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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1448-1455
Downregulation of Interleukin-12 (IL-12) Responsiveness in Human T
Cells by Transforming Growth Factor- : Relationship
With IL-12 Signaling
By
Cécile Pardoux,
Xiaojing Ma,
Stéphanie Gobert,
Sandra Pellegrini,
Patrick Mayeux,
Françoise Gay,
Giorgio Trinchieri, and
Salem Chouaib
From the Laboratoire Cytokines et Immunologie des Tumeurs Humaines,
INSERM U487, Institut Gustave Roussy, Villejuif; INSERM U363,
Hôpital Cochin, Paris; INSERM U276, Institut Pasteur, Paris,
France; and The Wistar Institute, Philadelphia, PA.
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ABSTRACT |
Interleukin-12 (IL-12) is a cytokine that plays a central role in
the control of cell-mediated immunity. We have previously shown that
transforming growth factor- 1 (TGF-) inhibitory effects on human
primary allogeneic cytotoxicity and proliferative responses interfere
with IL-12 pathway. The present study was undertaken to further
elucidate the biochemical basis of the functional interaction between
these two cytokines and to define the site of TGF- action on the
signaling pathway activated by IL-12. Our data indicate that TGF-
induced an inhibition of interferon- (IFN-) production without
affecting the IL-12R1 and IL-12R2 subunits mRNA expression by
activated T cells. We further show that TGF- has a significant inhibitory effect on the early signal transduction events following interaction of IL-12 with its receptor on activated T cells, resulting in the inhibition of both JAK2 and Tyk2 phosphorylation. In addition, TGF- was found to significantly inhibit IL-12-induced
phosphorylation of the STAT4 transcription factor. Electrophoretic
mobility shift assay indicated that TGF- induced a decrease in
IL-12-induced STAT4 DNA binding activity in T lymphocytes. This study
suggests that TGF- influences IL-12 responsiveness at least in part
by inhibiting early signaling events essential to gene induction in
IL-12-activated T cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTERLEUKIN-12 (IL-12), a potent
proinflammatory cytokine, plays a central role in the initiation and
control of cell-mediated immunity.1 It acts on T and
natural killer (NK) cells as costimulator of proliferation, inducer of
cytokine production,2-7 and by enhancing the generation as
well as the cytolytic activity of both cell types.8-13
IL-12 produced by accessory cells during early antigenic stimulation
has been shown to be a powerful inducer of Th1
responses,14,15 and a defect in its production has been
suggested to be a factor contributing to immune
depression.16,17 The biologic activity of IL-12 is mediated
by the binding of the IL-12 heterodimer to cell-surface receptors on
activated T and NK cells.18,19 The existence of multiple
forms of IL-12 receptor (IL-12R) has been reported.20,21
One component of the IL-12R, originally called IL-12 receptor chain
(IL-12R) and more recently designated IL-12R1, is a gp130-like
member of the hematopoietin receptor superfamily.21 When
expressed in COS cells, IL-12R1 binds IL-12 with low
affinity. Although the IL-12R1 chain per se is not
sufficient for IL-12 signaling, the inability of IL-12R1 knock-out
mice to respond to IL-12 suggests that this chain is an essential
component of the functional IL-12R.22 Recently, a second
component of the human IL-12 receptor has been cloned.23
This component, called IL-12R2, belongs to the same receptor family
as the IL-12R1 subunit. Several studies suggest that this subunit
acts as a high-affinity converter and is necessary for IL-12
signaling.23 To date, the JAK/STAT signal transduction
pathway is the best characterized signal transduction pathway used by
IL-12. IL-12 treatment of activated human T cells induces rapid
tyrosine phosphorylation of two members of the JAK (Janus kinases)
tyrosine kinases family, JAK2 and Tyk2, implicating these kinases in
the immediate biochemical response to IL-12.24,25 A number
of studies have characterized a family of transcription factors called
STATs (signal transducers and activators of transcription), which are
involved in the signal transduction cascades of many cytokines known to
activate JAK kinases.26 Evidence has been provided that
IL-12 induces STAT4 tyrosine phosphorylation and DNA binding
in phytohemagglutinin (PHA)-activated human T
cells.27,28 Given the role of IL-12 in determining the
nature of immune responses, understanding the regulation of its
production and its signaling is of major interest.
