A T cell-specific enhancer of the human CD40 ligand gene.

We observed that the human CD40 ligand (CD40L) gene 5'-flanking region conferred weak promoter activity in activated CD4 T cells, suggesting that additional regions are required for optimal CD40L gene transcription. We therefore examined a 3'-flanking segment of the CD40L gene, which contained a putative NF-kappaB/Rel cis-element, for its ability to enhance CD40L promoter function. This segment augmented CD40L promoter activity in an orientation-independent manner in CD4 T-lineage cells but not in human B cell or monocyte cell lines. Mapping of CD4 T-lineage cell nuclei identified a DNase I-hypersensitive site in the flanking region near the NF-kappaB/Rel sequence, suggesting a transcriptional regulatory role. This was further supported by truncation analysis and site-directed mutagenesis, which indicated that the CD40L 3'-flanking NF-kappaB/Rel cis-element was critical for enhancer function. Electrophoretic mobility shift assays showed that the cis-element preferentially bound the p50 form of the NF-kappaB1 gene contained in human T cell nuclear protein extracts. This binding also appeared to occur in vivo in CD4 T cells based on chromatin immunoprecipitation assays using NF-kappaB p50-specific antiserum. Together, these results suggest that the CD40L gene 3'-flanking region acts as a T cell-specific classical transcriptional enhancer by a NF-kappaB p50-dependent mechanism.

CD40 ligand (CD40L), 1 also known as CD154, is a member of the tumor necrosis factor (TNF) ligand superfamily that is transiently expressed on the cell surface of activated CD4positive T cells (1). It binds CD40, a TNF receptor superfamily molecule expressed on B cells, dendritic cells, and mononuclear phagocytes (2). The phenotype of humans with a genetic CD40L deficiency reveals that the CD40L/CD40 interaction is critical for T cell-and B cell-mediated immunity to protein antigens and for enhanced mononuclear phagocyte activation to certain opportunistic pathogens, such as Pneumocystis carinii (3).
The molecular events precipitating CD40L expression on activated CD4 T cells have similarities to those eliciting expression of the cytokines interleukin (IL)-2 and TNF. Cognate mRNAs for all three proteins are rapidly expressed de novo upon activation of antigenically naive human CD4 T cells (4 -7). Full T cell activation requires coordinate engagement of the ␣␤-T cell receptor-CD3 complex and the costimulatory molecule CD28, leading to an increased concentration of free intracellular calcium, [Ca 2ϩ ] i , and the activation of small GTP-binding proteins, such as Ras, and protein kinase C isoenzymes (8,9). These events lead to an increase in the activity of transcription factors implicated in cytokine and TNF ligand gene transcription including NF-B/Rel, nuclear factor of activated T cells (NFAT), and leucine zipper (Fos/Jun, CREB/ATF, and Maf) families of proteins (9 -12). T cell activation has been mimicked through direct intracellular activation of [Ca 2ϩ ] i -and Ras/protein kinase C-dependent signaling pathways by the combination of calcium ionophore, such as ionomycin, and phorbol esters, such as phorbol-12-myristate 13-acetate (PMA) (13). These combined stimuli potently induce de novo transcription of the genes encoding IL-2, TNF, and CD40L (4 -7, 14 -16).
Although the molecular events leading to CD40L expression appear to be similar to those eliciting expression of other cytokines (TNF and IL-2), transcriptional regulation of the gene encoding CD40L remains poorly understood. Previous studies suggest that both the IL-2 and TNF genes contain enhancerlike segments that include functional NF-B/Rel cis-activating elements important for their transcriptional activation. The 5Ј-flanking region of the human IL-2 gene consists of an inner core promoter immediately upstream of the 5Ј transcription start site and an adjacent 275-bp segment, located between bp Ϫ52 and Ϫ326 with respect to the transcription start site. This adjacent region behaves as a classic enhancer (i.e. it increases the activity of the core IL-2 promoter and does so in an orientation-independent manner) (17). This enhancer contains most of the cis-activating elements known to be required for optimal IL-2 gene transcription, including binding sites for NF-B/Rel, NFAT, and leucine zipper proteins (11,12).
A transcriptional enhancer active in mononuclear phagocyte cells has also been identified in the 3Ј-flanking region of the human TNF gene (18). This region includes a NF-B/Rel binding site that may contribute to increased TNF promoter activity (19). Currently, it is not known if this TNF 3Ј-flanking enhancer segment is active in T-lineage cells. Multiple putative NF-B/Rel cis-elements have been identified in the TNF gene's 5Ј-flanking region and appear to be important for TNF expression by mononuclear phagocytes (20). However, in contrast to the NF-B site of the IL-2 gene, the NF-B/Rel elements in the TNF promoter do not appear to regulate gene expression in T cells (21). Rather, the concerted binding of NFAT and leucine zipper proteins to the 5Ј-flanking region of the TNF gene appears to be essential for expression in activated CD4 T cells (21).
Like the TNF and IL-2 genes, CD40L gene transcription in activated T cells requires the cooperative binding of NFAT and leucine zipper proteins to the 5Ј-flanking region (22)(23)(24). A putative NF-B/Rel cis-element, GGGATTTCCA, has been identified in the 5Ј-flanking region of the CD40L gene based on sequence analysis (25) and has recently been reported to enhance promoter activity, possibly by binding NF-B p65 (26). Interestingly, a CD28 response element has also been identified in the CD40L promoter, identifying a role for CD28 engagement in the regulation of the CD40L promoter in CD4 T-lineage cells (27). However, no enhancer elements contributing to CD40L gene transcription by T cells or other cells have been described.
Here, we show that the human CD40L gene contains a T cell-specific, NF-B/Rel protein-dependent, classical enhancer in its 3Ј-flanking region that may be important in the regulation of CD40L transcription.
