(Received for publication, September 27, 1995; and in revised form, December 20, 1995)
From the
To study transcriptional regulation in normal human T cells, we
have optimized conditions for transient transfection. Interleukin-2
(IL-2) promoter-reporter gene behavior closely parallels the endogenous
gene in response to T cell receptor and costimulatory signals. As
assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal
AP-1 site and the NF-B site. Both primary activation, with
phytohemagglutinin or antibodies to CD3, and costimulation, provided by
pairs of CD2 antibodies or B7-positive (B cells) or B7-negative
(endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T
cell sites are much less important in normal T cells than in Jurkat T
cells. We conclude that IL-2 transcriptional regulation differs in
tumor cell lines compared with normal T cells and that different
costimulatory signals converge on the same cis-elements in the
IL-2 promoter.
The initiation of an immune response involves interaction
between foreign antigenic peptide bound to major histocompatibility
complex (MHC) ()molecules on the antigen-presenting cell
(APC) and a cognate antigen receptor on the T cell(1) . Under
physiological conditions, specific antigen-MHC complexes are usually
limiting, and T cells require additional costimulatory signals to be
fully activated(2) . The major source of such signals appears
to be direct interaction of cell surface ligands on the T cell and the
APC.
T cells that receive the correct combination of T cell receptor
(TCR)-mediated and costimulatory signals enter the cell cycle, express
activation antigens such as CD69 and CD25, and begin synthesis of
several cytokines, including the T cell autocrine/paracrine growth
factor interleukin-2 (IL-2)(2, 3) . IL-2 is an
essential factor required for progression of newly activated T cells
from G to S phase, and the quantity of IL-2 produced is a
major determinant of whether an effective response can be generated.
Moreover, inadequate IL-2 synthesis can lead to T cell death or
induction of a state of unresponsiveness known as clonal
anergy(4) .
Numerous T cell surface molecules have been suggested as mediators of costimulatory signals, the best characterized being CD2, interacting primarily with CD58 (LFA-3) and CD59 on the APC(5, 6, 7, 8) , and CD28, interacting with CD80 (B7-1) and CD86 (B7-2/B70) on the APC(9, 10, 11, 12, 13) . CTLA-4 is a second T cell molecule that interacts with the same ligands as CD28 and may also mediate costimulatory signals(14) , although this has been disputed(15, 16) . However, costimulatory activity is not limited to these molecules(17, 18) . Both the CD2 and CD28 pathways of costimulation are thought to increase the level of IL-2 transcription in human T cells, although this may not be the case for CD28 in murine T cell clones(19) . CD28-mediated signals may additionally stabilize IL-2 mRNA and affect post-transcriptional nuclear processing(19, 20) .
The IL-2 gene is not actively
transcribed in resting T cells. Transcription of IL-2 can be detected
as early as 40 min after activation, leading to peak levels of mRNA
around 6 h and a return to near zero levels by 12-18
h(2, 5) . Approximately 300 base pairs of the IL-2
promoter are sufficient to confer cell-specific, inducible expression
to reporter gene constructs(21) , although other regulatory
sequences may lie outside of this region(22) . Within these 300
base pairs, several transcription factor-binding sites have been
identified as positive regulatory elements in tumor T cells (see Table 2), including proximal and distal specific sequences for
the nuclear factor of activated T cells (NFAT) (21, 23, 24) and proximal and distal
sequences for AP-1(25, 26) , for
NF-B(27, 28) , for NIL-2A(29) , for
CD28-activated factors(30) , and for octamer
factors(31) . An additional AP-1 site has also recently been
identified just downstream of the dNFAT sequence(25) , which we
have designated as NFAP-1 as it appears to be functionally a part of
the ``NFAT'' binding sequence. Also, a binding site for SP1
and EGR-1 has been identified immediately upstream of the distal NFAT
site(32) . Many of these sites vary from consensus sequences in
other genes, and it appears that these differences account, at least in
part, for the T cell-specific expression of the IL-2 gene(33) .