Transforming growth factor-1 (TGF-) is arguably the most potent
immunosuppressive cytokine. It belongs to a family of pleiotropic polypeptide factors that regulate cell growth and
differentiation.29 Among its immune properties, TGF-
modulates proliferation, differentiation, and functions of macrophages,
T cells, B cells, and NK cells, thus regulating the innate,
non-Ag-specific as well as the Ag-specific immunity. Several studies
have indicated that TGF--induced functional inhibition involves
modulation of cytokine production (ie, tumor necrosis factor-
[TNF-], interferon- [IFN-], IL-1, IL-2) and cytokine-receptor surface expression cells.30-35 In this
context, we have previously shown that TGF- inhibits the development
of cytotoxic and proliferative allogeneic responses by a mechanism involving downregulation of IL-12 production following
allostimulation.36 Addition of exogenous IL-12 in the
primary mixed lymphocyte reaction (1° MLR) cultures in the presence
of TGF- did not result in reversal of CTL generation and T-cell
proliferation, suggesting that TGF- may interfere with IL-12R
expression or with the signal transduction pathway initiated through
the IL-12R. The present study was undertaken to further examine the
molecular and biochemical mechanism of TGF- inhibitory
effect on IL-12 pathway in human activated T cells. We show
that TGF- interferes with IL-12 responsiveness at least by a
mechanism involving an alteration of IL-12-induced tyrosine
phosphorylation of the kinases JAK2 and Tyk2 as well as the activation
of the transcription factor STAT4.
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MATERIALS AND METHODS |
Cytokines, antibodies (Abs), and reagents.
Recombinant human IL-12 (1.7 × 107 U/mg) was kindly
provided by S. Wolf (Genetics Institute, Cambridge, MA). Recombinant
human TGF-1 was purchased from R&D systems (Abingdon-Oxon, UK).
Polyclonal rabbit antiserum against JAK2 and monoclonal
antiphosphotyrosine Ab (clone 4G10) were purchased from Upstate
Biotechnology, Inc (Lake Placid, NY). Polyclonal rabbit Tyk2 antiserum
and the T10-2 monoclonal mouse Ab anti-human Tyk2, used for
immunoprecipitation and immunoblotting, respectively, have been
previously described.37 Polyclonal rabbit antisera against
human STAT4 (C-20, L-18) were purchased from Santa Cruz Biotechnology
Inc (Santa Cruz, CA). Goat anti-mouse and anti-rabbit Abs conjugated
with horseradish peroxidase were respectively purchased from Amersham
(Arlington Heights, IL) and Immunotech (Marseille, France).
Isolation and activation of T cells.
Normal human T cells (>90% CD3+) were isolated from the
peripheral blood of healthy donors (Banque du Sang, Hôpital
Saint-Louis, Paris) by Percoll gradient centrifugation as previously
described.38 Mixed lymphocyte reaction (MLR): T cells (0.5 × 106 cells/mL) were alloactivated with irradiated
(6,000 rads) stimulating B lymphoblastoid cells, E418 (0.125 × 106 cells/mL) at 37°C in 5% CO2 for 6 days
in RPMI 1640 medium (Biochrom KG, Berlin, Germany) supplemented with
15% heat-inactivated human serum (Institut J. Boy, Reims, France),
L-glutamine (2 mmol/L), penicillin (100 IU/mL), and streptomycin (100 µg/mL) (complete medium).
Before any stimulation with or without cytokines, 6-day alloactivated T
cells were acid-treated (RPMI, pH 6.4) for 1 minute and washed with
RPMI and resuspended in starvation medium (Dulbecco's modified
Eagle's medium [DMEM; Biochrom KG, Berlin, Germany]) for 4 hours.
IFN- assay.
Six-day alloactivated T cells were resuspended (1 × 106/mL) in RPMI 1640 + 10% human serum in medium alone,
IL-12 (2 U/106 cells), TGF- (2.5 ng/106 cells), or IL-12 plus TGF-. Supernatants were
collected and tested for INF- production after 48 hours of culture
(found to be optimal under our experimental conditions as determined by kinetics studies). IFN- secretion was measured by a specific enzyme-linked immunosorbent assay (ELISA; Genzyme; Cambridge, MA).
Ribonuclease protection assay for IL-12R mRNA expression.