Real Time PCR Analysis of mRNA-Total cellular RNA was isolated (32) from CD4 T cells after 3 h of culture with or without activating stimuli (1.5 M ionomycin (Calbiochem) plus 50 ng/ml PMA (Sigma)). Random hexamer-primed reverse transcription (2 g of total RNA/ sample) was performed using the reagents contained in a TaqMan Gold RT-PCR kit (PerkinElmer Life Sciences) following the manufacturer's instructions. Two l of each reverse transcription reaction was submitted to real time quantitative PCR in an ABI Prism 5700 Sequence Detection System (PerkinElmer Life Sciences). Amplifications were performed in 96-well plates using oligonucleotide primers for transcript amplification of human CD40L (sense, 5Ј-CCAGGTGCTTCGGTGTTT-GT-3Ј; antisense, 5Ј-ATGGCTCACTTGGCTTGGAT-3Ј) (6), human IL-2 (sense, 5Ј-AAACTCACCAGGATGC-TCACATT-3Ј; antisense, 5-Ј-TGT-GGCCTTCTTGGGCA-3Ј) (33), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (proprietary primers purchased from PerkinElmer Life Sciences). For all primer sets, the reverse-transcribed samples were amplified in triplicate using the reagents contained in a SYBR Green PCR Core Reagents Kit (PerkinElmer Life Sciences). Fluorescence signals were detected during each of 40 cycles (denaturing 15 s at 95°C, annealing/extension 1 min at 60°C), as determined by binding of SYBR green to double-stranded DNA products (34). Denaturing curves of CD40L, IL-2, and GAPDH products, performed at the end of the PCR, confirmed the homogeneity of the DNA products. A standard curve was included in each plate, consisting of serial dilutions of a reverse-transcribed sample of activated adult CD4 T cell RNA amplified for CD40L, IL-2, and GAPDH transcripts. The cycle threshold (Ct) value is the calculated PCR cycle at which products first become detectable and is inversely related to the abundance of transcripts in the initial RNA sample (34). This value was determined using software included as part of the ABI Prism 5700 Sequence Detector System, following the manufacturer's instructions. The Ct values for GAPDH transcripts in the stimulated and unstimulated RNA samples were used as a control, and results were normalized to GAPDH. Comparable GAPDH cycle threshold values indicated that the amount of mRNA reverse transcribed and potentially amplifiable by PCR was similar in unstimulated and stimulated samples.
Reporter Gene Plasmids-The human CD40L gene sequence has been previously published (35). The 1284-bp 3Ј-flanking region of the CD40L gene analyzed in this study is shown in Fig. 1A. Maps of the firefly luciferase reporter gene constructs, all derived from the pGL2 basic vector (Promega), are shown in Fig. 1B. To simplify the nomenclature, these constructs are named based on their CD40L promoter and 3Ј-flanking segments, omitting reference to the intervening luciferase reporter gene. pCD40L was created by subcloning a 1.3-kb Hin-dIII/HindIII fragment containing the human CD40L promoter 5Ј-flanking segment from the construct gp39-luc (22) immediately upstream of the luciferase cDNA segment of pGL2-Basic. The pIL-2, pIL-4, and pIL-13 constructs directed by the 5Ј-flanking segments of the human IL-2 (0.6-kb), IL-4 (0.6-kb), and IL-13 (1.1-kb) genes, respectively, were similarly derived from pGL2-Basic, as previously described (22,28,36). pSV40, in which the luciferase cDNA is directed by a 0.2-kb segment of the SV40 large T antigen promoter, was purchased (pGL2 Promoter Vector; Promega). The 1284-bp segment of the human CD40L gene's 3Ј-flanking region (3ЈFL) was subcloned into pCD40L at adjacent BamHI and SalI cloning sites, located 1 kb 3Ј of the luciferase gene, creating pCD40L.3ЈFL (Fig. 2B). Subcloning used a naturally occurring 5Ј BamHI site and a 3Ј SalI site generated by PCR using Vent polymerase (New England Biolabs) and a human CD40L genomic phage clone as template. This 1284-bp segment begins 103 bp 3Ј of the CD40L termination codon and ends 401 bp after the final residue of exon 5 ( Fig.  1, A and B (35)). Constructs containing the entire 1284-bp 3Ј-flanking segment or truncations of this region are numbered using the first residue of the BamHI site as position 1. A similar approach was used to produce a construct containing the 3Ј segment in the reverse orientation (pCD40L.R3ЈFL). An endogenous PstI site was utilized to generate truncations of the full-length segment. Both the 5Ј (BamHI/PstI site (bp 1-808)) and the 3Ј (PstI/SalI site (bp 809 -1284)) fragments were subcloned into the pBS/KS cloning vector (Stratagene). This was followed by secondary subcloning into pCD40L using BamHI and a polylinkerderived SalI site (5Ј fragment) or a polylinker-derived BamHI and SalI site. The construct containing the 5Ј fragment (pCD40L.3ЈFL:1-808) or the 3Ј fragment (pCD40L.3ЈFL.809 -1284) retained their original orientation with respect to the CD40L promoter. The 1284-bp CD40L 3Јflanking segment was also subcloned at the BamHI/SalI site 3Ј of the luciferase cDNA segment in pIL-2, pIL-13, and pSV40 constructs to create pIL-2.3ЈFL, pIL-13.3ЈFL, and pSV40.3ЈFL, respectively. All PCR generated segments were sequenced to ensure that no polymeraseinduced mutations occurred. In some experiments, transfection efficiency was assessed by co-transfection of a Renilla luciferase (Promega) reporter gene construct, in which expression was driven by either the human ␤-actin promoter (p␤-actin-RL, a gift of M. Sweetser, University of Washington) or by a minimal T7 bacteriophage promoter (Promega).