Activation of T cells through the TCR-CD3 signaling pathway leads to
the activation of transcription factors specific to several of these
sites. It has been proposed that costimulatory signals increase
transcription by either 1) altering the composition of transcription
factors that bind to sites targeted by TCR/CD3-derived signals or 2)
inducing new factors that bind to novel combinations of sites. We have
previously presented evidence that human umbilical vein endothelial
cell (EC) costimulation modifies the composition of the AP-1 complex
binding to the proximal AP-1 (pAP-1) site in mitogen-activated T cells (34) . Furthermore, in murine LBRM-33 cells, IL-1 induces
c-Jun, which combines with c-Fos protein induced by TCR-mediated
signals, and binds to the AP-1 sites(35) . IL-1 also induces
AP-1 proteins in mouse EL-4 cells(36) . However, normal human T
cells constitutively express high levels of c-Jun and do not respond to
IL-1 as a costimulator(34) . In Jurkat cells, CD28 has been
found to induce a transcription factor composed of NFKB1 (p50), RelA
(p65), and c-Rel (37, 38) that binds to a variant
NF-B site called the CD28 response element (CD28RE)(39) .
Recently, this site has been found to be also targeted by signals other
than CD28(40) . It is not known whether different accessory
cell types activate different combinations of nuclear factors.
Much
of the information on IL-2 transcription has been obtained from studies
of tumor cell lines such as Jurkat, LBRM-33, and EL-4, although
recently, the role of NF-B regulation in IL-2 transcription has
been studied in CD4
T cell clones(41) . Little
information is available on transcriptional control in freshly isolated
normal human T cells. The major barrier to such studies has been
technical; it is very difficult to measure the low transcriptional rate
of the IL-2 gene observed in normal T cells by conventional assays,
such as nuclear run-off. Also, such cells are generally resistant to
transfection by reporter genes. It has been reported that human
peripheral blood mononuclear cells can be made transfection-competent
by a brief period of suboptimal stimulation with the polyclonal
mitogenic lectin phytohemagglutinin (PHA)(42) . Unfortunately,
this approach has proven inconsistent and has not been widely used.
Here we describe an optimization of this technique that permits routine
measurement of IL-2 transcription, which is responsive to TCR and
costimulatory signals, in small numbers of normal human blood T cells.
In this study, we have used this model to characterize IL-2
transcription in normal human T cells and find that the nuclear
factor-binding sites that are important in these cells differ from
those required for optimal transcription in Jurkat cells. Furthermore,
we have characterized the sites required for responsiveness to various
costimulatory signals and accessory cell types.
Circulating human T cells are not transfectable. This has led most investigators to work with T cell tumor lines, cells that may not accurately reflect normal T cell behavior (see below). However, we have found that the T cells in a freshly isolated population of PBMC, upon stimulation with a concentration of PHA (1 µg/ml) that is insufficient to cause significant IL-2 secretion, pass through a window of transfection competency. The mitogen PHA polyclonally activates T cells by binding with high affinity to the TCR, but not to CD3, and inducing calcium fluxes in a similar manner to anti-CD3 or anti-TCR mAb (47) . Thus, presentation of antigen by MHC molecules is bypassed. In the two separate experiments shown in Fig. 1, PBMC were transfected by electroporation as described under ``Experimental Procedures'' at the indicated times after PHA stimulation; luciferase was assayed in all cases after restimulation for 6 h with PHA (5 µg/ml) plus PMA (50 nM). A sharp peak of luciferase activity was found for transfection at 20 h (Experiment 1) and 19.5 h (Experiment 2). We have found consistently high responses when cells are transfected during this window.
Figure 1:
Optimal time for
transfection of normal T cells. Cells were cultured with 1 µg/ml
PHA for the indicated times and then transfected with the wild-type
IL-2 promoter-luciferase reporter plasmid (60 µg/ml) as described
under ``Experimental Procedures.'' Cells were restimulated
with PHA (5 µg/ml) and PMA (50 nM) and harvested 6 h later
for assay of luciferase. Means of triplicate determinations are shown
for luciferase expression. Standard errors were <10% of the mean.
Data points indicate relative light units: , Experiment 1;
, Experiment 2. Each curve is an independent
experiment.