For analysis of IL-12R1 and IL-12R2 chains mRNA expression,
alloactivated T cells (day 6 of the MLR) were resuspended (1 × 106/mL) in RPMI 1640 + 10% human serum in the presence or
the absence of TGF- (2.5 ng/106 cells). After 24, 48, and 72 hours of incubation, T cells were obtained and total RNA was
extracted using a standard guanidium thiocyanate method. Ribonuclease
protection assays (RPA) were performed with 7 µg/lane total RNA using
the Pharmingen probe kit hCR-3 (San Diego, CA) according to the
company's protocol. Products were resolved on 6% denaturing
polyacrylamide gels and the protected fragments were visualized and
quantitated using a PhosphorImager 445 SI (Molecular Dynamics,
Sunnyvale, CA). Relative radioactivity values for IL-12R1 and
IL-12R2 transcripts were determined by normalizing to the values
obtained for L32, which was used as internal control for equal RNA loading.
Preparation of cytosolic cell extracts and immunoprecipitation.
Alloactivated T cells were resuspended (20 × 106
cells/mL) in DMEM and incubated for 20 minutes with IL-12 (5 U/106 cells), TGF- (1 ng/106 cells), or
IL-12 plus TGF- at 37°C. The reaction was terminated by addition
of ice-cold phosphate-buffered saline (PBS) containing 100 µmol/L
sodium orthovanadate (Na3VO4). After
centrifugation, cells were washed rapidly with
PBS/Na3VO4 and lysed in RIPA buffer containing
50 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, 1 mmol/L EDTA, 0.5% sodium
deoxycholate, 1% NP40, 0.05% sodium dodecyl sulfate (SDS), 1 mmol/L
phenyl-methyl-sulfonyl fluoride (PMSF), 1 mmol/L O-Phenantroline, 2 mmol/L sodium orthovanadate, 1 mmol/L NaF, 10 µg/mL aprotinin,
leupeptine, and 1 µg/mL pepstatin A. After 15 minutes of incubation
on ice, detergent insoluble materials were removed by centrifugation at
4°C for 20 minutes at 13,000g. The protein concentration
was determined using a BCA protein assay (Pierce Chemical Co, Rockford, IL).
For immunoprecipitations, cell lysates were precleared by incubation
with 20 µL of protein A-coupled Sepharose beads (Pharmacia Biotech,
Uppsala, Sweden) for 1 hour under rotation at 4°C.
After removal of the beads, the lysates (0.5 to 1 mg of protein) were incubated under rotation with an appropriate Ab (1 to 3 µg for each
sample) for 1 hour at 4°C, followed by addition of 30 µL of
protein A-Sepharose and incubation for 1 hour under the same conditions. The immunoprecipitates were then washed three times with
cold lysis buffer and prepared in Laemmli-reducing buffer.
Gel electrophoresis and immunoblotting.
SDS-polyacrylamide gels were prepared according to the Laemmli protocol
and used for immunoblotting. The concentration of polyacrylamide varied
from 7% to 10% depending on the molecular weight range
of the studied proteins. Equal amounts of protein were used in
immunoblotting experiments. All samples were prepared in the
Laemmli-reducing buffer and boiled for 5 minutes before application.
Gels were blotted onto protean nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) for 1 hour at 100 V. Blots were developed by
incubating in a blocking buffer containing 3% bovine serum albumin
(BSA) and 0.3% Tween 20 in Tris-buffered saline (TBS) for 2 hours,
followed by incubation in the primary Abs for 1 hour. After washing
three times in TBS-Tween, blots were incubated for 1 hour with the
secondary Ab conjugated with horseradish peroxidase. Detection was
performed by the use of enhanced chemiluminescence (ECL; Amersham).
When a membrane was reprobed, it was first stripped in acetic acid 0.1 mol/L at room temperature for 12 minutes.
Autoradiograms were scanned and intensity of each band was
densitometrically quantitated using the MacBAS image-analysing software
(v2.2, Fuji, France). Relative inhibition of the phosphotyrosine signal
was determined by first normalizing each signal with the values
obtained for the corresponding sample of immunoprecipitated protein.
Nuclear extracts and electrophoretic mobility shift assay (EMSA).