Site-directed Mutagenesis of the pCD40L.3ЈFL Plasmid-Site-directed mutagenesis was performed using a commercial kit (QuikChange; Stratagene) to introduce dinucleotide substitution mutations into the NF-B/ Rel site of the 3Ј-flanking segment of the pCD40L.3ЈFL from the wild type sequence of GGGAATTTTCCA to GTTAATTTCCA (pCD40L.3ЈFL.mut1) or to GTTAATTTAAC (pCD40L.3ЈFL.mut2). All mutated constructs were sequenced on both strands to verify these mutations and to confirm that no other alterations were introduced.
Transient Transfection and Reporter Gene Analysis-Ten g of firefly luciferase reporter gene plasmid DNA in complete RPMI medium was used for transient transfection by electroporation. The conditions used for the electroporation of primary CD4 T cells have been described (22,28,37). Jurkat cells (1.0 ϫ 10 7 cells/condition) were transiently transfected by electroporation at 260 V and 960 microfarads using a Genepulser electroporator (Bio-Rad). Electroporation conditions for 8.1.6 Epstein-Barr virus-transformed human B cells (2.5 ϫ 10 7 cells/condition) were 210 V and 960 microfarads, and for U937 human monocytelike cells (2.5 ϫ 10 7 cells/condition), conditions were 250 V and 960 microfarads. Following electroporation, cells were incubated for 20 -24 h at 37°C, harvested, and plated at a density of 1.0 ϫ 10 6 viable cells/well, in 96-well U bottom microtiter plates (Corning Glass). Cells were left untreated or stimulated with ionomycin (1.5 M; Calbiochem) plus PMA (25 ng/ml; Sigma) or with the combination of monoclonal antibodies (mAbs) directed against CD3-⑀ (mAb 64.1 (38)), 1:500 dilution of sterile ascites and CD28 (mAb 9.3 (39)), 1:200 dilution of sterile ascites, for 6 h prior to harvesting. CD3 and CD28 mAbs were purchased from Bristol-Myers Squibb Co. Cells were harvested and analyzed for firefly luciferase activity as previously described (28). For Jurkat cell lysates, a Monolight 1500 luminometer (Analytical Luminescence Laboratories, Ann Arbor, MI) was used. Primary human CD4 T cells have a relatively low transfection efficiency following electroporation (40), so a more sensitive luminometer, Berthold model LB9507, was used. For the data presented, at least one experiment was carried out in which either 0.1 g of p␤-actin-RL was included with transfections of Jurkat thymoma cells or 0.5 g of pRL-null was included with transfections of primary human CD4 T cells. Cell lysates isolated from these cells were assayed sequentially for firefly and Renilla luciferase activity using the reagents of the Dual Luciferase Assay Kit (Promega). Renilla activity between samples varied less than 10% in these experiments. Therefore, the data for firefly luciferase activity are presented without correction for transfection efficiency.
Nuclear Protein Extract Preparation and Electromobility Shift Assays (EMSAs)-Nuclear protein extracts were prepared as previously described (29) from CD4 T cells activated by incubation for 2 h with CD3 and CD28 mAb, and their protein concentration was determined by the Bradford method using a commercial kit (Pierce). For EMSAs, SDS-PAGE-purified double-stranded oligonucleotides (purchased from Invitrogen) were used as either 32 P-labeled probes or as unlabeled competitors. For each oligonucleotide, only the coding strand sequence is shown, with the NF-B binding sites underlined and mutated residues indicated in boldface type (see Table I). The CD40L 3Ј-flanking NF-B oligonucleotide, 5Ј-TGGAGGGAATTTTCCCAACC-3Ј (ϩ941 to ϩ960 bp of the 1284-bp 3Ј-flanking segment (Fig. 1A)), was radiolabeled using T4 polynucleotide kinase and [␥-32 P]ATP and then annealed. Competitors consisted of either the wild type CD40L 3Ј-flanking NF-B oligonucleotide and derivatives containing either a single dinucleotide substitution mutation, 5Ј-TGGAGTTAATTTTCCCAACC-3Ј (single mutant), or two dinucleotide mutations, 5Ј-TGGAGTTAATTTTAACAACC-3Ј (double mutant), or a 5Ј-flanking CD40L gene oligonucleotide containing a putative NF-B element, 5Ј-GGTAGGGATTTCCACAGCT-G-3Ј (bp Ϫ1203 to Ϫ1187 bp with respect to the transcription start site (22,25)). As a positive control, an oligonucleotide competitor, 5Ј-GAG-GGGACTTTCCGAG-3Ј, containing the NF-B site of the enhancer of the murine immunoglobulin (Ig) chain gene (41), was used. EMSAs were performed following the method of Ortiz et al. (42), using 1 g of nuclear protein and 1.0 ϫ 10 4 cpm of probe per reaction. Where indicated, unlabeled double-stranded oligonucleotides were preincubated with nuclear protein prior to the addition of probe. In some experiments, nuclear protein was preincubated with 1.0 l of supershifting antisera specific for individual NF-B/Rel family proteins or with a commercial supershifting mAb reactive with NFATc2 (NFAT1, NFATp) (Affinity Bioreagents, Golden, CO). The panel of NF-B/Rel-specific antisera (generously provided by Dr. N. R. Rice, NCI-Frederick Cancer Research and Development Center) included specific antiserum 1141 (N-terminal region of p50), 1267 (N-terminal region of p52), 1226 (Cterminal region of p65), and 1266 (C-terminal region c-Rel) as well as preimmune serum. The generation and characterization of these antisera have been described previously (43)(44)(45). In preliminary EMSAs, 1.0 l of antiserum resulted in maximal supershifting of DNA-protein complexes formed between a probe containing the Ig chain enhancer NF-B site and protein present in activated T cell nuclear extracts.
