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* Department of Cellular Injury, Walter Reed Army Institute of Research, Silver Spring, MD 20910; and
Department of Medicine, Washington Hospital Center, Washington, DC 20005
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-2 transcription is regulated by several factors, which bind to the
proximal promoter within 300 bp upstream of the ATG codon
(2). These transcription factors include NF-
B, NFAT,
AP1, Oct, and CREB, and there is evidence that all binding sites on the
IL-2 promoter need to be occupied to ensure maximal transcription and
production of IL-2 (3, 4). The CREB binding site, 180 bp
upstream of the start codon, is interesting for several reasons: first,
mutation of this site almost completely abolishes IL-2 production
(5, 6); second, T cells of mice expressing a dominant
negative form of CREB show a marked decrease in the production of
IL-2 (7); and third, it serves also as the binding site of
cAMP response element modulator
(CREM),3 which can act
as a transcriptional repressor (3, 5).
Systemic lupus erythematosus (SLE) patients often suffer and die from overwhelming infections, and decreased IL-2 production in response to antigenic stimuli represents one of the many contributing factors (8, 9). Inability of SLE T cells to produce IL-2 is important because on the one hand it diminishes the T cell response to Ags and on the other hand it fails to promote the activation-induced cell death of T cells, which is necessary to terminate the immune response (1). This can yield to a nonspecific activation of T cells, which may support the production of Abs by B cells.
The decreased production of IL-2 in response to antigenic stimulation
by SLE T cells is the result of altered transcriptional regulation of
the IL-2 gene in SLE T cells. Our group has shown decreased NF-B
activity due to a reduced expression of the p65 subunit in SLE T cells
(10). Furthermore, we have shown that T cells of SLE
patients express increased amounts of CREM protein. CREM can act as a
transcriptional repressor, and we have proposed that this is central
for decreased IL-2 production in these cells (3, 5).
Like CREB, inducible cAMP early repressor (ICER), and ATF1, CREM
belongs to the family of cAMP responsible factors, which share a high
level of sequence homology. CREM is present in many tissues and has
been shown to be constitutively active in spermatocytes and in the
brain (11, 12, 13). The expression of various isoforms of CREM
is regulated by four different promoters and alternative splicing
(14). These isoforms can function as either
transcriptional repressors or activators, depending on the presence or
the absence of the transactivating domains (
1 and/or 2)
(13, 15, 16, 17). The second promoter is responsible for the
regulation of an inducible CREM (ICER), which is expressed in the brain
(16) and has been suggested to play a role in T cells
(18, 19), but the CREM protein that we found up-regulated
in SLE T cells has a much higher molecular mass than that of the ICER
protein (36 vs 13 kDa).
We conducted experiments to determine whether the increased expression of CREM in SLE T cells is the result of increased transcriptional activity of the CREM gene and to establish that its binding to the IL-2 promoter is responsible for the decreased production of IL-2 by SLE T cells. To this end, we demonstrate that an antisense CREM plasmid not only decreases the expression of CREM mRNA and protein but also up-regulates the defective expression of IL-2 in SLE T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eighteen patients with SLE were studied (16 male and 2 female, mean age 43.4 ± 4.6 years, mean systemic lupus erythematosus disease activity index 4.2 ± 3.57) (20). All met the criteria of the American College of Rheumatologists for diagnosis of SLE. As controls, 16 healthy age- and gender-matched volunteers were used. Written permission was obtained from each patient.
Lymphocyte isolation
Heparinized peripheral venous blood was obtained from study subjects. T cells were separated by Rosette separation (Stemcell, Vancouver, Canada). Briefly, non-T cells are selected by a tetrameric Ab mixture against CD14, CD16, CD 19, CD56, and glyA and bound to erythrocytes. These complexes are separated from the T cells by a Lymphoprep gradient (Nycomed, Oslo, Norway). The purified T cells were >98% positive for CD3 as tested by flow cytometry.
Antibodies
Anti-CREM, anti-CREB, anti-Jun, anti-
-actin,
anti-CBF1, anti-E 47, goat anti-rabbit-HRP, and goat
anti-mouse-HRP-conjugated mAbs were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA).
