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* Committee on Immunology and
Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL 60637;
Department of Microbiology and Immunology, University of Miami, Miami, FL 33101; and
Department of Microbiology and Immunology, Vanderbilt University Medical School, Nashville, TN 37232
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, and was completely inhibited by the
expression of a TNFR-associated factor 2 (TRAF2) dominant-negative
transgene (TRAF2.DN-Tg), but not by that of an I-B
dominant-negative transgene. In parallel, expression of the TRAF2.DN-Tg
selectively prevented the induction of c-Jun N-terminal kinase and p38
MAPK, but not that of NF-B. Furthermore, IL-13 production was
reduced in a dose-dependent manner by the p38 MAPK inhibitor SB203580.
Together, these results suggest that TCR-independent CD30-mediated
production of IL-13 is triggered by association of CD30 with TRAF
family members and subsequent activation of p38 MAPK. Inasmuch as IL-13
can promote airway inflammation and cancer progression, production of
IL-13 in a TCR-independent manner has important pathological
implications in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vitro, CD30 expression peaks at days 4–5 following T cell activation (7) and its up-regulation requires signaling through the TCR in conjunction with either CD28 or the IL-4R (2). CD30 engagement augments proliferation of activated T cells at low levels of TCR stimulation in vitro (2, 8), but also triggers apoptosis of CD30-expressing tumor cells (8, 9), suggesting that the outcome of CD30 engagement may depend on cellular context. The role of CD30 in vivo is not well understood. CD30-deficient mice have been reported to have a defect in negative selection (10), and overexpression of CD30 on T cells results in augmented thymocyte depletion upon treatment with superantigen or antigenic peptide (11). However, a more recent study failed to confirm decreased negative selection in independently generated CD30-deficient mice (12), suggesting an alternative, as yet unidentified, role for CD30 in vivo.
The signaling mechanisms by which CD30 operates in normal T cells
remain to be established. TNFR family members signal, in part, through
TNFR-associated factors
(TRAFs)3
(13). There are six members of the TRAF family of adapter
proteins that all contain a conserved C-terminal TRAF domain required
for association with TNFRs. With the exception of TRAF1, all TRAF
proteins also contain an N-terminal RING finger domain, and
deletion of this domain results in generation of a dominant-negative
(DN) protein. TRAF proteins have been implicated in activation of
NF-B, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated
protein kinase (MAPK) (14). In transfected COS or 293
cells, CD30 associates with TRAF1, TRAF2, and TRAF3 (15, 16). There are two conserved C-terminal TRAF binding sites
in the CD30 cytoplasmic tail, which both can bind TRAF1 and TRAF2,
whereas TRAF3 can only bind to the upstream site (17).
Both conserved TRAF binding sites in CD30 are able to mediate NF-B
activation (18).
Biological functions of costimulatory receptors have been characterized
in the context of TCR engagement. In this study, we report that, in
contrast to CD28 and in addition to its costimulatory function, CD30
can signal in a completely TCR-independent fashion to induce IL-13
production by effector T cells. This TCR-independent function of CD30
correlates with activation of NF-B, JNK, and p38 MAPK and is
dependent on functional TRAF association. Our findings indicate that in
addition to its TCR-dependent costimulatory function, CD30 induces
TCR-independent biochemical signals that permit selective activation of
a transcription program in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DO11.10 transgenic (DO11.10-Tg) mice expressing a TCR specific
for OVA peptide (aa 323–339) were obtained from The Jackson Laboratory
(Bar Harbor, ME) and were on the BALB/c background. Transgenic animals
expressing a DN TRAF2 protein lacking the N-terminal RING finger
under the control of the H-2K promoter and IgH chain enhancer
(TRAF2.DN-Tg) (19) and animals expressing an I-B.DN
transgene driven by the proximal Lck promoter (20) were
intercrossed with DO11.10-Tg mice at Vanderbilt University and shipped
to the University of Chicago for acute use. Animals were kept in
specific pathogen-free facilities and used in agreement with the
Institutional Animal Care and Use Committee according to the National
Institutes of Health guidelines for animal use.
The anti-CD30 mAb (mCD30.1, Armenian hamster IgG1, -L chain) was
produced at the University of Miami (7) or purchased from
BD PharMingen (San Diego, CA). Isotype control-matched mAb (A19-3) was
purchased from BD PharMingen. Anti-CD28 mAb-producing hybridoma (PV-1,
Armenian hamster IgG1, -L chain) was generously provided by T.
