Full text of DOI:10.1078/0171-2985-00128

Immunobiol. (2002) 205, pp. 231–246
© 2002 Urban & Fischer Verlag
http://www.urbanfischer.de/journals/immunobiol
Department of Dermatology, University of Erlangen-Nürnberg, Erlangen, Germany
Cloning and Characterization of the Promoter Region of the
Human CD83 Gene
S
G
USANNE
EROLD
B
ERCHTOLD, PETRA
M
ÜHL-ZÜRBES, ELISABETH
MACZEK, ANTJE
GOLKA
,
S
CHULER, and ALEXANDER S
TEINKASSERER
Received August 8, 2001 · Accepted in revised form February 6, 2002
Abstract
Human CD83 (hCD83) is a glycoprotein expressed predominantly on the surface of dendritic cells
(DC). To get insight into the regulation of hCD83 expression, we cloned a 3037 bp fragment up-
stream of the translation initiation codon. Deletion mutants were constructed revealing highest
promoter activity in the –261 fragment containing four SP1 binding sites and one NF-ÍB element.
Electrophoretic mobility shift assays demonstrated the specific interaction of NF-ÍB factors with the
NF-ÍB element as well as specific binding of SP1 and SP3 to the SP1 binding site. The hCD83 pro-
moter was inducible by TNF-a. This inducibility was strictly dependent on the intact NF-ÍB ele-
ment.
Introduction
Dendritic cells (DC) are the most potent antigen presenting cells of the immune system.
In their immature stage, they reside in peripheral tissues and are highly phagocytic but
possess little T cell stimulatory capacity. Appropriate stimuli (microbial products,
inflammatory cytokines) induce the maturation of DC. They migrate to secondary lym-
phoid organs where they trigger a specific T cell response (1). In vitro, immature DC can
be generated from peripheral blood monocytes by culturing them in the presence of
GM-CSF and IL-4 (2, 3). These cells show high endocytic but low T cell stimulatory
capacity. Upon addition of monocyte conditioned medium (MCM) (4) or a cocktail
[IL-1b, IL-6, TNF-a, and prostaglandin E2 (PGE2)] that mimicks MCM (5), these cells
develop into fully mature, immunostimulatory cells. This maturation process is charac-
terized by functional changes as well as a change in the phenotypical expression pattern
of surface molecules. The expression of adhesion and costimulatory molecules is strong-
Abbreviations: TNF-a = tumor necrosis factor a; DC = dendritic cell; nt = nucleotide; bp = base pair;
IL-6 = interleukin 6; IL-4 = interleukin 4; IL-1b = interleukin 1b; GM-CSF = granulocyte-macro-
phage colony stimulating factor; PGE2 = prostaglandin E2.
0171-2985/02/205/03-231 $ 15.00/0

