Mutant CD83 promoter and use thereof

Abstract

The present invention provides a mutant CD83 promoter comprising the promoter/enhancer regions of human CD83 promoter and being dendritic cell-specific, and the use thereof, specifically for the treatment or prevention of diseases or medical conditions related to malignancy, autoimmunity or prevention of transplant rejections.

Claims

1. A CD83 promoter sequence comprising the CD83 Upstream promoter (UpP) sequence of SEQ ID NO:2, the CD83 Minimal Promoter (MP) sequence of SEQ ID NO:4 and the CD83 enhancer sequence of SEQ ID NO:5, or variants of said sequences having 98% homology over their entire length and/or being N- and/or C-terminally truncated by up to 10 nucleotides and having promoter activity, said UpP sequence being located 50 to 200 nucleotides upstream of said MP sequence and said enhancer sequence being located 200 to 4000 nucleotides upstream of the UpP sequence or 200 to 4000 nucleotides downstream of said MP sequence.

2. The CD83 promoter sequence of claim 1, wherein (i) the distance between the UpP sequence and the MP sequence is 70 to 120 nucleotides; and/or (ii) the distance between the UpP sequence/MP sequence and the enhancer is 250 to 750 nucleotides.

3. The CD83 promoter sequence of claim 1, wherein (i) the UpP sequence has SEQ ID NO:2; (ii) the MP sequence has SEQ ID NO:4; and/or (iii) the CD83 enhancer sequence has SEQ ID NO:5.

4. The CD83 promoter sequence of claim 3, wherein (i) the UpP sequence is linked to the MP sequence by the intermediate sequence of SEQ ID NO:3 or a variant thereof having 95% homology to SEQ ID NO:3 over its entire length; and/or (ii) the CD83 enhancer sequence is located 450 to 550 nucleotides upstream to the UpP sequence or 450 to 550 nucleotides downstream of the MP sequence.

5. The CD83 promoter sequence of claim 1, which (i) comprises the sequence of nucleotides 29-1259 of SEQ ID NO:28 or the sequence of nucleotides 27-1257 of SEQ ID NO:29; and/or (ii) is human dendritic cell-specific.

6. A vector or viral vector comprising the CD83 promoter sequence according to claim 1.

7. The vector or viral vector of claim 6, wherein (i) the CD83 promoter sequence is functionally linked to a gene of interest; and/or (ii) the viral vector is an adenoviral vector.

8. A cell, tissue culture or transgenic non-human organism, which comprises the CD83 promoter sequence according to claim 1.

9. The cell of claim 8, which is a dendritic cell.

10. A method for producing dendritic cells ex vivo, said method comprising introducing a vector or viral vector of claim 6 into a DC progenitor cell.

11. Transfection reagent comprising the vector or viral vector of claim 6.

12. A vector or viral vector comprising (i) the CD83 promoter sequence according to claim 1 functionally linked to (ii) a gene of interest.

13. A CD83 promoter sequence comprising the CD83 Upstream promoter (UpP) sequence of SEQ ID NO:2, the CD83 Minimal Promoter (MP) sequence of SEQ ID NO:4 and the CD83 enhancer sequence of SEQ ID NO:5, said UpP sequence being located 50 to 200 nucleotides upstream of said MP sequence and said enhancer sequence being located 200 to 4000 nucleotides upstream of the UpP sequence or 200 to 4000 nucleotides downstream of said MP sequence.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1: CD83 is upregulated during DC maturation and is not expressed by HFF cells.

(2) FIG. 2: Schematic depiction of the CD83 gene locus and the respective acetylation data resulting from the ChIP-chip™ microarray analysis.

(3) FIG. 3: Schematic depiction of constructs for gene reporter assays.

(4) FIG. 4: Schematic depiction of the subcloning strategy of the hyper-acetylated region of CD83 intron 2.

(5) FIG. 5: Fragment C enhances the MP-261 activity specifically in XS52 cells.

(6) FIG. 6: Schematic depiction of the deletion mutants C1-C14 of fragment C.

(7) FIG. 7: The 185 bp long deletion mutant C13 of fragment C enhances the MP-261 activity specifically in XS52 cells.

(8) FIG. 8: Fragment C and the 185 bp enhancer induce the MP-261 activity specifically in mDCs.

(9) FIG. 9: Neither fragments A, B, C nor the 185 bp enhancer induce the MP-261 in Raji and Jurkat cells.

(10) FIG. 10: Presumed bio-computational model for the interaction of the CD83 upstream promoter, the MP-261 and the 185 bp enhancer.

(11) FIG. 11: Schematic depiction of reporter constructs containing the UpP and the spacer sequence S1.

(12) FIG. 12: The spacer sequence S1 does not significantly affect the induction of the MP-261 and the P-510 in XS52 cells.

(13) FIG. 13: (A) The spacer sequence S1 does not significantly affect the induction of the MP-261 in mDCs. (B) The spacer sequence S1 does not significantly affect the induction of the P-510 in mDCs.

(14) FIG. 14: (A) The ternary complex of UpP, MP-261 and 185 bp enhancer shows a specific transcriptional induction in mDCs.

(15) FIG. 15: (A)-(C) The ternary complex of UpP, MP-261 and 185 bp enhancer shows no specific transcriptional induction in Raji, Jurkat and JCAM cells.

(16) FIG. 16: Schematic depiction of reporter constructs containing mutated IRF-sites used for luciferase assays.

(17) FIGS. 17: (A) and (B) Mutation of any of the three IRF-sites in the ternary complex significantly reduces the luciferase expression in XS52 cells.

(18) FIG. 18: Mutation of any of the three IRF-sites in the ternary complex significantly reduces the luciferase expression in mDCs.

(19) FIGS. 19: (A) and (B) Transcription factors of the NFκB-family and IRF-5 induce the MP-261 in 293T cells.

(20) FIG. 20: (A) p65 and IRF-5 induce the UpP in 293T cells. (B) The induction of the UpP by p65 and IRF-5 is abrogated when the IRF-site 3 is mutated.

(21) FIG. 21: Genomic sequences of the of functional human dendritic cell-specific CD83 promoter/enhancer regions CD83 Upstream promoter, Intermediate sequence, CD83 Minimal Promoter and 185 bp enhancer (NCBI genome build 37.2, contig GRCh37.p2). The 166 bp long CD83 upstream promoter (UpP) and the 261 bp long CD83 minimal promoter (MP-261) are separated by a 83 bp long intermediate sequence. Together these sequences constitute the CD83 promoter P-510. The P-510 cooperates with a 185 bp long enhancer element (185 bp enhancer) in the CD83 intron 2, located 4177 bp downstream of the CD83 transcription start. Referring to the NCBI human genome build 37.2, contig GRCh37.p2 (reference number NT_007592.15; SEQ ID NO:1), the UpP comprises the bp 14057533 to bp 14057698, the intermediate sequence bp 14057699 to Ser. No. 14/057,782, the MP-261 bp 14057783 to 14058042 and the 185 bp enhancer bp 14062220 to bp 14062404.

(22) FIG. 22: Overview of luciferase reporter constructs and PCR primers used for cloning fragments of CD83 intron 2.

DETAILED DESCRIPTION OF THE INVENTION

(23) The mutant CD83 promoter according to aspect (1) of the invention comprises the CD83 Upstream promoter (UpP) sequence of SEQ ID NO:2, the CD83 Minimal Promoter (MP) sequence of SEQ ID NO:4 and the CD83 enhancer sequence of SEQ ID NO:5, or variants of said sequences (i) having 90% homology over the entire length and having promoter activity and/or (ii) being N- and/or C-terminally truncated by up to 30 nucleotides and having promoter activity, said UpP sequences being located upstream of said MP sequence and said enhancer sequence being located 200 to 4000 nucleotides upstream of the UpP sequence or 200 to 4000 nucleotides downstream of said MP sequence.

(24) The feature “having promoter activity” of the variants refers to the promoter activity of the starting mutant CD83 promoter.

(25) The wording “being located upstream” and “being located upstream” according to the invention implies that the respective sequences are on the same nucleotide strand and are connected by linker/spacer nucleotide molecule (of a variable length, unless the length of the linker/spacer is specifically indicated.