Gajewski (University of Chicago). The hybridoma-producing anti-CD3
mAb (145-2C11) was a kind gift by J. Bluestone (University of
California, San Francisco, CA). All mAbs were purified by passage of
supernatant over a protein A column (Amersham Pharmacia, Arlington
Heights, IL).
Generation and restimulation of CD4+, CD30+ mouse T cells
Spleens were isolated from DO11.10-Tg, TRAF2.DN/DO11.10-Tg, and
I-B.DN/DO11.10-Tg mice, and RBCs were lysed using
ammonium/chloride/potassium buffer. Cells were plated at
106 cells/ml in DMEM (Life Technologies, Grand
Island, NY) supplemented with 10% FCS, penicillin (100 U/ml),
streptomycin (100 µg/ml), HEPES, 2-ME (50 µM), and additional amino
acids, in the presence of 1.3 ng/ml murine rIL-4 and OVA peptide (0.4
ng/ml, synthesized by the University of Chicago Peptide Core Facility).
After 5 days, live cells were collected and depleted of dead cells by
density gradient centrifugation through Ficoll-Hypaque (Cedarlane
Laboratories, Hornby, Ontario, Canada). Expression of CD4 and CD30 was
verified by flow cytometry. T cell purity at this stage was routinely
above 95%.
Bead-bound Abs were used to restimulate the CD4+CD30+ T cells. The beads (Dynal, Oslo, Norway) were coated with Abs in PBS supplemented with 0.5% BSA, in a rotator for 1–2 h at room temperature. A maximum of 2 x 107 beads/ml was used during the coating procedure. A 1:5 cell to bead ratio was used during the in vitro stimulation.
In some experiments, P815 cells (a mouse mastocytoma cell line) were used in place of beads. P815 cells were transfected with CD30 ligand (CD30L)-expressing construct, a control plasmid, or a B7.1-expressing construct. These cell lines were generously provided by Pfizer Laboratories (Groton, CT). P815 cells and T cells were incubated at a 1:1 ratio.
Cytokine production and proliferation assays
Primed DO11.10-Tg T cells were plated on flat-bottom 96-well plates at 50,000 cells/well in 200 µl complete DMEM in the presence of coated beads or P815 cells. For cytokine assays, supernatants were harvested at 24 h of restimulation, and concentration of cytokines in each sample was detected by ELISA using Ab pairs, as instructed by the manufacturer (BD PharMingen). In some experiments, a p38 inhibitor (SB203580) or a mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (PD98059) purchased from Calbiochem (San Diego, CA) was added at the indicated concentrations. Absorbance was detected in a 96-well spectrophotometer (Spectra Max 250; Molecular Devices, Sunnyvale, CA), and data were analyzed using Softmax software (Molecular Devices) by comparison with a standard curve generated using recombinant cytokines at known concentrations.
For proliferation assays, 1 µCi [3H]thymidine/well was added for the last 8 h of a 48-h culture. Cells were harvested using a Filtermate harvester, and incorporated [3H]thymidine was detected using a TopCount NXT microplate scintillation and luminescence counter with TopCount NXT software (Packard BioScience, Meriden, CT).
RNase protection assay
Total RNA was purified using TRIzol (Life Technologies), as
directed by the manufacturer. A total of 5 x
106 DO11.10-Tg
CD4+CD30+ T cells was used
for each sample, and the resulting RNA was dissolved in a final volume
of 50 µl diethyl pyrocarbonate-H2O. The RNA was
equalized, as previously described (21), and the
concentration also was determined by spectrophotometric analysis.
Approximately 2 µg total RNA was used for each sample. The levels of
IL-4, IL-5, IL-10, IL-13, IL-15, IL-9, IL-2, IL-6, and IFN-
mRNA
relative to the housekeeping genes L32 and GAPDH were determined using
the mCK-1 Mouse Cytokine MultiProbe Template Set (BD
PharMingen), according to the directions of the
manufacturer.
