CTLA-4 Ligation Suppresses CD28-induced NF-
B and AP-1 Activity
in Mouse T Cell Blasts*
Christina
Olsson
§,
Kristian
Riebeck
¶,
Mikael
Dohlsten
, and
Erik
Michaëlsson
From
Active Biotech Research AB and ¶ Department
of Medical Microbiology, Malmo University Hospital, and
Wallenberg Laboratory, Section of Tumor Immunology, Department
of Cell and Molecular Biology, University of Lund, SE-220 07 Lund
Sweden
 |
ABSTRACT |
The effects of cytotoxic lymphocyte antigen 4 (CTLA-4) on CD3/CD28 monoclonal antibody (mAb) activation of
CD4+/CTLA-4+ blastoid T cells were
studied in an in vitro model system. As previously
reported, coligation of CTLA-4 mAb results in suppression of T cell
proliferation and cytokine production. The proliferation but not the
interleukin 2 (IL-2) production could be restored by addition of
exogenous IL-2, suggesting that the inhibitory effect occurred at the
level of IL-2 production rather than at the regulation of the IL-2
receptor pathway. To study the effects on nuclear factors critical for
T cell activation, we analyzed the levels of the transcription factors
NF-
B and AP-1. These were potently induced in CD3/CD28
mAb-restimulated T cells. In contrast, CTLA-4 ligation strongly
suppressed the induction of both transcription factors. The
compositions of NF-
B and AP-1 family members were similar,
irrespective of stimulation conditions. Analyses of the NF-
B
regulator I
B-
revealed similar levels of I
B-
protein in the
preparations. However, a reduced phosphorylation of I
B-
in CTLA-4
coengaged T cell blasts compared with T cells ligated with CD3/CD28 was
found. Previous studies have concluded that CTLA-4 ligation regulates T
cell activation by inhibiting the T cell receptor-mediated signals.
However, the present findings propose that the major impact of CTLA-4
ligation is inhibition of signals mediated by CD28.
 |
INTRODUCTION |
The T cell surface receptors CD28 and CTLA-4 (CD152), and their
ligands CD80 and CD86 on professional antigen-presenting cells, regulate the activation of T cell receptor
(TCR)1-triggered T cells.
CD28 is expressed on both resting and activated cells, whereas CTLA-4
is only detectable on activated cells (1, 2). The CD28/B7 pathway is
known to be essential for the development and maintenance of T cell
responses (3). CTLA-4 was originally thought to have a similar function
as CD28. In agreement with this original thought,
B7-dependent costimulation of CD28-deficient T cells has
been demonstrated, which suggests that CTLA-4 ligation results in
costimulation (4). However, there is accumulating data showing that
CTLA-4 coligation has a negative regulatory effect on T cells, such as
down-regulation of interleukin 2 (IL-2) production and cell cycle
progression (5, 6). This negative regulatory effect is supported not
only by the phenotype of CTLA-4-deficient mice (7, 8) but also by the
effects observed after administration of CTLA-4 monoclonal antibody
(mAb) or Fab in animal models of autoimmune diseases and infection (6,
9-11).
The IL-2 gene is regulated by signals mediated by TCR as well as CD28
signaling that ultimately leads to DNA binding of nuclear factors such
as the prominent NF-
B and AP-1 transcription factors (12). After TCR
stimulation, the immunoreceptor tyrosine-based activation motifs within
the TCR/CD3
chain interact with intracellular signaling molecules.
The tyrosine residues within immunoreceptor tyrosine-based activation
motifs become phosphorylated and create binding sites for Src
homology-containing proteins (13). The activation of these
protein-tyrosine kinases results in phosphorylation of downstream
substrates, e.g. p21ras/mitogen-activated protein
kinases, phospholipase C
1 and Vav (13, 14). The TCR·CD3
complex
alone cannot stimulate proliferation, despite the activation of several
downstream pathways. The CD28 receptor provides additional signals,
including activation of p21ras/mitogen-activated protein
kinase, phospholipase C
1, and phosphatidylinositol 3-kinase (14).
This activation is attributable to tyrosine phosphorylation of the CD28
cytoplasmic domain after CD28 cross-linking. CD28·TCR signaling
causes I
B-
phosphorylation, whereupon the I
B-
is degraded,
and the I
B-
·NF-
B complex is resolved, allowing translocation of NF-
B into the nucleus (15, 16). Moreover, the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and jun
NH2-terminal kinase (JNK) are activated on signaling and
induce c-Fos and c-Jun transcription and the following formation of
AP-1 protein complexes (12).