(26) According to the invention it is preferred that the distance between the UpP sequence and the MP sequence is 50 to 200 nucleotides, preferably 70 to 120 nucleotides. Also it is preferred that the distance between the UpP sequence/MP sequence and the enhancer is 200 to 4000 nucleotides, preferably 250 to 750 nucleotides.

(27) According to the invention the variants of SEQ ID NOs: 2, 4 and 5 may have 90% homology, preferably 95% homology, most preferably 98% homology over the entire length of the respective basis sequence. Truncation by up to 30 nucleotides, or up to 20 nucleotides or up to 10 nucleotides are possible. Particularly preferred is that the UpP sequence has SEQ ID NO:2, the MP sequence has SEQ ID NO:4, and/or the CD83 enhancer sequence has SEQ ID NO:5.

(28) Further preferred is that the UpP sequence is linked to the MP sequence by the intermediate sequence of SEQ ID NO:3 or a variant thereof having 95% homology to SEQ ID NO:3 over its entire length; and/or that the CD83 enhancer sequence is located 450 to 550 nucleotides upstream to the UpP sequence or 450 to 550 nucleotides downstream of the MP sequence, preferably 450 to 550 nucleotides upstream to the UpP sequence.

(29) Particularly preferred mutant CD83 promoters comprises the sequence of nucleotides 29-1259 of SEQ ID NO:28 or the sequence of nucleotides 27-1257 of SEQ ID NO:29. Also preferred is that the mutant CD83 promoter is human dendritic cell-specific.

(30) To fully characterize the CD83 promoter, including all regulatory elements that provide for the cell type and maturation status specificity of CD83 expression in mature DC, areas containing potential regulatory elements like e.g. enhancers were identified by the analysis of differential H3K9 acetylation in iDCs, mDCs and HFF cells using a ChIP-chip™ microarray assay. The first 6 kb of CD83 Intron 2 were revealed to be exclusively H3K9 acetylated in mDCs, hinting at a cell type and maturation status-specific hotspot of transcriptional activity in this area.

(31) Next, the potential enhancer element within this area was narrowed down. This was achieved by mutation mutagenesis and luciferase assays. As a result, a 185 bp fragment (185 bp enhancer) of the H3K9 acetylated region within CD83 intron 2 has been shown to induce the CD83 minimal promoter (MP-261) in a cell type- and maturation status-specific manner in mDCs.

(32) Next, the molecular mechanism underlying the induction of the MP-261 by the 185 bp enhancer was unraveled. Therefore, a biocomputational analysis was performed by the co-inventor Dr. Werner. The Analysis predicted three NFκB- and five SP1-binding sites in the MP-261, two IRF- and one SP1-site in the 185 bp enhancer and additionally two NFκB- and one IRF-site in a presumed upstream promoter (UpP). Furthermore, the formation of a tripartite regulatory complex, consisting of UpP, MP-261 and 185 bp enhancer was foretold. The formation of this complex was supposedly mediated by the interaction of the IRF- and NFκB-transcription factors, thereby forming three NFκB-IRF-NFκB transcriptional modules in trans.

(33) To prove this model, the cooperation of all three predicted regulatory elements, namely UpP, MP-261 and 185 bp enhancer, as well as the functionality of the predicted NFκB- and IRF-sites had to be verified. Therefore, several different experimental approached were taken: (i) Adenoviral transduction with luciferase reporter vectors to prove the cooperation of the three regulatory elements in a chromosome-like configuration. (ii) Mutation of the three IRF-sites in the luciferase reporter plasmids to verify their functionality and significance for the formation of the tripartite regulatory complex. (iii) Induction of the predicted NFκB-sites by cotransfection of plasmids coding for members of the NFκB-transcription factor family and luciferase reporter plasmids to verify their functionality. Both the transcription factor binding sites as well as the cooperation of UpP, MP-261 and the 185 bp enhancer were thereby verified.

(34) The invention is further described in the following Examples, which are not to be construed to limit the invention.

EXAMPLES

(35) Methods

(36) Transient Transfection Methods and Luciferase Reporter Assay

(37) Transfection of DNA with the DEAE-Dextran Method:

(38) For promoter analyses, adherent XS52, NIH3T3 and HeLa cells were transfected with reporter plasmid DNA containing the promoter construct and a gene for the firefly luciferase by the DEAE-Dextran method in triplicates. Hence, 2×10.sup.5 cells per well were seeded in 12-well tissue culture plate (Falcon) and grown overnight at 37° C., 5% CO.sub.2. The next day, 2.5 μg of endotoxin-free plasmid DNA were diluted in 150 μl of TBS buffer and mixed with 50 μl 5 mg/ml DEAE-Dextran solution reaching a volume of 200 μl per transfection. Subsequently 0.2 μl Chloroquine solution (1 μg/μl, Roth) were added to the reaction mix. Cells were washed 1× with 500 μl/well TBS buffer. Then, the DNA-DEAE-Dextran-solution was added and plates were incubated on a rocker for 30 min to 1 h at RT. Afterwards, the DNA-DEAE-Dextran-solution was replaced by 500 μl of 10% DMSO for 2 min, cells were washed with 2×2 ml warm DPBS and finally 2 ml per well of warm cell culture medium was added. Transfected cells were cultured for 2 days at 37° C. in a humidified atmosphere of 5% CO.sub.2 before luciferase reporter assays were performed. Transfection efficiency was assessed by transfection of a GFP coding control plasmid and subsequent FACS analysis.

(39) Electroporation of Rail and Jurkat Cell Lines:

(40) For promoter analyses suspension Raji and Jurkat cells were transfected by electroporation with reporter plasmid DNA containing the promoter construct and a gene for the firefly luciferase.

(41) Therefore, 10×10.sup.6 cells were harvested, washed 1× in DPBS and resuspended in 250 μl fetal calf serum (PAA) containing 20 μg of DNA. The cell suspension was incubated 10 min at RT. Subsequently 250 μl of RPMI1640 without additives were added and cells were transferred to a 4 mm electroporation cuvette (Peqlab). Electroporation was performed with a Genepulser II (Bio-Rad) at 975 μF and 260 Volt. The cells were incubated for 3 min at RT and then transferred into 10 ml prewarmed growth medium and cultivated at 37° C., 5% CO.sub.2. After 24 h 10 ml of growth medium were added. Cells were applied to a luciferase assay 48 h after electroporation. The electroporation efficiency was assessed by the electroporation of a GFP coding control plasmid and subsequent FACS analysis.

(42) Lipofection of DNA with Lipofectamine™ LTX and PLUS™ (Invitrogen) Reagent:

(43) For the generation and assembly of adenoviruses, 293 cells were transfected with Lipofectamine™ LTX without PLUS™ reagent in a T25 tissue culture flask (Nunc) as follows: For optimal transfection efficiency the cells were transfected at 40-60% confluence. On the day of transfection, 4 μg of PacI-digested pAd-plasmid in 20 μl water was mixed with 13 μl Lipofectamine™ reagent in 500 μl OptiMEM (Gibco) and incubated for 30 min at RT. The cells were washed 1× with DPBS and covered with 2.5 ml OptiMEM. After the incubation the transfection mix was added directly to the cells and incubated for 6 h at 37° C. in a humidified atmosphere of 5% CO.sub.2. Afterwards the mixture was replaced by 7 ml of warm growth medium and cells were grown for 10-12 days until viral plaques could be detected. Viruses were then further amplified as described below.

(44) For co-transfection of luciferase reporter constructs and pCDNA3.1 vectors coding for transcription factors, the Lipofectamine™ LTX with PLUS™ reagent was used. Therefore, 6×10.sup.4 293T cells per well were seeded in a 24-well tissue culture plate (Falcon) one day prior to transfection in 500 μl antibiotic free growth medium. On the day of transfection 0.5 μg of total DNA, consisting of 0.05 μg reporter construct, 0.15 μg for each transcription factor construct and pCDNA 3.1 vector backbone to fill up to 0.5 μg, were diluted in 100 μl OptiMEM (Gibco). Then 0.5 μl per transfection mix PLUS reagent were added to the transfection mix, mixed vigorously and incubated for 5 min at RT. Subsequently, 1.25 μl Lipofektamin™ LTX was added to the transfection mix, vortexed and incubated 30 min at RT. After incubation the whole reaction mix was added drop wise directly to the cells. Transfected cells were cultured for 2 days at 37° C. in a humidified atmosphere of 5% CO.sub.2 before luciferase reporter assays were performed. Growth medium was changed after 24 h. The transfection efficiency was assessed by the lipofection of a GFP coding control plasmid and subsequent FACS analysis.

