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The Journal of Immunology, 2007, 178: 5454-5464.
Copyright © 2007 by The American Association of Immunologists, Inc.
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CD83 Knockdown in Monocyte-Derived Dendritic Cells by Small Interfering RNA Leads to a Diminished T Cell Stimulation1

Alexander T. Prechtel2, Nadine M. Turza, Alexandros A. Theodoridis and Alexander Steinkasserer

Department of Dermatology, University Hospital Erlangen, Erlangen, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mature human dendritic cells (mDCs) are the most powerful APCs known today, having the unique ability to induce primary immune responses. One of the best known surface markers for mDCs is the glycoprotein CD83, which is strongly up-regulated during maturation, together with costimulatory molecules such as CD80 and CD86. When CD83 surface expression was inhibited by interference with the messenger RNA export or by infection with certain viruses, DCs showed a dramatically reduced capability to induce T cell proliferation. However, in these cases side effects on other cellular functions cannot be excluded completely. In this study we present an efficient method to specifically influence CD83 surface expression by the use of RNA interference. We used small-interfering RNA targeted against CD83 and carefully evaluated an electroporation protocol for the delivery of the duplex into the cells. Furthermore, we identified freshly prepared immature DCs as the best target for the application of a CD83 knockdown and we were also able to achieve a long lasting silencing effect for this molecule. Finally, we were able to confirm that CD83 functions as an enhancer during the stimulation of T cells, significantly increases DC-mediated T cell proliferation, and goes hand in hand with clear changes in cytokine expression during T cell priming. These results were obtained for the first time without the use of agents that might cause unwanted side effects, such as low m.w. inhibitors or viruses. Therefore, this method presents a suitable way to influence DC biology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Antigen-presenting cells are characterized by specialized features such as pathogen recognition, Ag processing, migratory capacity, and surface expression of costimulatory molecules. Immune responses are initiated in the T cell areas of secondary lymphoid organs where APCs such as mature dendritic cells (mDCs)3 present processed Ags to naive T-lymphocytes (1). For the induction of proper immune responses, a direct contact between the dendritic cells (DC) and the T cell is necessary. Therefore, naive T cells form an immunological synapse with the APC where TCRs, MHC complexes, and costimulatory molecules get in close contact surrounded by a ring of adhesion molecules (2). The signals triggered inside the T cell by these interactions are influenced by three factors: 1) the duration of the cell-cell contact; 1) the number of MHC complexes that induce a signal; and 3) the number of costimulatory signals that enhance the message (3). The provision of costimulatory signals in the form of cytokines (such as IL-2 or IL-12) and membrane-bound ligands (such as CD80 (B7-1) and CD86 (B7-2)) by mDCs regulates T cell activation and differentiation (4, 5).

Among the many other surface molecules that are up-regulated during the maturation of DCs, one membrane-bound glycoprotein and member of the IgG superfamily is of extraordinary importance: CD83. This molecule is today’s best-known cell surface marker for fully mature human DCs that are able to induce immune responses (1). Although a preform of CD83 has been found inside monocytes, macrophages, and immature DCs (iDCs), the molecule is only stably expressed on mDCs (6) and some activated T cells and B cells (7). In the case of CD83, two different isoforms of this molecule have been reported in vivo to date: a membrane-bound form (mCD83) (8, 9, 10) and a soluble form (sCD83) (11, 12). The latter one is thought to be generated by proteolytic cleavage of the mCD83 (12). However, a precise mechanism for the generation of sCD83 has not yet been described.

Of utmost importance is the fact that several viruses use CD83 as a target to prevent the host’s immune response. The infection of iDCs with HSV-1 prevented the up-regulation of CD83 on the cell surface during maturation (13), while the infection of mDCs led to the down-modulation and intracellular degradation of mCD83 (14). In both cases, the loss of the membrane-bound isoform correlated with a significantly reduced capacity to stimulate T cells. For another member of the Herpesviridae family, the human CMV, Senechal et al. reported that the infection leads to a conversion of the mCD83 into the soluble form of CD83 (15). In this respect it is noteworthy that sCD83 has been described as impairing the activation of T cells by mDCs (15, 16, 17).

Furthermore, it has been shown that DC function can be modulated by interfering with the processing of the CD83 mRNA. A first hint came from the observation that the expression of CD83 goes hand in hand with the expression of eukaryotic initiation factor 5A (eIF-5A) (18), a protein involved in the export of cellular mRNAs from the nucleus to the cytoplasm (19). The expression level of eIF-5A is very low in iDCs but increases during DC maturation. For its biological function as a mRNA transporter, eIF-5A has to undergo posttranscriptional processing: i.e., hypusine modification. To date, eIF-5A is the only known cellular protein that contains the unusual amino acid hypusine (20). When this posttranscriptional modification is blocked by a low m.w. inhibitor (GC7), iDCs completely fail to up-regulate CD83 during maturation whereas other molecules like CD80 or CD86 are not influenced at all. The loss of CD83 surface expression is due to a block of CD83 mRNA export from the nucleus into the cytoplasm and is further correlated with the loss of T cell stimulatory capacity (18).