Preparation of mRNA and cDNA, PCR, and real-time PCR
One million T cells were used for extracting RNA (RNA Easy Mini kit; Qiagen, Valencia, CA). RNA was quantitated and 500 ng of total RNA was used for cDNA synthesis by reverse transcription (Reverse Transcription PCR kit; Promega, Madison, WI). A total of 25–50 ng of cDNA was used for each PCR. PCR primers were synthetized by Sigma Genosis (The Woodlands, TX).
PCR beads were used for amplification (Pharmacia, Piscataway, NJ).
RT-PCR was conducted in a conventional thermocycler. Real-time PCR was
conducted with a Cepheid Smart Thermocycler (Cepheid, Sunnyvale,
CA) by adding SYBR green to the reaction mixture. Primers used for PCR
were as follows: -actin, 5'-CATGGGTCAGAAGGATTCCT-3', reverse
5'-AGCTGGTAGCTCTTCTCCA-3'; IL-2,
5'-CACTACTCACATTAACCTCAACTCCTG-3', reverse
5'-CTGGGAAGCACTTAATTATCAAGTTAGTG-3'; CREM,
5'-GAAACAGTTGAATCCCAGCATGATGGAAGT-3', reverse
5'-TGCCCCGTGCTAGTCTGATATATG-3'. PCR products were separated on a
1.5% agarose gel and the OD was quantitated by using QuantityOne
software (Bio-Rad, Hercules, CA) after background subtraction from
each band.
For sequencing of CREM isoforms, PCR products were excised from the gel, extracted (gel extraction kit; Qiagen) and cloned into a TOPO cloning system (Topo TA; Invitrogen, Carlsbad, CA). Plasmids were isolated from recombinant clones and sequenced on an ABI Prism sequencer (PE Applied Biosytems, Foster City, CA).
For measurement of RNA stability, normal and SLE T cells were incubated for 4 h with actinomycin D (5 µg/ml) and RNA was extracted at different time points.
Preparation of nuclear extracts, EMSA, immunoblotting, and immunoprecipitation
Five to 10 million T cells were used for preparation of nuclear extracts as previously described (6). The dsDNA probe of the -180 site (-164 to 198 bp) on the IL-2 promoter was 5'-catccattcagtcagtctttgggggt-3' in shift and supershift assays as previously described (6). Nuclear extracts (5 µg) were separated electrophoretically on SDS gels and used in immunoblotting studies as previously described (6).
Chromatin immunoprecipitation analysis (CHIP)
Five million T cells were used per investigated Ab. The cells
were treated with formalin (1% final concentration) for 10 min,
washed, lysed, and sonicated. The DNA-protein complexes were
immunoprecipitated with a desired Ab and extracted by protein
A/G-Sepharose beads (Santa Cruz Biotechnology). After several washing
steps the crosslink between DNA and protein was reversed at 65°C,
followed by protein digestion with proteinase K, and the DNA was
extracted (QiaAmp DNA Extraction kit; Qiagen). The DNA was amplified
with primers flanking the IL-2 promoter, including the -180 site
(forward, 5'-CTAAGTGTGGGCTAATGTAAC-3'; reverse,
5'-TGTAAAACTGTGGGGGT-3'). DNA of 
1 million cells was used for each
PCR. PCR products were run on a 2% agarose gel and quantified with
QuantityOne software (Bio-Rad).
Transfection, luciferase assays, and quantitative determination of IL-2
Freshly isolated normal or SLE T cells were rested for 1 h
in RPMI, 10% FBS, and phytohem-agglutinin (1 µg/ml). Jurkat
cells were transfected without previous stimulation with PHA. Plasmids
encoding the IL-2 promoter luciferase construct (from -575 to +57 bp,
a kind gift from Dr. A. Rao, Department of Pathology, Harvard Medical
School, Boston, MA), CREM 
sense and antisense (a kind gift from Dr.
