sigma. The p38 inhibitor SB203580-Iodo was purchased from Calbiochem-Novabiochem (La Jolla, CA) and was dissolved in 0.1% DMSO.
Lymphocyte isolation
For T cell isolation, APC were depleted from spleen cell suspensions in HBSS (Life Technologies)/2.5% FCS/50 µM 2-ME, with a cocktail of Abs, anti-MHC class II, anti-B220, anti-heat stable Ag (M1/69), anti-MAC-1, and anti-CD11c (N418) each at a final concentration of 10 µg/ml at 4°C for 30 min. A 1:10 dilution of baby rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) was added and the cultures were incubated at 37°C for 40 min. To remove adherent cells, the cell suspensions were passed through a Sephadex G10/nylon wool column and then centrifuged through Percoll gradients consisting of 60, 70, and 80% Percoll layers. Small (high density) resting T cells were isolated from the 70/80% interface and used for subsequent experiments.
Mixed lymphocyte reactions
T cells were isolated from the spleen of CD28+ and CD28- mice using CD4 columns from Cytovax Biotechnologies (Edmonton, Alberta, Canada). Purity of the populations was assessed by flow cytometry and purity was found to be at least 88%. Stimulator B lymphomas were irradiated (10,200 rad) to prevent their proliferation. Before irradiation, M12.4.1 cells were treated overnight with dibutyryl-cAMP at a final concentration of 300 µM to induce B7 family molecules as previously described (16, 46). Primary MLR cultures were set up with 1 x 106 CD4+ T cells and 5 x 105 B lymphomas in a total volume of 1.5 ml for 5 days using 24-well plates. After 3 days, cultures were fed by removing 250 µl of supernatant, which was replaced with an equal volume of medium. After a total of 5 days of incubation, 1.0 ml supernatant from each culture was removed and frozen immediately at -70°C. For the inhibition of cytokine production, MLR cultures were stimulated as described above with the addition of soluble reagents (16).
For establishment of Th1 and Th2 effectors, an MLR was set up as described above in the presence of either IL-12 (0.5% v/v) plus anti-IL-4 (10 µg/ml) or IL-4 (0.5% v/v) plus anti-IL-2 (10 µg/ml) for Th1 vs Th2 differentiation, respectively. The generation and purification of rIL-12 and rIL-4 was previously described (16). On day 5, 1 ml of supernatant was removed and the cytokine profiles of the T cells evaluated. Cultures were replenished with 1 ml of fresh medium. Five days later, cultures were harvested and enriched for viable cells using Lympholyte-M (Cedarlane Laboratories). Recovered cells were restimulated with fresh APC as described above for the primary MLR.
Cytokine assays
IL-2 was detected using the indicator cell line CTLL and IL-4
was detected with CT.4S, as described (16). Previous
experiments have established the specificity of these cell lines for
the respective cytokines (16). Serial dilutions of the
supernatant were prepared in triplicate and 1 x
104 indicator cells were cultured for 24 h
in 100 µl in 96-well plates. During the final 8 h, the cells
were labeled with [3H]thymidine (Amersham Life
Science, Oakville, Ontario, Canada). Cultures were processed using the
Top Count 96-well harvester and analyzed on the Top Count 96-well
liquid scintillation counter (Canberra-Packard, Meriden, CT).
Supernatant levels of IFN- and IL-4 were measured by ELISA using the
pair of anti-murine IFN- mAbs or anti-murine IL-4 mAbs
purchased from PharMingen. ELISA was performed according to the
manufacturer’s instructions using the diluted supernatant.
T cell stimulation
For stimulation of the T cell hybrid, C8.A3, monoclonal anti-CD3 (145-2C11) was immobilized on the surface of a 96-well plate (Nunc, Gaithersburg, MD) by incubation overnight at 4°C. C8.A3 T cells (1 x 105) were stimulated overnight on wells that had been precoated overnight with 0.1 µg/ml anti-CD3 or were stimulated with 1 x 105 irradiated B cell lymphomas K46J or K46J 73.35. For the stimulation of primary resting T cells, monoclonal anti-CD3 (145-2C11) was immobilized on the surface of 96-well plates in the presence or absence of either immobilized s4-1BBL or anti-CD28.
Expression vectors and transfections
pcDNA3-HA-ASK1 and pcDNA3-HA-ASK1 K709-E have been previously described (56). C8.A3 T cells were transfected as previously described (31). Plasmids encoding GST-ATF2 and GST-cJun 5–89(5–89) bacterial fusion proteins were provided by Dr. J. Woodgett (University of Toronto). The generation of fusion proteins were previously described (31).
