4-1BB is a costimulatory member of the TNFR family, expressed on activated CD4+ and CD8+ T cells. Previous results showed that 4-1BB-mediated T cell costimulation is CD28-independent and involves recruitment of TNFR-associated factor 2 (TRAF2) and activation of the stress-activated protein kinase cascade. Here we describe a role for the p38 mitogen-activated protein kinase (MAPK) pathway in 4-1BB signaling. Aggregation of 4-1BB alone induces p38 activation in a T cell hybridoma, whereas, in normal T cells, p38 MAPK is activated synergistically by immobilized anti-CD3 plus immobilized 4-1BB ligand. 4-1BB-induced p38 MAPK activation is inhibited by the p38-specific inhibitor SB203580 in both a T cell hybridoma and in murine T cells. T cells from TRAF2 dominant-negative mice are impaired in 4-1BB-mediated p38 MAPK activation. A link between TRAF2 and the p38 cascade is provided by the MAPK kinase kinase, apoptosis-signal-regulating kinase 1. A T cell hybrid transfected with a kinase-dead apoptosis-signal-regulating kinase 1 fails to activate p38 MAPK in response to 4-1BB signaling. To assess the role of p38 activation in an immune response, T cells were stimulated in an MLR in the presence of SB203580. In a primary MLR, SB203580 blocked IL-2, IFN-γ, 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-γ 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.

Activation of resting T cells requires engagement of the TCR by MHC/peptide as well as additional costimulatory signals. CD28 ligation provides an important costimulatory signal during primary T cell activation. However, studies using CD28−/− mice indicate that CD28 is dispensable in some immune responses (1, 2, 3). Members of the TNFR family binding to their respective TNF family ligands (L),3 including 4-1BB/4-1BBL, OX-40/OX-40L, and CD27/CD70, can also enhance T cell activation and/or differentiation (4). During some immune responses, such additional interactions may be required to generate an optimal T cell response.

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-κB (4). CD28 costimulation also involves JNK/SAPK and NF-κB 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-κB activation following TNF-α 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.

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 κ-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.

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.

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 × 106 CD4+ T cells and 5 × 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.

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 × 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-γ and IL-4 were measured by ELISA using the pair of anti-murine IFN-γ mAbs or anti-murine IL-4 mAbs purchased from PharMingen. ELISA was performed according to the manufacturer’s instructions using the diluted supernatant.

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 × 105) were stimulated overnight on wells that had been precoated overnight with 0.1 μg/ml anti-CD3 or were stimulated with 1 × 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.

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).

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]γ-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 2× SDS sample buffer. Phosphoproteins were separated by SDS-PAGE and visualized by autoradiography.

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).

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.

FIGURE 1.

p38 MAPK activation following 4-1BB aggregation on a T cell hybrid, C8.A3. A, C8.A3 T cells (5 × 106) were stimulated with either 5 μg/ml anti-4-1BB followed by 20 μg/ml anti-rat Ig or 0.4 M sorbitol for 10 min in the presence of 0–50 μM of the p38 inhibitor SB203580 or DMSO control. p38 or JNK1 were immunoprecipitated from the lysates and kinase activity was assayed by an in vitro kinase assay with a substrate GST-ATF2 or GST-c-jun, respectively. p38 and JNK1 were detected in the lysates via Western blot using anti-p38 or anti-JNK1 Abs. B, C8.A3 T cells (5 × 105) were stimulated with either 1 μg/ml immobilized anti-CD3 or 5 × 105 irradiated K46J Ak cells in the presence of 0–50 μM SB203580 or DMSO control. After an overnight stimulation, culture supernatants were removed and serial dilutions of the supernatant were incubated with an IL-2-dependent cell line, CTLL, for 24 h. Similar results were obtained in five separate experiments.

FIGURE 1.

p38 MAPK activation following 4-1BB aggregation on a T cell hybrid, C8.A3. A, C8.A3 T cells (5 × 106) were stimulated with either 5 μg/ml anti-4-1BB followed by 20 μg/ml anti-rat Ig or 0.4 M sorbitol for 10 min in the presence of 0–50 μM of the p38 inhibitor SB203580 or DMSO control. p38 or JNK1 were immunoprecipitated from the lysates and kinase activity was assayed by an in vitro kinase assay with a substrate GST-ATF2 or GST-c-jun, respectively. p38 and JNK1 were detected in the lysates via Western blot using anti-p38 or anti-JNK1 Abs. B, C8.A3 T cells (5 × 105) were stimulated with either 1 μg/ml immobilized anti-CD3 or 5 × 105 irradiated K46J Ak cells in the presence of 0–50 μM SB203580 or DMSO control. After an overnight stimulation, culture supernatants were removed and serial dilutions of the supernatant were incubated with an IL-2-dependent cell line, CTLL, for 24 h. Similar results were obtained in five separate experiments.