(45) Electroporation of Dendritic Cells with DNA Using AMAXA Technology:

(46) For further promoter analyses iDC were electroporated using the AMAXA Human Dendritic Cell Nucleofector Kit (Lonza) and the Nucleofector I electroporation device (Lonza), according to the manufacturer's instructions. In brief: After harvesting and washing with warm DPBS, 2×10.sup.6 immature DCs were resuspended in 100 μl freshly prepared electroporation solution (provided with the Nucleofector Kit) containing 4 μg of plasmid DNA. Cells were transferred to the electroporation cuvette and electroporated with program U-2. Immediately after electroporation 500 μl of RPMI1640 without additives were added to the cuvette and the whole cell suspension was transferred in 12-well tissue culture plate (Falcon) containing 600 μl of prewarmed RPMI1640 supplemented with 2% autologous serum, 500 U/ml human recombinant IL-4 and 800 U/ml human recombinant GM-CSF (both CellGenix). Directly after the transfer, the cell suspension was divided into 2 wells by pipetting 600 μl in an empty well. After 4 h 1.4 ml RPMI1640 supplemented with 1% autologous serum, 250 U/ml IL-4, 400 U/ml GM-CSF and, for cells to be matured, with LPS to a final concentration of 0.1 ng/ml, was added to the cells, which were cultured 24 h at 37° C. in a humidified atmosphere of 5% CO.sub.2 before luciferase reporter assays were performed. The electroporation efficiency, generation and maturation of the dendritic cells were assessed by the electroporation of a GFP coding control plasmid and antibody staining of the appropriate lineage and maturation markers, respectively, by subsequent FACS analysis.

(47) Luciferase Reporter Assay:

(48) Measurements of luciferase activity of DNA-transfected or adenovirally transduced cell lines and moDCs were performed in triplicates with the Luciferase Assay System (Promega) 1-2 days after transfection. Adherent 293T, HeLa, XS52 and NIH3T3 cells were washed with warm DPBS and then 200 μl/well 1× Luciferase Cell Culture Lysis Reagent (Promega, diluted 1:5 with distilled water) were added before plates were frozen for at least 2 h to −80° C. Raji, Jurkat, JCAM and dendritic cells were first transferred to a reaction vial and then washed in 1 ml warm DPBS. Afterwards cells were collected by centrifugation (500 g, 5 min, RT) and lysed with 200 μl/vial 1× Luciferase Cell Culture Lysis Reagent and frozen to −80° C. for at least 2 h. Subsequently, plates or vials were thawed at RT and 10 μl (293T cells) or 20 μl (DC, Raji and Jurkat, JCAM) of cell lysate were mixed with 50 μl of Luciferase Assay Substrate (Promega) in a 96-well LumiNunc plate (Nunc). Determination of RLUs (relative luminescence units) was performed in a Wallac fluorometer (Perkin-Elmer, Rotgau) and normalized to the protein concentration of the lysate.

(49) Recombinant Adenoviruses

(50) Cloning of Plasmids Containing the Recombinant Adenoviral Genome:

(51) For the generation of plasmids containing the recombinant adenoviral genome the pAdEasy1-system was used. Resulting plasmids were transfected into 293 cells for virus assembly and amplification.

(52) Preparation of Recombinant Adenoviruses:

(53) All viruses were amplified in 293 cells and purified by two rounds of CsCl equilibrium density gradient ultracentrifugation. Therefore, transfected 293 cells which show viral plaques were harvested, centrifuged at 1100 rpm for 5 min at 4° C. and then resuspended in 5 ml RPMI1640/2% FCS. Virus was released from cells by 3 freeze-thaw cycles and cell debris was removed by centrifugation of the lysate at 4000 rpm for 15 min at 4° C. Supernatant (2 ml) containing the virus was used to infect new 293 cells in increasing numbers (up to 15×T175 flasks) to amplify the virus; the remaining supernatant was stored at −80° C. for further rounds of virus production. Viruses from 15×T175 flasks of 293 cells were prepared by 3 freeze-thaw cycles as described above and then loaded onto a CsCl gradient. For the CsCl equilibrium density gradient, 3 ml of CsCl at a density of 1.41 g/ml was overlaid with 5 ml of CsCl at a density of 1.27 g/ml and then the supernatant containing the enriched virus was filled up to 7 ml with DPBS and loaded onto the gradient. After ultracentrifugation at 32000 rpm for 2 h at 4° C., the virus band was harvested, diluted in HEPES buffer to 8 ml and loaded onto a second CsCl equilibrium density gradient as described above. Viruses were spun in an ultracentrifuge at 32000 rpm for 24 h at 4° C., before harvesting and purification with a PD-10 tip (GE Healthcare) according to the manufacturer's instructions. Finally, eluted virus (in DPBS) was mixed with 10% Glycerin and aliquots of 25 μl were frozen to −80° C. Verification of viral genomes and exclusion of wild-type contamination was performed by PCR. Physical particle concentration [viral particles (vp)/ml] was determined by OD.sub.260 reading and infectious particle concentration was determined by TCID.sub.50 assay on 293 cells.

(54) Determination of the Physical Particle Concentration:

(55) The physical particle concentration is defined by the amount of virus particles per ml [vp/ml]. To quantify the viral DNA, the virus preparation was diluted at different ratios (1:3, 1:5, 1:10, 1:50, 1:100) with viral lysis buffer (VLB [10 mM TE, 0.5% SDS]) and incubated at 56° C. in a thermomixer (Eppendorf) for 10 min. Cooled samples were measured with a spectrophotometer (Eppendorf) at OD.sub.260 and the mean value was calculated considering the dilution factors.

(56) Determination of the Infectious Particle Concentration:

(57) The concentration of infectious virus particles is determined by the “tissue culture infectious dose 50” (TCID.sub.50)-method. Therefore, 293 cells were seeded in a 96-well tissue culture plate (Falcon) at a density of 10.sup.5/well in RPMI1640/2% FCS. The virus was diluted from 10.sup.−1 to 10.sup.−12 in RPMI1640/2% FCS and cells were infected with two dilution series each. Cells were incubated at 37° C., 5% CO.sub.2 and 10 as well as 12 days after infection cell lysis was assessed microscopically. Wells that showed at least one plaque were considered “positive”.

(58) Adenoviral Transduction of Cells:

(59) Immature day 5 DCs were seeded in 12-well tissue culture plates (Falcon) at a concentration of 1×10.sup.6 cells/well in 250 μl medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4. Immediately, adenovirus at 500 TCID.sub.50/cell in a final volume of 250 μl medium without cytokines was added to the cells. After 1.5 h of incubation at RT, 2.5 ml of growth medium replenished with cytokines as described before was added per well. If mDCs were needed, LPS was added 4 h after transduction to final concentration of 0.1 ng/ml.

(60) Raji, Jurkat and JCAM cells were seeded in 12-well tissue culture plates (Falcon) at a concentration of 1×10.sup.6 cells/well in 250 μl RPMI1640 medium supplemented with 2% FCS. Immediately, adenovirus at 500 TCID.sub.50/cell (Jurkat and JCAM cells) or 50 TCID.sub.50/cell (Raji) in a final volume of 250 μl RPMI1640 medium supplemented with 2% FCS was added to the cells. After 1.5 h of incubation at RT on the rocker, 2.5 ml of growth medium replenished with 10% FCS were added per well. To determine transduction efficacy, cells were transduced with Ad5TL and the percentage of living green fluorescent cells was assessed by flow cytometric analysis with a “FACScan” flowcytometer (BD Biosciences, Heidelberg). Only experiments that yielded transduction efficiencies of more than 70% were evaluated and are shown.

(61) Polymerase Chain Reaction (PCR):

(62) PCR was used to (i) amplify DNA fragments for cloning from either chromosomal- or plasmid DNA and (ii) to screen adenoviral DNA for insertions.