After cytokine stimulation as above, nuclear extracts were prepared
from cytokine-stimulated T cells as previously described.39 EMSAs were done using a 32P end-labeled double-stranded oligonucleotide (5'-GTATTTCCCAGAAAAAG-3') corresponding to the IFN-
response element (GRR) of the human Fc receptor I (FcRI)
gene.39,40 Briefly, 5 µg of nuclear extracts were
incubated with end-labeled probe (50,000 cpm/sample) and 1 µg of
poly(dI-dC) in binding buffer (20 mmol/L HEPES, pH 8, 0.2 mmol/L EDTA,
100 mmol/L NaCl, 100 mmol/L KCl, 10 mmol/L MgCl2, 8 mmol/L
spermidine, 4 mmol/L DTT, 200 µg/mL BSA, 5% glycerol, 8% Ficoll
400) for 30 minutes at 4°C before electrophoresis on 6%
polyacrylamide gels, drying, and autoradiography. For supershift
experiments, nuclear extracts were preincubated with polyclonal rabbit
anti-STAT4 (L18X, 3 to 4 µg/sample) for 60 minutes at 4°C before
the start of the binding reaction. Autoradiograms were scanned and
intensity of each band was densitometrically quantitated using the
MacBAS image-analyzing software (v2.2).
| |
RESULTS |
TGF- inhibits IL-12-induced IFN-
production by activated T cells.
We have recently reported that IL-12 added exogenously at the
initiation of the 1° MLR failed to restore CTL generation and T-cell proliferation in the presence of TGF-.36 In the
present study, we asked whether TGF- intereferes with IL-12
responsiveness of activated T cells. Because IL-12 is a potent inducer
of IFN-, we have examined the effect of TGF- on the ability of
IL-12 to induce IFN- production by alloactivated T cells.
Figure 1 shows that IL-12 induced IFN-
production by these cells upon 48 hours of incubation. In the presence
of exogenous TGF- (2.5 ng/106 cells), a 50% inhibition
(obtained after substracting the amount of IFN- produced by the T
cells in response to medium alone) of IL-12-induced IFN- production
was observed, suggesting that TGF- inhibits IL-12 T-cell
responsiveness.
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| Fig 1.
Effect of TGF- on IL-12-induced IFN- production of
alloactivated T cells. Alloactivated T cells were incubated at
1.106/mL for 48 hours in medium alone, IL-12 (2 U/106 cells), TGF- (2.5 ng/106 cells), or
IL-12 plus TGF-. Culture supernatants were then collected and tested
for IFN- production. Results are presented as mean ± SD of
duplicate determinations. IFN- maximal production represents the
IFN- production obtained with IL-12. Similar results were obtained
with TGF- ranging from 1 to 5 ng/106 cells. Equivalent
results were obtained in three separate experiments.
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Effect of TGF- on IL-12R1 and
IL-12R2 subunits expression in alloactivated T cells.
To determine whether the TGF--induced alteration of IL-12
responsiveness was associated with modulation of IL-12R expression, we
examined by the RPA IL-12R1 and IL-12R2 mRNA expression in alloactivated T cells incubated for 24, 48, and 72 hours in the presence or absence of TGF- (2.5 ng/106 cells). As shown
in Fig 2A, IL-12R2 mRNA transcription
was slightly affected by incubation with TGF- for 48 and 72 hours.
In a study with 4 different donors (Fig 2B), this modest inhibition
(20% to 25%) was found to be not significant, as judged by a
nonparametric Wilcoxon test, thus indicating that TGF- had no
significant effect on the regulation of the IL-12R2 subunit gene
expression in activated T lymphocytes. As seen in Fig 2, TGF- had no
effect on the mRNA expression of the IL-12R1 subunit nor on its
protein surface expression, observed by immunofluorescence analysis
(data not shown). These data suggest that TGF--induced inhibition
of IL-12 responsiveness is not associated with modulation of IL-12R
expression. Thus, TGF- may act downstream of the IL-12R.
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| Fig 2.
Effect of TGF- on IL-12R expression in alloactivated T
cells. Six-day (d6) alloactivated T cells were incubated at
1.106/mL in the presence or absence of TGF- (1 ng/106cells) at 37°C. Total RNA was extracted after 24, 48, and 72 hours of incubation, and transcripts encoding the IL-12R1
and IL-12R2 subunits, L32 and GAPDH, as loading control were
quantitated by ribonuclease protection assays as described in Materials
and Methods. (A) RPA bands of selected mRNA accumulation from a
representative donor. Relative radioactivity values for IL-12R1 and
IL-12R2 transcripts (upper row), after normalization against L32
(lower row) values, expressed in arbitrary units for lanes 1 through 7, respectively, were: 37143, 33773, 28275, 30560, 23923, 19729, and
16587. (B) Relative expression of IL-12R1 and IL-12R2 mRNA
obtained from four donors. Data are given as mean (± SD) relative
radioactivity values (expressed in arbitrary units) for IL-12R1 and
IL-12R2 transcripts from four donors after normalization against the
corresponding L32 values.