DNase I-hypersensitive Site Mapping-Techniques used for purification of nuclei and DNase I digestions were modifications from Siebenlist et al. (46). Briefly, human thymoma CD4 T-lineage cells of the D1.1 Jurkat cell line, which constitutively expresses CD40L (47), were grown in RPMI medium containing 10% fetal bovine serum. Fifty million cells were harvested and centrifuged three times in ice-cold Hanks' balanced salts solution. The final pellet was resuspended in ice-cold nuclear isolation buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl 2, 0.1 mM EGTA, 15 mM Tris-HCl (pH 7.4), 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 0.3 M sucrose). Nuclei were then prepared by lysis with 0.1% Nonidet P-40 and layering onto an equal volume of nuclear isolation buffer, containing 1.7 M sucrose as a cushion. Nuclei were centrifuged for 30 min at 30,000 ϫ g in an ultracentrifuge using a SW-40.1 rotor, and the nuclear pellet was resuspended in 2.5 ml of nuclear isolation buffer, containing 5% glycerol, and aliquoted into 0.5-ml samples. Graded amounts of DNase I (Worthington) were added to each nuclear sample for 3 min at room temperature. Reactions were stopped by the addition of 50 l of 5% SDS, 100 mM EDTA. Samples were then digested with proteinase K at 37°C overnight, and DNA was subsequently isolated by repeated organic extraction and ethanol precipitation. DNase I-treated or -untreated DNA samples were digested to completion with BamHI, electrophoresed on 1% agarose gels (10 g of sample/lane), and blotted onto Hybond Plus nylon membrane (Amersham Biosciences) using 20ϫ SSC. A 289-bp 32 P-labeled probe, corresponding to the extreme 3Ј-end of the 2.2-kb BamHI fragment that contains the 3Ј-untranslated region of the human CD40L gene and downstream sequences, was generated by PCR using primers 5Ј-ATT-TCATCTCCTGCTGCCACC-3Ј and 5Ј-GGGCCAATTCAATGCTA-3Ј derived from publicly available human genome sequence. Hybridization was performed in roller bottles using QuickHyb (Stratagene, La Jolla, CA) solution with 1.25 ϫ 10 7 cpm of spun column-purified probe per 10 ml of hybridization solution at 68°C. Blots were washed, with the last wash of high stringency (0.05ϫ SSC with 0.1% SDS at 68°C for 30 min).
Real Time PCR-based Chromatin Immunoprecipitation (ChIP) Assays-ChIP assays were carried out using a commercial kit following the manufacturer's instructions (Upstate Biotechnology, Inc., Lake Placid, NY). Briefly, 20 ϫ 10 6 CD4 T cells were isolated from the peripheral blood of healthy adult donors and stimulated for 2 h at 37°C with CD3 and CD28 mAbs. Cross-linking with 1% formaldehyde for 15 min at 37°C was followed by cell lysis and sonication of DNA into 200 -800-base pair fragments. Proteins linked to DNA were immunoprecipitated with preimmune sera or antiserum 1141 (reactive with the N terminus of NF-B p50) (both generously provided by Dr. Nancy Rice) using salmon sperm DNA-protein A-agarose beads. Immunoprecipitated protein-DNA complexes were washed, eluted, and unlinked, and proteins were then degraded by proteinase K digestion for 1 h at 45°C. DNA was phenol/chloroform-extracted, precipitated with glycogen carrier, and used as a template for real time PCRs following an approach similar to that of Christenson et al. (48). Genomic sequence primers encompassing the NF-B/Rel cis-element (902F, 5Ј-CCTAAAGCTCCC-AGCCAGGT-3Ј; 972R, 5Ј-AAACATCACAAAGGTTGGGAAAAT-3Ј) were used to amplify immunoprecipitated DNA as template. Samples were done in triplicate as described above for the real time PCR analysis of mRNA levels using the reagents contained in a SYBR Green PCR Core Reagents kit (PerkinElmer Life Sciences). Fluorescence signals were detected during each of 40 cycles (denaturing for 15 s at 95°C, annealing/extension for 1 min at 60°C) as determined by binding of SYBR green to double-stranded DNA products.

CD40L 5Ј-Flanking Promoter Activity Is Lower than That of the IL-2 Promoter in Polyclonally Activated CD4 T Cells-The
promoter activity of the 1.3-kb 5Ј-flanking segment of the CD40L gene (pCD40L) or a 0.6-kb 5Ј-flanking segment of the IL-2 gene (pIL-2) was assessed using luciferase reporter constructs. After transfection of these constructs into peripheral blood CD4 T cells, cells were either treated with medium alone (unstimulated) or polyclonally activated using ionomycin and PMA at concentrations that yield maximal levels of CD40L and IL-2 gene transcription and mRNA accumulation (5,14,15). Transfection with the promoterless parent plasmid, pGL2, served as a negative control and produced barely detectable luciferase expression ( Fig. 2A). Transfection of pCD40L or pIL-2 resulted in reporter gene activity that was markedly enhanced by CD4 T cell stimulation with ionomycin and PMA. We consistently observed that pCD40L-mediated luciferase expression was considerably lower than reporter gene expression driven by pIL-2 in both freshly isolated T cells ( Fig. 2A) and in CD4 T cells that had previously been primed in vitro (data not shown). The relatively low promoter activity of the 1.3-kb 5Јflanking CD40L gene segment does not appear to be due to strong negative regulatory elements, since we have previously shown that truncation of the 5Ј region of this segment does not increase promoter activity (22).