232 · S. BERCHTOLD et al.
ly upregulated. Currently the best marker for mature DC is the human CD83 (hCD83)
molecule, a glycoprotein which belongs to the immunoglobulin superfamily. It is com-
posed of an extracellular Ig-like domain, a transmembrane region and a cytoplasmic tail.
Meanwhile the mouse homologue has also been cloned (6, 7). CD83 is expressed pre-
dominantly on the surface of dendritic lineage cells, including skin Langerhans’ cells,
circulating dendritic cells (DC) and interdigitating reticulum cells present in the T cell
areas of lymphoid organs (8–10). hCD83 is also expressed on monocyte-derived DC
after stimulation with inflammatory cytokines (11). In addition, weak hCD83 expressi-
on was found on several B and T cell lines and on mitogen activated T cells (9). The
gene encoding CD83 was localized on chromosome 6p23 (12).
Although the function of hCD83 is still unknown, its selective expression and the
upregulation of CD83 expression during DC maturation together with the costimula-
tory molecules suggest an important role during the immune response. Moreover, it was
shown that inhibition of CD83 expression by interfering with a specific RNA export
pathway as well as downregulation of CD83 expression after infection with Herpes
Simplex Virus leads to a reduction of the DC-mediated T cell stimulation (13, 14). In
addition, recently we demonstrated that a soluble CD83-molecule inhibits DC-media-
ted T cell stimulation in a concentration-dependent manner (15)
Here we report for the first time the characterization of the promoter region of the
hCD83 gene. We show the influence of the transcription factors SP1 and NF-ÍB on the
promoter activity and its inducibility by TNF-a. The hCD83 promoter represents a
potential tool to express heterologous genes in a specific cell subset.
Materials and Methods
Cloning of the hCD83 promoter region
A 3037 bp fragment corresponding to the genomic region upstream of the translational start site was
amplified by polymerase chain reaction (PCR) using the PAC clone 380E11 (Acc. Nr. AL022396) as
template. The following oligonucleotides were used: hCD83 promoter sense 5¢-GGCTTAAGCGAT-
CCTCCTGCCTCA-3¢, hCD83 promoter antisense 5¢-GGCTGGAGCG-CTGCGCCGCTG-3¢.
PCR reaction was performed using the Advantage GC Genomic PCR kit (Clontech, Palo Alto, CA,
USA) according to the manufacturer’s instructions. The PCR fragment was cloned into the vector
pCR2.1TOPO (Invitrogen, San Diego, CA, USA) and sequenced (Sequiserv, Vaterstetten, Germany).
Plasmid constructions
To assay for promoter activity, the genomic fragment was cloned as an Asp718-XhoI fragment into
the pGL3-Basic vector (Promega, Madison, WI, USA). This vector lacks eukaryotic promoter and
enhancer sequences and contains the luciferase gene. To generate deletion mutants of the hCD83 pro-
moter, unique restriction sites or PCR amplification reactions were used. Deletion mutants containing
the following fragments were constructed: phCD83 (–578), phCD83 (–3037/–578), phCD83
(–261), phCD83 (–215), phCD83 (–123), phCD83 (–100), phCD83 (–50). Plasmid phCD83
(–215)mut contains a mutation in the SP1 site located at position –208/–198 and was constructed by
PCR using phCD83 (–215) as a template. PhCD83 (–123)mut contains a mutation in the NF-ÍB
site located at position –115/–106. PhCD83 (–123) was used as a template for PCR. All PCR reac-
tions were performed using the Advantage GC Genomic PCR kit (Clontech). Constructs were num-
bered with reference to the translation initiation site (+1). Correct orientation and sequence was veri-
fied in each construct (Sequiserv).