Immunoprecipitation of CD30-TRAF2 complexes
Primed DO11.10-Tg T cells (2 x 107) were incubated with control or anti-CD30 Ab (5 µg) on ice for 5 min and then placed at 37° for 15 min in the presence or absence of cross-linking Ab. The cells were pelleted and lysed in 0.5% Nonidet P-40 lysis buffer (10 mM Tris, pH 8, 1 mM MgCl2, 50 mM NaCl, 0.5% Nonidet P-40, 2 µg/ml aprotinin, and 1 mM PMSF). Lysate supernatants were added to protein G-Sepharose (Amersham Pharmacia) and incubated at 4°C for a minimum of 2 h or overnight. The Sepharose beads were washed four times with ice-cold lysis buffer, and immunoprecipitated proteins were resolved on 10% SDS-polyacrylamide gels. Western blots were performed using anti-CD30 (BD PharMingen) or anti-TRAF2 Abs (Santa Cruz Biotechnology, La Jolla, CA).
Electromobility shift assays
Primed DO11.10-Tg T cells were plated at 0.5–1 x 106 cells/ml in 24-well plates in the presence of coated beads, as described above. At the indicated time points, cells were harvested and nuclear lysates were prepared, as previously described (20). Nuclear extracts were kept frozen at -80°C until use.
For the gel shift assays, double-stranded probes containing the NF-B
binding site (5'-CAACGGCAGGGGAATTCCCCTCTCCTT-3') of the IL-2R promoter
or an Oct1 consensus binding site (5'-TGTCGAATGCAAATCACTAGAA-3')
were end labeled using T4 polynucleotide kinase and
[-32P]ATP. Typically, 0.2 ng radiolabeled
probe and 4 µg nuclear protein were used for each gel shift reaction.
DNA-binding reactions were performed as described (20) and
resolved on a 6% native acrylamide gel. Protein-DNA complexes were
visualized by autoradiography.
Determination of MAPK activation
JNK kinase assay.
Primed T cells (5 x 106) in 0.5 ml complete
DMEM were used for each sample and incubated at 37°C with Ab-coated
beads. At the indicated time points, cells were washed by addition of 1
ml PBS at room temperature and pelleted by 10-s centrifugation in a
microcentrifuge. The cells were lysed in 300 µl ice-cold lysis buffer
(20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton
X-100, 2.5 mM sodium pyrophosphate, 1 mM 
-glycerolphosphate, 1 mM
Na3VO4, 1 mM PMSF, and 1
µg/ml leupeptin), and lysates were incubated with 10 µg
GST-c-jun fusion protein coupled to glutathione Sepharose
beads on a rotator for 3 h at 4°C. The beads were pelleted, and
lysates were kept for Western blotting analysis. The beads were washed
four times with 0.75 ml HEPES-binding buffer (20 mM HEPES, pH 7.7, 50
mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, pH 8.0,
0.05% Triton X-100, 0.1 mM
Na3VO4, 1 mM PMSF, and 1
µg/ml leupeptin). A total of 30 µl kinase buffer (20 mM HEPES, pH
7.6, 20 mM MgCl2, 20 mM -glycerolphosphate, 20
mM p-nitrophenylphosphate, 0.1 mM
Na3VO4, 2 mM DTT, 20 mM
ATP, and 5 µCi [-32P]ATP) was added to
each sample, and kinase reactions were performed for 15 min at 30°C.
The samples were washed three times with 0.5 ml HEPES-binding buffer,
and proteins were resolved by SDS-PAGE. The GST-c-jun fusion
protein was visualized by staining with Coomassie blue, and the amount
of phospho-GST-c-jun was detected using autoradiography.
p38 activity. Protein lysates saved from the GST-c-jun pull-down experiments described above were resolved by SDS-PAGE. Western blots were performed using Abs specific for phospho-p38 and total p38 (New England Biolabs Cell Signaling Technology, Beverly, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Splenocytes from DO11.10-Tg animals were cultured with OVA peptide
and mouse rIL-4 for 5 days and analyzed by flow cytometry for
expression of CD30. As shown in Fig. 1
A, DO11.10-Tg T cells
uniformly expressed high levels of CD30. To confirm the costimulatory
role of CD30 on these CD30-expressing T cells, primed DO11.10-Tg T
cells were stimulated with beads previously coated with anti-CD30
mAb or an isotype control mAb and increasing concentrations of
anti-CD3 mAb. Both IFN- and IL-5 were augmented by CD30
cross-linking at low concentrations of anti-CD3 mAb (Fig. 1B). CD30 cross-linking also augmented TCR-dependent
induction of T cell proliferation, as inferred by increased
[3H]thymidine incorporation (Fig. 1C). To determine whether the augmentation of cytokine
production by CD30 engagement was due to CD30-mediated signals or to
increased avidity for the Ab-coated beads, experiments were repeated in
the presence of Abs coated on separate beads. CD30 cross-linking
augmented cytokine production similarly whether coated on the same
beads (in cis) or on separate beads (in trans) as
the anti-CD3 mAb (Fig. 1D), indicating that these
functional effects are dependent on signaling events mediated by CD30
cross-linking and confirming a costimulatory function for
CD30.