(63) (i) DNA fragments amplified for cloning were generated in a final reaction volume of 50 μl using a DNA polymerase containing proof-reading activity (Platinum Pfx DNA Polymerase, Invitrogen) according to the manufacturer's instructions.

(64) (ii) PCR reactions for screening of adenoviral DNA were performed in a final reaction volume of 25 μl with a recombinant Taq DNA Polymerase from Invitrogen according to the manufacturer's instructions.

(65) TABLE-US-00001 TABLE 1 PCR program for amplification of DNA fragments Step Temperature Time Cycles Initial denaturation 95° C. 5 min  1x Denaturation 95° C. 1 min 25x Annealing 56° C.-62° C. 1 min to Extension 68° C. 0.5 min-2 min 30x Final extension 68° C. 10 min   1x Pause  4° C. ∞
Materials
Schematic Depiction of CD83 Intron 2 Fragments for Luciferase Assay:

(66) Intron 2 was divided into 3 fragments (A, B, C) and subcloned in the pGL3/MP-261 reporter construct. Subsequently fragment C was narrowed down via deletion mutagenesis in the fragments C1-C14.

(67) FIG. 22 provides an overview of luciferase reporter constructs and PCR primers used for cloning fragments of CD83 intron 2.

(68) Adenoviruses

(69) TABLE-US-00002 TABLE 3 Adenoviruses Adenovirus Specification Ad5TL Replication deficient adenovirus serotype 5; E1 region replaced by a CMV-GFP cassette Kindly provided by D.T. Curiel, Birmingham, AL, USA Ad5Luc1 Replication deficient adenovirus serotype 5; E1 region replaced by a CMV-luciferase cassette Kindly provided by D.T. Curiel, Birmingham, AL, USA Ad261/S1 Replication deficient adenovirus serotype 5; E1 region replaced by a MP −261/S1-luciferase cassette Ad510/S1 Replication deficient adenovirus serotype 5; E1 region replaced by a P −510/ S1-luciferase cassette Ad261/S1 + Es Replication deficient adenovirus serotype 5; E1 region replaced by a MP −261/S1/185 bp enhancer sense-luciferase cassette Ad261/S1 + Eas Replication deficient adenovirus serotype 5; E1 region replaced by a MP −261/S1/185 bp enhancer antisense-luciferase cassette Ad510/S1 + Es Replication deficient adenovirus serotype 5; E1 region replaced by a P −510/ S1/185 bp enhancer sense-luciferase cassette Ad510/S1 + Eas Replication deficient adenovirus serotype 5; E1 region replaced by a P −510/ S1/185 bp enhancer antisense-luciferase cassette AdBasic/S1 + Es Replication deficient adenovirus serotype 5; E1 region replaced by a 185 bp enhancer sense-luciferase cassette AdBasic/S1 + Eas Replication deficient adenovirus serotype 5; E1 region replaced by a 185 bp enhancer sense-luciferase cassette
Cell Lines and Cell Culture Media:

(70) If not indicated otherwise, all cell culture media and reagents were purchased by Lonza (Basel, Switzerland) and PAA (Cölbe).

(71) TABLE-US-00003 TABLE 4 Cell lines and cell culture media Cell type Short definition Culture medium 293(T) Human embryonic kidney cell Dulbecco's Modified Eagle Medium (DMEM) (Lonza) line, transformed with sheared without L-Glutamine, 4.5 g glucose/I (Lonza), 10% adenovirus genome (stably FCS (PAA), 1% Penicillin/Streptomycin/L- transfected with large T Glutamine (PAA) antigen) HeLa Human cervix carcinoma cell Dulbecco's Modified Eagle Medium (DMEM) (Lonza) line without L-Glutamine, 4.5 g glucose/I (Lonza) 10% FCS (PAA)1% Penicillin/Streptomycin/L-Glutamine (PAA) HFF Human foreskin fibroblast Minimum Essential Medium (DMEM) (Lonza) primary cells without L-Glutamine (Gibco), 7.5% FCS (PAA), 1% L-Glutamine (PAA), 0.001% Gentamycin Jurkat T cell leukemia derived T RPMI1640 without L-Glutamine (Lonza), 10% FCS lymphocyte cell line (PAA), 1% Penicillin/Streptomycin/L-Glutamine (PAA) NIH3T3 BALB/c derived embryonic Dulbecco's Modified Eagle Medium (DMEM) (Lonza) fibroblast cell line without L-Glutamine, 4.5 g glucose/I (Lonza), 10% FCS (PAA), 1% Penicillin/Streptomycin/L- Glutamine (PAA) NS47 BALB/c derived Iscove's Modified Dulbecco's Medium (IMDM) fibroblast cell line (Lonza) without L-Glutamine (Lonza), 10% FCS (PAA), 1% Penicillin/Streptomycin/L-Glutamine (PAA), 1% Sodium pyruvate (PAA) PBMC (Human) Peripher blood RPMI1640 without L-Glutamine (Lonza), 1% human mononuclear cells autologous serum, 1% Penicillin/Streptomycin/L- Glutamine (PAA), 1% Hepes (Lonza) Raji Burkitt's lymphoma derived RPMI1640 without L-Glutamine (Lonza), 10% FCS lymphoblastoid B cell line (PAA), 1% Penicillin/Streptomycin/L-Glutamine (PAA), 1% Hepes (Lonza) XS52 BALB/c derived DC-like cell line Iscove's Modified Dulbecco's Medium (IMDM) (Lonza) without L-Glutamine (Lonza), 10% FCS (PAA), 1% Penicillin/Streptomycin/L-Glutamine (PAA), 1% Sodium pyruvate (PAA), 10% Supplement NS47, 10 ng/ml murine GM-CSF JCAM T cell leukemia derived T RPMI1640 without L-Glutamine (Lonza), 10% FCS lymphocyte cell line, similar to (PAA), 1% Penicillin/Streptomycin/L-Glutamine Jurkat, but lacking the LCK (PAA) kinase
DNA Modifying Enzymes

(72) TABLE-US-00004 TABLE 5 DNA modifying enzymes Enzyme Supplier DNA restriction enzymes New England Biolabs (NEB [Frankfurt/Main]) Alkaline Phosphatase, Calf Intestinal (CIP) New England Biolabs (NEB [Frankfurt/Main]) T4 DNA ligase (Rapid ligation kit) Roche (Mannheim) Platinum Pfx polymerase Invitrogen (Karlsruhe) T4 Poly nucleotide kinase (T4-PNK) New England Biolabs (NEB [Frankfurt/Main]) Klenow fragment of DNA Polymerase I New England Biolabs (NEB [Frankfurt/Main])
Human Cytokines and Maturation Agents

(73) TABLE-US-00005 TABLE 6 Human cytokines and maturation agents Stock Cytokine/Agent concentration Purity Company Lipopolysaccharide 1 mg/ml Sigma-Aldrich, (LPS) München Recombinant GM-CSF 4 × 10.sup.4 U/ml >95% Cell Genix, Freiburg Recombinant IL-4 2 × 10.sup.5 U ml >95% Cell Genix, Freiburg Recombinant IL-6 1 × 10.sup.6 U/ml >95% Cell Genix, Freiburg
Cloning
Luciferase Reporter Constructs
Notes and Abbreviations:

(74) s: sense; as: antisense; CDS: coding sequence; enh: enhancer; UPP: upstream promoter; S1: spacer sequence 1.