Although the downstream consequences of CTLA-4 signaling are still
unclear, two distinct models have been proposed to explain the
inhibitory activity of CTLA-4 (17). The first model suggests that
CTLA-4 antagonizes CD28 function, either by competing for B7 binding
(2, 18, 19) and/or actively blocking CD28 signal transduction (5, 6,
20). The second model suggests that CTLA-4 interferes with TCR
signaling. An effect on TCR signaling was recently proposed because of
identification of hyperactive kinases associated with TCR observed in
CTLA-4-deficient T cells (21), suggesting that CTLA-4 counteracts the
hyperphosphorylation in a normal situation. Two recent studies using
CD28-deficient mice found that CTLA-4 in the absence of CD28
antagonized the TCR-mediated signal, arguing that it is sufficient to
explain the inhibitory effect of CTLA-4 (17, 22). A recent study using preactivated T cells showed that CTLA-4 selectively inhibited TCR
induced ERK-2 activity and TCR·CD28-induced JNK activity (20). Furthermore, an intracellular activation motif in the CTLA-4
cytoplasmic domain has been identified, which shows homology to the
CD28 cytoplasmic tail (24, 25). This common domain has been shown to be
the binding site for p85 phosphatidylinositol 3-kinase; however
controversy regarding its role in costimulation exists (26,
27).
In the present study, we examined the effect of CTLA-4 coligation on
nuclear transcription factors in mouse CD4+ T cell blasts.
We found that CTLA-4 ligation had profound effects on the transcription
factors NF-
B and AP-1. Moreover, the effects of CTLA-4 ligation were
only evident when CD28 was also coligated, supporting the hypothesis
that the major function of CTLA-4 is suppression of CD28 signaling.
 |
EXPERIMENTAL PROCEDURES |
Medium and Reagents--
RPMI 1640 medium supplemented with 2 mM L-glutamine, 0.01 M HEPES, 1 mM NaHCO3, 0.1 mg/ml gentamicin sulfate, 1 mM sodium pyruvate, 10% fetal bovine serum, and 50 µM
-mercaptoethanol was used throughout for cell cultures.
UC10-4F10-11 (hamster IgG anti-murine CTLA-4) hybridoma was a kind gift
from Dr. J. Bluestone (University of Chicago, Chicago, IL). Anti-CTLA-4
mAbs were produced from the hybridoma and purified on a protein G
column (Amersham Pharmacia Biotech).
Hamster anti-mouse CD3 (145-2C11), CD28-PE and unconjugated (37.51)
CTLA-4-PE (UC10-4F10-11), hamster IgG-PE/unconjugated (G235-2356,
isotype control), and rat anti-mouse CD4-PE/fluorescein isothiocyanate
(GK1.5) were purchased from PharMingen (San Diego, CA).
Preparation of Mouse CD4+ T Cell Blasts--
Spleens
from 8-12-week-old female C57BL/6 mice (H-2b) purchased
from M & B (Ry, Denmark) were used as a source of CD4+ T
cells. Erythrocytes were depleted from spleen cells by hypotonic lysis,
and the remaining cells were cultured in the presence of soluble
anti-CD3 mAb (0.5 µg/ml). After 48 h, the cells were harvested and live cells were enriched by density centrifugation over
Ficoll-Paque (Amersham Pharmacia Biotech). The enrichment procedure was
followed by positive selection using magnetic beads coated with
anti-CD4 mAb (Miltenyi Biotec, Sunnyvale, CA) according to the
manufacturer's descriptions. The purity of the CD4+ T
cells (
96%) was routinely checked by fluorescence-activated cell
sorting (FACS) analyses. Although not separated by size, they are
denoted as T cell blasts throughout this paper for simplicity.
T Cell Blast Proliferation and Cytokine Production--
Latex
beads (Interfacial Dynamics Corp., Portland, Oregon) were coated with
mAbs toward CD3, CD3/CD28; CD3/CTLA-4; CD3/CD28/CTLA-4; or hamster IgG
(isotype control) as described by Krummel and Allison (5). The
concentrations of mAbs used for coating were anti-CD3, 0.5 µg/ml;
anti-CD28, 1 µg/ml; and anti-CTLA-4, 4.5 µg/ml. The total amount of
mAb was compensated for by adding control mAb (trinitrophenol-specific
hamster IgG) to a final Ig concentration of 6 µg/ml.