Kinase assays
Protein kinase assays were performed as previously described
(31). C8.A3 T cells or primary murine T cells were
stimulated and the lysates were immunoprecipitated with either
anti-p38 or anti-JNK1 (for the p38 assay) or with anti-HA
(ASK1 assay; 2 h rotating at 4°C). Immune complexes were
collected on protein A-Sepharose beads (2 h rotating at 4°C). Immune
complexes were washed three times with PBS-Triton X-100. Kinase assays
were performed in 20 µl of kinase buffer (10 mM
MgCl2, 50 mM Tris-HCl (pH 7.5), 1 mM EGTA (pH
7.5)) in the presence of 1.2 µCi [32P]-ATP
(Amersham Life Science, Oakville, ON) and 2 µg of either GST-c-Jun
(5–89) or GST-ATF2 as the in vitro substrate (30°C for
30 min). The reaction was stopped by the addition of 2x SDS sample
buffer. Phosphoproteins were separated by SDS-PAGE and visualized by
autoradiography.
Western blot
T cells were stimulated and lysed as previously described. Lysates were immunoprecipitated with either anti-p38, anti-JNK1, or anti-HA Ab and protein-A Sepharose. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bedford, MA). p38 proteins were detected by anti-p38 Ab, JNK was detected by anti-JNK1 Ab, and ASK1 was detected with anti-HA Ab. Bound Abs were detected with Ig HRP and detected by chemiluminescence according to the manufacturer’s protocol enhanced chemiluminescence system (Amersham Life Science).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the requirement for activation of the p38 MAPK
during 4-1BB mediated costimulation, we used a T cell hybridoma, C8.A3,
previously shown to respond to 4-1BB signaling. C8.A3 T cells express
low levels of 4-1BB constitutively and up-regulate 4-1BB expression
following TCR ligation (15). Previous results have shown
that engagement of 4-1BB by Ab without aggregation fails to induce
TRAF2 recruitment (8). Thus, C8.A3 cells were stimulated
with anti-4-1BB followed by secondary cross-linking in all
experiments. As a positive control for p38 MAPK activation, T cells
were treated with sorbitol to induce osmotic shock. Fig. 1
shows that phosphorylated GST-ATF2,
indicative of p38 MAPK activation, could be detected following
treatment of C8.A3 cells with sorbitol or following 4-1BB ligation.
Phosphorylation of GST-ATF2 was significantly reduced when T cells were
stimulated in the presence of increasing concentrations of the specific
p38 inhibitor, SB203580 (57, 58) (Fig. 1). C8.A3 T cells
were also stimulated with anti-ICAM plus secondary Ab as a negative
control, and no detectable p38 MAPK activation was observed (data not
shown). At concentrations 
10 µM, SB203580 may lose its selectivity
and also blocks JNK/SAPK activation (59). However, Fig. 1
shows that under the same conditions of T cell activation used for the
p38 MAPK assay, JNK activity, as determined by an in vitro kinase
assay, remained unaffected by SB203580.
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B). When stimulated with anti-CD3, the C8.A3 T cell
response is costimulation independent. In contrast, C8.A3 T cells are
costimulation dependent when stimulated by Ag/MHC on an APC
(15). C8.A3 T cells were stimulated with either
immobilized anti-CD3 or with an APC (K46J73.75) expressing
transfected Ak. This APC expresses high levels of
4-1BBL, but little or no B7 molecules (15). The parental
cell line (K46J), lacking Ak was used as a
negative control (data not shown). Fig. 1B demonstrates that
SB203580 inhibited IL-2 secretion in response to anti-CD3 as well
as in response to stimulation with APC expressing Ag/MHC and 4-1BBL.
Thus, p38 MAPK is required for 4-1BB/4-1BBL-dependent T cell
activation.
To verify that the results obtained for the T cell hybridoma were valid
for primary T cells, resting T cells were isolated as described in
Materials and Methods. Primary T cells do not express 4-1BB
constitutively and respond poorly to anti-4-1BB Ab unless treated
first with anti-CD3 to induce 4-1BB expression (17).
In contrast to the result with anti-4-1BB Ab, we have shown that
primary T cells respond without prior stimulation when stimulated with
immobilized anti-CD3 together with immobilized soluble 4-1BBL
(8). Therefore, for experiments using primary T cells we
used s4-1BBL immobilized on plastic to signal via 4-1BB. Fig. 2A demonstrates that
phosphorylated GST-ATF2 could be detected following T cell treatment
with sorbitol or anti-CD3, but not with immobilizeds4-1BBL.