(75) TABLE-US-00006 TABLE 7 Cloned luciferase reporter constructs Donor/template Excision method Insert Recipient Result pEGFP-N1 Restriction GFP CDS pGL3/CMV_luc; pGL3/CMV_GFP digest; HindIII/XbaI (luc HindIII/XbaI CDS replaced) RP1-258E1 PCR; KpnI CD83 intron 2 pGL3/MP-261; KpnI pGL3/fragment A fragment A s_as/MP-261 RP1-258E1 PCR; KpnI CD83 intron 2 pGL3/MP-261; KpnI pGL3/fragment B fragment B s_as/MP-261 CMVβL/cRel Restriction cRel CDS pCDNA 3.1; Hind pCDNA 3.1/cRel digest; Hind III/XbaI III/XbaI CMVβL/p50 Restriction p50 CDS pCDNA 3.1; Hind pCDNA 3.1/p50 digest; Hind III/XbaI III/XbaI CMVβL/p65 PCR; Hind P65 CDS pCDNA 3.1; pCDNA 3.1/p65 III/XbaI HindIII/XbaI pGL3/185bp enh. Restriction 185 bp pGL3/Basic; KpnI pGL3/185bp enh. s/MP-261 digest; KpnI enhancer s_as pGL3/fragment PCR; KpnI 185 bp pGL3/MP-261; KpnI pGL3/185bp enh C/MP-261 enhancer s_as/MP-261 Geneart standard Restriction UPP pGL3/Basic; pGL3/UPP vector/CD83_-510 digest; NheI/ NheI/XhoI (blunt) Smal (blunt) Geneart standard Restriction UPP pGL3/185bp enh pGL3/185bp enh. vector/CD83_-510 digest; NheI/ s_as/MP-261; s_as/UPP SmaI (blunt) NheI/XhoI (blunt) RP3-380E11 PCR; SacI/MluI S1 pGL3/UPP SacI/MluI pGL3/UPP + S1 RP3-380E11 PCR; SacI/MluI S1 pGL3/185bp enh. pGL3/185bp enh. s_as/UPP SacI/MluI s_as/UPP + S1 Geneart standard Restriction P-510 pGL3/MP-261 pGL3/P-510 vector/CD83_-510 digest; NheI/XhoI NheI/XhoI Geneart standard Restriction P-510 pGL3/185bp enh pGL3/185bp enh. vector/CD83_-510 digest; s_as/MP-261 s_as/P-510 NheI/XhoI NheI/XhoI RP3-380E11 PCR; SacI/MluI S1 pGL3/P-510; pGL3/P-510 + S1 SacI/MluI RP3-380E11 PCR; SacI/MluI S1 pGL3/185bp enh. pGL3/185bp enh. s_as/P-510; s_as/P-510 + S1 SacI/MluI RP3-380E11 PCR; SacI/MluI S1 pGL3/MP-261; pGL3/MP-261 + S1 SacI/MluI RP3-380E11 PCR; SacI/MluI S1 pGL3/185bp enh pGL3/185bp enh s_as/MP-261; s_as/MP-261 + S1 SacI/MluI RP3-380E11 PCR; KpnI CD83 intron 2 pGL3/MP-261; KpnI pGL3/fragment C fragment C s_as/MP-261 RP3-380E11 PCR; KpnI CD83 intron 2 pGL3/MP-261; KpnI pGL3/fragment A fragment A s_as/MP-261 RP3-380E11 PCR; KpnI CD83 intron 2 pGL3/MP-261; KpnI pGL3/fragment B fragment B s_as/MP-261 pGL3/fragment PCR; KpnI CD83 intron 2 pGL3/MP-261; KpnI pGL3/fragment C1-C14 C/MP-261 fragments C1-C14 s_as/MP-261
Luciferase Reporter Constructs with Mutated IRF-Sites
Notes and Abbreviations:

(76) Via PCR mutagenesis the IRF-Sites 1 and 2 in the 185 bp enhancer were mutated by PCR mutagenesis. The 3.sup.rd IRF-site in the P-510 promoter (or rather in the UpP) was mutated by GENEART and provided in the “GENEART standard vector/CD83_-510mut 3.sup.rd IRF-Site”. Combinations of the 3 IRF-site mutations resulted in constructs, bearing 1, 2 or 3 mutated IRF-sites:

(77) TABLE-US-00007 mut: mutated pGL3/UPPmut 3.sup.rd IRF-Site pGL3/P-510mut 3.sup.rd IRF-Site These constructs pGL3/185 bp enh. s_as 1.sup.st IRF-site mut/ were subsequently P-510 {close oversize brace} extended with the pGL3/185 bp enh. s_as 2.sup.nd IRF-site mut/ spacer sequence “S1” P-510 pGL3/185 bp enh. s_as 1.sup.st/2.sup.nd IRF-site mut/P-510 pGL3/185 bp enh. s_as 1.sup.st/2.sup.nd mut/ P-510mut 3.sup.rd IRF-Site

(78) TABLE-US-00008 TABLE 8 Cloned luciferase reporter constructs with mutated IRF-sites Donor/template Excision method Insert Recipient Result GENEART standard Restriction digest; P-510mut pGL3/MP-261 pGL3/P-510mut 3.sup.rd vector/CD83_- NheI/XhoI 3.sup.rd IRF-Site NheI/XhoI IRF-Site 510mut 3.sup.rd IRF- Site GENEART standard Restriction digest; UPPmut pGL3/Basic; pGL3/UPPmut 3.sup.rd vector/CD83_- NheI/SmaI (blunt) 3.sup.rd IRF-Site NheI/XhoI IRF-Site 510mut 3.sup.rd IRF- (blunt) Site pGL3/185bp enh. PCR; KpnI 185 bp pGL3/P-510; pGL3/185bp enh. s/MP-261 enhancer 1.sup.st KpnI s_as 1.sup.st IRF-site IRF-site mut mut/P-510 pGL3/185bp enh. PCR; KpnI 185 bp pGL3/P-510; pGL3/185bp enh. s/MP-261 enhancer 2.sup.nd KpnI s_as 2.sup.nd IRF-site IRF-site mut mut/P-510 pGL3/185bp enh. PCR; KpnI 185 bp pGL3/P-510; pGL3/185bp enh. s/MP-261 enhancer 1.sup.st/ KpnI s_as 1.sup.st/2.sup.nd IRF-site 2.sup.ndIRF-site mut/P-510 mut pGL3/185bp enh PCR; KpnI 185 bp pGL3/P- pGL3/185bp enh. s/MP-261 enhancer 1.sup.st/ 510mut 3.sup.rd s_as 1.sup.st/2nd mut/P- 2.sup.ndIRF-site IRF-Site; KpnI 510mut 3.sup.rd IRF-Site mut RP3-380E11 PCR; SacI/MluI S1 pGL3/P- pGL3/P-510mut 3.sup.rd 510mut 3.sup.rd IRF-Site + S1 IRF-Site; SacI/MluI RP3-380E11 PCR; SacI/MluI S1 pGL3/185bp pGL3/185bp enh. enh. s_as 1.sup.st s_as 1.sup.st IRF-site IRF-site mut/P- mut/P-510 + S1 510; SacI/MluI RP3-380E11 PCR; SacI/MluI S1 pGL3/185bp enh. pGL3/185bp enh. s_as 1.sup.st/2.sup.nd IRF- s_as 1.sup.st/2.sup.nd IRF-site site mut/P-510; mut/P-510 + S1 SacI/MluI RP3-380E11 PCR; SacI/MluI S1 pGL3/185bp enh. pGL3/185bp enh. s_as 1.sup.st/2.sup.nd s_as 1.sup.st/2.sup.nd mut/P- mut/P-510mut 510mut 3.sup.rd IRF- 3.sup.rd IRF-Site; Site + S1 SacI/MluI
8. Primers

(79) All primers were purchased from MWG Eurofins Operon (Ebersberg).

(80) Notes and Abbreviations:

(81) fw, for: DNA oligonucleotides in forward orientation

(82) rev: DNA oligonucleotides in reverse orientation

(83) mt: mutant

(84) wt: wild type

(85) aa: amino acid

(86) The original IRF-2 PCR template (EF1α/IRF-2) provided by Kay Childs (Division of Basic Medical Sciences St. George's, University of London, U.K.) contained 3 defective aa (3, 4, 5). First the defective sequence was subcloned via PCR (primers: IRF-2 fwd HindII; IRF-2 rev XbaI) in pCDNA 3.1 and from that template a PCR product with the corrected sequence was generated by using PCR mutagenesis (primers: IRF-2mut; fwd IRF-2mut rev;).