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TGF- effect on IL-12-induced JAK2 and Tyk2
phosphorylation.
To determine the molecular and biochemical basis of TGF--induced
attenuation of IL-12 responsiveness, we asked whether this was
associated with the inhibition of IL-12-induced tyrosine
phosphorylation of JAK2 and Tyk2. Western blot analysis of JAK2 and
Tyk2 immunoprecipitates from alloactivated T cells stimulated with
IL-12 (5 U/106 cells) for 20 minutes exhibited an induction
of tyrosine phosphorylation of JAK2 and Tyk2
(Fig 3A and B). Treatment with TGF- (1 ng/106 cells) led to a significant decrease in the
IL-12-induced phosphorylation of Tyk2 kinase (75% of inhibition as
evaluated by densitometric quantitation, Fig 3B), and to a lesser
extent (34% of inhibition) of JAK2 kinase (Fig 3A). The TGF- effect
on IL-12-induced tyrosine phosphorylation of Tyk2 and JAK2 can be seen
as early as 20 minutes after incubation with TGF-, suggesting that
this effect is independent from protein synthesis. When the blots were
reprobed with anti-Tyk2 or anti-JAK2 Abs, no difference was observed in
protein levels following treatment with TGF-. Although the level of
TGF--induced inhibition varied from donor to donor (mean ± SD
on 5 different donors = 55 ± 20% for Tyk2 and = 38 ± 16% for
JAK2), it was found to be very significant by a nonparametric Wilcoxon
test (P = .0036 and .0069 for Tyk2 and JAK2, respectively, data
not shown).
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| Fig 3.
Inhibition of IL-12-induced tyrosine phosphorylation of
JAK2 and Tyk2 kinases by TGF-. Western blot analysis of 6-day
alloactivated T cells incubated in medium alone, IL-12 (5 U/106 cells), TGF- (1 ng/106 cells), or
IL-12 + TGF- at 37°C for 20 minutes. The JAK2 (A) and Tyk2 (B)
immunoprecipitates were resolved on 7% SDS-PAGE, transferred to
nitrocellulose membrane, and probed with anti-phosphotyrosine Ab 4G10
(upper panels). The blots were stripped and reprobed with anti-JAK2 or
anti-Tyk2 Abs (lower panels). Similar results were obtained in five
separate experiments.
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Inhibition of IL-12-induced STAT4 phosphorylation in
alloactivated T cells by TGF-.
Activation of JAKs leads to tyrosine phosphorylation of STAT
factors.26,41 In both human and murine T cells, the
transcription factor STAT4 appears to be quite specifically activated
by IL-12 and is essential for IL-12-mediated responses in
lymphocytes.42,43 To determine whether TGF- inhibits
IL-12-induced STAT4 tyrosine phosphorylation, alloactivated T cells
were stimulated with IL-12 in the presence or absence of TGF-. Cell
lysates were then immunoprecipitated with STAT4 Abs, resolved by
SDS-polyacrylamide gel electrophoresis (PAGE), and analyzed by
antiphosphotyrosine immunoblotting. As shown in
Fig 4, stimulation of T cells with IL-12
induced tyrosine phosphorylation of STAT4. Treatment of these cells
with TGF- partially inhibited IL-12-induced tyrosine
phosphorylation of STAT4 protein (45% inhibition as evaluated by
densitometric quantitation). This inhibition was observed after 20 minutes of treatment with TGF-, suggesting that this phenomenon was
also independent of protein synthesis. As seen on reprobed blots with
anti-STAT4 Ab, neither TGF- nor IL-12 altered STAT4 expression level
under our stimulating conditions. The TGF--induced inhibition (mean ± SD on 6 different donors = 50 ± 28%) was found to be
significant by a nonparametric Wilcoxon test (P = .0081, data
not shown).
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| Fig 4.
