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*
Sidney Kimmel Cancer Center, San Diego, CA 92121;
The Cancer Center, University of California at San Diego, La Jolla, CA 92093;
Department of Cell and Molecular Biology, Section for Tumor Immunology, University of Lund, Lund, Sweden; and
§
Laboratory of Signal Transduction, La Jolla Cancer Research Center, The Burnham Institute, La Jolla, CA 92037
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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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, IFN-
, and IL-4
(10, 11, 12). These CD28-induced effects promote an efficient
T cell response, T cell proliferation and effector functions
(9, 10, 11, 12). Signaling via the CD28 costimulatory pathway also
prevents the development of T cell anergy (13) and
protects cells from programmed cell death (14). Our
preliminary studies suggest that LFA-3/CD2 interaction primarily
supports autocrine production of IL-2, allowing the induction of clonal
expansion and the production of cytokines such as TNF- and IFN-,
while B7-1/CD28 interaction supports paracrine production of IL-2
necessary to activate other cells of the immune response and to support
long lasting T cell proliferation (6).
The CD28 molecule is a 44-kDa, disulfide-linked homodimer expressed on
most T lineage cells (11, 12) and is a member of the Ig
superfamily. Mature thymocytes have higher levels of CD28 than the
immature cells and among peripheral T cells, 95% of
CD4+ T cells and 50% of
CD8+ T cells express CD28. Activation of T cells
leads to enhanced CD28 expression (11). CD28 has also been
detected on plasma cells and NK cells. The ligands for CD28 are the two
B7 proteins, B7-1 or CD80 and B7-2 or CD86, which display a restricted
pattern of expression on APCs, including on activated B cells (5, 16). Although ligation of CD28 alone does not cause measurable
biochemical events, coligation of CD28 with the TCR leads to increased
and sustained tyrosine phosphorylation of several cellular proteins
compared with ligation of the TCR alone (17). The
biochemical events triggered or facilitated by CD28 induce the
formation of transcription factor complexes that recognize specific
response elements in a number of genes (11, 12, 13, 14, 15, 16, 17, 18).
Accordingly, these elements are referred to as CD28 response elements
(CD28RE)3, even if
other costimulatory molecules or pharmacological agents can also induce
binding of nuclear proteins to this DNA element (18). The
transcription factor complexes that bind CD28REs are typically composed
of members of the NF-
B/Rel family of NFs and members of the AP-1
family of transcription factors, like c-Fos, c-Jun, and JunD as well as
with other nuclear proteins. The CD28RE in the IL-2 promoter
(nucleotides -162 to -153) is crucial for transcriptional activation
of the IL-2 gene (19, 20, 21, 22, 23, 24) and probably works in
conjunction with an adjacent AP-1 site (20, 25, 26).
We have studied the role of CD2 and CD28 costimulation in the transcriptional activation of the human CD40 ligand (CD40L) gene. Surface expression of CD40L is inducible on CD4+ and CD8+ T cells (27, 28, 29, 30) and can be detected as soon as 16 h after TCR ligation. CD40L plays an important role in the development and maintenance of a T cell response (31, 32, 33, 34, 35, 36, 37, 38). It appears that ligation of CD40L on the T cell by its APC-bound receptor, CD40, may affect the T cell both directly, by generating costimulatory signals, and indirectly through enhanced expression of B7-1 and B7-2 on the APC (27, 38). We have confirmed that CD2 and especially CD28 costimulation induced CD40L gene transcription. CD2 induced 8- to 10-fold lower levels of transcriptional activity of the IL-2 gene promoter compared with CD28. This suggests that the differences between CD2 and CD28, with respect to IL-2 production, may reside at the level of transcription.