(87) Cloning Primers for Luciferase Reporter Constructs

(88) TABLE-US-00009 TABLE 9 Cloning primers for luciferase reporter constructs Primer (SEQ ID NO:) Sequence Description A-1 (6) 5′-GGCGGTACCAGCTGGGGCTCTTCTCAATATTATAAAG-3′ Fragment C  deletion; rev; wt A-10 (7) 5′-GGCGGTACCAGATGATTTCCAAAGGAAGGGAG-3′ Fragment C  deletion; fw; wt A-1b (8) 5′-GGCGGTACCCAATATTATAAAGTCTATTTATAG-3′ Fragment C  deletion; 185 bp  enhancer; rev; wt A-2 (9) 5′-GGCGGTACCAGGTGCCAATGGGGACAGTACG-3′ Fragment C  deletion; rev; wt A-3 (10) 5′-GGCGGTACCAGAAGGCATTGCAACTCTGG-3′ Fragment C  deletion; A-4 (11) 5′-GGCGGTACCGATGCTTCACTCTCCTCACC-3′ Fragment C  deletion; rev; wt A-8 (12) 5′-GGCGGTACCAGTACTTTGGGCCTGGTTGATAATC-3′ Fragment C  deletion; fw; wt A-9 (13) 5′-GGCGGTACCCCTATGGGTGATGCAAAACGAAAG-3′ Fragment C  deletion; 185 bp  enhancer; fw; wt C-forward  5′-CCAGGGTACCGAGGAGGTATTTTGAGAAAATATG-3′ CD83 intron  (14) 2 fragment  C fw; wt C-forward 2  5′-CCAGGGTACCACAATATCATGTCTGTGAGGAGTAAAGC-3′ Fragment C  (15) deletion; fw; wt C-Kurz 1  5′-GGCGGTACCTATAATATTGAGAAGAGCCC-3′ Fragment C  (16) deletion; fw; wt C-Kurz 2  5′-GGCGGTACCATTGGCACCTATAGTACTTG-3′ Fragment C  (17) deletion; fw; wt C-Kurz 3rev  5′-GGCGGTACCCTTACGCCTGTAATCCCAGC-3′ Fragment C  (18) deletion; rev; wt C-reverse  5′-GCAGGGTACCTTCCTCTTCTTTGTGTAGTG-3′ CD83 intron  (19) 2 fragment  C rev; wt Intron2-A_for 5′-TTAAGGTACCGTAGGTGCTGCGATACC-3′ CD83 intron  (20) 2 fragment A; fw; wt Intron2-A_rev 5′-CCGGGGTACCAATGAAGTAGGAATATTTAAC-3′ CD83 intron  (21) 2 fragment  A; rev; wt Intron2-B_for 5′-CATTGGTACCTTACTTACTGTGGGATCAGAG-3′ CD83 intron  (22) 2 fragment  B fw; wt Intron2-B_rev 5′-GCAAGGTACCAAAGAACCACATCTATTACAAC-3′ CD83 intron  (23) 2 fragment  B rev; wt
Cloning Primers for the Mutated 185 bp Enhancer

(89) TABLE-US-00010 TABLE 10 Cloning primers for the mutated 185 bp enhancer luciferase reporter constructs. Primer (SEQ ID NO:) Sequence Description 185 bp 5′-GGCGGTACCCCTATGGGTGATGCAGGACTATAGAGG 185 bp enhancer; Enh.fw/MT ATATATGGTG-3′ fw; 1.sup.st IRF-site (24) mt 185 bp 5′-GGCGGTACCCCTATGGGTGATGCAAAACGAAAGAGG 185 bp enhancer; Enh.fw/WT ATATATGGTG-3′ fw; wt (25) 185 bp 5′-GGCGGTACCCAATATTATAAAGTCTATTTATAGTAGAC 185 bp enhancer; Enh.rev/MT TTTTATATGAAGTCACACTCTTATTCCTCTCCTCTCCC-3′ rv; 2.sup.nd IRF-site (26) mt 185 bp 5′-GGCGGTACCCAATATTATAAAGTCTATTTATAGTAGA 185 bp enhancer; Enh.rev/WT CTTTTATATGAAGTCACACTCTTACTTCCCTTTTCTCCC-3′ rev; wt (27)

Example 1: CD83 is Upregulated During DC Maturation and is not Expressed by HFF Cells

(90) FIG. 1, (A): One hundred thousand day 5 iDCs, 20 h LPS-matured DCs and HFF cells for each setup were harvested and stained with PE-labeled mouse anti-human CD83 mAB or mouse IgG1 isotype-control for flow cytometric analyses. Each quadrangle represents 1×10.sup.4 living cells determined by a gate on forward and side light scatter and PI-staining. In each histogram plot the black line represents mean fluorescence intensity of the isotype-control stained cells and the red line represents mean florescence intensity of the anti-CD83 stained cells. (B): One million day 5 iDCs or HFF cells per well were seeded in a 12-well tissue culture plate. Immature DCs were either matured for 3 h, 6 h, 10 h and 24 h or left untreated as the HFF cells. Cells were lysed in RIPA buffer at the indicated time points. For Western blot 20 μg per lane of whole cell lysates from either immature DCs, maturing DCs (3-24 h after addition of LPS) or HFF cells were loaded on a denaturing 12.5% SDS polyacrylamide gel and blotted afterwards onto a nitrocellulose membrane. The membranes were incubated with a rat anti-human CD83 and a mouse anti-human GAPDH primary antibody and subsequently with an anti-rat HRP-conjugated or an anti-mouse HRP-conjugated secondary antibody, respectively. The signal was detected via chemiluminescence that was visualized on a photo film. (C) Total RNA was isolated from iDCs, 20 h LPS-matured DCs and HFF cells using a QIAshredder and RNeasy Mini Kit (Quiagen) according to the manufacturer's instructions and reversely transcribed with an Oligo (dT).sub.12-18 primer. Afterwards PCR was performed with an intron spanning primer pair for CD83 and a control primer pair for GAPDH. Additionally, one PCR reaction was prepared with water instead of DNA as a negative control.

Example 2: Schematic Depiction of the CD83 Gene Locus and the Respective Acetylation Data Resulting from the ChIP-Chip™ Microarray Analysis

(91) FIG. 2, (A): Schematic depiction of the CD83 genelocus and nucleotide positions on chromosome 6. Coding sequence depicted in blue (CDS), exons in grey (E1-E5) and introns in red (I1-I4). (B) Data for the CD83 hyperacetylation were provided by NimbleGen Systems and interpolated subsequently over a space of 500 bp using the Signal Map software. (C) Overlay of the interpolated data for mature DCs aligned with the positions of the CD83 exons (black; E1-E5) and introns (red; I1-I4)

Example 3: Schematic Depiction of Constructs for Gene Reporter Assays

(92) FIG. 3: A pGL3 vector backbone only coding for the luciferase gene and lacking any regulatory element (pGL3/Basic) was used for background control. The pGL3 backbone containing the luciferase gene driven by a CMV promoter (pGL3/CMV/luc) was generated for its usage as a positive control and for internal normalization. The same plasmid coding for GFP instead of luciferase (pGL3/CMV/GFP) was used to determine transfection efficiency. As reference control a pGL3 plasmid coding for the luciferase gene driven by the CD83 minimal promoter MP-261 (pGL3/MP-261) was generated. Finally, plasmids containing a putative regulatory element in both orientations upstream of the MP-261 (pGL3/Put. reg./MP-261) were generated to study its effects on the MP-261.

Example 4: Schematic Depiction of the Subcloning Strategy of the Hyperacetylated Region of CD83 Intron 2

(93) FIG. 4: The first 6 kb of CD83 intron 2 were subdivided into 3 fragments A (1239 bp), B (2359 bp), and C (2220 bp). After PCR amplification each fragment was subcloned in sense and antisense orientation upstream the MP-261 into the pGL3/MP-261 plasmid. Hyperacetylated regions are depicted as blue bars, exons as black bars (E1-E5) and introns are marked in red (I1-I5). The red boxes indicate the base pair position relatively to the transcription start (bp 1).