Cloning and characterization of the promoter region of the human CD83 gene · 233
Cell culture and transfections
Jurkat and U937 cells were maintained in RPMI 1640 medium (Bio Whittaker, Verviers, Belgium)
supplemented with 10 % heat-inactivated fetal calf serum (FCS) (PAA, Cölbe, Germany), 2 mM
L-glutamine (Life Technologies, Karlsruhe, Germany), 100 IU/ml penicillin (Sigma, Deisenhofen,
Germany) and 100 mg/ml streptomycin (Sigma). DC2.4 cells (mouse dendritic cell line, kindly pro-
vided by K. ROCK) (16) were cultured in the same medium containing only 5 % FCS. Human DC
were generated as described (17). Maturation was induced with a cytokine cocktail of PGE2 (1 mg/ml,
Sigma),
TNF-a (25 ng/ml), (Boehringer Ingelheim, Ingelheim, Germany), IL-1b (10 ng/ml), (Sigma).
Jurkat and U937 cells were transfected using the DEAE-dextrane method (18). Briefly, 107 cells were
resuspended in 1 ml STBS (137 mM NaCl, 25 mM Tris-HCl pH 7.4, 0.6 mM Na2HPO4, 0.7 mM
CaCl2, 0.5 mM MgCl2, 5 mM KCl) containing 2 mg of pHM75 encoding the b-galactosidase gene
driven by the CMV promoter, 5 mg of the reporter plasmid and 500 mg/ml of DEAE-dextrane. Cells
were incubated for 90 minutes at 37 °C. Afterwards cells were exposed to 10 % DMSO for 2.5 minu-
tes. After several rounds of washing cells were plated and incubated for 48 hours before lysis. Where
indicated, Jurkat cells were stimulated with TNF-a (25 ng/ml) for 24 hours before lysis. DC2.4 cells
were also transfected using the DEAE-dextrane method. In brief, cells were incubated for 30 min at
room temperature with 2 mg of pHM75 and 5 mg of reporter plasmid in TBS (137 mM NaCl, 5 mM
KCl, 1 mM Na2HPO4, 25 mM Tris-HCl pH 7.4, 1 mM CaCl2, 0.5 mM MgCl2) supplemented with
250 mg/ml of DEAE-dextrane. After a DMSO shock (10 % DMSO in PBS (136 mM NaCl, 8 mM
Na2HPO4, 1.5 mM KH2PO4, 2.7 mM KCl pH 7.4)) cells were washed twice with PBS and then
incubated for 48 hours before lysis. Mature DC were electroporated with 4 mg of pHM75 and 16 mg
of reporter plasmid at 260 V, 1050 mF and lysed after 24 h.
Luciferase activity was determined using the Luciferase Reporter Gene Assay (Roche Diagnostics,
Mannheim, Germany) according to the manufacturer’s instructions. The activity was quantified in a
Victor 1420 multilabel counter (Wallac, Gaithersburg, MD, USA). To normalize for transfection effi-
ciency, a b-galactosidase gene driven by the CMV promoter was always cotransfected and b-galacto-
sidase values were determined using the b-Gal Reporter Gene Assay (Roche Diagnostics). In addition
luciferase values were normalized for protein content (Biorad Assay, BioRad, München, Germany).
Electrophoretic mobility shift assays (EMSAs)
Probes for EMSAs were generated by annealing corresponding oligonucleotides derived from the
hCD83 promoter. The oligonucleotides were end-labeled using (g-32P)ATP (3000 Ci/mmol,
Amersham-Pharmacia Biotech, Freiburg, Germany) and T4 polynucleotide kinase (New England
Biolabs, Beverly, MA, USA). To analyze the binding activity of NF-ÍB, nuclear extracts were prepa-
red from DC2.4 as described (19). The activity of NF-ÍB was determined as described (20). Where
indicated unlabeled oligonucleotide was added in a 100 fold molar excess immediately prior to the
labeled oligonucleotide. For supershift experiments cell extracts were preincubated with antibodies
directed against the p50, p65 or c-Rel subunits of NF-ÍB (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) for 30 minutes on ice. To determine the binding activity of SP1, DC2.4 cells were lysed in a
hypertonic buffer (20 mM Hepes pH 7.9, 350 mM NaCl, 20 % Glycerol, 1 % Nonidet P 40, 1 mM
MgCl2, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 0.5 mM Pefabloc) and incubated for 15
minutes on ice. Lysates were cleared by centrifugation and EMSAs were performed as described (21).
For supershift experiments antibodies directed against SP1 and SP3 (Santa Cruz Biotechnology) were
used. The following oligonucleotides derived from the hCD83 promoter were used: NFÍB WT
(–123/–94) 5¢-GCCCGCCGGGGAATCCCCCG-GGCTGGCGCG-3¢, NFÍB mut 5¢-GCCCG-
CCGGCCAATCCCCCGGGCTGGCGCG-3¢, SP1 WT (–215/–186) 5¢-CGGGGACGGGG-
GCGGGGACGGGGGCGAAGG-3¢, SP1 mut 5¢-CGGGGACGGGCCCGGGGACGGGGGC-
GAAGG-3¢. For competition experiments, a NF-ÍB consensus oligonucleotide (Promega, Mann-
heim, Germany) was used: 5¢-AGTTGAGGGGACTTTCCCAGGC-3¢.