Polyclonally Activated CD4 T Cells Accumulate Similar or Higher Levels of CD40L Transcripts than IL-2 Transcripts-The observation that pCD40L-mediated reporter gene expression was lower than pIL-2-driven reporter expression suggested that the level of CD40L transcripts would also be reduced in comparison with IL-2 mRNA levels. This assumed that the half-lives of CD40L and IL-2 mRNA are similar in ionomycin and PMA-stimulated CD4 T cells, which has been verified. 2 To address this, total RNA was isolated from peripheral blood CD4 T cells 3 h after culture, with or without ionomycin and PMA stimulation, and reverse-transcribed RNA was analyzed for CD40L, IL-2, and GAPDH mRNA levels using real time PCR. A lower Ct value for CD40L and IL-2 transcripts in stimulated versus unstimulated CD4T cells indicated that activation increased CD40L and IL-2 gene transcription in agreement with previous results (Fig. 2B and Refs. 5 and 15). The high Ct value and low level of CD40L and IL-2 transcripts in total RNA from unstimulated cells was not due to degraded template, since similar levels of GAPDH transcripts were detected in unstimulated and stimulated T cells (data not shown). In contrast to the low level of activity of the CD40L promoter in transient transfection experiments, the Ct values in activated CD4 T cells indicated that CD40L transcripts were present in equal or greater amounts than IL-2 transcripts (Fig. 2B). This is unlikely to be an artifact resulting from more efficient amplification of the CD40L product, since both CD40L and IL-2 PCR products were 50 bp in size and were amplified using identical reaction conditions. Together, these findings suggest that regions of the CD40L gene other than the 1.3-kb 5Ј-flanking region are necessary for optimal transcription in polyclonally activated CD4 T cells.
A 1284-bp Segment of the CD40L 3Ј-Flanking Region Acts as an Enhancer of CD40L Promoter Activity in CD4 T Cells-Because the 3Ј-flanking region of many genes expressed by T lymphocytes, such as the genes encoding CD2 and IL-4 (49), contain transcriptional enhancers, we investigated whether the 3Ј-flanking region of the CD40L gene might contribute to its transcriptional activation in CD4 T-lineage cells. Examination of the 3Ј-flanking region DNA sequence revealed a potential NF-B/Rel cis-element, GGAATTTTCCC, at bp 946 -956 with respect to the BamHI site of exon 5, which matched 9 of 10 residues of the ideal NF-B/Rel consensus sequence, GGGRNYYYCC (50) (see Table I). Since NF-B/Rel family proteins have been implicated in the activation-dependent expression of several T cell-derived cytokines, including IL-2 and IL-3 (50), we tested the ability of a 1284-bp segment containing this putative NF-B/Rel binding site to modulate the activity of the CD40L promoter. This segment was subcloned downstream (ϳ1.0 kb) of a luciferase reporter gene cDNA segment driven by the human CD40L proximal promoter (pCD40L), maintaining the normal orientation between the 3Ј-flanking segment and pCD40L to yield pCD40L.3ЈFL (Fig. 1B). In order to evaluate orientation dependence, a reporter gene construct with the 3Ј segment subcloned in the reverse direction (pCD40L.R3ЈFL) was generated.
The 3Ј-flanking segment enhanced basal CD40L promoter activity. However, in Jurkat CD4 cells stimulated with ionomycin and PMA, or with CD3 mAb alone or in combination with CD28 mAb (51), the 3Ј-flanking segment greatly increased the CD40L promoter independent of the orientation of the 3Ј segment (Fig. 3A). These data suggest that this 3Ј-flanking segment acted as a classical (orientation-independent) enhancer (52).
Transformed human CD4 T-lineage cell lines, such as Jurkat, have been reported to differ from primary human CD4 T cells in their transcriptional regulation of activation-dependent genes, such as IL-2 (37). Therefore, we tested whether the 3Ј-flanking segment could enhance CD40L promoter activity in  freshly isolated CD4 T cells. In the absence of stimulation, luciferase expression was not detected for any of the CD40L promoter-directed constructs or for the promoterless pGL2 parent vector in CD4 T cells ( Fig. 2A, and data not shown). However, similar to Jurkat T cells, the 3Ј-flanking segment increased CD40L promoter activity in stimulated CD4 T cells in an orientation-independent manner (Fig. 3B).
The CD40L 3Ј-Flanking Region Enhances the Activity of Heterologous Cytokine Promoters in a T Cell-specific Manner-We next assessed the specificity of the transcriptional enhancer activity for CD40L versus other activation-dependent genes in CD4 T-lineage cells by inclusion of the CD40L 3Ј segment in firefly luciferase reporter constructs directed by the promoters for cytokine genes IL-2 and IL-13 and for the large T antigen of the SV40 virus. The 3Ј-flanking segment of the CD40L gene augmented ionomycin-and PMA-stimulated activity of the IL-2 and IL-13 promoters in both Jurkat cells and in freshly isolated peripheral blood mononuclear cells, used as a source of T cells in this experiment (Fig. 4). In contrast, this segment failed to enhance SV40 promoter activity in either source of T cells, suggesting that the 3Ј-flanking segment is not a generic enhancer of promoters that are active in CD4 T-lineage cells.
To evaluate the tissue specificity of the transcriptional enhancer activity of the 3Ј-flanking region of the CD40L gene, a human Epstein-Barr virus-transformed B cell line, 8.1.6, or the monocyte-like cell line, U937, was transfected with pCD40L or pCD40L.3ЈFL and evaluated for reporter gene activity. Low amounts of CD40L promoter activity were detectable in both of these cell lines in the absence of stimulation, but this increased ϳ1.5-2-fold in response to ionomycin and PMA treatment (data not shown). However, there was no significant increase in ionomycin-and PMA-stimulated transcriptional activity mediated by the 3Ј-flanking segment (Fig. 4) in the context of either the CD40L promoter or the heterologous cytokine promoters.
Similar to Jurkat and nontransformed T cells, the CD40L 3Ј-segment did not augment SV40-promoter activity in 8.1.6 or U937 cells. These results suggest that activation-induced enhancer activity of the 3Ј-flanking region of the CD40L gene is relatively T cell-specific.