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To determine whether CD30 engagement regulated cytokine gene
expression, RNase protection assays were performed using probes for a
panel of cytokine genes. In the presence of anti-CD3 mAb
stimulation, IL-4, IL-5, and IFN- mRNA gene expression was augmented
by cross-linking of either CD30 or CD28 mAbs (Fig. 2A), consistent with the
increased secretion of these cytokines at the protein level, as
determined by ELISA (Fig. 1
). Unexpectedly, cross-linking of CD30 for
6 h, in the absence of TCR stimulation, consistently resulted in a
specific up-regulation of IL-13 mRNA gene expression when compared with
treatment with control mAb (Fig. 2A). This was not observed
with cross-linking of CD28 alone. Of the nine cytokines analyzed in
this RNase protection assay, IL-13 was the only gene induced by CD30 in
a TCR-independent manner. In particular, TCR-independent CD30-mediated
induction was not observed on expression of IL-10 mRNA, despite
detectable levels of transcripts in unstimulated samples, or on
expression of IL-4, IL-5, and IFN- mRNA, despite effective
CD30-mediated augmentation of TCR-dependent gene induction. Differences
in gene expression were quantitated by densitometry. At 6 h, the
mean ratio of IL-13/L32 mRNA expression following CD30 cross-linking
was 3.2 ± 0.9-fold higher than that following control IgG
treatment in two independent experiments. IL-13 mRNA was also induced
at 3 h of CD30 cross-linking alone (data not shown).
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B)
were at or below the level of detection of the assay. However, IL-13
protein levels were significantly induced following stimulation with
anti-CD30 mAb. In fact, the amount of IL-13 induced by
anti-CD30 mAb alone was almost comparable with that observed
following anti-CD3 stimulation (1.2 ± 0.3 ng/ml at 0.1
µg/ml anti-CD3). Interestingly, engagement of anti-CD30 also
further augmented anti-CD3-mediated production of IL-13 (3.1
± 0.4 ng/ml).
It has been suggested that receptor engagement by mAbs may lead to
different signaling effects than binding by natural ligands
(22). To verify the physiological relevance of
CD30-mediated IL-13 production in the absence of TCR stimulation,
primed DO11.10-Tg T cells were incubated in the presence of syngeneic
(H-2d) P815 cells transfected with control-,
B7-1-, or CD30L-encoding vectors. P815 cells are negative for MHC class
II and are therefore not expected to present Ag to DO11.10-Tg T cells.
As observed following Ab cross-linking of CD30, engagement of CD30 by
CD30L resulted in a significant induction of IL-13 production (Fig. 2C). In contrast, no IL-13 was detected when T cells were
incubated with control- or B7-1-expressing P815 cells. Together, these
results indicate that CD30 engagement alone on effector T cells can
induce the specific release of IL-13, in contrast to the coordinate
release of IL-4, IL-5, IL-13, and IFN- induced by coligation of CD30
and the TCR.
Cross-linking of CD30 results in rapid nuclear translocation of
NF-B
The signaling properties of CD30 in normal T cells are not known.