One hundred thousand CD4+ T cell blasts were cocultured
with 105 coated beads in round-bottom 96-well plates (Nunc,
Roskilde, Denmark). After 24 h of restimulation, the cells were
pulsed for 4 h with 0.5 µCi of [3H]thymidine, and
the amount of incorporated [3H]TdR was determined by
liquid scintillation counting (28). The amount of IL-2 was quantified
by a sandwich enzyme-linked immunosorbent assay using specific Ab pairs
from PharMingen (JES6-1A12 and JES6-5H4) and recombinant murine IL-2
(Roche Molecular Biochemicals). The amount of IFN-
was quantified by
a sandwich enzyme-linked immunosorbent assay using recombinant murine
IFN-
and specific Ab pairs from PharMingen (R4-6A2 and XMG1.2).
Preparation of Protein Extracts and Electrophoretic Mobility
Shift Assay--
Nuclear and cytoplasmic protein extracts were
prepared, with minor modifications, according to the method described
by Schreiber et al. (29). Aliquots of protein were stored at
70 °C until required, and protein concentrations of extracts were
measured with a Bio-Rad protein assay kit.
[
-32P]ATP-labeled probes (30,000 cpm) were prepared as
described previously (30). The oligonucleotides used contained
the following sequences: AP-1 consensus, 5'-CTAGTGATGAGTCAGCCGGATC-3'; NF-
B consensus, 5'-GATCGAGGGGACTTTCCCTAGC-3'; and Oct binding site,
5'-CGTCTCATGCGATGCAAATCACTTGAGATC-3' (31). Binding reactions were
performed with the same amount of protein in each mixture (0.5-2
µg), and the samples were electrophoresed on a 6.5%
polyacrylamide-Tris borate-EDTA gel as described previously (30). For
supershift analyses, the mixture of nuclear protein extract and labeled
oligonucleotide was incubated with 1 µg of Abs against various
transcription factors for 1 h on ice. Polyclonal Abs directed to
Rel family proteins p50 (sc-114), p65 (sc-372), c-Rel (sc-70), and
c-Rel (sc-272, pan-c-Rel) and to AP-1 gene family proteins c-Jun/AP-1
(sc-44, pan-Jun), c-Jun/AP-1 (sc-45), Jun B (sc-046), Jun D (sc-74),
c-Fos (sc-253), c-Fos (sc-052), Fra-1 (sc-183), and Fra-2 (sc-171) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Western Blot--
The cytoplasmic protein extracts were analyzed
for content of total as well as phosphorylated I
B-
protein by
Western blotting. Protein extracts were separated on 10%
SDS-polyacrylamide gel electrophoresis and electroblotted onto
nitrocellulose membranes (Millipore, Sundbyberg, Sweden).
Phosphorylated I
B-
and total I
B-
protein were analyzed with
the PhosphoPlus I
B-
antibody kit according to the instructions
from the manufacturer (New England Biolabs, Beverly, MA).
 |
RESULTS |
Coligation of CTLA-4 Down-regulates T Cell Proliferation and
Cytokine Production--
C57BL/6 spleen cells were prestimulated for
various time points, after which the expression of both CD28 and CTLA-4
was analyzed by FACS. CD28 was expressed on nonstimulated
CD4+ T cells, but no CTLA-4 was detectable on the surface
(Fig. 1A). After 48 h of
stimulation the CD4+ T cells contained higher levels of
CD28 (Fig. 1B), expressed the activation marker IL-2
receptor
(IL-2R
) (data not shown), and 40-60% of the activated
CD4+/CD28+ T cells also expressed CTLA-4 (Fig.