A DNase I-hypersensitive Site Is Present in the 3Ј-Flanking Region of CD40L-For many genes, key transcriptional regulatory regions are identified based on their hypersensitivity to digestion with DNase I (53). To determine whether this applied to the CD40L 3Ј-flanking region, we isolated nuclei from a CD40L-expressing CD4 T-lineage cell line, subjected these to DNase I treatment, and evaluated the 3Ј-flanking region for hypersensitive sites using Southern blotting and an appropriate probe. In the absence of DNase I treatment, a full-length 2.2-kb BamHI fragment was detected (Fig. 5). Treatment of nuclei with increasing concentrations of DNase I revealed a 1.4-kb band (Fig. 5, lane 4), which mapped to the 3Ј-flanking region that contained the putative NF-B/Rel site. These findings indicated that the NF-B/Rel site was contained within a region having an open chromatin configuration, consistent with this region playing a role in transcriptional regulation of the CD40L gene.

A Functional NF-B/Rel Site in the 3Ј-Flanking Region Is Required for Enhancement of CD40L Promoter Activity in CD4
T Cells-To determine whether the putative NF-B/Rel site was important for 3Ј-flanking region enhancement of CD40L transcription, we compared the enhancement of the CD40L promoter by truncations of the 1284-bp segment that either lacked (bp 1-809 segment) or contained (bp 809 -1284) this site (Fig. 1B). While the presence of the 1-809 bp segment did not consistently increase CD40L promoter activity by CD4 T cells (Fig. 6A), additional truncations within this region suggested the presence of both positive and negative regulatory elements capable of affecting CD40L promoter activity (data not shown). In contrast, the 809 -1284-bp segment was sufficient to substantially augment CD40L promoter activity in CD4 T cells, indicating that it contained an important transcriptional activation element.
We next tested whether the putative NF-B/Rel-binding segment of the 1284-bp 3Ј-flanking region was essential for enhancement of CD40L promoter activity. This segment AGG-GAATTTTCCC includes not only a 9 of 10 match (underlined) for the consensus NF-B decamer-binding site (GGGRN-TYYCC (50)) but also contains a 7 of 7 match (italic type) on the noncoding strand for an NFAT binding site (HGGAAAA (54)). This finding could be of functional importance, since there are examples where NF-B and NFAT transcription factors can independently contribute to transcriptional activation by binding to such NF-B/NFAT composite cis-elements (e.g. the human immunodeficiency virus-1 long terminal repeat promoter (55,56).
To distinguish the role of these two transcription factor families we used site-directed mutagenesis. Two full-length mutant enhancers were created. We compared the enhancement of CD40L promoter activity by the wild type 1284-bp segment with that conferred by either single (GTTAATTTCC) or double dinucleotide mutations (GTTAATTTAAC). The single dinucleotide mutation was expected to effectively abrogate binding by most NF-B/Rel proteins, based on the results of in vitro studies using recombinant NF-B/Rel proteins to define ideal binding sites (57), while leaving the NFAT binding site intact. In contrast, the double mutant was expected to abrogate both NF-B/Rel and NFAT binding (54,57). We found that the single dinucleotide mutation resulted in more than 50% loss of the enhancement of CD40L activity by the 1284-bp 3Ј-flanking region in CD4 T cells (Fig. 6B). On the other hand, the double dinucleotide mutation led to only a slight additional decrease in enhancement, suggesting that NFAT binding at this site made a minor contribution to enhancer activity. These results suggested that NF-B/Rel proteins, rather than NFAT, were crucial for the enhancer activity mediated by the NF-B/Rel site.
The NF-B/Rel Site Binds CD4 T Cell p50 NF-B1 in Vitro and in Vivo-Using EMSAs, we found that an oligonucleotide probe containing the NF-B/Rel site of the CD40L 3Ј-flanking region formed a complex with nuclear protein from CD3 and CD28 mAb-activated CD4 T cells (Fig. 7A, lane 1). Formation of this complex was inhibited by either a 20-or 200-fold molar excess of unlabeled oligonucleotide (Fig. 7A, lanes 2 and 3,  Self). In contrast, a 20-or 200-fold molar excess of oligonucleotides containing mutations of the NF-B/Rel site were ineffective in competing complex (Fig. 7A, lanes 4 -7). This indicated that complex formation at the NF-B/Rel site with nuclear protein correlated with the ability of this site to contribute to CD40L enhancer activity. The specific complex was also effectively competed by an oligonucleotide containing the canonical NF-B/Rel site of the murine immunoglobulin chain, indicating that it might bind NF-B/Rel proteins (Fig. 7A, lanes 8 and  9). In contrast, the putative NF-B/Rel element located in the 5Ј-flanking region of the human CD40L gene (24) was ineffective as a competitor (Fig. 7B, lanes 1-4), suggesting that it did not specifically bind the same proteins as the 3Ј-flanking site.
The inclusion of specific antiserum reactive with the p50 form of the NF-B1 gene product resulted in a virtually complete supershift of the specific complex formed with the CD40L 3Ј-flanking NF-B oligonucleotide, suggesting that p50 was a major component (Fig. 7B, lanes 5 and 6). In contrast, antisera to p52 (NF-B2), p65 (RelA), c-Rel, or NFAT1, the major NFAT protein contained in these extracts (28), had little or no effect on the formation or the mobility of the complex (Fig. 7B, lanes  7-10). We also failed to obtain supershifts using a variety of commercial antisera or mAbs specific for p65 and c-Rel. Together, these results indicated that p50 homodimers were likely to be the predominant NF-B/Rel species in the DNAbinding complex and that the CD40L 3Ј-flanking NF-B/Rel site was an important cis-element for the enhancement of CD40L promoter activity by binding NF-B p50.
To address whether binding of NF-B1 p50 to the 3Ј-flanking NF-B/Rel site occurred in intact CD4 T cells, ChIP assays were performed. Primary CD4 T cells were briefly stimulated with CD3 and CD28 mAbs, and, after formaldehyde crosslinkage and sonication, proteins were immunoprecipitated with preimmune antiserum or an antiserum specific for the N-terminal region of NF-B p50. CD40L 3Ј-flanking region primers encompassing the NF-B/Rel site were then used to amplify immunoprecipitated DNA in a real time PCR assay. The Ct value (Ct ϭ 27.6) obtained with NF-B p50 antiserum was consistently lower than that obtained with preimmune antiserum (Ct ϭ 29). This indicated that the NF-B p50 antiserum specifically immunoprecipitated the CD40L 3Ј DNA template containing the NF-B/Rel site (Fig. 8) and that NF-B p50 binds to this site in vivo in human CD4 T cells.