To determine the time course of
transcription mediated by the IL-2 promoter in transfected normal T
cells, we transfected cells as described, stimulated them with optimal
doses of PHA and PMA, and then assayed luciferase activity and secreted
IL-2 over the next 48 h. As shown in Fig. 2, onset of
transcription is rapid, being easily detectable at 2 h. Transcription
peaks between 4 and 8 h and declines rapidly to 20% of peak levels
by 18 h and then more slowly to near basal levels by 48 h. These
kinetics are consistent with previously reported kinetics of mRNA
appearance following the activation of resting human blood T cells (5) and parallel the behavior of IL-2 promoter-reporter genes
in the widely used T cell tumor line, Jurkat (data not shown and (2) ). More important, no transcription was detectable in
transfected PBMC in the absence of additional stimulation, despite the
prior exposure to low concentrations of PHA.
Figure 2:
Time course of IL-2 promoter activity and
IL-2 synthesis in transfected normal T cells. Normal T cells were
transfected with the wild-type IL-2 promoter-luciferase reporter
plasmid (60 µg/ml) as described under ``Experimental
Procedures'' and cultured in the presence of PHA (5 µg/ml) and
PMA (50 nM) for the indicated times before harvest of the
supernatant for IL-2 bioassay and of the cells for luciferase assay.
Means and standard errors are as described for Fig. 1. No
luciferase activity above background levels or IL-2 was detected in the
absence of PHA plus PMA. Data are representative of one of three
experiments with similar results. , relative light units;
, units/milliliter IL-2.
Secreted IL-2 was
measured as an indicator of the activity of the endogenous gene in the
medium of the same cultures. No secreted IL-2 is detectable in the
absence of stimulation, indicating that the native IL-2 gene is not
active as a result of the prior manipulations to render the cells
transfection-competent. IL-2 is first measurable in the medium at
3-6 h and then rapidly increases over the next 20 h. Levels
begin to plateau after
24 h. These data are also consistent with
previously published reports of resting T
cells(34, 48) .
Figure 3:
The transfected IL-2 promoter is only
active in T cells. Total PBMC were transfected with the wild-type IL-2
promoter-luciferase reporter plasmid (60 µg/ml) as described under
``Experimental Procedures'' and then sorted by FACS using
mAbs specific for CD4 (T cells), CD8 (T cells), CD19 (B cells), and
CD14 (monocytes). Sorting produced populations of >99.5% purity.
IL-2 secretion for the purified stimulated populations was as follows:
CD4 cells, 29 units/ml; CD8
cells, 2
units/ml; and B cells and monocytes, not detectable. Means and standard
errors are as described for Fig. 1. Data are representative of
one of two similar experiments. RLU, relative light units; open bars, unstimulated; shaded bars, PHA +
PMA.
As shown in Fig. 4A, both B cells and EC augmented reporter gene
transcription in PHA-activated normal T cells above the level induced
by PHA alone. Over several experiments, augmentation ranged from 2- to
7-fold, with EC and B cells augmenting to approximately the same level.
Expression of the endogenous gene was also augmented, but B cells
seemed to be much more effective at augmenting secreted IL-2 than were
EC (Fig. 4B). Both B cells and EC also costimulated T
cells purified by FACS. In this case, little or no luciferase or
secreted IL-2 was detectable in the absence of accessory cells. IL-2
promoter-dependent transcription was detectable in both highly purified
CD4 and CD8
T cells stimulated by
accessory cells, and both cell types secreted IL-2. CD4
T cells were severalfold more responsive than CD8
T cells (data not shown).
Figure 4:
Different accessory cells costimulate
through different surface ligands. Normal T cells were transfected with
the wild-type IL-2 promoter-luciferase reporter plasmid (60 µg/ml)
as described under ``Experimental Procedures'' and cultured
for 12 h before harvest of the supernatant for IL-2 bioassay and of the
cells for luciferase assay. Transfected cells were stimulated with PHA
(5 µg/ml) and cultured with EC (1 10
/well) or
with BJAB cells (1
10
/well) in the presence of
control mAb K16/16 (10 µg/ml), anti-CD2 mAb TS2/18 (10 µg/ml),
control Ig (10 µg/ml), or CTLA-4-Ig (10 µg/ml). Cells were also
cultured with PHA in the absence of accessory cells or blocking
reagents. A, luciferase expression; B, IL-2
secretion. Means ± S.E. (luciferase) of three wells are shown.
Means ± S.D. (IL-2) are calculated as described under
``Experimental Procedures.'' Data are representative of one
of two similar experiments. RLU, relative light
units.