Attenuation of IL-12-induced tyrosine phosphorylation of
STAT4. Western blot analysis of 6-day alloactivated T cells incubated
in medium alone, IL-12 (5 U/106 cells), TGF- (1 ng/106 cells), or IL-12 + TGF- at 37°C
for 20 minutes. The STAT4 immunoprecipitates were resolved on 10%
SDS-PAGE, transferred to nitrocellulose membrane, and blotted
sequentially with anti-phosphotyrosine (upper panel) and anti-STAT4
(C-20) Abs (lower panel). Similar results were obtained in six separate
experiments.
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Alteration of IL-12-induced STAT4 DNA binding in alloactivated T
cells by TGF-.
Because tyrosine phosphorylation of STAT proteins is associated with
and is required for the binding of STATs transcription factors to
conserved promoter elements,44 we asked whether the inhibition of STAT4 tyrosine phosphorylation by TGF- observed in our
model was associated with an inhibition of IL-12-induced DNA binding
activity. Nuclear cell extracts from 6-day alloactivated T cells
stimulated with IL-12 in the presence or absence of TGF- were
examined by EMSA for binding to an oligonucleotide probe corresponding
to the GRR of the human Fc receptor I gene. This probe is known to
contain a high-affinity binding site for various STAT factors,
including STAT4. As shown in Fig 5,
treatment of T cells with IL-12 induced a strong binding activity
(IL-12-stimulated factor, IL-12 SF) at the FcRI probe in 20 minutes
(lane 3 compared with lane 1). In contrast to the constitutive complex
migrating under the IL-12 SF, the IL-12 SF complex could be displaced
with 100-fold excess of unlabeled FcRI oligonucleotide indicating that this protein/DNA interaction was specific (data not shown). Supershift experiments confirmed that the IL-12 complex contained STAT4, because rabbit antiserum to STAT4 but not normal rabbit serum
(data not shown) interfered with formation of the IL-12-induced protein/DNA complex (lane 4 compared with lane 2). As shown in lanes 5 and 6, IL-12 stimulation of T cells in the presence of TGF-
exhibited a lower induced DNA binding activity of STAT4 (36% of
inhibition as evaluated by densitometric quantification), as compared
with IL-12 alone (lanes 3 and 4), indicating that TGF- downregulates
IL-12-induced STAT4 activation.
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| Fig 5.
TGF- inhibits IL-12-induced DNA binding of
STAT4-containing complexes. Six-day alloactivated T cells were
incubated in medium alone (lanes 1 and 2), IL-12 (5 U/106
cells, lanes 3 and 4), TGF- (1 ng/106 cells, lanes 7 and
8), or IL-12 + TGF- (lanes 5 and 6) at 37°C for 20 minutes.
Nuclear cell extracts were prepared and an EMSA was done using the GRR
oligonucleotide probe. Where indicated, antiserum against STAT4 (lanes
2, 4, 6, and 8) was incubated with the cell extracts for 60 minutes
before addition of the probe. Similar results were obtained in three
separate experiments.
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DISCUSSION |
Cytokines form a network of regulatory signals with considerable
overlaps in their activities that lead to unexpected patterns of
synergism or antagonism resulting in the modulation of the clonal
expansion and differentiation of Ag-reactive cells. It is well
established that IL-12 has a major role in the cytokine network and is
an immunoregulatory cytokine important in the promotion and control of
cell-mediated immunity.45 This cytokine represents an
important link between innate immunity and specific immune responses.1 Therefore, the elucidation of its interaction
with the immunosuppressive cytokine TGF- may be of considerable
importance in the understanding of T-cell activation and the
development of T-cell immune response.
We showed in a recent study that addition of exogenous TGF- at the
sensitizing phase of the 1° MLR resulted in the inhibition of both
allogeneic cytotoxic and proliferative human T-cell
responses.36 This inhibitory effect of TGF- involved an
abrogation of IL-12/p70 production. Based on the failure of exogenous
IL-12 to restore the allogeneic response, we postulated that TGF-
may exert its inhibitory effect by additional mechanisms distinct from
inhibition of IL-12 production. When alloactivated T cells, expressing
both IL-12R1 and IL-12R2 subunits, were examined for their
ability to produce IFN- in response to exogenous IL-12 in the
presence of TGF-, a significant decrease in IFN- production was
observed (Fig 1), suggesting that TGF- treatment resulted in an
alteration of the ability of T cells to respond to IL-12.
Because TGF- has been reported to modulate the expression of surface
receptors important in cell activation and growth,29 we
investigated further whether the effect of TGF- on IL-12
responsiveness was associated with regulation of IL-12R expression.