234 · S. BERCHTOLD et al.
Results
Cloning of the promoter region of the hCD83 gene
A 3037 bp fragment upstream of the translational start site of hCD83 was amplified by
PCR (accession number AJ427415) and cloned into the vector pGL-3 Basic encoding
the luciferase gene and lacking eukaryotic promoter and enhancer sequences. In additi-
on two complementary deletion mutants were constructed: phCD83 (–3037/–578)
comprising the upstream part and phCD83 (–578) comprising the downstream part of
the putative promoter region (Fig. 1A). All constructs were transfected in U937 (human
promonocytic), Jurkat (T-cell leukemia) and DC2.4 (murine dendritic) cells. After 48
hours, cells were lysed and luciferase activity was determined (Fig. 1B–D). The empty
vector served as a negative control (Fig. 1B–D, lane 2). A plasmid encoding the luci-
ferase gene under the control of the CMV immediate early enhancer/promoter
(pCMVluc) was used as a positive control (Fig. 1B–D, lane 1). Whereas the constructs
phCD83 (–3037) and phCD83 (–578) displayed promoter activity reflected by luci-
ferase activity (Fig. 1B–D, lanes 3, 5), the luciferase activity of phCD83 (–3037/–578)
was even lower than the activity of the empty vector (Fig. 1B–D, lane 4). These data
indicate that the hCD83 promoter activity is contained in the 578 bp fragment upstre-
am of the ATG.
Analysis of transcription factor binding sites
Since the –578 bp fragment displays promoter activity, we analyzed the region close to
the translation initiation site for potential transcription factor binding sites. As shown
in Fig. 2A, four SP1 binding sites were found from nt –260 to –177 as well as a NF-ÍB
binding site from nt –115 to –106. To analyze the functional role of these transcription
factor binding sites, deletion mutants were generated (Fig. 2B) containing all sites
(phCD83 (–261)), the two downstream SP1 sites and the NFÍB site (phCD83 (–215)),
only the NF-ÍB site (phCD83 (–123)) or lacking all sites (phCD83 (–100), phCD83
(–50)). In addition, point mutations were introduced destroying the SP1 binding site
located at position –207 to –198 (phCD83 (–215)mut) or the NF-kB element
(phCD83 (–123)mut).
Figure 1A

Cloning and characterization of the promoter region of the human CD83 gene · 235
Figure 1. Structure and function of phCD83 (–3037) and deletion mutants. (A), Structure of
phCD83 (–3037) and of the deletion mutants phCD83 (–578) and phCD83 (–3037/–578).
Nucleotide positions refer to the translation initiation side (+1). The constructs were cloned into a vec-
tor encoding the luciferase gene. (B–D), Activity of the hCD83 promoter constructs in different cell
lines. DC2.4 (B), U937 (C), and Jurkat (D) cells were transfected with the indicated promoter con-
structs. A b-galactosidase gene was included to normalize for transfection efficiency. After 48 hours,
cells were lysed and luciferase activity was determined. One representative experiment out of four is
shown.

236 · S. BERCHTOLD et al.
Figure 2. Putative transcription factor binding sites in the hCD83 promoter sequence. (A), Putative
binding sites for NF-ÍB and SP1 are indicated. Numbers at the left margin refer to the translation
initiation codon (+1). Binding sites were identified using the proscan program version 1.7. (B),
Structure of phCD83 variants. Deletion mutants containing intact or mutant (mut) binding sites for
SP1 or NF-ÍB were generated and inserted into a vector containing the luciferase gene. Nucleotide
positions refer to the translational start site.