Moreover, we find that CD28 and, to a lesser extent, CD2 costimulation greatly enhances TCR-induced transcriptional activation of luciferase reporter constructs under the control of the CD40L promoter. Deletion analyses, mutation studies, and gel-shift assays all indicate that the CD40L promoter contains a functional CD28RE localized between nucleotides -170 and -164 upstream of the start site and adjacent to an AP-1 site. Finally, we find that the transcription factor complex bound to this site contains the AP-1 factors JunD, c-Fos, p50, p65, and c-Rel, but not c-Jun. The sum of observations indicate a new mechanism of regulation of CD40L by the CD28 pathway and suggest a potential cooperation between the CD28RE and AP-1 site similar to that observed in the IL-2 promoter.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Staphylococcal enterotoxin E (SEE) was purchased from Toxin
Technology (Madison, WI). The protease inhibitors PMSF, leupeptin,
pepstatin, aprotinin, and bestatin were obtained from Boehringer
Mannheim (Indianapolis, IN). [-32P]ATP was
obtained from ICN Pharmaceutical (Costa Mesa, CA). T4 polynucleotide
kinase and poly(dI-dC)2 were obtained from
Pharmacia Biotech (Piscataway, NJ). Tris-borate-EDTA buffer and
acrylamide-bisacrylamide (29:1) were obtained from Bio-Rad (Richmond,
CA). Polyclonal Abs against c-Jun, c-Fos, JunD, p50, p65, and
c-Rel were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA). Luciferase assay reagent, lysis buffer, and the pGL-2
luciferase vector were purchased from Promega (Madison, WI). The
transformed site-directed mutagenic kit was purchased from Clontech
Laboratories (Palo Alto, CA).
Transfected cell lines
Chinese hamster ovary (CHO) cells stably transfected with the cDNAs encoding the human HLA-DR, B7-1, and LFA-3 cell surface molecules have been described in detail elsewhere (5). Single and double transfectants expressing similar levels of the transfected molecules were established by repeated cell sorting (5) and they were periodically reanalyzed.
Cell culture and stimulation
The human T leukemia cell line Jurkat was maintained at logarithmic growth in RPMI 1640 supplemented with 2 mM L-glutamine and 10% FCS. The transfected CHO cells were maintained in the same medium plus G418 and/or L-methionine sulfoximine. Stimulation of the T cells with SEE was performed at a concentration of 1 x 106 cells/ml in the presence of 0.1 x 106 cells/ml CHO cell transfectants at 37°C as previously described (5, 6, 19).
Plasmid construction and mutagenesis
A 1294-bp 5' noncoding fragment of the human CD40L gene, corresponding to nucleotides -1227 to +67, was kindly provided by David Lewis (University of Washington, Seattle, WA). This DNA was subcloned at the HindIII site of the luciferase reporter plasmid pGL-2 to yield a plasmid we call pGL1.2. Shorter pieces of 600, 300, or 95 bp from the same DNA were PCR amplified with a 3' reverse primer containing a HindIII site lying just 5' of the start codon and a 5' primer containing a BglII site. These products were subsequently cloned into the pGL-2 to yield plasmids pGL0.6, pGL0.3, and pGL0.095, respectively. The minimal human IL-2 promotor-enhancer fragment (20, 39, 40), nucleotides -500 to +60, was also subcloned into pGL-2. Mutant CD40L-luciferase constructs were created by incorporating the mutagenic primer carrying a 2-bp mutation using the Transformer site-directed mutagenesis kit from Clontech Laboratories. The 2-bp mutagenic primer for specific site in the CD40L is 5'-CAAAAAGcAcAGCCTGGAAG-3'. The identity and integrity of the resulting plasmid, pGL-2-CD40LM, was verified by restriction enzyme digests and by sequence analysis.
DNA transfection and luciferase activity analyses
Jurkat cells (8 x 106) were transfected with 10 µg of luciferase reporter plasmid by electroporation using an electro cell manipulator 600 (BTX, San Diego, CA) at 130 V and 1700 µF capacitance. After incubation for 24 h at 37°C in RPMI 1640 medium with 10% FCS, the cells were incubated with the different CHO transfectants (see Transfected cell lines) in the presence or absence of SEE for another 8 h (or as indicated in the figures). Subsequently, the cells were washed twice in PBS and suspended in lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N', N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100), kept on ice for 5–10 min, and clarified by centrifugation at 13,000 x g for 1 min. The supernatants were mixed with luciferase reagent and measured in duplicate in a luminometer (MicroLumat LB 96 P; Berthold, Bad Wildbad, Germany) for 5 s. A background measurement was subtracted from each duplicate and experimental values are expressed either as recorded light units of luciferase activity or as activity relative to extracts from unstimulated cells (19).