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To evaluate the role of p38 MAPK activation in 4-1BB/4-1BBL-mediated cytokine production, C8.A3 T cells were stimulated in the presence or absence of SB203580 and IL-2 secretion was analyzed (Fig. 1,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. 1 B 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. 2,A 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. 2,B 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. 2 C).

FIGURE 2.

4-1BB aggregation on the surface of primary T cells activates p38 MAPK. A, Primary T cells (5 × 106) were stimulated with 0.4 M sorbitol for 0 and 30 min as a positive control. T cells were stimulated with either 1 μg/ml immobilized anti-CD3, 5 μg/ml s4-1BBL, or anti-CD3 plus s4-1BBL for 0, 24, and 48 h. p38 was immunoprecipitated from the lysate and its activity was assayed in an in vitro kinase reaction with 5 μg GST-ATF2 as a substrate. B, T cells (5 × 106) were stimulated as previously described for 48 h in the presence of either 10 μg/ml AP control or 10 μg/ml 4-1BB.AP. p38 was immunoprecipitated from the lysate and its activity determined in an in vitro kinase assay with 5 μg GST-ATF2 as a substrate. C, T cells (5 × 106) were stimulated with immobilized anti-CD3, s4-1BBL, or anti-CD3 plus s4-1BBL for 48 h in the presence of 0–20 μM SB203580 or DMSO control. p38 and JNK1 were immunoprecipitated from the lysates and kinase activity determined by an in vitro kinase assay using GST-ATF2 or GST-c-jun as a substrate, respectively. p38 and JNK1 were detected in the lysate by Western blot using anti-p38 and anti-JNK1 Abs. This experiment is representative of four experiments.

FIGURE 2.

4-1BB aggregation on the surface of primary T cells activates p38 MAPK. A, Primary T cells (5 × 106) were stimulated with 0.4 M sorbitol for 0 and 30 min as a positive control. T cells were stimulated with either 1 μg/ml immobilized anti-CD3, 5 μg/ml s4-1BBL, or anti-CD3 plus s4-1BBL for 0, 24, and 48 h. p38 was immunoprecipitated from the lysate and its activity was assayed in an in vitro kinase reaction with 5 μg GST-ATF2 as a substrate. B, T cells (5 × 106) were stimulated as previously described for 48 h in the presence of either 10 μg/ml AP control or 10 μg/ml 4-1BB.AP. p38 was immunoprecipitated from the lysate and its activity determined in an in vitro kinase assay with 5 μg GST-ATF2 as a substrate. C, T cells (5 × 106) were stimulated with immobilized anti-CD3, s4-1BBL, or anti-CD3 plus s4-1BBL for 48 h in the presence of 0–20 μM SB203580 or DMSO control. p38 and JNK1 were immunoprecipitated from the lysates and kinase activity determined by an in vitro kinase assay using GST-ATF2 or GST-c-jun as a substrate, respectively. p38 and JNK1 were detected in the lysate by Western blot using anti-p38 and anti-JNK1 Abs. This experiment is representative of four experiments.

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Primary T cells were also stimulated with immobilized anti-CD3 in the presence or absence of either immobilized s4-1BBL or anti-CD28 and the pattern of cytokine secretion was evaluated. Anti-CD3 in conjunction with s4-1BBL resulted in detectable secretion of IL-2 and IL-4 (Fig. 3), but no detectable IFN-γ (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.

FIGURE 3.

Role for p38 MAPK in T cell cytokine secretion. T cells (1 × 105) were stimulated with 1 μg/ml immobilized anti-CD3 plus or minus either 10 μg/ml immobilized anti-CD28 or 10 μg/ml s4-1BBL for 48 h in the absence or in the presence of increasing concentrations of SB203580 (0–10 μM) or DMSO control. This experiment is representative of three individual experiments.

FIGURE 3.

Role for p38 MAPK in T cell cytokine secretion. T cells (1 × 105) were stimulated with 1 μg/ml immobilized anti-CD3 plus or minus either 10 μg/ml immobilized anti-CD28 or 10 μg/ml s4-1BBL for 48 h in the absence or in the presence of increasing concentrations of SB203580 (0–10 μM) or DMSO control. This experiment is representative of three individual experiments.

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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.

FIGURE 4.