Cloning and characterization of the promoter region of the human CD83 gene · 237
Functional role of SP1 and NF-ÍB binding sites in the hCD83 promoter
To analyze the functional role of the SP1 and NF-ÍB binding sites, the different reporter
constructs were transfected into Jurkat, U937 and DC2.4 cells. After 48 hours, cells were
lysed and luciferase activity was determined (Fig. 3). In all cell lines, highest luciferase
activities were obtained with phCD83 (–261) containing four SP1 binding sites and the
NF-ÍB element (Fig. 3A–C, lane 5). This construct displayed even higher promoter
activity than the larger constructs phCD83 (–3037) and phCD83 (–578) (Fig. 3A–C,
lanes 3, 4). Deletion of the two upstream SP1 binding sites (phCD83 (–215)) resulted in
a clear decrease in promoter activity (Fig. 3A–C, lane 6) which was further enhanced by
mutation of one of the remaining SP1 sites (phCD83 (–215)mut) (Fig. 3A–C, lane 7).
Deletion of the last SP1 binding site again led to a strong decrease in luciferase activity
(Fig. 3A–C, lane 8). Mutation (phCD83 (–123)mut) or deletion of the NF-ÍB (phCD83
(–100)) element reduced the promoter activity to almost baseline levels (Fig. 3A–C, lines
9, 10).The activity of the shortest construct phCD83 (–50) was identical to the empty
vector (Fig. 3A–C, line 11). The relative strength of the hCD83 promoter varied in the
different cell lines when compared to the CMV promoter. Whereas in DC2.4 cells
pCMVluc displayed a higher activity than phCD83 (–261) (Fig. 3B, lanes 1, 5), in U937
cells phCD83 (–261) showed a higher activity than pCMVluc (Fig. 3A, lanes 1, 5) indi-
cating cell type specific differences in hCD83 promoter activity.
Binding of NF-ÍB to the hCD83 promoter
To corroborate the functional results and to prove that a member of the NF-ÍB family
is able to interact with the NF-ÍB element at position –115/–106 in the hCD83 pro-
moter, EMSA experiments were performed using the oligonucleotide –123/–94 as a
probe. Nuclear extracts were prepared from DC2.4 cells and introduced into EMSAs.
Two DNA binding complexes were observed which can be supershifted with antibodies
directed against p50 (Fig. 4, lane 2) and p65, respectively (Fig. 4, lane 3), demonstra-
ting that the p50 and p65 NF-ÍB subunits are part of the complex. The specificity of
DNA binding was demonstrated by introducing non-radioactive and the mutant versi-
on of the oligonucleotide from the hCD83 promoter as well as a NF-ÍB consensus oli-
gonucleotide into the binding reaction. DNA binding of the NF-ÍB complex was sup-
pressed by a 100-fold molar excess of the unlabeled oligonucleotide (Fig. 4, lane 5) and
of a NF-ÍB consensus oligonucleotide (Fig. 4, lane 7), but not by the mutant version
(Fig. 4, lane 6). These results suggest that members of the NF-ÍB family bind to the
hCD83 promoter and regulate gene expression.
Binding of SP1 to the hCD83 promoter
To investigate whether or not the transcription factor SP1 is able to bind to the hCD83
promoter, EMSAs were performed. The radiolabeled oligonucleotide –215/–186 was
used as a probe. Since SP1 is constitutively active, whole cell extracts were prepared from
unstimulated DC2.4 cells and bandshift assays were performed (Fig. 5). Several strong
DNA protein complexes were observed (Fig. 5, lane 1). Supershifts were observed when
antibodies against SP1 (Fig. 5, lane 2) or SP3 (Fig. 5, lane 3) were included in the bind-
ing reaction demonstrating that SP1 and SP3 are part of the complexes. Binding was

238 · S. BERCHTOLD et al.
Figure 3. Promoter activity of phCD83 constructs in different cell lines. U937 (A), DC2.4 (B) and
Jurkat (C) cells were transfected with the indicated promoter constructs. A b-galactosidase gene was
cotransfected to normalize for transfection efficiency. After 48 hours cells were lysed and luciferase
activities were determined. The empty vector pGL-3 Basic served as a negative control, pCMVluc as
a positive control. The activity of the construct phCD83 (–261) was arbitrarily set 100. For each cell
line, one representative experiment out of four is shown.

Cloning and characterization of the promoter region of the human CD83 gene · 239
Figure 4. Identification of a NF-ÍB motif within the –123/–94 fragment of the hCD83 promoter.
Nuclear extracts of DC2.4 cells were prepared and bandshift experiments were performed using the
32P-labeled –123/–94 fragment as a probe. A p50-, p65- and c-Rel-specific antiserum was included in
the binding reaction in lanes 2,3 and 4, respectively. Unlabeled (lane 5), mutant (lane 6) or a NF-ÍB
consensus (lane 7) oligonucleotide in a 100-fold molar excess was introduced into the binding reac-
tion.

240 · S. BERCHTOLD et al.
Figure 5. Specific binding of SP1 to the –215/–186 fragment of the hCD83 promoter. Whole cell
extracts were prepared from DC2.4 cells and bandshift experiments were performed using the 32P-
labeled –215/–186 fragment as a probe. A SP1- and SP3-specific antiserum was included in the bind-
ing reaction in lanes 2 and 3, respectively. Unlabeled (lane 4) or mutant (lane 5) oligonucleotide in a
100-fold molar excess over the radioactive probe was introduced into the binding reaction.