Preparation of nuclear extracts
Nuclear extracts were prepared as previously described (19). Briefly, 3–5 x 107 Jurkat T cells were stimulated as above. After stimulation, the CHO cells were removed by plastic adherence. The T cells were washed once in ice-cold PBS, once in buffer A (10 mM HEPES (pH 7.9), 15 mM KCl, 2 mM MgCl2, 6 mM DTT, 0.1 mM EDTA, and 1 mM PMSF) and resuspended in buffer A with 0.2% Nonidet P-40. The pelleted nuclei were resuspended in buffer B (50 mM HEPES (pH 7.6), 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 10% glycerol) in the presence of 0.3 M (NH4)2SO4 (pH 7.9) and rocked for 30 min at 4°C. The broken nuclei were then centrifuged for 10 min at 100,000 x g. A 125-µl aliquot of supernatant was transferred to a second tube and more (NH4)2SO4 was added to a final concentration of 1.5 M followed by a second centrifugation at 100,000 x g for 10 min. The supernatant was removed and the pellet was resuspended in 50 µl of buffer B and stored at -70°C. The protein concentration in each extract was estimated using the Bio-Rad stain protein assay kit with bovine albumin as standard.
EMSAs
Three double-stranded oligonucleotides corresponding to elements in the CD40L promoter were synthesized. Their sense strand sequences are: oligonucleotide 1 (-254 to -214) 5'-CCA TTG TCT GTT AAG AAG TCT ATG ACA TTT CAA GGC AAG A-3'; oligonucleotide 2 (-213 to -173): 5'-TGA ATA TAT GGA AGA AGA AAC TTG TTT CTT CTT TAC TTA C-3'; oligonucleotide 3 (-172 to -144): 5'-AAA AGG AAA GCC TGG AAG TGA ATG ATA T-3'. Three mutant and 5-bp shorter versions of oligonucleotide 3 were prepared as follows (changed nucleotides in lower case): oligonucleotide 3, mutant 1 (changed at -168 to -165): 5'-AAA Acc ggA GCC TGG AAG TGA ATG-3'; oligonucleotide 3, mutant 2 (changed at -163 to -160): 5'-AAA AGG AAA ctt aGG AAG TGA ATG-3'; oligonucleotide 3, mutant 3 (changed at -157 to -154): 5'-AAA AGG AAA GCC TGG cct aGA ATG-3'. As a control, we used a 33-mer taken from the CD28RE of the human IL-2 promoter, 5'-GAT CGC CCA AAG AGG AAA ATT TGT TTC ATA CAG-3'. The AP-1 consensus sequence oligonucleotides was: 5'-CTA GTG ATG AGT CAG CCG GAT C-3'.
Two pmols of sense DNA were mixed with 25 µCi
[-32P]ATP, 1.5 µl 10 x kinase
buffer, 8 units of T4-polynucleotide kinase and water to a final volume
of 15 µl and incubated at 37°C for 1 h. After heating the
mixture at 95°C for 10 min to denature the enzyme, 2 pmols of
corresponding antisense oligonucleotide was added. After another 5 min
at 95°C, the mixture was placed in a 65°C water bath, which was
turned off, and incubated until the water bath was to room temperature.
The labeled double-strand DNA was purified on 5% polyacrylamide gels
in 89 mM Tris, 89 mM boric acid, and 2 mM EDTA. The gel was exposed to
film and the band containing the double strand was excised and
extracted for 
5 h. The labeled DNA was transferred to a mixture of
50% chloroform/isoamyl alcohol (25:1) and 50% of phenol, mixed, and
centrifuged briefly. The upper aqueous layer was transferred to another
tube containing only chloroform/isoamyl alcohol, mixed, and
centrifuged. The upper phase was transferred to another tube containing
2.5 volume 95% alcohol and 0.1 volume 3 M sodium acetate, mixed, kept
at 70°C for 20 min, and centrifuged for 15 min, and the supernatant
was removed. The pellet was air dried for 15 min at 37°C and
dissolved in 100 µl Tris-EDTA buffer. One microliter was counted in a
scintillation counter.
A total of 1–2 µl of nuclear protein extract corresponding to 5–10 µg of protein was added to 4 µl binding buffer containing 2–3 µg of poly(dI-dC)2 as a nonspecific competitor. The reaction mixtures were incubated at 37°C for 30 min with 15,000 cpm of double-stranded 32P-labeled oligonucleotides in a final volume of 15 µl. The samples were electrophoresed on 5% polyacrylamide gels in 89 mM Tris, 89 mM boric acid, 2 mM EDTA. The gels were fixed in 40% methanol and 10% acetic acid for 15 min, dried, and exposed to film.