To begin to investigate the biochemical pathways by which CD30
engagement alone on T cells could lead to IL-13 production, we studied
pathways activated by TNFR family members in transfection systems or in
TCR-stimulated T cells. CD30 cross-linking of spontaneously
proliferating tumor cell lines has been reported to result in nuclear
translocation of NF-B (18, 23). Therefore, primed
DO11.10-Tg T cells stimulated with beads coated with combinations of
mAbs were lysed at various time points, and EMSAs were
performed using radioactively labeled NF-B- and Oct-1-binding
probes. Supershift analysis indicated that the upper band revealed by
the NF-B-binding probe was composed of heterodimers of the p50 and
p65 members of the NF-B/Rel family, whereas the lower band mostly
comprised p50-p50 homodimers (data not shown). Stimulation with beads
coated with low concentrations of anti-CD3 mAb (0.1 µg/ml)
induced a weak increase in NF-B activation at 6 h that was
further augmented at the 24-h time point when compared with treatment
with control mAb-coated beads (Fig. 3A). Both CD30 and CD28
stimulation marginally enhanced TCR-mediated NF-B activation at
these time points. Interestingly, increased NF-B activation was
observed at much earlier time points when CD30 was cross-linked in the
absence of TCR stimulation, when compared with samples incubated with
control mAb-coated beads (Fig. 3A). Maximal NF-B
activation in this setting was observed 1 h after CD30
cross-linking, although it was already detectable at the 30-min time
point, and was sustained up to 6 h. DNA-binding activity was
particularly enhanced for the NF-B p50/p65 heterodimeric complex,
which contains a DNA transactivation domain. In contrast, cross-linking
of CD28 had no reproducible effect on NF-B DNA-binding activity in
the absence of TCR ligation, confirming that CD28 engagement alone does
not signal NF-B activation in T cells. Levels of Oct1 complexes were
comparable for each set of control/anti-CD30/anti-CD28 mAbs
stimulating conditions. These results indicate that CD30 ligation alone
delivers a signal in normal T cells independent from the TCR.
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A, anti-CD30 treatment
for 1 h resulted in TCR-independent up-regulation of NF-B
activation when compared with control mAb treatment (Fig. 3B, last two lanes). Basal levels of nuclear NF-B
proteins in P815 cells were very low (Fig. 3B, first three
lanes). In contrast, robust activation of NF-B was observed in
nuclear extracts from T cells incubated with CD30L-transfected P815
cells, when compared with T cells cultured with either control- or
B7-1-transfected P815 cells. Thus, CD30-mediated TCR-independent
signaling can occur not just as a result of Ab cross-linking, but also
as a consequence of interacting with the natural ligand for
CD30. Cross-linking of CD30 results in activation of JNK and p38
Engagement of other TNFR family members has been shown to augment
TCR-mediated MAPK induction in T cells (24). Therefore, we
next determined whether ligation of CD30 alone activated MAPKs. Primed
DO11.10-Tg T cells were stimulated with Ab-coated beads and lysed at
different time points. As shown in Fig. 4A, cross-linking with
anti-CD30 (with or without anti-CD3 mAb) resulted in a dramatic
induction of JNK kinase activity within 15 min of treatment when
compared with control mAb-treated samples. The CD30-mediated
augmentation of JNK activation was also significant at 30 min of
stimulation, and was in some experiments sustained up to 60 min (data
not shown). In contrast, cross-linking of CD28 induced JNK activity
only when the TCR was coligated. CD28 ligation has been shown to
potently induce JNK activation following stimulation of normal T cells
with low concentration of TCR stimuli (25). Therefore,
absence of JNK activity following CD28 engagement alone confirmed that
JNK induction by anti-CD30 mAb alone was not due to carry-over of
Ag from the priming conditions.
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B). CD28 cross-linking
alone had no detectable effect on p38 activation. Thus, in contrast to
CD28, CD30 ligation was able to activate both JNK and p38 in a manner
independent of TCR coengagement. A proliferation assay performed in
parallel revealed similar background levels of
[3H]thymidine incorporation in T cells
stimulated with control, anti-CD30-, and anti-CD28-coated beads
(98 ± 33, 153 ± 40, and 351 ± 73 cpm, respectively),
whereas T cells stimulated with anti-CD3-coated beads (0.03
µg/ml) exhibited 100-fold more [3H]thymidine
incorporation (11,782 ± 1,026), further suggesting that the
biochemical effects observed following CD30 engagement were not due to
Ag carry-over. Induction of JNK and p38 MAPK activity could not be verified following engagement of CD30 with its natural ligand using CD30L transfectants, as high basal levels of active endogenous JNK and p38 in P815 cells obscured any detectable effect on T cells. In contrast to JNK and p38 MAPK, ERK was not induced following ligation of CD30 alone (data not shown).
Expression of the TRAF2.DN-Tg prevents CD30-mediated JNK and p38 MAPK activation
Some signals mediated by TNFR family members are transmitted
through the TRAF family of adapter proteins. In transfected 293 cells,
CD30 has been shown to associate with TRAF1 and TRAF2
(18). To determine whether CD30 could associate with TRAF
family members in normal T cells, CD30 was immunoprecipitated from
primed DO11.10-Tg T cells in the presence or absence of an
anti-hamster cross-linking Ab, and Western blot analysis was
performed using anti-CD30 or anti-TRAF2 Abs. TRAF2 was
identified in anti-CD30- but not control mAb-immunoprecipitated
samples, indicating that CD30 and TRAF2 interacted specifically in
normal T cells (Fig. 5A). In
addition, the association was not enhanced by the presence of the
cross-linker.