P. Sassone-Corsi, Laboratoire de Génétique Moleculaire des
Eucaryotes, Institute National de la Santé et de la Recherche
Médical, Strasbourg, France) (21), and corresponding
empty vector plasmid (pSG5) were used for
transfection. Five micrograms of each plasmid were used per
transfection. About 5–10 x 106 T cells
were transfected by electroporation at 250 mV and 1000 µF in Opti-MEM
(Life Technologies, Rockville, MD) and resuspended in AIMV medium (Life
Technologies) containing 10% autologous plasma. After 20 h, T
cells were harvested and the luciferase assay was conducted as
described previously (5). IL-2 production was measured in
culture supernatants by ELISA (R&D Systems, Minneapolis, MN). For
stimulation of T cells, CD 3 Ab (final concentration, 10 µg/ml), CD28
Ab (final concentration, 2.5 µg/ml), and a goat anti-mouse
crosslink Ab (final concentration, 25 µg/ml) were used and T cells
were stimulated beginning 18 h after transfection for 6
h.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have found that SLE T cells express increased amounts of the
transcriptional repressor CREM protein, which binds specifically at the
-180 site of the IL-2 promoter, associates with the transcriptional
cofactor p300, and actively suppresses the transcription of the
IL-2 gene (5). To determine whether CREM was
up-regulated at the gene transcription level in SLE T cells, we
designed appropriate oligonucleotide primers and determined the level
of IL-2 and CREM mRNA by real-time PCR or RT-PCR. -actin primers
were used as internal control. As shown in Fig. 1
, A and B, we
found a statistically significant difference of the CREM:-actin mRNA
ratio between T cells from 18 SLE patients and T cells of 16 healthy
controls. The RNA stability in both SLE and normal T cells are
comparable (Fig. 1C) and, apparently, it does not contribute
to the increased amount of CREM mRNA found in SLE T cells.
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(data not
shown). These data show that CREM is transcriptionally up-regulated
in SLE T cells. As with a previously studied cohort of SLE patients
(5), no relationship was detected between disease activity
and treatment status. Levels of CREM mRNA correlate inversely with the IL-2 mRNA in SLE T cells
To determine whether there is a relationship between CREM mRNA
levels and IL-2 mRNA, which would imply the involvement of CREM in
the regulation of the expression of IL-2, we plotted the ratio of
IL-2:-actin against that of CREM :-actin mRNA, all of which
had been generated by using RT-PCR and visualized and quantitated on an
agarose gel. As shown in Fig. 1
C, we noted an inverse
correlation (r = 0.56; p = 0.014)
between the levels of IL-2 and CREM mRNA in unstimulated SLE T
cells. This indicates that increased expression of CREM mRNA is
associated with decreased expression of IL-2 mRNA.
CREM binds to the IL-2 promoter in vivo
The above data, as well as those published previously (3, 5), have provided only indirect evidence on the significance of
CREM in the repression of IL-2 expression. To determine whether CREM
binds to the IL-2 promoter in live T cells we performed CHIP analysis.
The cells were fixed with formalin, sonicated to break DNA to 200- to
300-bp fragments, and incubated with appropriate Abs to precipitate
DNA-protein complexes. The immunoprecipitated DNA was extracted and
detected with appropriate primers. As shown in Fig. 2, we detected increased binding of CREM
to the IL-2 promoter in SLE T cells compared with normal T cells. The
CREB and c-Jun binding is decreased compared with normal cells. An
anti-E 47 Ab was used as control, because E 47 does not have any
known binding site on the IL-2 promoter. Thus, CREM binds to the IL-2
promoter in live SLE T cells and corroborates previous conclusions made
by applying shift assays (5).
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up-regulates the activity of the IL-2 promoter
To prove that CREM is indeed responsible for the suppression
of IL-2 transcription we used sense CREM and antisense CREM
plasmids in SLE T cells, normal T cells, and Jurkat cells. An empty
vector plasmid served as control. We first used these constructs in
Jurkat T cells and, as can be seen in Fig. 3, antisense plasmid caused increased
activity of a reporter construct driven by the proximal IL-2 promoter.
In contrast, the sense CREM plasmid resulted in decreased activity
compared with cells transfected with the empty vector (Fig. 3).
Subsequently, we asked whether transfection of SLE T cells with the
antisense CREM plasmid would block the increased expression of CREM and
restore the production of IL-2. Transfection of antisense CREM plasmid
into SLE T cells led to decreased production of CREM protein in these
cells (Fig. 4A). CREB and
-actin protein levels were not affected. We noted an 8-fold
up-regulation of IL-2 mRNA in these cells compared with the cells
transfected with an empty vector plasmid using real-time PCR technique
(Fig. 4B). As a result of the mRNA up-regulation, IL-2
protein was also increased. The effect was especially remarkable after
6 h of stimulation with CD3 and CD28 Ab (Fig. 5).