For supershift analysis, the mixtures containing nuclear extracts were incubated with 1–2 µg of Abs against various transcription factors for 20 min before addition of 32P-labeled oligonucleotides. Purified rabbit IgG was used as a control Ab. Control experiments performed with the various Abs and DNA probes in the absence of nuclear extract demonstrated that none of the Abs bound directly to the target sequences (data not shown).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presentation of the superantigen SEE by CHO cells engineered
to express human MHC class II (HLA-DR), alone or together with
costimulatory molecules, is a close approximation of the normal
presentation of Ag to T cells. This model system allows us to vary the
presence of defined costimulatory molecules, such as B7-1, LFA-3, and
ICAM (5, 6, 19, 26), and to use readily transfectable
TCR-V
+ T leukemia line Jurkat. These cells
express CD28 and require CD28 costimulation for the transcriptional
activation of several genes that contain a CD28RE. We have used this
model system extensively to study the role of regulatory elements such
as CD28RE in the control of the IL-2 gene promoter (5, 6, 19, 25).
Fig. 1
shows schematically the
5'-noncoding region of the human CD40L gene that we used as starting
material and the location of the previously identified putative NFAT
sites (41, 42) as indicated. First, we cloned the entire
1.2-kb region of the CD40L promoter into the pGL-2 plasmid upstream of
the luciferase gene. This construct (pGL1.2) was transiently
transfected into Jurkat T cells and the cells were stimulated with SEE
presented by CHO-HLA-DR cells with or without costimulatory molecules.
When the cells were lysed after various times and analyzed for
luciferase activity, we observed that increased activity was detectable
as early as 2 h after stimulation, greatly increased to maximum
values at 8 h, and then slowly declined (Fig. 2). This time-course is similar to that
observed with reporter genes driven by the IL-2 promoter (19, 25). Importantly, the magnitude of the response was dependent on
the expression of costimulatory molecules on the CHO cells with B7-1
being consistently more efficient than LFA-3 (Fig. 2). Thus, the 1.2-kb
region contains sufficient regulatory elements to drive
receptor-induced transcription in a CD28-dependent manner.
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Next we made three shorter constructs of the 5'-noncoding DNA from
the CD40L gene having 600 bp (pGL0.6), 300 bp (pGL0.3), or 95 bp
(pGL0.095) upstream of the start site. Transient transfection of the
two former constructs into Jurkat T cells revealed that the luciferase
activity could be induced to the same magnitude as the larger pGL1.2
and with the same dependence on B7-1 or LFA-3 costimulation (Fig. 3). In marked contrast, in parallel
experiments the shortest construct having only 95 bp upstream of the
start site could not be induced to exhibit luciferase activity at all
(Fig. 3). We conclude that the most important transcriptional control
elements in the CD40L gene must lie between nucleotides -300 and
-95.
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A comparison of the luciferase activities induced by the minimal
CD40L promoter (pGL0.3) and the minimal 500-bp IL-2 gene promoter
(20) showed that the latter displayed a stronger
dependence on B7-1 than the CD40L promoter (Fig. 4). LFA-3 expressing CHO cells induced
>50% as much CD40L-driven luciferase activity compared with B7-1
expressing CHO cells, while induction of the IL-2 reporter activity
showed little response to APCs having LFA-3 instead of B7-1 (Fig. 4).
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It has been reported that the CD40L promoter contains several
putative NFAT sites (41, 42) (see Fig. 1
). Only one of
these lies in the minimal 300-bp promoter region defined above. To
evaluate whether this putative site plays any role in TCR plus
CD28-induced activation, we introduced a 4-bp mutation at position
-264 to -261. This mutation resulted in a small (< 20%) reduction
in the induction of transcriptional activity (Fig. 5) and this decrease is not significant
(0.06 < p < 0.46). The dependence on
costimulation was not affected. We conclude that, while the NFAT site
may moderately affect luciferase activity, it is not a crucial
regulator of CD40L transcription at least under the condition of our
assays.