Recent evidence indicates that the IL-12R subunits play a distinct
functional role in the triggering of IL-12 responsiveness. The
expression of a functional IL-12R (high affinity) requires the
IL-12R1 chain and the IL-12R2 chain, which acts as a
high-affinity converter crucial in IL-12
signaling.18,19,21,23 Several studies have clearly shown
that the pattern of IL-12 responsiveness correlated with the expression
of this IL-12R2 subunit.46-49 Although it has been
reported by Wu et al50 that TGF- can inhibit the
upregulation of the IL-12R1 and IL-12R2 expression during
anti-CD3 activation of naive T cells, our data clearly show that
TGF- has no significant effect on the regulation of IL-12R1 and
IL-12R2 subunits gene expression in alloactivated T lymphocytes (Fig
2). However, when added at the initiation of the allostimulation,
TGF- selectively interferes with the upregulation of the IL-12R2
mRNA expression (44% to 61% of inhibition at day 6 of the
allostimulation) during alloactivation of naive T cells (data not
shown). Thus, it appears that TGF- inhibitory effect on IL-12R2
mRNA expression depends on the experimental conditions, more precisely
on the state of activation of T cells. Although TGF- exerts an
inhibitory effect on the primary induction of the IL-12R2
expression, it is unable to downregulate IL-12R2 expression on
activated T cells. Under our experimental conditions, TGF- did not
significantly inhibit the expression of IL-12R1 mRNA or its protein
expression during alloactivation of naive T cells.36 This
discrepency could be explained by the nature of the stimuli delivered
to the T cells (anti-CD3 v B-cell line expressing costimulatory
molecules). We show in the present study that TGF- has no
significant effect on the regulation of IL-12R1 and IL-12R2
subunits mRNA expression in alloactivated T lymphocytes, thus we can
not exclude the possibility that TGF- may regulate IL-12R2
expression by a posttranscriptional mechanism. Based on the report of
Rogge et al49 indicating the existence of a correlation
between the expression of the transcript encoding the 2 component of
the IL-12 receptor and the presence of high-affinity IL-12 binding
sites on Th1 cells, it is reasonable to assume that the lack of TGF-
effect on IL-12R2 mRNA expression observed in alloactivated T
lymphocytes also reflects a lack of TGF- effect on IL-12R2
subunit protein surface expression. This suggests in our model that
TGF- may act presumably downstream the IL-12R to alter IL-12
responsiveness and prompted us to investigate the effect of TGF- on
IL-12 signaling. Our data clearly indicate that TGF- inhibits the
IL-12-induced tyrosine phosphorylation of Tyk2 and JAK2, without
altering the expression of these kinases (Fig 3). It is well
established that the transcriptional factor STAT4 is essential for
mediating responses to IL-12 in lymphocytes and regulating the
differentiation of both Th1 and Th2 cells.42,43 Our data
show that TGF- exerts an inhibitory effect on the IL-12-induced tyrosine phosphorylation and DNA binding activity of STAT4 (Figs 4 and
5). Again, the TGF- inhibitory effect is exerted rapidly and is not
due to a lack of STAT4 protein. Our data are consistent with previous
studies showing that a defect in IL-12 signaling via the JAK/STAT
pathway can be asssociated with an absence of STAT4
activation.47,51 It has been shown that T and NK cells from
STAT4 knock-out mice do not produce IFN- in response to IL-12,
suggesting that STAT4 is directly involved in IFN- gene expression.22 Furthermore, in a recent study, Barbulescu et al52 identified a STAT4 binding site at the IFN-
promoter and showed its functional importance for mediating IL-12
effects in primary human T cells.52 This fits with our data
demonstrating a correlation between inhibition of STAT4 and IFN-
production. It should be noted that TGF- inhibited only partially
IL-12 signaling, which resulted in a partial inhibition of IL-12 T-cell
responsiveness (proliferation36 and IFN- production).