ASK1 activates the p38 MAPK cascade. C8.A3 T cells (2 × 107) were transiently transfected with either vector (pcDNA3) control, HA-ASK1 or HA-ASK1 K709E. Thirty-eight hours following transfection, 8 × 106 transfected C8.A3 T cells were stimulated with 5 μg/ml anti-4-1BB plus 20 μg/ml second step anti-rat or 0.4 M sorbitol. p38 was immunoprecipitated from the lysate and its activity was assayed in an in vitro kinase reaction with 5 μg GST-ATF2 as a substrate. p38 and the HA-tagged proteins were present in the lysate as detected by Western blot analysis with anti-p38 and anti-HA (12CA5). This experiment is representative of three experiments.

FIGURE 4.

ASK1 activates the p38 MAPK cascade. C8.A3 T cells (2 × 107) were transiently transfected with either vector (pcDNA3) control, HA-ASK1 or HA-ASK1 K709E. Thirty-eight hours following transfection, 8 × 106 transfected C8.A3 T cells were stimulated with 5 μg/ml anti-4-1BB plus 20 μg/ml second step anti-rat or 0.4 M sorbitol. p38 was immunoprecipitated from the lysate and its activity was assayed in an in vitro kinase reaction with 5 μg GST-ATF2 as a substrate. p38 and the HA-tagged proteins were present in the lysate as detected by Western blot analysis with anti-p38 and anti-HA (12CA5). This experiment is representative of three experiments.

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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.

FIGURE 5.

TRAF2 is required for 4-1BB-mediated p38 MAPK activation. Primary T cells (5 × 106) isolated from control or TRAF2DN mice were stimulated with either 1 μg/ml immobilized anti-CD3, 10 μg/ml anti-CD28, 5 μg/ml s4-1BBL, anti-CD3 plus anti-CD28 or anti-CD3 plus s4-1BBL for 48 h. T cells were incubated with 0.4 M sorbitol for 30 min as a control. p38 was immunoprecipitated from the lysate and the kinase activity was determined by an in vitro kinase assay with GST-ATF2 as a substrate. p38 was detected in the lysate by Western blot. This experiment is representative of three experiments.

FIGURE 5.

TRAF2 is required for 4-1BB-mediated p38 MAPK activation. Primary T cells (5 × 106) isolated from control or TRAF2DN mice were stimulated with either 1 μg/ml immobilized anti-CD3, 10 μg/ml anti-CD28, 5 μg/ml s4-1BBL, anti-CD3 plus anti-CD28 or anti-CD3 plus s4-1BBL for 48 h. T cells were incubated with 0.4 M sorbitol for 30 min as a control. p38 was immunoprecipitated from the lysate and the kinase activity was determined by an in vitro kinase assay with GST-ATF2 as a substrate. p38 was detected in the lysate by Western blot. This experiment is representative of three experiments.

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In response to pathogens, naive CD4+ T cells can differentiate into Th1 and Th2 effector cells. Th1 cells produce IFN-γ 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. 6,A). T cells responding to K46J in a primary MLR produced IL-2, IL-4, and low levels of IFN-γ. 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. 6,B). 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. 6 B). These data establish that K46J and cAMP-treated M12 cells provide T cell costimulation via the 4-1BB vs CD28 costimulatory pathways, respectively.

FIGURE 6.

Costimulatory molecule requirements and expression during an MLR. A, Flow cytometric analysis of B cell lymphomas K46J and dibutyryl-cAMP-treated M12 stained with either isotype controls (thin lines), anti-B7-1, anti-B7-2 or 4-1BB.AP (bold lines). B, CD4+ T cells (1 × 106) were stimulated with irradiated K46J cells or cAMP-treated M12 cells (5 × 105) in the presence of either CTLA4-Ig, Ig control, 4-1BB.AP or AP control. After 5 days, supernatant levels of IL-2, IFN-γ, and IL-4 were determined as described in Materials and Methods. This experiment is a representation of four individual experiments.

FIGURE 6.

Costimulatory molecule requirements and expression during an MLR. A, Flow cytometric analysis of B cell lymphomas K46J and dibutyryl-cAMP-treated M12 stained with either isotype controls (thin lines), anti-B7-1, anti-B7-2 or 4-1BB.AP (bold lines). B, CD4+ T cells (1 × 106) were stimulated with irradiated K46J cells or cAMP-treated M12 cells (5 × 105) in the presence of either CTLA4-Ig, Ig control, 4-1BB.AP or AP control. After 5 days, supernatant levels of IL-2, IFN-γ, and IL-4 were determined as described in Materials and Methods. This experiment is a representation of four individual experiments.

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Fig. 7 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. 6,B, T cells stimulated with K46J (4-1BBLhigh) secreted IL-2, IFN-γ, and IL-4. Addition of the p38 inhibitor, SB203580, resulted in a dose-dependent decline in secretion of IL-2, IFN-γ, 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.