1B). To study the role of CTLA-4 on activated T cells, we
stimulated T cell blasts expressing CTLA-4 as described by Krummel and
Allison (5). We chose not to rest the T cell blasts before stimulation,
because we noted that this caused a rapid down-regulation of surface
CTLA-4 (data not shown). After stimulation with mAbs toward CD3/CD28,
the T cell blasts produced considerable amounts of IL-2 and IFN-
and were shown to proliferate (Fig. 2). On
the contrary, when CTLA-4 was coligated, T cell blasts did not produce
IL-2 or proliferate, and IFN-
production was reduced to the level
observed for blasts restimulated with anti-CD3 only (Fig. 2). In
cultures restimulated with control mAb only, none of the cytokines
analyzed could be detected. The effects of CTLA-4 ligation on IL-2
production was also apparent at the mRNA level. At 40 and 90 min
after restimulation, T cell blasts restimulated with anti-CD3/anti-CD28
showed a marked up-regulation of IL-2 mRNA, whereas the mRNA
levels in T cell blasts restimulated with coligated CTLA-4 were
comparable with the nonstimulated T cell blasts. Similar
glyceraldehyde-3-phosphate dehydrogenase levels were detected in all
samples (data not shown).

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Fig. 1.
CTLA-4 and CD28 expression on nonstimulated
CD4+ T cells and CD4+ T
cell blasts. Spleen cells from C57BL/6 mice were stimulated with
soluble anti-CD3 for 48 h, whereafter the CD4+ T cells
were collected by positive selection according to magnetic cell
separation and analyzed by FACS. A, nonstimulated
CD4+ T cells; B, CD4+ T cell blasts.
Dashed line, isotype control shown in gray,
CTLA-4 expression; shown in black. CD28 expression. The
graph is representative of three independent experiments.
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Fig. 2.
CTLA-4 coligation down-regulates T cell
immune response. Bulk spleen cells from C57BL/6 mice were
stimulated with soluble anti-CD3 for 48 h, whereafter the
CD4+ T cell blasts were collected by positive selection by
magnetic cell separation. T cell blasts were then stimulated with the
indicated mAb immobilized on beads. A, IL-2; B,
IFN- production; C, proliferation as determined by
[3H]thymidine incorporation after 24 h of
stimulation. Shown is one representative experiment of three similar
performed.
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T cell blast proliferation, but not the IL-2 production, could be
restored by the addition of recombinant human IL-2 in cultures stimulated with coligated CTLA-4 (Fig. 2, A and
C). Because the IL-2R
expression was only slightly
influenced by CTLA-4 coligation, this suggested that the influence of
CTLA-4 was at the level of IL-2 protein production rather than
regulation of the IL-2R pathway. These results are in agreement with
others (6, 32).
CTLA-4 Ligation Abrogates Expression of the NF-
B and AP-1
Transcription Factors--
We further dissected the impact of CTLA-4
ligation on intracellular signaling events by studying NF-
B and AP-1
binding activity in nuclear protein preparations from restimulated
CD4+ T cell blasts. Nuclear protein extracts were analyzed
using gel shift analysis and oligonucleotides encoding an NF-
B or an
AP-1 consensus motif. The protein preparations from T cell blasts, restimulated with anti-CD3 mAb only, produced a weak signal of NF-
B
and AP-1 binding activity (Fig. 3). The
inclusion of CD28 mAb led to a substantial increase in both NF-
B and
AP-1 binding activity, whereas the concomitant presence of CTLA-4
brought the NF-
B and AP-1 binding activity down to levels seen in T
cell blasts restimulated with anti-CD3 mAb only (Fig. 3).
Interestingly, there was no effect of CTLA-4 ligation in the absence of
CD28 ligation, suggesting that CTLA-4 mainly inhibited CD28 and not the
CD3 signaling. Thus, the observed abrogated IL-2 production in
CTLA-4-ligated T cells (Fig. 2A) correlated with markedly
reduced amounts of NF-
B and AP-1 DNA binding activity.

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Fig. 3.
Low NF- B and AP-1
transcription factor binding activity in nuclear extracts from T cell
blasts after CTLA-4 engagement. Expression of NF- B, AP-1, and
Oct-1 binding proteins after stimulation of mouse CD4+ T
cell blasts was determined using the appropriate Ab, as indicated.
Nuclear protein extracts were prepared as described under
"Experimental Procedures" and used for gel shift analysis with
32P-labeled consensus oligonucleotides specific for
NF- B, AP-1, or Oct-1 sites. One of three representative independent
experiments is shown.
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We also analyzed DNA binding of the ubiquitous octamer binding
proteins. Constitutive expression of two complexes, which most likely
corresponded to Oct-1 and Oct-2, was seen in all nuclear extracts
irrespective of stimulation conditions (Fig. 3).