Whether an additional transductional pathway activated by IL-12 that is
not inhibited by TGF- exists remains to be determined. Taken
together, our observations clearly provide evidence for another site of
TGF- action on IL-12 responsiveness of T cells by its ability to
interfere, at least in part, with IL-12 signaling and suggest the
existence of a direct cross-talk between IL-12R and TGF-R signal
transduction pathways in T cells. The ability of TGF- to interfere
with other cytokine-induced signaling via the JAK/STAT pathway has been
reported. Bright et al53 have recently shown that TGF-
inhibited IL-2-induced tyrosine phosphorylation and activation of JAK1
and STAT5 in murine T lymphocytes. In addition, Pazdrak et
al54 have reported that in human eosinophils, TGF- can
interfere with IL-5 signaling by inhibiting tyrosine phosphorylation of
JAK2 kinase and STAT1 nuclear factor. These observations suggest that
the mechanisms that down-modulate signaling after ligand binding and
JAK activation are critical to the regulation of cytokine action.
Dephosphorylation events are likely to occur upon recruitement and
activation of protein-tyrosine phosphatases, thus playing an important
role in the regulatory control of JAK kinases in T cells.26
In this regard, the activation of phosphatases by TGF- has been
already reported in keratinocytes.55 Furthermore, Bright et
al53 have shown that the inhibition by TGF- of
IL-2-induced tyrosine phosphorylation and activation of JAK1 could be
rapidly (within 10 minutes) restored following treatment with vanadate,
a known tyrosine phosphatase inhibitor, suggesting the involvement of
protein tyrosine phosphatase in the regulation of the JAK/STAT pathway
by TGF- in T cells. The activation of phosphatases may explain how
TGF- exerts its inhibitory effect on tyrosine kinases JAK2 and Tyk2
phosphorylation within 20 minutes in our model. It should also be noted
that a TGF--inhibitory element consensus sequence GNNTTGGtGA has
been found in a number of genes that are inhibited by
TGF-.56 Whether this sequence is found in IL-12-induced
gene promoters would be of major interest.
It has become clear that cytokines play an important role in the
conflict between tumor and immune system.57 Evidence has been provided that tumor cells produce immunosuppressive cytokines that
may contribute to immune dysregulation. TGF- is arguably the
prototype of immunosuppressive cytokines reported to drive a shift
toward Th2 type responses in tumor bearing mice.58
Therefore, it is tempting to speculate that the presence of TGF- in
the T-cell microenvironment during Ag presentation and initiation of
T-cell responses may alter IL-12 responsiveness of these cells, thus
interfering with the development toward a Th1 phenotype and consequently with the induction of a specific cell-mediated immunity. Whether TGF- facilitates the emergence of Th2 response in humans remains to be determined. Immunoregulatory cytokine-mediated
immunotherapy aimed at manipulating the immune system still represents
a potential strategy in immunologic intervention. Therefore,
understanding the functional interaction between TGF- and these
cytokines may ultimately assist in the optimization of cytokine-based
therapeutic intervention against human malignancies.
| |
ACKNOWLEDGMENT |
We are grateful to the volunteers (Banque du Sang, Hopital Saint Louis
and Centre Transfusion Sanguine de Créteil) without whom this
study would not have been possible. We wish to thank Genetics Institute
for the generous gift of rhIL-12, and J. Chehimi for reading this
manuscript and helpful comments. We specially wish to acknowledge Y. Lecluse for helpful technical advice concerning Western-blot.
| |
FOOTNOTES |
Submitted July 21, 1998; accepted September 23, 1998.
Supported by grants from the Institut National de la Santé et de
la Recherche Médicale, from the Association pour la Recherche sur
le Cancer (ARC Contract no. C6227-C2036), from the Ligue Nationale contre le Cancer and from the Institut Gustave-Roussy. C.P. is supported by the Comité Val de Marne of the Ligue Nationale
contre le Cancer.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Salem Chouaib, PhD,
Laboratoire Cytokines et Immunologie des Tumeurs Humaines, INSERM U487,
Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif
Cedex, France; e-mail: chouaib{at}igr.fr.
| |
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B. Cipriani, G. Borsellino, F. Poccia, R. Placido, D. Tramonti, S. Bach, L. Battistini, and C. F. Brosnan
Activation of C-C beta -chemokines in human peripheral blood gamma delta T cells by isopentenyl pyrophosphate and regulation by cytokines
Blood,
January 1, 2000;
95(1):
39 - 47.
[Abstract]
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C.G. Begley and N.A. Nicola
Resolving Conflicting Signals: Cross Inhibition of Cytokine Signaling Pathways
Blood,
March 1, 1999;
93(5):
1443 - 1447.
[Full Text]
[PDF]
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Top
Abstract
Introduction