Cloning and characterization of the promoter region of the human CD83 gene · 241
Figure 6. Effect of TNF-a treatment on the activity of the hCD83 promoter. Jurkat cells were trans-
fected with the indicated reporter constructs. A b-galactosidase gene was always included to normali-
ze for transfection efficiency. Cells were left untreated (lanes 1, 3, 5, 7, 9, 11, 13) or stimulated with
TNF-a (lanes 2, 4, 6, 8, 10, 12, 14) for 24 hours. After 48 hours, luciferase activities were determi-
ned. One representative experiment out of four is shown.
completely suppressed when the unlabeled oligonucleotide was added in 100 fold molar
excess to the binding reaction (Fig. 5, lane 4). In contrast, addition of the mutant oli-
gonucleotide had no effect demonstrating the specificity of the binding reaction (Fig. 5,
lane 5). This finding confirms the results from transfection experiments indicating an
important role for SP1 in regulating hCD83 promoter activity.
Effect of TNF-a on the activity of the hCD83 promoter
Since TNF-a is a known inducer of NF-ÍB and also leads to an increase in surface
expression of CD83 in DC, we investigated the influence of TNF-a on the activity of
the hCD83 promoter. Jurkat cells were transfected with different hCD83 promoter con-
structs and stimulated with TNF-a for 24 hours or left untreated. As shown in Fig. 6,
stimulation with TNF-a led to an increase in promoter activity in the constructs con-
taining an intact NF-ÍB site (phCD83 (–3037) (Fig. 6, lanes 3, 4), phCD83 (–261)
(Fig. 6, lanes 5, 6), phCD83 (–215) (Fig. 6, lanes 7, 8), phCD83 (–215)mut, (Fig. 6,
lanes 9, 10), phCD83 (–123) (Fig. 6, lanes 11, 12)). In contrast, no induction was
observed when the NF-ÍB site was mutated (phCD83 (–123)mut) (Fig. 6, lanes 13, 14)
or deleted (phCD83 (–100)) (data not shown). The empty vector was not induced by
TNF-a either (Fig. 6, lanes 1, 2). These data indicate that the increase in promoter
activity after stimulation with TNF-a is mediated by NF-ÍB.

242 · S. BERCHTOLD et al.
Figure 7. Activity of the hCD83 promoter in mature human DC. Mature human DC were trans-
fected with the indicated promoter constructs. A b-galactosidase gene was cotransfected to normalize
for transfection efficiency. After 24 hours cells were lysed and luciferase activities were determined.
Activity of the hCD83 promoter in human DC
As hCD83 is predominantly expressed on the surface of mature DC, we investigated the
activity of the hCD83 promoter in DC (Fig. 7). DC were generated from peripheral
blood monocytes. Maturation was induced with a cytokine cocktail (PGE2, IL-1b,
TNF-a). Forty-eight hours later mature DC were transfected with different hCD83
promoter constructs. Again phCD83 (–261) showed the highest activity (Fig. 7, lane 3)
which was decreased by deletion of two and four SP1 sites, respectively (Fig. 7, lanes 4,
5). Mutation (Fig. 7, lane 6) and deletion (Fig. 7, lane 7) of the NF-ÍB site decreased
the activity to almost background levels (Fig. 7, lane 2). These data show that the
hCD83 promoter is also highly active in mature DC.
Discussion
In the present study, we report the characterization of the promoter of the hCD83 gene.
Starting with a 3037 bp fragment upstream of the translational start ATG, we generated
a number of deletion mutants and found that the fragment –261 displayed highest pro-