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The dependence on CD28 costimulation for maximal induction of
CD40L transcription suggests the presence of a CD28RE in the 300-bp
minimal promoter. To examine this notion, we employed an EMSA to
determine whether CD28 costimulation would induce the specific binding
of nuclear proteins to CD40L promoter DNA and, if so, to localize the
DNA region. First, we prepared three oligonucleotides corresponding to
distinct candidate CD28REs within the 300 bp of the minimal CD40L
promoter. As shown in Fig. 6A,
a positive result was obtained with one of the oligonucleotides
(oligonucleotide 3) corresponding to -172 to -144, but not with the
other two oligonucleotides. Furthermore, a specific complex was
detected only in cells stimulated through both TCR and CD28
but only weakly in cells stimulated through TCR plus CD2, and not in
response to TCR stimulation alone or in the absence of stimulation. The
binding of this complex was lost in samples containing excess unlabeled
oligonucleotide 3, but not in samples with a 50-fold excess of a
consensus AP-1 binding oligonucleotides (Fig. 6B). These
results indeed suggest that a CD28RE is present in the CD40L promoter
between nucleotides -172 and -144.
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C. Two of
these sequences still bound a protein complex in extracts from cells
stimulated through the TCR plus CD28, while the most 5' mutation
essentially abrogated any binding. These experiments suggest that
nucleotides -168 to -165 are involved in binding and, therefore, are
wholly or partly included in the CD28RE. The CD28RE is required for B7-1- and LFA-3-induced CD40L transcription
Kinetic studies demonstrated that the CD40L promoter-luciferase
reporter gene activity peaked at 8 h after B7-1 and LFA-3
costimulation (Fig. 2). To verify that the putative CD28RE plays a
significant role in B7-1- and LFA-3-induced CD40L promoter
transcription, we introduced a 2-bp mutation in the putative CD28RE
sequence of the CD40L promoter (5'-168 to -165–3'). The mutation of
the CD28RE site reduced the induction of CD40L transcription by 80% in
CHO-DR/B7-1 and 70% in CHO-DR/LFA-3 costimulated Jurkat T cells (Fig. 7). These decreases are significant
(p < 0.01) and the observations strongly
indicate that the CD28RE sequence is specifically required for B7-1-
and LFA-3-induced transcription.
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To begin to characterize the complex of nuclear proteins that
binds to the CD28RE, we repeated the EMSAs using oligonucleotide number
3 (-172 to -144) in the presence of Abs against known transcription
factors. In these assays, the addition of Abs can cause
"supershifts" or can block or inhibit DNA-protein complex formation
as observed previously (43). Here, Abs specific for c-Fos,
JunD, c-Rel, p65, and p50 all caused a decrease in the
intensity of the DNA-protein complex band (Fig. 8). Indeed, digitization of the
autoradiographs (e.g., Fig. 8) showed that the addition of most Abs
(anti-c-Rel, anti-p65, and anti-p50) lead to a
very similar integrated band intensities with an average of 48% of
that of the complete complex (Fig. 8, lane 3). Addition of
anti-c-Fos and anti-p50 lead to only slightly less reductions
in band intensity to 54% and 57% respectively. No diminution or shift
in position was observed upon addition of anti-c-Jun, a polyclonal
sera that we have previously characterized (20). This
results suggests that both, the NF-B/Rel family proteins
c-Rel, p65, and p50, and the AP-1 components c-Fos and JunD
are parts of the CD28-induced complex that binds to the oligonucleotide
corresponding to nucleotides -172 to -144 of the CD40L promoter.
These results support the cross-talking between these two families of
NFs as a requirement for inducing optimal transcriptional activity of
the CD40L gene.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A comparison of the CD28RE in the CD40L promoter (within nucleotides
-172 to -160: AAAaggaaaGCCT, probable binding site
underlined) with the CD28RE in the IL-2 promoter (nucleotides -166 to
-150: TTTAAagaaattcCAAA, binding site underlined) shows
both similarities and interesting differences. Although a similar set
of Fos, Jun, and Rel family proteins bound to the CD28RE in the CD40L
as in the IL-2 promoter (20, 25, 26), the nucleotide
sequences of each response element differ beyond the GAAA core. We also
noted that the activation of the IL-2 promoter showed a much stricter
requirement for CD28 costimulation while a partial CD40L activation can
be achieved by coligation of CD2. The reason for this less strict
requirement for CD28 costimulation remains unknown, but may be related
to the overall structure of the promoter and the proximity of NF-AT or
AP-1 sites to the CD28RE in the CD40L promoter (41, 42).