To identify the surface molecules involved in costimulation by B cells and EC, we used blocking antibodies and fusion proteins. mAb to CD2 blocked transcription and IL-2 secretion induced by B cells or EC (Fig. 4, A and B). Consistent with our previous findings(5) , anti-CD2 mAb could not completely inhibit EC costimulation. Blocking of B cell costimulation with anti-CD2 mAb was also incomplete, ranging from 40 to 90%. Most experiments were performed with anti-CD2 mAb TS2/18; however, we obtained identical results using anti-CD2 mAbs 35.1 and TS1/8 or anti-LFA-3 mAb TS2/9 (data not shown).
To investigate the role of CD28-mediated signals in this system, we used CTLA-4-Ig fusion protein, which binds to B7-1 and B7-2 and blocks interaction with their ligands, CD28 and CTLA-4. Consistent with our previous data demonstrating the absence of CD28 ligands on human EC and the lack of effect of CTLA-4-Ig or anti-CD28 mAb Fab fragments on EC costimulation of IL-2 secretion(49) , CTLA-4-Ig did not block EC costimulation of IL-2 promoter-dependent transcription (Fig. 4A). Similarly, this reagent did not block IL-2 secretion in response to EC. In sharp contrast, however, CTLA-4-Ig was very effective at blocking the costimulatory effects of CD28 ligand-bearing B cells on both transcription (Fig. 4A) and IL-2 secretion (Fig. 4B). Indeed, CTLA-4-Ig completely inhibited the augmented secretion of IL-2 in response to B cell costimulation. We have found substantially similar, although somewhat more variable, results using anti-CD28 mAb Fab fragments (data not shown).
Figure 5:
Electrophoretic mobility shift assay
analysis of nuclear factor binding to mutant NF-B IL-2
promoter-binding site. The inside PCR primers used to create specific
mutations were annealed to form a double-stranded probe for
competition. The wild-type probe was end-labeled with
[
-
P]ATP and incubated with 1-2 µg
of nuclear extract for 10 min at room temperature in the presence of
100 ng of poly(dI
dC) and a 50-fold molar excess of unlabeled
wild-type (WT) or mutant (MT) competitor probe. Bound
and unbound probes were resolved on 6% nondenaturing polyacrylamide
gels, which were then dried and exposed to storage phosphor screens and
analyzed on a Molecular Dynamics
PhosphorImager.
Each of the
mutant promoters was cloned upstream of the luciferase reporter gene
and transfected into normal T cells along with either pXGH (a growth
hormone-expressing plasmid) or pCMV--gal (a
-galactosidase-expressing plasmid) to normalize for transfection
efficiency. Transfected cells were then stimulated with PHA or with PHA
plus PMA and harvested 12 h later. Levels of secreted IL-2 produced by
the transfected cells varied by <10% between the different construct
pools (data not shown). As shown in Fig. 6, mutations in several
sites reduced transcription in PHA-activated normal T cells, including
the dNFAT, NF-
B, and pAP-1 sites. Over several experiments, the
pAP-1 site was consistently the most important single site. Mutation of
this element reduced transcription by 85-95%. Mutating the NFAP-1
or dAP-1 sites generally had small effects (0-30% reduction), and
in several experiments, mutating the dAP-1 site had no effect.
Figure 6:
Effect of nuclear factor-binding site
mutations on IL-2 transcription in normal T cells. Cells were
transfected with the wild-type (WT) and mutant (mt)
IL-2 promoter-luciferase reporter plasmids (60 µg/ml) along with a
-galactosidase-expressing control plasmid (10 µg/ml) as
described under ``Experimental Procedures'' and cultured for
12 h before harvest of the supernatant for IL-2 bioassay and of the
cells for
-galactosidase and luciferase assay. Transfected normal
T cells (pretreated with 1 µg/ml PHA) were restimulated with PHA (5
µg/ml) and PMA (10 ng/ml) (closed bars) or with PHA alone (open bars). Luciferase expression for the different
constructs was normalized to
-galactosidase, and then corrected
expression of each construct was normalized to the wild type.
Uncorrected wild-type expression for normal T cells was 245 relative
light units (RLU). Data are representative of one of three
similar experiments. d/pNFAT is a construct with both NFAT
sites mutated.