However, anti-CD3 together with s4-1BBL resulted in greater
phosphorylation of GST-ATF2 than observed with anti-CD3 alone. As a
control for the specificity of the interaction, the T cells were also
stimulated with anti-CD3 plus 4-1BBL in the presence of a soluble
form of 4-1BB (4-1BB.AP) to block the interaction of 4-1BB with its
ligand. Fig. 2B shows a decline in p38 MAPK activation
following the addition of the soluble receptor, 4-1BB.AP, but not after
addition of control AP. Stimulation of primary T cells in the presence
of 0–20 µM SB203580 resulted in a significant decrease in p38 MAPK
activation while JNK1 activity remained unaffected (Fig. 2C).
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), but no detectable IFN-
(not shown). The p38 inhibitor was found to inhibit this
4-1BB-dependent cytokine secretion confirming the result with the T
cell hybridomas (Fig. 3). The above data indicate that 4-1BB
aggregation on the surface of a T cell hybridoma or on primary T cells
leads to p38 MAPK activation and that this activation is required for
4-1BB-dependent cytokine production.
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Previous studies have shown that the MAPK kinase kinase (MAPKKK)
ASK1 can provide a link between TRAF2 and the JNK/SAPK cascade
(31, 33, 34). To investigate whether 4-1BB-mediated
costimulation also results in ASK1-dependent activation of p38 MAPK,
C8.A3 T cells were transfected with ASK1 or a dominant-negative variant
(ASK1 K709E). Following stimulation of the transfected C8.A3 T cells,
p38 was immunoprecipitated and the immune complexes were subject to an
in vitro kinase assay with GST-ATF2 as the substrate. The mock
transfectant demonstrates that an endogenous MAPKKK is sufficient to
activate p38 MAPK, although there was an increase in p38 MAPK
activation when ASK1 was overexpressed. RT-PCR analysis and Western
blot analysis demonstrate that ASK1 is present in C8.A3 T cells (Ref.
31 and data not shown). Overexpression of ASK1 K709E
resulted in a significant decrease in p38 MAPK activity (Fig. 4). These data suggest that following
4-1BB aggregation, recruitment (31) and activation of ASK1
can lead to p38 MAPK activation.
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Transient transfection systems using overexpressed epitope-tagged
proteins have revealed that ASK1 can interact with TRAF2, TRAF5, and
TRAF6 resulting in JNK/SAPK activation (33, 34).
TRAF2-/- and TRAF2DN mice have revealed that
TRAF2 is essential for JNK/SAPK activation in response to TNF, CD40
(27, 28), or 4-1BB (31). To determine whether
TRAF2 is required for activation of the p38 MAPK pathway we used
lymphocytes from TRAF2DN mice. Phosphorylation of the p38 MAPK
substrate GST-ATF2 was detected in an in vitro kinase assay following T
cell treatment with either anti-CD3, anti-CD28 or sorbitol for
both wild-type and TRAF2DN T cells (Fig. 5). In contrast, T cells from TRAF2DN
mice fail to activate p38 MAPK following stimulation with anti-CD3
plus s4-1BBL (Fig. 5), whereas the wild-type mice retained
4-1BB-dependent p38 MAPK activation. To verify the necessity of TRAF2
for 4-1BB-mediated cytokine production, resting T cells from control
and TRAF2DN mice were stimulated with immobilized Abs. TRAF2DN T cells
failed to secrete IL-2 or IL-4 in response to anti-CD3 plus
s4-1BBL, but retained their ability to secrete cytokines in response to
anti-CD3 plus anti-CD28 (data not shown). These data imply that
TRAF2 is required for p38 MAPK activation in response to costimulation
via 4-1BB. Fig. 4 also shows that anti-CD28 alone induced p38 MAPK
activation, confirming other recent reports (37, 38, 39, 60).
We did not detect a synergistic activation of p38 MAPK following
anti-CD3 plus anti-CD28 stimulation as previously reported for
murine T cells (36, 38), although the latter experiments
evaluated this synergy at much earlier time points than were
examined here.