Ligation of CD28 and CTLA-4 Induces Similar Composition of Either
NF-
B or AP-1 Protein Complexes--
The NF-
B/Rel family of
transcription factors are composed of homo- or heterodimers of p50,
p65, and c-Rel, whereas the AP-1 transcription factors are homo- or
heterodimers of different members of the Fos (e.g. c-Fos,
Fra-1, and Fra-2) and Jun (e.g. c-Jun, JunB, and JunD)
family of proteins. Supershift analyses with Abs to a panel of
different Rel, Fos, and Jun family proteins were used to determine
whether the CTLA-4 inhibitory effect was attributable to differences in
compositions of the prominent transcription factors NF-
B and AP-1.
Because our findings propose that the major impact of CTLA-4 ligation
is inhibition of signals mediated by CD28, we focused on studying T
cell blasts stimulated with CD3/CD28 in the presence or absence of
coligated CTLA-4.
The components of the Rel family of transcription factors revealed two
complexes binding to the NF-
B element. The main constituent in T
cell blasts stimulated with either mAb combination was a faster-migrating p50-p50 homodimer (Fig.
4A). Addition of Ab against p65 to nuclear protein preparations stimulated with either mAb combination reduced the upper complex, which indicated participation of
p65 in the NF-
B·Rel complex. Because the upper complex was also
shifted by Ab against p50, it is reasonable to assume that it
represents a p50-p65 heterodimer. Also, supershift analyses of AP-1
components revealed a similar pattern irrespective of stimulation
protocols, with the major Jun component present being Jun D. The AP-1
complex was reduced in intensity when Ab against Fra-2 was added,
indicating the participation of Fra-2 components in the AP-1 complexes
(Fig. 4B). No shifts were observed with control IgG (data
not shown). Control experiments performed with a mixture of Abs and DNA
probe in the absence of protein extract showed that none of the Abs
used bound directly to the DNA target sequences (data not shown).

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Fig. 4.
Similar composition of either
NF- B or AP-1 protein complexes was induced
irrespective of restimulation protocol. Shown is a supershift
analysis of NF- B and AP-1 binding complexes. CD4+ T cell
blasts were stimulated for 20 h with immobilized
anti-CD3/anti-CD28 with or without anti-CTLA-4. Nuclear protein
preparations were used for gel shift analysis with
32P-labeled oligonucleotides specific for NF- B
(A) and AP-1 (B) together with subunit-specific
Abs indicated. One of two similar experiments is shown.
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Because a similar composition of either NF-
B/Rel or AP-1 family
members appeared in the absence or presence of CTLA-4 coligation, it
appears that CTLA-4-mediated down-regulation of T cell activation was
attributable to interference with CD28 signal transduction rather than
interference with a specific nuclear factor.
CTLA-4 Coligated T Cell Blasts Contain Low Levels of Phosphorylated
I
B-
--
NF-
B is present in the cytoplasm bound to the
I
B-
protein; I
B-
retains NF-
B in the cytoplasm by
masking the nuclear localization signal (16, 33). Phosphorylation of
I
B-
at serines 32 and 36 leads to the release and nuclear
translocation of active NF-
B/Rel transcription factors (16, 33) and
represents the major regulatory step in NF-
B activation. Examination
of cytoplasmic protein extracts by Western blot revealed that CD3/CD28 and CD3/CD28/CTLA-4 coligated T cell blasts contained similar levels of
total I
B-
protein (Fig.
5A). Blotting with
phosphospecific I
B-
Ab showed that CD4+ T cell blasts
activated with CD3/CD28 mainly contained the slower-migrating phosphorylated form of I
B-
(Fig. 5B), which suggested
that the majority of NF-
B has been translocated into the nucleus and
is active in transcriptional events. Interestingly, cytoplasmic protein extracts from CTLA-4-coligated CD4+ T cell blasts
demonstrated reduced levels of the phosphorylated I
B-
protein
(Fig. 5B). The difference in phosphorylation status was more
pronounced at 20 than at 4 h. Note that cytoplasmic protein extracts from T cell blasts that were not stimulated exhibited no
detectable phosphorylated I
B-
protein (Fig. 5B),
indicating that restimulation was required to induce phosphorylated
I
B-
proteins.