Interestingly, we noted a striking difference between normal T cells
activated with PHA plus PMA and those activated with PHA alone.
Specifically, the NF-B site appeared to be much more important in
the absence of the strong protein kinase C activator PMA (Fig. 6). We have repeated this experiment using normal T cells
activated with anti-CD3 mAb OKT3 and find the same pattern as with PHA
alone. Under these conditions, the pAP-1 and NF-
B sites appear to
be the dominant sites, and there are significant contributions from the
NFAT and NFAP-1 sites as well as the pOCT site (data not shown).
Figure 7:
Effect of nuclear factor-binding site
mutations on IL-2 transcription in normal T cells costimulated by EC,
JY B cells, or costimulatory antibodies. Cells were transfected with
the wildtype (WT) and mutant (MT) IL-2
promoter-luciferase reporter plasmids (50 µg/ml) along with pXGH or
pCMV--gal (10 µg/ml) as described under ``Experimental
Procedures'' and cultured for 12 h before harvest. A,
transfected T cells were cultured with 5 µg/ml PHA either alone or
in the presence of EC (1
10
/well) or JY cells (1
10
/well). Data are representative of one of six
similar experiments. B, transfected T cells were cultured
either alone or with anti-CD2 mAbs CB6 and GD10 (1 µg/ml) or
anti-CD3 mAb OKT3 (0.5-1 µg/ml) plus mAb 9.3 (1 µg/ml).
Data are representative of one of two similar experiments. C,
transfected T cells were cultured either alone or with OKT3
(0.5-1 µg/ml) in the presence of EC (1
10
/well) or JY cells (1
10
/well). Data
are representative of one of two similar experiments. Shown are means
± S.E. of triplicate determinations. IL-2 in the medium did not
vary by more than 10% between pools of cells transfected with each of
the constructs. RLU, relative light
units.
Finally, we repeated these experiments to compare defined
costimulatory antibodies with accessory cells. We used an activating
pair of anti-CD2 mAbs or an anti-CD28 mAb in conjunction with an
anti-CD3 mAb (Fig. 7B) and compared these to EC and B
cells (Fig. 7C). Again, the same combination of cis-regulatory elements was critical in each case. The pAP-1
and NF-B sites were most important for CD2-, CD28-, or accessory
cell-mediated costimulation. The pOCT and NFAT sites also contributed,
along with the NFAP-1 site. Interestingly, although the CD28RE mutation
did not affect CD2-, B cell-, or EC-mediated costimulation, it did have
a variable effect on anti-CD28 mAb-mediated transcription: in three of
five experiments, we saw a 25-50% reduction in transcription from
the mutant CD28RE promoter compared with the wild-type promoter (Fig. 7B and data not shown).
Figure 8: Comparison of nuclear factor-binding site mutations on IL-2 transcription in normal T cells versus Jurkat tumor T cells. The normal T cell results are the same as those shown in Fig. 6for cells activated by PHA plus PMA. The conditions for transfection and activation of Jurkat cells are the same as for normal T cells. Uncorrected wild-type expression for normal T cells was 245 relative light units (RLU), and that for Jurkat cells was 67,599 relative light units. Data are representative of one of three similar experiments. d/pNFAT is a construct with both NFAT sites mutated. WT, wild-type construct; mt, mutant construct; open bars, normal T cells + PHA + PMA; closed bars, Jurkat tumor T cells (JK) + PHA + PMA.
Transfected normal human T cells provide a sensitive and physiologically relevant model for studying cytokine transcription. We have used this system to analyze the regulation of IL-2 synthesis and find that the human IL-2 promoter is inducible in these cells and responds to the same primary and costimulatory signals as the endogenous promoter. Furthermore, we find that the regulation of IL-2 transcription in normal T cells differs from that in the tumor cell line Jurkat. Finally, we have shown that transfected normal human T cells respond to costimulation by different APC and that B cells and EC signal through different costimulatory ligands, but target the same cis-acting elements in the IL-2 promoter.
In the
experiments described here, we have transfected a mixed population of
cells. However, by use of FACS of transfected cells, we have determined
that only T cells express the luciferase reporter gene and that only T
cells secrete IL-2. CD4 T cells are severalfold more
active in this respect than CD8
T cells. Thus, in this
system, the IL-2 promoter is highly T cell-specific.