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In response to pathogens, naive CD4+ T cells
can differentiate into Th1 and Th2 effector cells. Th1 cells produce
IFN- thereby promoting cell-mediated immunity, whereas Th2 cells
secrete IL-4, IL-5, and IL-10 to activate a humoral response
(61). Previous studies have established that both Th1 and
Th2 responses can develop in the absence of a signal through CD28 if
costimulation is provided by 4-1BB/4-1BBL interaction
(16). To determine the role of costimulation-mediated p38
MAPK activation in the development of Th1 and Th2 responses, we
analyzed the ability of T cells from wild-type and
CD28- mice to secrete cytokines in an MLR in the
presence of SB203580. We used two B cell lymphomas as APC for these
experiments. K46J expresses high levels of 4-1BBL but little or no B7
family molecules (15), whereas a variant of the M12.4.1 B
cell lymphoma, upon treatment with cAMP was found to express CD80 and
CD86 but little or no 4-1BBL (Fig. 6A). T cells responding to
K46J in a primary MLR produced IL-2, IL-4, and low levels of IFN-.
The response to K46J cells was sensitive to inhibition with a soluble
form of 4-1BB, 4-1BB.AP, but was insensitive to CTLA4.Ig (Fig. 6B). T cell activation by cAMP-treated M12 cells resulted in
a similar cytokine profile and was inhibited by CTLA4.Ig but not by
4-1BB.AP (Fig. 6B). These data establish that K46J and
cAMP-treated M12 cells provide T cell costimulation via the 4-1BB vs
CD28 costimulatory pathways, respectively.
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shows the analysis of cytokine
production in a primary MLR in which purified
CD4+ T cells from C57BL/6
CD28+/+ and CD28-/- mice
were incubated with irradiated BALB/c B cell lymphomas expressing
either 4-1BBL or B7 family members in the presence of different
concentrations of SB203580. As in Fig. 6B, T cells
stimulated with K46J (4-1BBLhigh) secreted IL-2,
IFN-, and IL-4. Addition of the p38 inhibitor, SB203580, resulted in
a dose-dependent decline in secretion of IL-2, IFN-, and IL-4 by the
T cells responding to 4-1BBL-mediated costimulation. Similar results
were observed for CD4+ T cells stimulated with
APC expressing B7 family members (cAMP-treated M12 cells; Fig. 7).
These data suggest that p38 MAPK activation is critical for the
development of a Th1 or Th2 response whether costimulation is mediated
via 4-1BB or CD28.
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A shows a dose-dependent
inhibition of IL-2 and IFN- production by Th1 cells stimulated in
the presence of SB203580, regardless of whether the T cells were
restimulated with APC expressing 4-1BBL or CD80/86. IL-4 production by
Th2 cells was not impaired even at the highest concentration of
SB203580. IL-4 production was also evaluated by ELISA with similar
results (data not shown). Nevertheless, IL-4 production by the Th2
cells remained costimulation dependent (Fig. 8B). These
results imply that costimulatory signals are required following Th1 or
Th2 commitment; however, the requirements for p38 MAPK activation
diverge following T cell differentiation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The data presented in this report demonstrate a critical role for
p38 MAPK in the development of Th1 or Th2 responses involving 4-1BB or
CD28-mediated costimulation. The p38 MAPK can be activated by multiple
stimuli including TNF family ligands, proinflammatory cytokines, and
environmental stimuli (35). Recent data have suggested a
role for p38 MAPK in CD28-mediated costimulation of primary murine and
human T cells (36, 37, 38, 39, 40, 41). In the present study, we found
that 4-1BB aggregation on the surface of a T cell hybridoma induced p38
MAPK activation (Fig. 1). In primary T cells, CD3 ligation alone
activated the p38 MAPK pathway with enhanced p38 MAPK activation when
anti-CD3 was coimmobilized with 4-1BBL (Fig. 2). The requirement of
anti-CD3 for 4-1BB-mediated p38 MAPK activation is likely due to
the requirement for anti-CD3 to induce 4-1BB expression on the
primary T cells. In contrast, anti-4-1BB alone induced p38 MAPK
activation on a T cell hybridoma that constitutively expressed 4-1BB
(Fig. 1). For the experiments with normal murine T cells the time
course for detection of p38 MAPK (24 h) raises the possibility that
other factors secreted during T cell activation could lead to p38 MAPK
activation by an indirect route. However, on a T cell line 4-1BB
aggregation leads to rapid TRAF2 and ASK1-dependent p38 MAPK activation
within 10 min of stimulus, making it less likely that the effects of
4-1BB on p38 MAPK activation are indirect.