DISCUSSION
CD40L gene expression is crucial in both the initiation and progression of various immune responses, particularly for those involving T cells and B cells, as demonstrated by the severe immunological consequences of CD40L genetic deficiency. Ligation of CD40 is an important step for regulation of expression of cytokines, adhesion molecules, apoptotic mediators, and microbicidal activities by a number of cell types. In addition, CD40L plays a key role in pathogenesis of chronic inflammatory diseases such as autoimmune disorders, graft versus host disease, atherosclerosis, and neurodegenerative disorders (1, 58 -61). Definition of the critical regulatory regions of the CD40L gene is therefore not only of great importance in understanding the events invoked in the generation of the adaptive immune response, but may allow the development of novel therapies that target this important protein in a variety of diseases.
We found that the 3Ј-flanking region of the CD40L gene substantially enhanced the activity of the CD40L promoter in transformed CD4 T-lineage cell lines as well as in primary peripheral blood CD4 T cells. This DNA segment enhanced transcription in either orientation, indicating that it acted as a classic enhancer (52). The enhancement of CD40L promoter activity by the 3Ј-flanking region was observed with activation using CD3 mAb alone or a combination of CD3 and CD28 mAbs, indicating that it was not unique to pharmacological stimulation. These results suggest that such enhancement is likely to apply to physiological activation of CD4 T cells following engagement of the ␣␤-T cell receptor-CD3 complex by antigenic peptide bound to class II MHC molecules or by microbe-derived superantigens.
The core 475-bp CD40L enhancer segment contained a decameric sequence, GGAATTTTCC, that is similar to well characterized and functional NF-B/Rel cis-elements of other genes (see Table I and references therein). In addition, a DNase I hypersensitivity site that corresponded to the NF-B/Rel element within the enhancer was identified. Consistent with the functional importance of the NF-B/Rel site, an oligonucleotide containing the NF-B/Rel element formed a specific complex with nuclear protein contained in CD4 T cells, of which NF-B p50 was a prominent component. ChIP assays confirmed the association of NF-B p50 with the 3Ј-enhancer site. Importantly, the ability of this element to form an NF-B-containing protein complex with CD4 T cells strictly correlated with the ability of the 3Ј-flanking region to enhance CD40L promoter activity in this cell type. In contrast, the presence of an additional mutation in the 3Ј region of the element, which would be expected to also prevent binding of NFAT proteins (12), only led to a modest additional decrease in enhancer activity. This suggests that the binding of NF-B/Rel proteins, rather than NFAT proteins, is necessary for most of the enhancer activity of this element in CD4 T cells and is consistent with our inability to detect NFAT proteins in CD4 T cell nuclear protein complexes formed with the CD40L 3Ј-flanking NF-B/Rel element (data not shown). In support of our findings, NF-B p50 was noted to be required for the induction of CD40L expression, based on studies using mice with genetic disruption of the NF-B1 gene (63). However, these results do not formally exclude the possibility that NFAT proteins may not act independently but only cooperatively with NF-B/Rel proteins in mediating enhancer activity. While there are clear examples of composite NF-B/Rel and NFAT cis-elements in which both transcription factor families independently contribute to transcriptional activation in T-lineage cells (e.g. the human immunodeficiency virus long terminal repeat promoter (55,56) and the first intron interferon-gamma gene enhancer (64)), we conclude that this does not appear to apply to the CD40L 3Јflanking enhancer. The active DNA-binding forms of NF-B/Rel proteins are dimers, which may consist of any two members of the NF-B/ Rel family, including p50 (a derivative of the NF-B1 gene), p52 (a derivative of the NF-B2 gene), p65, c-Rel, and RelB (50,57,65). With the exception of RelB, all of these NF-B/Rel proteins are expressed by T cells (65). We found that the p50 product of the NF-B1 gene was the predominant type of NF-B/Rel protein contained in the nuclear extracts of human CD4 T cells that bound to the 3Ј-flanking enhancer site in vitro and that p50 antisera immunoprecipitated the enhancer region of the CD40L 3Ј-flanking segment. No detectable binding of p52, p65, or c-Rel was found, although the nuclear protein extracts we employed contain substantial amounts of these proteins (data not shown). p50 homodimers have a tendency to bind with high affinity to palindromic NF-B/Rel sites (57), as defined by using in vitro binding site selection assays (66). It is also interesting to note that, with the exception of one nucleotide indicated in italics, the NF-B/Rel element consists of an 11-bp palindromic sequence, GGGAATTTTCCC, and that a similar 11-bp palindromic sequence, found in the enhancer of the class I MHC gene (see Table I), has previously been shown to preferentially bind p50 homodimers (67). These results, taken together, suggest that p50, probably in the form of homodimers, enhances CD40L promoter activity by binding to a 3Ј-flanking cis-activation element.