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Fig. 5.
Reduced cytoplasmic levels of phosphorylated
I B- in CTLA-4
coligated T cell blasts. Shown are Western blot analyses of
cytoplasmic protein extracts from CD4+ T cell blasts
stimulated for 4 and 20 h with immobilized mAb anti-CD3/anti-CD28
with or without anti-CTLA-4. A, detection of phosphorylated
I B- ; B, total I B- protein. Shown is one
representative experiment of two similar experiments.
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 |
DISCUSSION |
It is well established that CD28 strongly costimulates T cells
(3). Initially, it was assumed that a positive signal was also
transduced through the CTLA-4 receptor. However, results have
accumulated that suggest CTLA-4 mediates down-regulation of the ongoing
immune response. It has been shown that cross-linked anti-CTLA-4 mAb
inhibits proliferation in T cells stimulated with anti-CD3/anti-CD28
mAb (6, 9), whereas soluble anti-CTLA-4 mAb or Fab enhances T cell
proliferation and cytokine production. The observations in CTLA-4
deficient mice have strengthened the hypothesis that a negative signal
is mediated by CTLA-4 coligation (7, 8). In support of this, it has
also been shown that costimulation with B7-1 inhibits the response of
primed CD28-deficient T cells (17, 22). On the other hand, another
study could demonstrate B7-dependent costimulation of
CD28-deficient T cells, which suggested that CTLA-4 ligation led to T
cell activation rather than down-regulation (4). These contradictory
results indicate the importance of studying the effects of CD28 and
CTLA-4 in a relevant model with both players present.
In attempts to identify intracellular targets involved in CTLA-4
signaling, a motif containing a consensus binding site of phosphatidylinositol 3-kinase was identified (26). Several studies have
reported activation of a phosphatidylinositol 3-kinase after CD28 (34,
35) and CTLA-4 ligation (36). However, the functional importance of the
CD28-CTLA-4 interaction with phosphatidylinositol 3-kinase required for
costimulation of T cells remains unclear (36). The tyrosine phosphatase
SYP was demonstrated to specifically associate with the CTLA-4
cytoplasmic tail, and showed significant phosphatase activity toward
the RAS regulator p52SHC, indicating that the Ras pathway
may be regulated by CTLA-4 (21). Marengere and co-workers (21) were
also able to show that CTLA-4-deficient T cells had hyperactivated
protein-tyrosine kinases Fyn, Lck, and ZAP70. This argues for
CTLA-4-negative regulation being mediated via the TCR signal pathway.
That CTLA-4 ligation inhibits activation of CD28-deficient T cells has
also been taken as an argument for CTLA-4 having its effect(s) on TCR
signaling-associated molecules. On the other hand, it cannot be ruled
out that the CD28 signal pathway is the main target, and that the
effects on TCR signaling represent additional substrates for CTLA-4
interference, which are revealed by the genetic removal of CD28.
In the present study, we examined the effects of CTLA-4 ligation in
CD3/CD28-triggered CD4+ T cell blasts. The experimental
model was designed to mimic the role of CTLA-4 on activated T cells.
The rationale for doing the experiments on T cell blasts expressing
both CD28 and CTLA-4 on the surface was that we believe that CTLA-4
exerts its main function by down-regulating these activated cells. We
found a clear inhibitory effect on IL-2 mRNA induction, T cell
growth, and cytokine production. Our results are in agreement with
previous studies, which show a down-regulation of T cell activation by
cross-linking CTLA-4 with CD3/CD28 (5). T cell blasts stimulated with
CD3/CD28/CTLA-4 mAb and human recombinant IL-2 proliferated strongly,
indicating that these cells expressed functional IL-2R. However, the
cells produced no detectable IL-2, which suggested that the blockade of
proliferation by CTLA-4 was mainly a consequence of its interference with IL-2 protein production.