A potential
limitation of these studies is that we must culture normal T cells with
1 µg/ml PHA to induce transfection competence. Under these
conditions, there is no significant IL-2 production. IL-2 is required
for progression of T cells from G to S phase(2) .
This treatment, therefore, probably moves the T cells from G
to G
. However, several observations demonstrate that
transfected normal T cells still respond similarly to freshly isolated
resting T cells. 1) Once transfected, the cells do not secrete IL-2 or
transcribe the reporter gene unless further activated with mitogen. 2)
The kinetics of the transcriptional response parallel those of the
endogenous IL-2 gene in resting T cells. 3) OKT3 signals are weak and
must be costimulated by anti-CD28 mAb to activate optimal IL-2
transcription. 4) The cells can receive costimulation from APC. 5)
Costimulation can be blocked by mAb to CD2 or fusion proteins that bind
CD28 ligands.
The IL-2 promoter contains numerous transcription factor-binding sites. Several of these have been shown by genomic footprinting to bind proteins, and some have been shown to be functional by transfection of mutant reporter gene constructs (21, 24, 28, 35, 39, 41, 50). However, nearly all of these studies have been done in tumor cell lines, with the assumption that gene regulation in these cells would be identical to that in normal T cells, and there is little or no direct information as to which sites are relevant for IL-2 transcription in normal nontransformed T cells. A step toward analysis of the IL-2 promoter in vivo has been made by the generation of transgenic mice with the IL-2 promoter driving lacZ expression(51) .
This study suggests that differences
between normal cells and tumor cells do exist. In Jurkat cells, for
example, we confirm that the dNFAT site is critical for inducible
transcription regulated by the IL-2 promoter(50) . The nearby
sequence we have designated as NFAP-1 was equally important. In normal
T cells, however, these sites were far less important. We found instead
that the pNFAT site is more important than the dNFAT site and that the
NF-B and pAP-1 sites are quantitatively most important. Mutation
of either element almost completely blocked transcription in
PHA-stimulated normal T cells. The recent generation of an NF-
B
p50 knockout mouse suggests that the NF-
B site may be important in vivo as T cells from these mice failed to proliferate to
mitogenic signals, suggesting a defect in the generation of
IL-2(52) . Surprisingly, we found a difference in the
importance of the NF-
B site in normal T cells in the presence and
absence of PMA. Specifically, in the presence of PMA, mutation of the
NF-
B site reduced transcription by <50%. However, when normal T
cells were activated in the absence of PMA, by PHA, by OKT3 plus
anti-CD28 mAb, or by pairs of anti-CD2 mAbs, mutation of the NF-
B
site consistently reduced transcription by 75-90%. Most studies
of Jurkat cells have used PHA plus PMA or ionomycin plus PMA to
activate the cells. This suggests that studies of tumor cells requiring
the use of nonphysiologic activators such as PMA may additionally
complicate interpretation of results.
APC activate T cells by presenting antigen in the context of self-MHC molecules and providing costimulatory signals such as those mediated by CD2 and CD28. We compared B cells, bone marrow-derived ``professional'' APC expressing B7, with B7-negative EC, which have been described as ``semiprofessional'' APC for their ability to costimulate IL-2 transcription and secretion from mitogen-activated normal T cells(49, 53) . We have previously shown that EC can stimulate allogenic T cell proliferation (54) and do so because they provide costimulatory signals that result in augmented IL-2 transcription and secretion(34) . EC signal T cells through LFA-3-CD2 and CD59-CD2 interaction and through a second, unidentified pathway(55) . In this study, CD2 was found to be critical in mediating signals that resulted in enhanced IL-2 transcription and secretion when either B cells or EC were used as costimulators. In contrast, the CD28 pathway proved to be critical for B cell costimulation of transcription and IL-2 secretion, but was not productively engaged by EC. Thus, costimulation by two different accessory cells can be distinguished by the surface ligands through which they signal T cells. It is likely that both B cells and EC also signal through other ligands, not addressed in this report. An interesting finding was that transcription induced by B cells or EC was usually of comparable magnitude, whereas secreted IL-2 was usually greater from T cells costimulated by B cells. One explanation for this finding is that B cells do increase transcription beyond that registered by the transfected promoter, but do so through a site outside of the 600 base pairs we used. It is more likely that B cells, probably acting through CD28, are affecting IL-2 levels post-transcriptionally. Indeed, the increased secretion of IL-2 in response to B cell costimulation can be almost completely blocked by CTLA-4-Ig (Fig. 4). It has been reported that CD28 signaling has effects on IL-2 stability as well as on processing of nuclear transcripts and export from the nucleus(19, 20) . A similar mechanism has also been described for regulation of IL-2 transcripts in the absence of CD28 signaling(56) . These effects are likely specific for IL-2 mRNA and presumably would not affect the reporter gene.