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down-regulates the binding of CREM to the IL-2
promoter in vivo
To exclude nonspecific effects of the antisense plasmid, we
determined the levels of CREM binding to the IL-2 promoter after
transfection of T cells with sense and antisense plasmids. As shown in
Fig. 6A, transfection of
normal T cells with antisense CREM resulted in decreased binding of
CREM to the IL-2 promoter compared with cells transfected with empty
vector plasmid; in contrast, transfection of T cells with sense CREM
plasmid increased the binding to the IL-2 promoter in live
cells.
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B). After transfection of T cells with the antisense CREM
, the binding of nuclear protein to the -180 site in these assays
was reduced, while transfection with sense CREM clearly increased
the binding to the -180 site compared with nuclear extracts from cells
transfected with empty vector. The binding to the -180 site was
specific, because it was abolished by a CREM Ab but not by an
irrelevant Ab against CBF1 (Fig. 6C). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous work from this laboratory has established that decreased IL-2
production by human SLE T cells is the consequence of defective IL-2
gene transcription due to decreased NF-B activity (10, 27) as well as increased expression of the repressor CREM
(5). Increased quantities of CREM protein were found in
the nuclei of SLE T cells and the protein was shown to bind to a site
180 bp upstream of the transcription initiation point (5).
This site had been previously recognized as an AP1 binding site, but
later it was found to bind CREM/CREB in T cells that fail to produce
IL-2 (3).
The present study has established that increased CREM expression in SLE
T cell is controlled at the gene transcription level and mainly
represents the CREM isoform. The RNA stability of CREM does not
differ between SLE and normal T cells. CREM was found to bind to the
IL-2 promoter in live cells and therefore unambiguously participates in
the regulation of IL-2 gene expression. The most significant finding of
this study is the demonstration that an antisense CREM plasmid
suppresses the levels of CREM mRNA and protein and its binding to the
IL-2 promoter, and, more importantly, reverses the suppressed
expression of IL-2 mRNA and protein. In particular, the marked increase
of IL-2 protein after stimulation of SLE T cells transfected with
antisense CREM plasmid shows the physiologic relevance of this finding
(Fig. 5).
The complexity of the pathogenesis of human SLE is fascinating
(23). The diverse molecular abnormalities that have been
identified in SLE T cells may simply represent the expression of a
limited number of central defects, the nature of which remains at
large. Alternatively, they may reflect the fact that SLE is a
heterogeneous disease (23, 26). In reference to the
transcriptional repression of the IL-2 gene, a number of
defects have been identified. First, the activity of NF-B is
decreased because SLE T cells lack the p65 subunit (10),
which, after forming heterodimers with the p50 subunit, accounts for
increased expression of a number of genes including IL-2. SLE T cells
express sufficient amounts of p50, which may homodimerize and bind to
the NF-B site of the IL-2 promoter and repress its transcriptional
activity (27). The origin of decreased p65 expression in
SLE T cells is not known, but increased caspase 8 activity, associated
with the increased spontaneous apoptotic rate of SLE cells (28, 29), may contribute to the degradation of the p65 chain.
Interestingly, forced expression of p65 reverses the decreased
production of IL-2 in SLE T cells (10). Second, the
activity of AP1 is decreased in SLE T cells because of decreased
expression of c-fos, a component of the AP1 heterodimers
(30). The IL-2 promoter defines a number of AP1 binding
sites. Last, the increased expression of the repressor CREM, which
binds to the -180 site of the IL-2 promoter, represents a central
culprit in the decreased expression of IL-2. The -180 site
binds CREM as shown in shift assays (Refs. 3 and
5 and Fig. 6) and in vivo (Fig. 3), and two tandem sites
in front of a luciferase reporter gene are active in normal but not in
SLE T cells (5). It should be noted that, in normal T cell
nuclear extracts, unlike SLE T cell extracts, CREB binds to the -180
site of the IL-2 promoter. SLE T cells have decreased protein kinase A
(PKA) activity (22) and PKA is responsible for the
activation of CREB. Therefore, it is possible that defective activation
of CREB permits the expression of CREM and indirectly contributes to
the suppression of the expression of IL-2. Forced expression of PKA RI
subunit caused increased expression of IL-2 in SLE T cells
(31). Furthermore, it is known that c-fos
contains cAMP response element sites in its promoter, and forced
expression of CREM down-regulates c-fos activity
(13). This means that CREM can act directly on the IL-2
promoter in SLE T cells but it also influences other transcription
factors that bind to the IL-2 promoter.