Analysis of enhancer activity in peripheral blood CD4 T cells revealed that most of the activity of the 3Ј-flanking segment was localized to a 475-bp subregion. This truncation, in which only 75 bp of the 3Ј-untranslated region remained, actually further increased enhancer activity compared with the initial 1248-bp segment, suggesting the presence of inhibitory regions in the 3Ј-untranslated region (see Figs. 1B and 5A). Determination of whether the 3Ј-untranslated region also contains transcriptional activation regions that contribute to enhancer activity will require additional study, but our preliminary results with additional truncation constructs are consistent with this possibility.  Table I Our results contrast with several well described transcriptional contexts in which p50 homodimer binding correlates with the inhibition rather than the activation of transcription (e.g. in the 5Ј-flanking promoters of the class I MHC (67) and IL-2 genes (68)). However, p50 homodimers have been shown in other contexts to act as transcriptional activators, such as in the induction of transcription of the long terminal repeat promoter of the human immunodeficiency virus, type 1 (69). Although p50 lacks the Rel homology domain found in p65, c-Rel, and RelB (65), which associates with co-activator proteins (70), p50 has been shown to contain other domains that mediate transcriptional activation (71,72). p50 can also associate with other non-NF-B/Rel DNA-binding proteins in mediating transcription (65), such as members of the CCAAT enhancer-binding protein, CREB/ATF, HMG, and SP-1 families, and with the proto-oncogene product, bcl-3, which can act as a transcriptional co-activator (73). Therefore, it is plausible that p50 homodimers could contribute to increased CD40L gene transcription in T-lineage cells. This is particularly the case, since such homodimers are a major component of the nuclear extracts of freshly isolated T cells (74), which have a high capacity to rapidly express the CD40L gene (6,7).
Both basal and activation-induced transcription were increased by the 3Ј-flanking enhancer in Jurkat cells, while in primary cells, such as purified T cells or unfractionated peripheral blood mononuclear cells, enhancer function was strictly activation-dependent. Since CD40L mRNA and transcription by primary T cells are usually undetectable in the absence of activation (6,7,14,15), these results suggest that the transcriptional regulatory environment of Jurkat cells may not fully replicate the normal inhibition of CD40L gene transcription that occurs in the absence of T cell activation. Further, although selected Jurkat cell lines have been identified that constitutively express CD40L at high levels (58), we are unaware of any Jurkat or other transformed T-lineage cell lines in which most CD40L gene expression is regulated in a physiological, activation-dependent manner. For these reasons, as well as previous studies documenting substantial differences between Jurkat cells and primary T cells in the transcriptional regulation of the IL-2 gene (37), we relied on primary circulating leukocyte populations containing T cells to further define regions of the CD40L 3Ј flanking segment that were necessary for enhancer activity.
Studies of the IL-2 gene as well as the genes encoding other proteins, such as granulocyte-macrophage colony-stimulating factor and TNF, have shown that CD28 engagement increases both the rate of cytokine gene transcription and cytokine mRNA stability, resulting in marked increases in cytokine production (75,76). Our data using reporter gene constructs is consistent with CD28 engagement having a modest effect on CD40L gene transcription but does not exclude the possibility that CD40L mRNA stability may also be enhanced in this context. The modest increase in CD40L promoter activity that occurred with the combination of CD3 and CD28 mAbs compared with CD3 mAb alone is also consistent with previous reports finding only a significant but modest increase in CD40L surface expression by CD4 T cells achieved by the combination of CD3 and CD28 engagement (77). These results also suggest that the 5Ј-flanking region of the CD40L gene contains a functional CD28 response element, as recently reported (27), but does not rule out the possibility that the 3Ј-flanking enhancer may also contain such a cis-element with such responsiveness.
There is precedence both for 3Ј-flanking enhancers of genes expressed by T lymphocytes, such as the CD2 protein (45) and IL-4 (49), and for 3Ј-flanking region enhancers utilizing NF-B/ Rel cis-elements (e.g. immunoglobulin chain genes (78)) in Blineage cells. However, to the best of our knowledge, the enhancer of the CD40L gene described here is the first in which a NF-B/Rel element located in the 3Ј-flanking region has been implicated in activation-dependent gene expression by T cells. This is in contrast to previously reported NF-B/Rel sites utilized by T cell activation-dependent genes, which have been identified either in the 5Ј-flanking promoter (e.g. the IL-2 (79) and granulocyte-macrophage colony-stimulating factor genes (80)) or the first intron, such as for the interferon-␥ gene (64). As discussed above, the 3Ј-flanking NF-B/Rel site we identified in the CD40L gene also differs from these other sites in that it appears to preferentially bind p50 homodimers rather than other NF-B Rel complexes commonly found in human T cells, such as heterodimers of p50 or p52 with Rel A (p65) or with c-Rel (74).
Our results suggest that the 3Ј-flanking region of other activation-dependent genes expressed by T cells, such as other members of the TNF ligand superfamily, could play a role in transcriptional activation. Interestingly, TNF has been reported (18) to contain a 3Ј-flanking transcriptional enhancer that is active in monocyte lineage cells stimulated with lipopolysaccharide or the exotoxin, toxic shock syndrome toxin-1. Like the CD40L enhancer, this TNF enhancer segment includes a NF-B/Rel cis-element in the 3Ј genomic region immediately flanking the last exon, and this element may augment lipopolysaccharide-induced TNF transcription by mononuclear phagocyte lineage cells (19). This suggests the possibility that such NF-B/Rel-dependent 3Ј-flanking enhancers may be a previously unappreciated feature of other members of the TNF ligand superfamily. Whether this 3Ј-flanking TNF enhancer and its NF-B/Rel cis-element also have activity in T-lineage cells is not known but, given our results, is of potential interest.
Finally, the observation that a relatively small region of the CD40L gene 3Ј-flanking region acts in a relatively T cell-specific and activation-specific manner may have usefulness as a means to augment activation-dependent transcription by T cells in a number of contexts. Such an approach might be considered for gene therapy treatment of inherited CD40L deficiency (3,81) or other immune deficient states in which expression of TNF ligands or hematopoietin cytokines by T cells is reduced, such as during early postnatal life (7). However, the importance of maintaining the normal physiologic pattern of CD40L gene expression in such therapy is suggested by the development of T cell lymphomas in mice following the administration of retroviruses in which CD40L expression is directed by a constitutively active promoter (62). Therefore, it will be of interest to determine whether including authentic transcriptional regulatory elements, such as the CD40L 3Јflanking enhancer region, in gene expression vectors results in higher levels of T cell activation-dependent expression of CD40L in vivo.