Example 5: Fragment C Enhances the MP-261 Activity Specifically in XS52 Cells

(94) FIG. 5: Two hundred thousand XS52, NIH3T3 or HeLa cells per well were seeded in a 12-well tissue culture plate and were grown overnight at 37° C., 5% CO.sub.2. The next day, the cells were transfected with 2.5 μg of reporter plasmids containing fragment A, B or C of the CD83 intron 2 in sense (s) or antisense (as) orientation upstream of the MP-261 or the pGL3/MP-261 as reference control using the DEAE-Dextran method. As controls the pGL3/Basic and the pGL3/CMV/luc vector were transfected to assess background activity and for internal normalization, respectively. The promoter activity was determined by luciferase reporter assays 48 h after transfection. The resulting RLUs were further normalized to the protein concentration of each lysate. The plasmids containing fragments A, B and C were compared to the pGL3/MP-261 vector for each cell line and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

Example 6: Schematic Depiction of the Deletion Mutants C1-C14 of Fragment C

(95) FIG. 6: The deletion mutants C1-C14 of the CD83 intron 2 fragment C were generated by PCR amplification and cloned in sense (s) and antisense (as) orientation into the pGL3/MP-261 reporter plasmid upstream of the MP-261. Induction of the minimal promoter activity was determined in XS52, NIH3T3 and HeLa cell lines by luciferase reporter assays. Induction is depicted relatively to the activity of the pGL3/MP-261. −−− no induction of the MP-261, + weak induction of the MP-261, ++ medium induction of the MP-261; +++ strong induction of the MP-261.

Example 7: The 185 bp Long Deletion Mutant C13 of Fragment C Enhances the MP-261 Activity Specifically in XS52 Cells

(96) FIG. 7: Two hundred thousand XS52, NIH3T3 or HeLa cells per well were seeded in a 12-well tissue culture plate and were grown overnight at 37° C., 5% CO.sub.2. The next day, cells were transfected with 2.5 μg of either pGL3/MP-261 reporter plasmids containing the deletion mutants C1-C14 of the fragment C of the CD83 intron 2 in sense (s) or antisense (as) orientation upstream of the MP-261 or the pGL3/MP-261 as reference control using the DEAE-Dextran method. As controls the pGL3/Basic and the pGL3/CMV/luc vector were transfected to assess background activity and for internal normalization, respectively. The promoter activity was determined by luciferase reporter assays 48 h after transfection. The resulting RLUs were further normalized to the protein concentration of each lysate. The plasmids containing the different fragments were compared to the pGL3/MP-261 vector for each respective cell line and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

Example 8: Fragment C and the 185 bp Enhancer Induce the MP-261 Activity Specifically in mDCs

(97) FIG. 8: Two million day 5 iDCs were electroporated with 4 μg of either pGL3/Basic, pGL3/MP-261, pGL3/FragmentC/MP-261 s/as or pGL3/185 bp Enh. (C13)/MP-261 s/as luciferase reporter plasmid using the AMAXA technology. Afterwards, cells were transferred to a 12-well tissue culture plate and split into two equal fractions. One of the fractions was matured 3 h after electroporation with LPS to a final concentration of 0.1 ng/ml and the other fraction was replenished with growth medium without LPS. Cells were then grown at 37° C., 5% CO.sub.2 for 20 h. The pGL3/Basic and the pGL3/CMV/luc vector were electroporated to assess background activity and for internal normalization, respectively. The promoter activity was determined by luciferase reporter assays and the resulting RLUs were normalized to the protein concentration of each lysate. The plasmids containing the different fragments were compared to the pGL3/MP-261 vector and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001. Results depict the means (±SEM) of four independently performed experiments representing four different donors in (A) and three experiments representing three different donors in (B).

Example 9: Neither Fragments A, B, C Nor the 185 bp Enhancer Induce the MP-261 in Raji and Jurkat Cells

(98) FIG. 9: Ten million Raji or Jurkat cells were electroporated with 20 μg of reporter plasmids containing fragment A, B, C or the 185 bp enhancer in sense (s) or antisense (as) orientation upstream of the MP-261 or the pGL3/MP-261 as reference control using a Genepulser II. As controls the pGL3/Basic and the pGL3/CMV/luc vector were electroporated to assess background activity and for internal normalization, respectively. After electroporation, cells were transferred into 10 ml prewarmed growth medium and cultivated at 37° C., 5% CO.sub.2 for 24 h. The promoter activity was determined by luciferase reporter assays and the resulting RLUs were further normalized to the protein concentration of each lysate. The plasmids containing the different fragments were compared to the pGL3/MP-261 vector for each respective cell line and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

Example 10: Presumed Biocomputational Model for the Interaction of the CD83 Upstream Promoter, the MP-261 and the 185 bp Enhancer

(99) FIG. 10, (A): Depiction of the three regulatory elements with the predicted transcription factor binding sites regulating CD83 transcription: (i) The upstream promoter (UpP) containing NFκB-sites 1 and 2, IRF-site 3 and one SP1 site, (ii) the 261 bp long CD83 minimal promoter (MP-261) containing NFκB-sites 3, 4 and 5 and four SP1-sites and (iii) the 185 bp enhancer containing IRF-sites 1 and 2 as well as one SP1-site. The UpP and the MP-261 including the 82 bp naturally occurring spacer sequence are termed 510 bp promoter (P-510). Previously confirmed and published transcription factor binding sites are marked with a red arrow. (B): Designated interaction of the 185 bp enhancer with the MP-261 mediated by the interaction of IRF-sites 1 and 2 with NFκB-sites 3 and 5, respectively. (C): Ternary complex formation with the UpP as third interaction partner. The interaction between the UpP and the MP-261 is mediated by IRF-site 3 and NFκB-site 4.

Example 11: Schematic Depiction of Reporter Constructs Containing the UpP and the Spacer Sequence S1

(100) FIG. 11: The P-510 as well as the CD83 upstream promoter (UpP) were cloned upstream of the luciferase gene into the pGL3/Basic plasmid, thus generating pGL3/P-510 and pGL3/UpP, respectively. The UpP comprised in the P-510 is highlighted in purple. Furthermore, a 500 bp long spacer sequence (S1) was inserted in between MP-261 and P-510 generating the plasmids pGL3/185 bp Enh./S1/MP-261 and pGL3/185 bp Enh./S1/MP-261, respectively. The spacer sequence was also introduced into the plasmids pGL3/MP-261 and pGL3/P-510, resulting in the plasmids pGL3/S1/MP-261 and pGL3/S1/P-510.

Example 12: The Spacer Sequence S1 does not Significantly Affect the Induction of the MP-261 and the P-510 in XS52 Cells

(101) FIG. 12: Two hundred thousand XS52, NIH3T3 and HeLa cells per well were seeded in a 12-well tissue culture plate and were grown overnight at 37° C., 5% CO.sub.2. The next day, cells were transfected with 2.5 μg of either pGL3/MP-261 or pGL3/P-510 with or without the 185 bp enhancer in sense (s) or antisense (as) orientation either containing the spacer sequence 51 or not, using the DEAE-Dextran transfection method. As controls the pGL3/Basic and the pGL3/CMV/luc vector were transfected to assess background activity and for internal normalization, respectively. The promoter activity was determined by luciferase reporter assays 48 h after transfection. The resulting RLUs were further normalized to the protein concentration of each lysate. The plasmids containing the spacer sequence S1 were compared to the corresponding plasmids without the spacer sequence for each respective cell line. The resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

Example 13A: The Spacer Sequence S1 does not Significantly Affect the Induction of the MP-261 in mDCs

(102) FIG. 13, (A): Two million day 5 iDCs were electroporated with 4 μg of either pGL3/MP-261 with or without the 185 bp enhancer in sense (s) or antisense (as) orientation upstream of the MP-261 or with their respective counterparts comprising the spacer sequence S1, using the AMAXA technology. Afterwards, cells were transferred to a 12-well tissue culture plate and split into two equal fractions. Cells were matured 3 h after electroporation with LPS to a final concentration of 0.1 ng/ml and were then cultured at 37° C. in a humidified atmosphere at 5% CO.sub.2 for 20 h. As controls the pGL3/Basic and the pGL3/CMV/luc vector were electroporated to assess background activity and for internal normalization, respectively. The promoter activity was determined by luciferase reporter assays. The resulting RLUs were further normalized to the protein concentration of each lysate. The plasmids containing the spacer sequence S1 were compared to the corresponding plasmids lacking the spacer sequence. The resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of five independently performed experiments representing different donors.

Example 13B: The Spacer Sequence S1 does not Significantly Affect the Induction of the P-510 in mDCs

(103) FIG. 13, (B): The cells were treated and electroporated in the same way as in (A), but instead of plasmids bearing the MP-261, plasmids comprising the P-510 were used. The resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments representing different donors.