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B was mediated by TRAF signaling, T cells from
TRAF2.DN/DO11.10-Tg mice were used. The truncated DN form of TRAF2 is
unable to mediate downstream signaling events, but can bind to TNFR
family members and presumably blocks binding of endogenous TRAF
proteins to the CD30 cytoplasmic sites that can bind TRAF1, 2, and 3.
We verified that primed TRAF2.DN-Tg exhibited similar levels of surface
CD30 as control DO11.10-Tg CD4+ T cells (data not
shown). To control for the effect of the TRAF2.DN transgene,
CD30-expressing primed T cells were stimulated with TNF for 10 min and
JNK activation was examined, as this was reported to be inhibited in
TRAF2.DN-Tg lymphocytes (19). As expected, TNF-mediated
JNK activation was blocked in TRAF2.DN-Tg when compared with wild-type
(WT) T cells, confirming the DN effect of the transgene (data not
shown).
When CD30 was cross-linked alone, significant induction of JNK
activation was observed in wild-type (WT) DO11.10-Tg T cells. In
contrast, no augmentation of JNK activation was detectable in the
TRAF2.DN-Tg cells treated with anti-CD30 mAb (Fig. 5B).
This was not due to an inability of TRAF2.DN-Tg T cells to
up-regulate JNK activity, as CD28 cross-linking effectively
resulted in up-regulation of JNK in anti-CD3-treated cells when
compared with cells treated with anti-CD3 and control mAb (Fig. 5B).
To determine whether CD30-mediated activation of p38 was TRAF
dependent, Western blot assays were performed using the lysates from
the JNK kinase assay following the GST-c-jun pull-down. As
shown for JNK activation, the presence of the TRAF2.DN transgene
completely blocked TCR-independent CD30-mediated activation of p38
(Fig. 5C). In contrast, when CD28 was cross-linked in
combination with TCR stimulation, p38 activation was increased,
indicating that other conditions could induce p38 activation in
TRAF2.DN-Tg T cells. Taken together, these results indicate that
association of CD30 with TRAF family members is necessary for
CD30-mediated TCR-independent activation of JNK and p38.
To address whether NF-B activation downstream of CD30 depended on
CD30 association with TRAF family members, nuclear extracts were
prepared from bead-stimulated primed DO11.10-Tg T cells. CD30
cross-linking in the absence of TCR stimulation resulted in robust
activation of NF-B in both the control and the TRAF2.DN-Tg T cells
(Fig. 5D). Thus, in contrast to JNK and p38 activation, the
presence of the TRAF2.DN transgene did not prevent CD30-mediated
activation of NF-B, suggesting that NF-B activation induced by
CD30 engagement can occur in a TRAF-independent manner.
IL-13 production following CD30 ligation is TRAF and p38 dependent
IL-13 production upon CD30 engagement correlated with TRAF
association and activation of JNK, p38, and NF-B. In addition,
normal TRAF signaling correlated with activation of JNK and p38, but
not of NF-B. To determine whether IL-13 production was dependent on
TRAF association and/or NF-B activation, experiments were repeated
using primed T cells from DO11.10-Tg, TRAF2.DN/DO11.10-Tg, and
DO11.10-Tg mice crossed with mice expressing an I-B.DN-Tg under
the control of a T cell-specific promoter/enhancer pair. T cells from
I-B.DN-Tg exhibit dramatically reduced NF-B activation upon T
cell stimulation (20). EMSAs confirmed a 70–80%
reduction of nuclear NF-B activity, both in naive and stimulated
CD4+ I-B.DN/DO11.10-Tg T cells when
compared with control DO11.10-Tg T cells (data not shown). Primed
I-B.DN/DO11.10-Tg CD4+ T cells exhibited
similar levels of surface CD30 as control DO11.10-Tg and
TRAF2.DN/DO11.10-Tg CD4+ T cells (data not
shown). Primed T cells were stimulated with P815 cells transfected with
CD30L- or B7-1-encoding vectors. As shown in Fig. 6A, similar IL-13 induction
was observed in control and NF-B activation-defective T cells
following CD30 engagement alone. Importantly, IL-13 was not induced by
CD30 engagement in TRAF2.DN-Tg T cells, indicating that TCR-independent
IL-13 induction by CD30 is absolutely TRAF dependent, but appears to be
NF-B independent.