A major conclusion of this study is that
although different accessory cells costimulate T cells through
different surface ligands, the same sites in the IL-2 promoter appear
to be targeted. Thus, for both B cell and EC costimulation, the
NF-B and pAP-1 sites and, to a lesser extent, the NFAT, NFAP-1,
and pOCT sites are critical. This same combination of sites was also
important for signaling by pairs of anti-CD2 mAbs. A surprising finding
in these studies was the failure of the CD28RE mutation to reduce
transcription induced by B cell costimulation, given that the mutation
muted the effects of anti-CD28 mAb. T cell CD28 was productively
engaged by B cell CD28 ligands in these experiments as CTLA-4-Ig fusion
protein blocked augmentation of secreted IL-2. The most likely
interpretation is that natural ligands for CD28 may deliver signals
that differ from those produced by some anti-CD28 mAbs and that these
signals do not induce activation of factors that bind to the CD28RE.
Indeed, a recent report suggests that not all signaling through CD28 is
identical. Using a panel of anti-CD28 mAbs, Nunès et al.(57) found that whereas a given pair of
anti-CD28 mAbs may induce similar calcium fluxes in T cells, they can
induce very different levels of IL-2 secretion. The mAbs recognize
several different epitopes on CD28, and the implication is that
signaling through CD28 (perhaps involving different epitopes) activates
more than one signaling pathway. Alternatively, B cells (and
potentially EC), unlike single mAbs, provide multiple costimulatory
signals that result in expression of functionally redundant sets of
transcription factors, obviating the role of factors bound to the
CD28RE. This interpretation presupposes that CD28-mediated signals
target other sites in addition to the CD28RE. Interestingly, the CD28RE
has recently been shown to bind members of the NF-
B family of
proteins, specifically NFKB1 (p50), RelA (p65) and c-Rel(38) ,
suggesting that CD28 may also activate the NF-
B site. However, in
normal T cells, the NF-
B site and the CD28RE do not appear to be a
functionally redundant pair since mutation of the NF-
B site cannot
be compensated for by a functional CD28RE.
Although the same
combination of IL-2 promoter elements is targeted by different
costimulatory molecules, we have not ruled out the possibility that
different combinations of nuclear factors may bind to these sites.
Indeed, we have previously shown that when T cells are costimulated by
EC, they express enhanced levels of c-Fos mRNA, and the AP-1 complex
that binds to the pAP-1 site contains severalfold higher levels of Fos
protein compared with cells activated in the absence of
EC(34) . Interestingly, a c-Fos knockout mouse has recently
been generated that displays normal IL-2 induction, suggesting that
other Fos proteins can substitute for c-Fos in these mice(58) .
There are also multiple NFAT proteins that may have differing roles
under different conditions. Thus, it is possible that in response to
different accessory cells, sequential expression of nuclear factors,
expression of different family members, or expression of factors with
longer half-lives, for example, may affect transcription. Indeed, IL-2
mRNA levels remain elevated longer in the presence than in the absence
of EC costimulation. ()The data presented in this study
define the sites most relevant for future study of nuclear factor
binding.
In conclusion, our findings suggest that the IL-2 promoter in normal T cells integrates signals by using a combination of several sites to activate transcription. This model is in agreement with a previous proposal describing fine tuning of IL-2 transcription(33) . Furthermore, these studies emphasize the feasibility of working with normal nontransformed cells and the possibility that such cells may differ in significant ways from the tumor cells that have become ``standard'' models for the study of transcriptional regulation.