Evidence for a critical role for p38 MAPK in 4-1BB-mediated T cell
costimulation was derived using SB203580, a highly specific inhibitor
of p38 MAPK (57, 58). p38 MAPK activation in response to
4-1BB ligation was dramatically impaired by addition of SB203580 while
JNK1 activation remained unaffected (Figs. 1 and 2). SB203580
effectively blocked cytokine secretion (IL-2 and IL-4) following
primary T cell stimulation with immobilized anti-CD3 in the
presence of either s4-1BBL or anti-CD28 (Fig. 3). These results
support a critical role for p38 MAPK in the stimulation of primary T
cells regardless of whether the costimulatory signal was provided by
CD28 or 4-1BB. Furthermore, a T cell hybridoma responding to
anti-CD3 alone was also inhibited by SB203580, consistent with a
critical role for p38 MAPK in cytokine production in
costimulation-independent as well as costimulation-dependent T cell
responses (Fig. 1B). The fact that p38 MAPK is also
activated in response to anti-CD3 stimulation alone suggests that
the effects of SB203580 on primary T cell activation are likely due to
inhibition of signaling downstream of both the TCR and the
costimulatory receptors.
The effects of 4-1BB ligation on p38 MAPK activation were compared with those of anti-CD28 treatment in the same experiments. On normal T cells, 4-1BB-mediated p38 MAPK activation required ligation of both CD3 and 4-1BB. In contrast, anti-CD28 alone induced p38 MAPK activation with no further enhancement when anti-CD3 and anti-CD28 were combined. Similar results were found for CD28-mediated costimulation of human T cells (37, 39, 60). In contrast, murine T cells stimulated with anti-CD3 plus anti-CD28 induced synergistic activation of p38 MAPK activation (36, 38). The lack of synergy between anti-CD3 and anti-CD28 observed in our experiments may be due to the differences in the kinetics of the analysis or due to different conditions of stimulation.
Although the membrane proximal events leading to signal transduction
via CD28 and 4-1BB are quite distinct, both CD28 and 4-1BB aggregation
lead to activation of the JNK/SAPK and p38 MAPK pathways and to NF-B
activation (22, 23, 24, 25, 26, 29, 31) and this report). We
previously reported that osmotic shock, a known stimulator of the
JNK/SAPK and p38 MAPK pathways, can replace the costimulatory signal
for primary resting T cells (31), consistent with a key
role for these pathways in T cell costimulation. A major difference
between CD28 and 4-1BB in their costimulatory effects seems to be their
expression patterns; CD28 is expressed on resting cells, whereas 4-1BB
is absent from resting T cells and its expression peaks at about
72 h following TCR engagement.
How does 4-1BB aggregation result in p38 MAPK activation?
TRAF2 serves as an adapter protein linking the activation of TNFR
family members to downstream signaling events. Overexpression studies
have implicated TRAF2 in TNF-induced NF-B, JNK/SAPK, and p38 MAPK
activation (35). 4-1BB aggregation on the surface of C8.A3
T cells induces TRAF1 and 2 recruitment (8), TRAF2
association with ASK1, and activation of ASK1 (31). In
this report we showed that T cells from TRAF2DN mice fail to activate
p38 MAPK in response to 4-1BB aggregation (Fig. 5), suggesting a
critical role for TRAF2 in 4-1BB-mediated p38 MAPK activation. T cell
stimulation assays have shown that TRAF2 is necessary for
4-1BB-dependent IL-2 production (8). Thus, both JNK/SAPK
and p38 MAPK cascades are activated and are likely to be important in
4-1BB-mediated costimulation.
The present studies demonstrate that overexpression of a
dominant-negative ASK1 impairs 4-1BB-induced p38 MAPK activation and
that p38 MAPK activation is enhanced by overexpression of wild-type
ASK1 (Fig. 4). Recent experiments using overexpression systems have
shown that the association of TRAF2 with the cytoplasmic tail of TNFR
is upstream of ASK1 (33, 34). Previous results showed that
ASK1 is also involved in 4-1BB-mediated JNK/SAPK activation as well as
IL-2 production in response to MHC/Ag and 4-1BBL (31).