This confirms earlier results that have linked NF-AT activation to the
TCR signal transduction pathway (19). It has been
demonstrated that up-regulation of CD40L is TCR dependent (44, 45) and several studies have demonstrated that the CD2 signal
synergizes with TCR to increased activation signaling via the TCR
(46, 47). These results suggest that strong signaling can
occur in the absence of a CD28 signal, perhaps via a redundant signal
transduction through other costimulatory molecules such as CD2. These
data suggest that CD28 induces a quantitatively stronger signal along
CD28 signal pathway, but that no major qualitative differences occur in
the NF controlling the CD40L promoter. In both the CD40L and IL-2
promoters there is an adjacent potential AP-1 binding site suggesting a
further cooperative effect between members of the AP-1 and Rel/NF-B
families in the regulation of transcription. In the case of the IL-2
promoter, transcriptional activation is reduced in the absence of this
adjacent AP-1 site (20, 25, 26). We suggest that this is
likely to be the case for the CD40L promoter where the putative AP-1
binding sequence TGATATG is located at nucleotides -148 to -142.
Both the IL-2 and CD40L promoters contain several NFAT elements. In the case of the CD40L, only one NFAT binding site (at -265 to -258) lies in the 300-bp fragment that is sufficient to drive a full transcription in our assays. Mutation of this site had a minor effect, suggesting that it is not crucial for the transcriptional activity of the 300-bp promoter. The exact molecular signaling events induced by CD28 coligation that subsequently result in the formation of the protein complex that binds to the CD28RE remains partly speculative. The transcription of the CD40L gene does not reach its peak activity until several hours after T cell stimulation. Thus, there is sufficient time for changes in the transcription, translation, and posttranslational regulation of many factors that may be involved in binding to the CD28RE and the adjacent AP-1, and perhaps to other parts of the promoter. Within this window of time, the genes for both c-fos and c-jun (and perhaps junD) are activated, resulting in increased levels of the corresponding proteins. Our experiments confirm the presence of c-Fos and JunD in the CD28RE binding complex. For transcriptional activity, these proteins also need to be phosphorylated by members of the mitogen- and stress-activated protein kinase families, including extracellular signal-regulated kinase and c-Jun N-terminal kinase 48, 49). Thus, early activation of these kinases may be required for fos and jun gene activation, while kinase activity at later time points may be important for activation of the transactivating capacity of the Fos and Jun proteins. In our system, elevated extracellular signal-regulated kinase and c-Jun N-terminal kinase activity can still be detected 2 h after SEE presentation to T cells, particularly in the presence of B7-1 on the APC (data not shown).
Our experiments also revealed the presence of NF-B/Rel family
members in the protein complex that bound the CD28RE. The presence of
these proteins in a functional complex following CD28 costimulation
implies the activation of the protein kinases that phosphorylate IB,
IKK, and IKK-, and induce the proteolytic breakdown of IB
(21). It is currently not known how CD28 signals to these
kinases. However, TCR ligation alone is not sufficient to activate the
IKKs (21). Thus, our finding that these factors bind the
CD28RE in the CD40L promoter provides a molecular explanation for the
requirement for CD28 or LFA-3 costimulation for the induction of CD40L
expression.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eduardo Parra, Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121.
3 Abbreviations used in this paper: CD28RE, CD28 response element; CD40L, CD40 ligand; CHO, Chinese hamster ovary; SEE, staphylococcal enterotoxin E. LFA-3, leukocyte function-associated Ag-3.
Received for publication August 2, 2000. Accepted for publication December 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B/IB family: intimate tales of association and dissociation. Genes Dev. 9:2723.B family. c-Rel, p50, and p65. Proc. Natl. Acad. Sci. USA 90:1696.B like response element. J. Biol. Chem. 266:1479.
1, fibronectin, and plasminogen activator inhibitor-1. J. Biol. Chem. 274:4400.This article has been cited by other articles:
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J. F. McDyer, Z. Li, S. John, X. Yu, C.-y. Wu, and J. A. Ragheb IL-2 Receptor Blockade Inhibits Late, But Not Early, IFN-{gamma} and CD40 Ligand Expression in Human T Cells: Disruption of Both IL-12-Dependent and -Independent Pathways of IFN-{gamma} Production J. Immunol., September 1, 2002; 169(5): 2736 - 2746. [Abstract] [Full Text] [PDF]
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L. A. Schubert, R. Q. Cron, A. M. Cleary, M. Brunner, A. Song, L.-S. Lu, P. Jullien, A. M. Krensky, and D. B. Lewis A T Cell-specific Enhancer of the Human CD40 Ligand Gene J. Biol. Chem., February 22, 2002; 277(9): 7386 - 7395. [Abstract] [Full Text] [PDF]
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