FIGURE 7.

Role of p38 MAPK activation in the MLR. CD4+ T cells (1 × 106) isolated from CD28+ and CD28 mice were stimulated with either irradiated K46J or cAMP-treated M12 (5 × 105) cells for 5 days in the presence of 0–20 μM SB203580 or DMSO control. Supernatant levels of IL-2, IFN-γ, and IL-4 were determined as described in Materials and Methods and are shown in 1:4 dilution of supernatant. This experiment is representative of four individual experiments.

FIGURE 7.

Role of p38 MAPK activation in the MLR. CD4+ T cells (1 × 106) isolated from CD28+ and CD28 mice were stimulated with either irradiated K46J or cAMP-treated M12 (5 × 105) cells for 5 days in the presence of 0–20 μM SB203580 or DMSO control. Supernatant levels of IL-2, IFN-γ, and IL-4 were determined as described in Materials and Methods and are shown in 1:4 dilution of supernatant. This experiment is representative of four individual experiments.

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To examine the potential role of p38 MAPK activation in the regulation of cytokine production by committed Th1 and Th2 cells, Th1 and Th2 cells were generated as described in Materials and Methods and restimulated in the presence of the p38 inhibitor. Fig. 8,A shows a dose-dependent inhibition of IL-2 and IFN-γ 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. 8 B). 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.

FIGURE 8.

Divergent requirement for p38 MAPK following T cell differentiation. A, CD4+ T cells isolated from CD28+ and CD28 were developed into Th1 or Th2 effector cells as described in Materials and Methods. T cells were restimulated with either irradiated K46J or cAMP-treated M12 cells in the presence of 0–20 μM of the p38 inhibitor SB203580 or DMSO control. Supernatant levels of IL-2, IFN-γ, and IL-4 were determined as previously described and are shown as a 1:4 dilution. B, CD4+ T cells Th2 biased were restimulated with irradiated K46J or cAMP M12 in the presence of AP, 4-1BB.AP Ig, or CTLA4.Ig (10 μg/ml) and IL-4 secretion was evaluated. A 1:4 dilution of the supernatant is shown. This experiment is representative of four experiments.

FIGURE 8.

Divergent requirement for p38 MAPK following T cell differentiation. A, CD4+ T cells isolated from CD28+ and CD28 were developed into Th1 or Th2 effector cells as described in Materials and Methods. T cells were restimulated with either irradiated K46J or cAMP-treated M12 cells in the presence of 0–20 μM of the p38 inhibitor SB203580 or DMSO control. Supernatant levels of IL-2, IFN-γ, and IL-4 were determined as previously described and are shown as a 1:4 dilution. B, CD4+ T cells Th2 biased were restimulated with irradiated K46J or cAMP M12 in the presence of AP, 4-1BB.AP Ig, or CTLA4.Ig (10 μg/ml) and IL-4 secretion was evaluated. A 1:4 dilution of the supernatant is shown. This experiment is representative of four experiments.

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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. 1 B). 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-κB 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.

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-κB, 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.

FIGURE 9.

4-1BB-mediated signaling events. 4-1BB aggregation results in TRAF1 and TRAF2 recruitment. TRAF2 can interact with and activate ASK1. ASK1 can then activate downstream signaling cascades including JNK/SAPK and p38 MAPK. The thick arrows represent pathways defined for 4-1BB-mediated signaling events. For further details, see text.

FIGURE 9.

4-1BB-mediated signaling events. 4-1BB aggregation results in TRAF1 and TRAF2 recruitment. TRAF2 can interact with and activate ASK1. ASK1 can then activate downstream signaling cascades including JNK/SAPK and p38 MAPK. The thick arrows represent pathways defined for 4-1BB-mediated signaling events. For further details, see text.

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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-γ, 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-γ (Fig. 8,A). In contrast, Th2 cells did not require p38 MAPK activation (Fig. 8,A) but were still dependent on costimulation for the secretion of IL-4 (Fig. 8 B). 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-γ 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-γ, 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-γ. 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.

We thank Jim Woodgett and Klaus Hoeflich for provision of HA-ASK1, HA-ASK1 K709E, and GST-ATF2 constructs, and Tak Mak for providing CD28−/− mice. We thank Birinder Ghumman for the purification ofs4-1BBL, members of the Watts lab for their helpful discussion, and Nancy Berg for critically reviewing the manuscript.

1

This research was supported by a grant from the Medical Research Council of Canada (to T.H.W.). J.L.C. was funded by a Medical Research Council of Canada doctoral award.

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.

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