Several transcription factors, such as NF-
B, AP-1, and Oct, have
been described as critical for transcriptional activation of the IL-2
gene promoter (14, 37). Analysis of CTLA-4-ligated T cell blasts showed
only small amounts of NF-
B and AP-1 binding activity in nuclear
protein extracts. However, large amounts of NF-
B and AP-1 DNA
binding activity were induced in CD3/CD28-stimulated T cell blasts. TCR
signaling alone can lead to recruitment of NF-
B and AP-1; however,
the magnitude of these factors is superinduced by CD28-mediated
costimulation. In our studies, an inhibition of the analyzed
transcription factors could not be observed after CTLA-4 and CD3
ligation alone but required CD28 ligation. Moreover, we found that
coengagement of CTLA-4 induced only low levels of I
B-
phosphorylation in the cytosol, compared with a significant amount of
phosphorylated I
B-
in CD3/CD28-stimulated T cell blasts. In
contrast, similar levels of total I
B-
protein level were found in
the cytoplasmic protein preparations. The existence of mainly
unphosphorylated I
B-
may represent inactivation of residual low
amounts of NF-
B in the cytosol of CTLA-4-ligated T cell blasts but
could also be attributable to a CTLA-4-mediated reduction of synthesis
of NF-
B/Rel family proteins.
Our findings concerning AP-1 are in concordance with a study by Calvo
et al. (20), who showed that CTLA-4 selectively turns off
activation of downstream TCR/CD28 signaling events, i.e.
activation of ERK-2 and JNK (20). Interestingly, the effects shown on
ERK-2 and JNK activity occurred very rapidly after stimulation, which is in contrast to the present results. In the present study a much
later effect by CTLA-4 coligation was detected. Only small quantitative
differences were exhibited at earlier time points (1-8 h; data not
shown). The observed differences between the studies may be explained
by the divergence in our experimental protocols. Calvo et
al. (20) studied lymph node T cells prestimulated with soluble
anti-CD3/anti-CD28 for 40 h and rested before restimulation. Furthermore, they focused on kinase activity in their experiments.
A recent report concluded that I
B kinase-
and -
could mediate
the NF-
B-inducing activity of mitogen-activated protein kinase/ERK
kinase 1 (MEKK1), which supports a general model of sequential
involvement of mitogen-activated protein kinase/ERK kinase 1, I
B
kinase, and I
B in NF-
B activation (38). Mitogen-activated protein
kinase/ERK kinase 1 has also previously been shown to be central for
the JNK pathway and was proposed to be a coordinate activator of both
NF-
B and AP-1 pathways (23). Our findings, with profound effects
on both NF-
B and AP-1, support the idea that CTLA-4 interferes with
a common coordinator of these pathways, e.g.
mitogen-activated protein kinase/ERK kinase 1. Moreover, ERK2 activity
was shown to be down-regulated by CTLA-4 coligation in the absence of
CD28 coligation (20). TCR signaling was effected and the ERK2 activity
induced was lower than in the absence of CTLA-4. Together these data
indicate that CTLA-4 down-regulates T cells by affecting both the CD28
and TCR signaling pathway. Importantly, in our system with
CD3/CD28/CTLA-4 present on activated T cells, the main down-regulatory
effects seem mainly mediated through effects on the CD28 signaling pathway.
In conclusion, one could speculate that in the T cell cap facing toward
the antigen-presenting cell, phosphatases activated through binding to
the CTLA-4 cytoplasmic tail act on substrates in its vicinity.
Depending on the strength of the signal through TCR versus
CD28, one could find an impact on the respective pathways. We have used
anti-CD3 mAb as a model antigen, which gives rise to a strong T cell
response. Despite this, the inhibition by CTLA-4 coligation could be
demonstrated only in the presence of coligated CD28, which suggests
that the main function of CTLA-4 is suppression of the T cell response
by modulating signals delivered through CD28 ligation.
 |
ACKNOWLEDGEMENTS |
The contributions of Jan Nilsson, Ann-Sofie
Thornqvist, Agnethe Henriksson, Christa Edvardsson, Madelein
Jacobsson-Andén, and Dr. Vicky Avery are greatly appreciated.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Active Biotech Research
AB, P. O. Box 724, SE-220 07 Lund, Sweden. Tel.: 46-46-191160; Fax: 46-46-191105; E-mail: christina.olsson{at}activebiotech.com.
 |
ABBREVIATIONS |
The abbreviations used are:
TCR, T cell
receptor;
IL, interleukin;
IL-2R, IL-2 receptor;
mAb, monoclonal
antibody;
ERK, extracellular signal-regulated kinase;
JNK, jun
NH2-terminal kinase;
FACS, fluorescence-activated cell
sorting;
CTLA-4, cytotoxic T lymphocyte antigen 4.
 |
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