Cloning and characterization of the promoter region of the human CD83 gene · 243
moter activity. Within this fragment four SP1 elements and one NF-ÍB binding site
have been identified. The hCD83 promoter is inducible by TNF-a. This is in accor-
dance with previous findings showing that TNF-a is a maturation stimulus for DC.
This maturation process is accompanied by the upregulation of the hCD83 molecule
(3). The induction of the hCD83 promoter by TNF-a is dependent on the intact NF-
ÍB site at position –115/–106. A member of the NF-ÍB family has been shown to be
critical for DC function. Mice deficient for RelB still possess immature DC but lack
mature DC in lymphoid organs (22, 23). Moreover RelB has been shown to be requi-
–
+
red for the development of myeloid CD8a DC, but not of lymphoid CD8a DC (24).
NF-ÍB factors have been shown to be differentially expressed during the maturation of
DC. Whereas the level of p65 remains unchanged, there is a strong upregulation of p50,
RelB and especially c-Rel after stimulation with maturation-inducing cytokines resulting
in an increased DNA binding activity which leads to an enhanced transcription of tar-
get genes (25). This upregulation of NF-ÍB factors together with the NF-ÍB-dependent
TNF-a inducibility of the hCD83 promoter suggests that NF-ÍB factors play a key role
in the upregulation of the hCD83 molecule during DC maturation. NF-ÍB has also
been shown to play an important role in the transcription of the costimulatory molecu-
les CD80 and CD86 which are also upregulated during DC maturation (26, 27).
Besides NF-ÍB we found SP1 binding activity to be critical for hCD83 promoter
activity since deletion of four neighbouring SP1 binding elements strongly decreased
promoter activity. In contrast to previous findings (28), we could detect SP1 binding
activity in human DC (data not shown). This difference might be due to different cul-
ture conditions. We used mature DC stimulated with a cocktail consisting of TNF-a,
IL-1b, IL-6 and PGE2 whereas in the other study unstimulated DC were used. For
macrophages, LPS stimulation was shown to strongly enhance SP1 binding activity
(28). In unstimulated DC SP1 binding activity might be below detection limit.
We found differential hCD83 promoter activity in different cell lines. In the mono-
cytic cell line U937 and in Jurkat cells, both expressing CD83 on their mRNA level or
cell surface, the promoter displayed high activity. Noteworthy, also in the murine den-
dritic cell line DC2.4 the hCD83 promoter was active. Furthermore, the CD83 pro-
moter was also active in mature human DC, where CD83 shows the highest expression,
underlining that this promoter might represent a very useful tool in order to express spe-
cific gene products in mature DC. Interestingly, in all cell types tested the –261 frag-
ment, containing the four SP1 elements and one NF-ÍB binding site, displayed the hig-
hest CD83 promoter activity.
In a previous study, the transcriptional start sites of the hCD83 gene have been map-
ped using RNA from Staphylococcus aureus-activated B lymphocytes (29). Several start
sites ranging from 38 to 41 bp upstream of the translation initiation site were found. In
that study, however, no additional experiments to characterize the hCD83 promoter
were performed.
DC have been shown to be a potent tool in immunotherapy. DC loaded with pepti-
de or transfected with RNA are able to induce specific T cell responses (30-32).
Although these approaches have shown promising results, peptide loading itself has
some inherent disadvantages, including the uncertainty regarding the longevity of anti-
gen presentation, the restriction by the patient’s HLA haplotype and the relatively low
number of known MHC related epitopes. An alternative approach would be the gene-
tic modification of DC expressing full length antigens. Potential advantages of this

244 · S. BERCHTOLD et al.
approach include the presentation of MHC class I and II epitopes, the inclusion of pre-
viously unknown epitopes of a given tumor antigen in any MHC context and the abili-
ty to include sequences of immunomodulators (for example cytokines such as IL-12), or
costimulatory molecules.
In this respect the cloning and characterization of the hCD83 promoter represents a
promising tool for the specific in vivo expression of tumor antigens and immunomodu-
lators in genetically modified DC. In addition, since DC are also involved in the induc-
tion of tolerance one could envisage to express tolerance-inducing cytokines (e. g. IL-
10) under the control of the hCD83 promoter with the aim to develop a new thera-
peutic approach for diseases including allergies, autoimmunity and transplantation
rejection.
Acknowledgements
S. B. and P. M.-Z. are supported by the Deutsche Forschungsgemeinschaft Grant STE 432/2-3 and
by the Fifth (EC) Framework Programme 1998-2002, Specific Programme – “Quality of Life and
Management of Living Resources”, Contract No QLK2-2000-00470. E. M. and A. G. are supported
by the ELAN-Program funded by the ‘Klinikum of the University of Erlangen-Nürnberg’. We would
like to thank Manfred Lutz for critical reading of the manuscript.
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Dr. SUSANNE
BERCHTOLD, (present address:) Institute of Physiology, University of Tübingen,
Gmelinstr. 5, 72076 Tübingen, Germany, Tel. ++49-7071-2977356