Example 14: The Ternary Complex of UpP, MP-261 and 185 bp Enhancer Shows a Specific Transcriptional Induction in mDCs

(104) FIG. 14, (A): One million day 5 iDCs per well were seeded in a 12-well tissue culture plate in 250 μl medium supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4. Recombinant adenovirus as indicated at 500 TCID.sub.50/cell in a final volume of 250 μl medium without cytokines was added to the cells. After 1.5 h of incubation at RT, 2.5 ml of growth medium replenished with cytokines as described before was added per well. For the generation of mDCs LPS was added 3 h after transduction to final concentration of 0.1 ng/ml. The promoter activity was determined by luciferase reporter assays 24 h after transduction. As controls the Ad5luc1 was transduced for internal normalization. The resulting RLUs were further normalized to the protein concentration of each lysate. The transduction setups were compared to the transduction of Ad261/S1 and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001. Results represent the means (±SEM) of three independently performed experiments representing different donors.

Example 15: The Ternary Complex of UpP, MP-261 and 185 bp Enhancer Shows No Specific Transcriptional Induction in Raji, Jurkat and JCAM Cells

(105) FIG. 15, (A)-(C): One million Raji, Jurkat and JCAM cells per well were seeded in a 12-well tissue culture plate in 250 μl RPMI1640 supplemented with 2% FCS. Recombinant adenovirus as indicated at 500 TCID.sub.50/cell for Jurkat and JCAM and 50 TCID.sub.50/cell for Raji in a final volume of 250 μl RPMI1640 supplemented with 2% FCS was added to the cells. After 1.5 h of incubation at RT, 2.5 ml of appropriate full growth medium was added per well. Raji cells were either stimulated 3 h after transduction with LPS to a final concentration of 0.1 ng/ml or left untreated. Jurkat and JCAM cells were either stimulated 3 h after transduction with PGE.sub.2 and TNF-α to a final concentration of 1 μg/ml and 10 ng/ml, respectively, or left untreated. The promoter activity was determined by luciferase reporter assays 24 h after transduction. As additional controls Ad5luc1 was transduced for internal normalization. The resulting RLUs were further normalized to the protein concentration of each lysate. The transduction setups were compared to the transduction of Ad261/S1 and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001. Results represent the means (±SEM) of three independently performed experiments.

Example 16: Schematic Depiction of Reporter Constructs Containing Mutated IRF-Sites Used for Luciferase Assays

(106) FIG. 16: To generate the pGL3/UpP plasmid, the CD83 upstream promoter a 244 bp fragment of the P-510 containing the UpP was isolated by enzymatic digestion and cloned upstream of the luciferase gene into the pGL3/Basic plasmid. The individual IRF-sites in the pGL3/UpP, the pGL3/S1/P-510 and the pGL3/185 bp Enh./S1/P-510 plasmids were mutated by PCR mutagenesis. The IRF-sites were mutated in different combinations in concordance with the point mutations in the EMSA control oligonucleotides.

Example 17: Mutation of any of the Three IRF-Sites in the Ternary Complex Significantly Reduces the Luciferase Expression in XS52 Cells

(107) FIGS. 17, (A) and (B): Two hundred thousand XS52 cells per well were seeded in a 12-well tissue culture plate and were grown overnight at 37° C., 5% CO.sub.2. The next day, cells were transfected with 2.5 μg of the wildtype pGL3/UpP or the pGL3/S1/P-510 with and without the 185 bp enhancer in sense (s) or antisense (as) orientation upstream of the P-510 or with the analogical plasmids with one, two or three mutated IRF-sites using the DEAE-Dextran transfection method. The promoter activity was determined by luciferase reporter assays 48 h after transfection. As controls the pGL3/Basic and the pGL3/CMV/luc vector were transfected to assess background activity and for internal normalization, respectively. The resulting RLUs were normalized to the protein concentration of each lysate. The plasmids containing the different mutations were compared to the respective wildtype control and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. The mutated IRF-sites are indicated by their respective numbers. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

Example 18: Mutation of any of the Three IRF-Sites in the Ternary Complex Significantly Reduces the Luciferase Expression in mDCs

(108) FIG. 18: Two million day 5 iDCs were electroporated with 4 μg of either wildtype pGL3/S1/P-510 with and without the 185 bp enhancer in sense (s) or antisense (as) orientation upstream of the P-510 or with the analogical plasmids with one, two or three mutated IRF-sites, using the AMAXA technology. Afterwards, cells were transferred to a 12-well tissue culture plate and split into two equal fractions. Cells were matured 3 h after electroporation with LPS to a final concentration of 0.1 ng/ml and were then cultured at 37° C. in a humidified atmosphere at 5% CO.sub.2 for 20 h. The promoter activity was determined by luciferase reporter assays 48 h after transfection. As controls the pGL3/Basic and the pGL3/CMV/luc vector were transfected to assess background activity and for internal normalization, respectively. The resulting RLUs were further normalized to the protein concentration of each lysate. The plasmids containing the different mutations were compared to the respective wildtype control and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. The mutated IRF-sites are indicated by their respective numbers. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments from different donors.

Example 19: Transcription Factors of the NFκB Family and IRF-5 Induce the MP-261 in 293T Cells

(109) FIGS. 19, (A) and (B): Sixty thousand 293T cells per well were seeded in a 24-well tissue culture plate and grown overnight at 37° C., 5% CO.sub.2. The next day, cells were cotransfected with 0.05 μg of pGL3/MP-261 reporter plasmid and 0.15 μg of each plasmid coding for the indicated transcription factors (p50, p65, cRel or IRF-5 in pCDNA3.1) or empty vector backbone as control. The total amount of DNA was adjusted with pCDNA3.1 empty vector backbone to 0.5 μg. For transfection the Lipofectamine™ LTX system was used. The promoter activity was determined by luciferase reporter assays 48 h after transfection. As controls the pGL3/Basic and the pGL3/CMV/luc vector were transfected to assess background activity and for internal normalization, respectively. The resulting RLU were further normalized to the protein concentration of each lysate. The cotransfections were compared to the pGL3/MP-261 control and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

Example 20A: p65 and IRF-5 Induce the UpP in 293T Cells

(110) FIG. 20, (A): Sixty thousand 293T cells per well were seeded in a 24-well tissue culture plate and grown overnight at 37° C., 5% CO.sub.2. The next day, cells were cotransfected with 0.05 μg of pGL3/UpP reporter plasmid and 0.15 μg of each plasmid coding for the indicated transcription factors (p50, p65 and IRF-5 in pCDNA3.1) or empty vector backbone as control. The total amount of DNA was adjusted with pCDNA3.1 vector backbone to 0.5 μg. The promoter activity was determined by luciferase reporter assays 48 h after transfection. As controls the pGL3/Basic and the pGL3/CMV/luc vector were transfected to assess background activity and for internal normalization, respectively. The resulting RLU were further normalized to the protein concentration of each lysate. The cotransfections were compared to the pGL3/UpP control and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

Example 20B: The Induction of the UpP by p65 and IRF-5 is Abrogated when the IRF-Site 3 is Mutated

(111) FIG. 20, (B): The cells were transfected as described in (A) either with 0.05 μg pGL3/UpP or pGL3/UpP 3.IRFmut reporter plasmid alone or cotransfected with 0.15 μg of each indicated transcription factor (p65 and IRF-5 in pCDNA3.1). The total amount of DNA was adjusted with pCDNA3.1 vector backbone to 0.5 μg. The pGL3/UpP and pGL3/UpP 3.IRFmut setups were compared and the resulting p-values were determined via one way ANOVA and Bonferroni's Multiple Comparison post hoc test. *p<0.05, **p<0.01, ***p<0.001 and n.s. not significant (p>0.05). Results represent the means (±SEM) of three independently performed experiments.

(112) TABLE-US-00011 Sequence Listing, Free Text SEQ ID NO: Description 1 Genomic sequence of the CD83 promoter of NT_007592.15; UpP bp 2531-2696, intermediate sequence bp 2697-2779, MP-261 bp 2780-3040, and 185 bp enhancer bp 7218-7402 2 Upstream promoter (UpP) 3 Intermediate sequence 4 CD83 Minimal Promoter (MP-261) 5 185 bp enhancer  6-27 Primers 28-29 Promoter regions of vectors pGL3/-510/S1/Es and pGL3/-510/S1/Eas, respectively