These data are consistent with a role for ASK1 in 4-1BB mediated
activation of the JNK/SAPK and p38 MAPK cascades (Fig. 9). However, it is possible that the
overexpression of the dominant-negative ASK1 interferes with the
activation of another endogenous MAPKKK. Other MAPKKK’s, such as
MAP/extracellular signal-related kinase kinase 1 (MEKKI), germinal
center kinase, germinal center like kinase, and germinal center kinase
related (GCKR) can link TNF to the JNK/SAPK (62) and/or
p38 MAPK pathways (63, 64, 65). The relative importance of
these MKKK’s in signaling via TNFR family members may depend on tissue
distribution as well the specific TNFR family member involved.
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The polarization of T cell cytokine profiles toward Th1 or
Th2 responses is one of the mechanisms by which the immune system
responds to microbial challenge. The development of an inappropriate
immune response can lead to ineffective immunity and immune pathology.
Therefore, understanding the signal transduction mechanisms leading to
Th1 vs Th2 responses is important. In this report, we found that the
p38 specific inhibitor, SB203580, caused a dose-dependent decrease in
IL-2, IFN-, and IL-4 production by CD4+ T
cells stimulated with APC expressing either CD80/CD86 or 4-1BBL (Fig. 7). These results are consistent with the results of Zhang et al.
(38) who showed that cytokine production during
CD28-dependent responses is inhibited by SB203580. Thus, p38 MAPK
activation is required for the development of both Th1 and Th2 cells in
response to either 4-1BB or CD28-dependent costimulation. However, once
CD4+ T cells have developed into Th1 or Th2
effector cells, the requirement for p38 MAPK activation is altered. We
found that Th1 cells require costimulation and p38 MAPK activation for
the continued secretion of the Th1 cytokines, IL-2, and IFN- (Fig. 8A). In contrast, Th2 cells did not require p38 MAPK
activation (Fig. 8A) but were still dependent on
costimulation for the secretion of IL-4 (Fig. 8B). Our
results regarding the p38 MAPK-independence of IL-4 production by
committed Th2 cells are in agreement with those of Rincon et al.
(42). Rincon et al. used SB203580 inhibition as well as
mice expressing a dominant-negative p38 MAPK to show that IFN- but
not IL-4 production by preestablished Th1 and Th2 cells responding to
Con A was dependent on p38 MAPK activation (42). These
authors did not assess the role of p38 MAPK in the primary production
of IL-4 by T cells, as they added IL-4 to the cultures during the
initial stimulation of wild-type or p38 dominant-negative T cells
(42). Taken together, the present and previous results
indicate that p38 MAPK is not required for the response of T cells to
IL-4 (44) or the production of IL-4 by Th2 cells (Ref.
42 and this report) but is required for initial IL-4
production by primary T cells (Ref. 38 and this
report).
Human T cells also activate p38 MAPK following anti-CD28 ligation;
however, inhibition of this pathway has a different effect on cytokine
secretion profiles (66). Following stimulation of human T
cells, SB203580 results in a dose-dependent decline in IL-4, IL-5, and
IFN-, but not IL-2 production (39, 60). Furthermore,
following T cell polarization, p38 MAPK inhibition dramatically
impaired IL-4 but only partially impaired IL-2 secretion
(60). Interestingly, Mori et al. (41) have
conflicting data indicating that T cell clones from asthmatic donors
require p38 MAPK for IL-5 synthesis but not for IL-2, IL-4, and
IFN-. These differing results might reflect the use of particular T
cell clones (41) vs bulk T cell cultures (39, 60).
In summary, the results presented here suggest that p38 MAPK activation
is crucial for 4-1BB-mediated costimulatory signals in primary T cells.
Based on these and previous experiments, it is now established that
4-1BB aggregation recruits TRAF2, which in turn can activate ASK1,
resulting in the activation of the JNK/SAPK and p38 MAPK pathways (Fig. 9). In addition we show that p38 MAPK activation of
CD4+ T cells is required for the development of
Th1 and Th2 cells and for the maintenance of Th1 effector cells
regardless of whether the cells are stimulated in a CD28- or a
4-1BB-dependent manner. However, the re-activation of Th2 cells is p38
MAPK independent while maintaining costimulation dependence. Thus,
essential signaling processes leading to cytokine production diverge
following CD4+ T cell differentiation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tania H. Watts, Department of Immunology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8.
3 Abbreviations used in this paper: L, ligand; ASK1, apoptosis-signal-regulating kinase 1; JNK, c-jun N-terminal kinase; SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; s4-1BBL, soluble 4-1BBL; TRAF, TNFR-associated factor; TRAF2DN, dominant-negative TRAF2; HA, hemaglutinin; AP, alkaline phosphatase.
Received for publication April 26, 2000. Accepted for publication September 1, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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