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B). Taken together, these results indicate that the
production of IL-13 induced by CD30 cross-linking alone is dependent
upon TRAF association and p38 MAPK kinase activation, but can occur in
the absence of complete NF-B activation. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. These findings provide evidence for a mechanism whereby
TCR-independent CD30 engagement may function to modulate the profile of
cytokines released by T cells at sites of inflammation.
TCR-independent T cell function has recently been described in a few
systems. Type I IFN and IL-15 have been shown to induce proliferation
of memory CD8+ T cells in vivo, in an
NF-B-dependent, TCR-independent manner (26). IL-12 and
IL-18 synergize to induce transcription of IFN- by
CD4+ T cells in a TCR-independent manner
(27). Cross-linking of TNFR family members such as Fas and
TNFRI results in complex signaling events in T cells leading to
apoptosis (13). Signaling by these proapoptotic receptors
appears to require a death domain present in their cytoplasmic tail
that is not shared by CD30. In this study, we show that CD30 engagement
leads to IL-13 production by T cells in a TCR-independent manner. Thus,
CD30 represents a TNFR family member that activates effector T cell
function via a death domain-independent pathway.
TCR-independent selective cytokine production has not been described in
normal T cells for the prototypic costimulatory receptor CD28. In fact,
TCR-independent production of IL-2 following CD28 cross-linking only
occurred when normal human T cells were transfected with constructs
encoding both VAV and SLP76 adaptors (28). Overall, the
effects of CD28 engagement in normal T cells appear to be different
from those of CD30: in contrast to CD30, ligation of CD28 did not
result in activation of JNK, p38 MAPK, NF-B, or in cytokine
production in primed DO11.10-Tg T cells.
Other TNFR family members such as 4-1BB and Ox40 that are up-regulated
following initial T cell activation also augment TCR-mediated
biochemical signals and cytokine production. 4-1BB in particular has
been shown to increase TCR-mediated activation of JNK and p38 in normal
T cells at 24 and 48 h of stimulation (24). Whether
Ox40 or 4-1BB would also have TCR-independent effects in normal T cells
remains to be demonstrated. In a T cell hybridoma, 4-1BB engagement led
to rapid activation of JNK and p38 in the absence of TCR stimulation
(24). In Jurkat cells, aggregation of the Ox40 or 4-1BB
cytoplasmic tail alone resulted in NF-B activation
(29). However, these signaling events were not reproduced
in normal T cells (24), and no correlation with a
functional T cell outcome was reported. This may be because experiments
were performed using naive T cells that do not express TNFR family
members, and perhaps experiments performed in primed effector T cells
might reveal such an effect. Thus, whether similar events as we
describe for CD30 would be observed following ligation of other TNFR
family members in normal T cells, or whether this is a unique feature
of CD30, remains to be established.
Several differences between CD30 and 4-1BB are already apparent. First,
the TCR-dependent costimulatory function of 4-1BB has been found to
require TRAF association, as both p38 activation and the enhancement of
cytokine production induced by 4-1BB engagement during TCR stimulation
were shown to be inhibited in TRAF2.DN-Tg T cells (24, 30). In our system, although TRAF association appeared necessary
for TCR-independent induction of p38 and JNK activation by CD30
ligation, the presence of the TRAF2.DN transgene did not prevent the
enhancement by CD30 ligation of TCR-mediated IL-5 or IFN- production
(data not shown).
Second, coupling of 4-1BB to TRAF1 and TRAF2 has been reported to require cross-linking of 4-1BB (30). In the primed CD30-expressing normal T cells used in our study, TRAF2 association with CD30 was detected in the absence of secondary Ab cross-linking. Although it is conceivable that CD30 molecules were already aggregated from the priming conditions, our results suggest that TRAF2 may be constitutively associated with CD30 in normal T cells.
TCR-independent CD30-mediated activation of JNK and p38 was TRAF dependent. Activation of these MAPKs was specific, as no induction of ERK activation was detected under similar TCR-independent stimulating conditions (data not shown). Our results also indicate that IL-13 production mediated by CD30 engagement is p38 dependent, as production of IL-13 following CD30 ligation was significantly reduced in a dose-dependent manner by SB203580. However, it is known that high concentrations of SB203580 can also inhibit JNK activation in addition to p38 activation. As no specific JNK inhibitor was commercially available at the time of these experiments, it is possible that JNK activation may also be involved in CD30-dependent TCR-independent IL-13 production. Interestingly, p38 MAPK was also identified as a mediator of IL-12- and IL-18-induced TCR-independent T cell proliferation (27), suggesting that p38 MAPK may be a key regulator of TCR-independent T cell functions.