The demonstration (Fig. 1) of a significant, inverse correlation
between IL-2 and CREM mRNA and the demonstration of direct binding of
CREM to the IL-2 promoter endows CREM with a central role in the
repression of the IL-2 gene expression. The ability to reestablish the
expression of IL-2 in SLE T cells by suppressing the expression of CREM
with an antisense plasmid is of particular importance. Notwithstanding
the fact that electroporation, which was used to insert the antisense
plasmid into primary SLE T cells, cannot be used currently in clinical
practice, the idea of eliminating transcriptional repressors to
increase the expression of a gene that is important in many clinical
conditions is appealing.
IL-2 infusions have been used to treat cancers including melanoma
(32) and renal cell carcinoma (33), and they
have been limited by unwanted side effects such as capillary leak
syndrome (32). T cells from patients with AIDS fail to
produce IL-2 (34), and reconstitution of IL-2 production
is desirable to increase the ability to generate cytotoxic responses
(35, 36). It is possible that oligonucleotides with
antisense CREM activity that can enter readily T cells will be designed
and will be used to increase the production of IL-2 when desired. CpG
oligonucleotides have been used in humans without side effects
(37, 38), and additional modes of delivery, including
liposomes, could be considered. The fact that CREM is expressed in
various tissues may limit its controlled suppression, but antisense
oligonucleotides that target their effect to CREM expressed in lymphoid
cells would be desirable. Herein we showed that SLE T cells mainly
express the isoform CREM .
In conclusion, in this study we have shown that the isoform CREM
binds to the IL-2 promoter and down-regulates IL-2 production in SLE T
cells. Targeting the increased expression of CREM using antisense
plasmid approaches represents a modality to reverse decreased IL-2
production. Because IL-2 production is central for the ignition and
termination of the immune response, the development of means to control
its expression in T cells is important.
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Acknowledgments |
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sense
and antisense plasmid. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. George C. Tsokos, 503 Robert Grant Avenue, Building 503, Room 1A32, Silver Spring, MD 20910. E-mail address: gtsokos{at}usuhs.mil
3 Abbreviations used in this paper: CREM, cAMP response element modulator; CHIP, chromatin immunoprecipitation; PKA, protein kinase A; SLE, systemic lupus erythematosus; ICER, inducible cAMP early repressor.
Received for publication June 18, 2002. Accepted for publication August 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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participates in the activation of cyclic AMP-responsive element-binding protein and its subsequent binding to the -180 site of the IL-2 promoter in normal human T lymphocytes. J. Immunol. 166:5665.B activity in T lymphocytes from patients with systemic lupus erythematosus is associated with decreased p65-RelA protein expression. J. Immunol. 163:1682.-cells. Diabetes 49:1681.[Abstract]
isoforms expressed by two newly identified cAMP-responsive promoters active in the testis. Endocrinology 141:3923.. Clin. Immunol. Immunopathol. 81:293.[Medline]
B/Rel family, in T cells from patients with systemic lupus erythematosus. J. Rheumatol. 27:116.[Medline]
mRNA expression in bone marrow cells from systemic lupus erythematosus patients. Scand. J. Immunol. 48:551.[Medline]
subunit deficiency in lupus T lymphocytes: bypassing a block in RI translation reconstitutes protein kinase A activity and augments IL-2 production. J. Immunol. 166:7600.This article has been cited by other articles:
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  M. Ahlmann, G. Varga, K. Sturm, R. Lippe, K. Benedyk, D. Viemann, T. Scholzen, J. Ehrchen, F. U. Muller, M. Seidl, et al. The Cyclic AMP Response Element Modulator {alpha} Suppresses CD86 Expression and APC Function J. Immunol., April 1, 2009; 182(7): 4167 - 4174. [Abstract] [Full Text] [PDF]
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 J. C Crispin, V. Kyttaris, Y.-T. Juang, and G. C Tsokos Systemic lupus erythematosus: new molecular targets Ann Rheum Dis, November 1, 2007; 66(suppl_3): iii65 - iii69. [Abstract] [Full Text] [PDF]
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 K. Tenbrock, Y.-T. Juang, V. C. Kyttaris, and G. C. Tsokos Altered signal transduction in SLE T cells Rheumatology, October 1, 2007; 46(10): 1525 - 1530. [Abstract] [Full Text] [PDF]
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V. C. Kyttaris, Y. Wang, Y.-T. Juang, A. Weinstein, and G. C. Tsokos Increased Levels of NF-ATc2 Differentially Regulate CD154 and IL-2 Genes in T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., February 1, 2007; 178(3): 1960 - 1966. [Abstract] [Full Text] [PDF]
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 J. Bodor, Z. Fehervari, B. Diamond, and S. Sakaguchi Regulatory T cell-mediated suppression: potential role of ICER J. Leukoc. Biol., January 1, 2007; 81(1): 161 - 167. [Abstract] [Full Text] [PDF]
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K. Tenbrock, Y.-T. Juang, N. Leukert, J. Roth, and G. C. Tsokos The Transcriptional Repressor cAMP Response Element Modulator {alpha} Interacts with Histone Deacetylase 1 to Repress Promoter Activity J. Immunol., November 1, 2006; 177(9): 6159 - 6164. [Abstract] [Full Text] [PDF]
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O. Barabitskaja, J. S. Foulke Jr., S. Pati, J. Bodor, and M. S. Reitz Jr. Suppression of MIP-1{beta} transcription in human T cells is regulated by inducible cAMP early repressor (ICER) J. Leukoc. Biol., February 1, 2006; 79(2): 378 - 387. [Abstract] [Full Text] [PDF]
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 E. S. Hwang, J.-H. Hong, and L. H. Glimcher IL-2 production in developing Th1 cells is regulated by heterodimerization of RelA and T-bet and requires T-bet serine residue 508 J. Exp. Med., November 7, 2005; 202(9): 1289 - 1300. [Abstract] [Full Text] [PDF]
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K. Tenbrock, V. C. Kyttaris, M. Ahlmann, J. M. Ehrchen, M. Tolnay, H. Melkonyan, C. Mawrin, J. Roth, C. Sorg, Y.-T. Juang, et al. The Cyclic AMP Response Element Modulator Regulates Transcription of the TCR {zeta}-Chain J. Immunol., November 1, 2005; 175(9): 5975 - 5980. [Abstract] [Full Text] [PDF]
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V. C. Kyttaris, Y.-T. Juang, K. Tenbrock, A. Weinstein, and G. C. Tsokos Cyclic Adenosine 5'-Monophosphate Response Element Modulator Is Responsible for the Decreased Expression of c-fos and Activator Protein-1 Binding in T Cells from Patients with Systemic Lupus Erythematosus J. Immunol., September 1, 2004; 173(5): 3557 - 3563. [Abstract] [Full Text] [PDF]
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 V C Kyttaris, Y-T Juang, and G C Tsokos Gene therapy in systemic lupus erythematosus Lupus, May 1, 2004; 13(5): 353 - 358. [Abstract] [PDF]
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M. P. Nambiar, C. U. Fisher, A. Kumar, C. G. Tsokos, V. G. Warke, and G. C. Tsokos Forced Expression of the Fc Receptor {gamma}-Chain Renders Human T Cells Hyperresponsive to TCR/CD3 Stimulation J. Immunol., March 15, 2003; 170(6): 2871 - 2876. [Abstract] [Full Text] [PDF]
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K. Tenbrock, Y.-T. Juang, M. Tolnay, and G. C. Tsokos The Cyclic Adenosine 5'-Monophosphate Response Element Modulator Suppresses IL-2 Production in Stimulated T Cells by a Chromatin-Dependent Mechanism J. Immunol., March 15, 2003; 170(6): 2971 - 2976. [Abstract] [Full Text] [PDF]
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