In contrast to activation of JNK and p38, NF-B activation by CD30
alone was not affected by the presence of the TRAF2.DN transgene. It is
possible that residual association of CD30 with endogenous TRAF
molecules was sufficient for NF-B activation, but not for JNK and
p38 activation. However, in transfection systems using cell lines, both
TRAF-dependent and TRAF-independent sites for NF-B activation have
been described in the cytoplasmic tail of CD30 (18).
Furthermore, one report has indicated that CD30 could also associate
with TRAF5 in addition to TRAF1, TRAF2, and TRAF3, and that TRAF5 may
bind to an adjacent, but separate region from the two conventional
binding sites (16). If this occurs in normal T cells and
TRAF5 can also mediate NF-B activation, the presence of a TRAF2.DN
transgene may not prevent this association. TRAF5-deficient lymphocytes
have been reported to have defects in CD27 and CD40 signaling
(31), suggesting that TRAF5 may play a role in signaling
by TNFR family members in normal T cells. In preliminary experiments,
we have examined TRAF5-deficient T cells and found that CD30-mediated
induction of JNK, p38, and NF-B is intact (data not shown).
The finding of a mechanism by which CD30/CD30L interactions alone, in the absence of TCR stimulation, can selectively promote IL-13 release has important implications for the pathophysiology of several diseases. IL-13 plays an important role in elimination of the parasite Nippostrongylus brasiliensis (32, 33). However, IL-13 also has an unwanted role in promoting allergic asthma, probably as a result of eotaxin induction and recruitment of eosinophils into the lungs (34, 35, 36). Interestingly, eosinophils express high levels of CD30L that could engage CD30 on primed T cells, inducing further IL-13 production even in the absence of Ag, and thus possibly amplifying the allergic response. Furthermore, IL-13 has recently been shown to promote cancer progression by two independent mechanisms. First, IL-13 appears to limit immune surveillance by T cells, and blockade of IL-13 has been shown to enhance tumor elimination in vivo (37) by a mechanism dependent on STAT6 (38). Second, IL-13 may act as a growth factor for certain tumors, promoting tumor expansion (39). In Hodgkin’s disease, CD30+ Reed-Sternberg (H/RS) cells are surrounded by nonneoplastic cells, many of which are mast cells expressing CD30L (40). IL-13 production following engagement of CD30 on T cells by CD30L on mast cells could potentially both directly promote growth of certain tumor types and reduce tumor elimination by T cells. Thus, our findings raise the possibility that CD30 on T cells may play a role in down-regulating immune surveillance and promoting tumor expansion, and in exacerbating airway inflammation through the ability of CD30L+ eosinophils or mast cells to evoke TCR-independent IL-13 release. Furthermore, CD30 represents an immune regulator the ligation of which dictates distinct T cell effector responses depending on whether the TCR is concurrently engaged or not.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Maria-Luisa Alegre, University of Chicago, 5841 South Maryland Avenue, MC 0930, Chicago, IL 60637. E-mail address: malegre{at}midway.uchicago.edu
3 Abbreviations used in this paper: TRAF, TNFR-associated factor; CD30L, CD30 ligand; DN, dominant negative; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein/ERK kinase; Tg, transgenic; WT, wild type.
Received for publication May 9, 2002. Accepted for publication July 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation. J. Biol. Chem. 272:2042.B by the CD30 receptor is mediated by TRAF1 and TRAF2. Mol. Cell. Biol. 17:1535.[Abstract]
B activation and regulates lymphocyte proliferation and survival. Immunity 7:703.[Medline]
B. J. Exp. Med. 185:1897.B activation in human T cell lines. Eur. J. Immunol. 25:2870.[Medline]
required in cytokine-induced, but not TCR-induced, IFN- production. Nat. Immun. 2:157.[Medline]
B. Mol. Cell. Biol. 18:558., and Stat6 are required for the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity 8:255.[Medline]
-deficient and IL-4-deficient mice reveal a role for IL-13 in the regulation of Th2 responses. Curr. Biol. 8:669.[Medline]
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