Thus, both forms of CD83 seem to have completely different functions; whereas sCD83 seems to act as an immunosuppressive molecule, the main function of which is to interfere with DC-mediated immune responses (12, 21, 22, 23), the membrane-associated CD83 plays an important role during the induction of cellular immune responses. To simulate the membrane-bound form of CD83, Scholler et al. fused the extracellular domain of CD83 to an Ig domain (i.e., CD83-Ig) and coimmobilized it with anti-CD3 Abs. Interestingly, only this coimmobilized protein induced strong proliferation of PBMCs while CD83-Ig alone failed to do so (17). Furthermore, the ratio of CD8+ to CD4+ T cells increased by factor 2.5, indicating a specific function of mCD83 in the induction of T lymphocyte differentiation and the enhancement of immune responses (17, 24). The identification of CD137 as a cofactor for increased antitumor immunity of CD83-expressing cells essentially confirmed this observation (25). However, CD83 seems not only to be important during T cell activation but is also of particular importance during T cell development in the thymus. Fujimoto et al. demonstrated by using CD83-deficient (CD83–/–) knockout mice that without CD83 significantly less CD4 single-positive T cells developed (26). The specific modulation of CD83 surface expression would therefore be a very useful tool for further investigating its role during the induction of immune responses and tolerance as well as during the prevention of autoimmune diseases and control of allergy.

A common and well established way to specifically control gene activity is the method of RNA interference (RNAi). RNAi by small interfering RNA (siRNA) is a mechanism for posttranscriptional gene silencing in which the steady-state level of a specific mRNA is specifically decreased (27, 28). siRNA duplexes of 21 nucleotides, which can be easily produced in large scale, represent an efficient way to induce sequence-specific gene silencing (29, 30). Modification of DC function by siRNA technology has been proven to be an important and efficient way to investigate DC’s biology (for examples, see Refs. 31, 32, 33).

In this study we describe for the first time an efficient method to knock down CD83 surface expression in monocyte-derived DCs by the use of siRNA. Using real-time PCR we identified one duplex of a group of five—all directed against the CD83 message—that led to the most significant reduction of the total level of cellular CD83 mRNA. Furthermore, we were able to show that the time point of siRNA application during DC differentiation and maturation is critical for an efficient knockdown and that the only reasonable target to knock down CD83 efficiently is the population of iDCs. Finally, we compared siRNA treated with untreated DCs in their capacity to activate T cells in a MLR as well as in their capacity to secrete cytokines. Our data provide evidence that CD83 has an enhancing effect on the T cell stimulatory capacity of mDCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Generation of DCs

PBMCs were isolated from a single healthy donor by sedimentation using Lymphoprep (Nycomed Pharma) and cultured in RPMI 1640 (BioWhittaker) supplemented with 1% autologous serum, 10 mM HEPES (pH 7.5) (Sigma-Aldrich), 2 mM L-glutamine (Cambrex), 100U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich). Mononuclear cells were seeded into standard tissue culture flasks for 1 h (Nunc). The nonadherent fraction was washed off after 1 h with pure RPMI 1640. iDCs were generated in RPMI 1640 supplemented with 1% autologous serum, 10 mM HEPES, 2 mM L-glutamine, 800U/ml GM-CSF (Wyeth), and 250U/ml IL-4 (Strathmann). The nonadherent cells (i.e., iDCs) were collected after 4 days of cultivation, counted, and transferred into new flasks. Maturation was induced by adding 10 ng/ml TNF-{alpha} (Strathmann), 1 µg/ml PGE2 (Sigma-Aldrich), 200 U/ml IL-1beta (Strathmann), 40U/ml GM-CSF, and 250U/ml IL-4 to the medium. Maturation was complete 2 days later.

To ensure the purity of the achieved mDCs, the cell population was analyzed by FACS for contamination with T cells (CD3 Ab; BD Biosciences), B cells (CD19 Ab, clone S725-C1; Caltag Laboratories), and NK cells (CD56 Ab, clone MEM-188; ImmunoTools) together with the respective isotype-controls IgG1 and IgG2a (clone G155-178; BD Biosciences). Normally, the preparation showed a purity of 85–90% for mDCs.

siRNA duplexes

All siRNA duplexes were obtained from MWG Biotec, dissolved in a siRNA suspension buffer (Qiagen) to a final concentration of 1 µg/µl, heated up for 1 min to 90°C, and incubated at 37°C for 60 min. Resolved duplexes were stored in aliquots at –80°C. For target sequences and sequences of the sense and antisense oligonucleotides, please see Table I.


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  		Table I. CD83 siRNA duplexes

 
Electroporation of DCs

DCs were harvested, washed once with pure RPMI 1640, and once with PBS (all at room temperature). The cells were resuspended in Opti-MEM without phenol red (Invitrogen Life Technologies) at a concentration of 4 x 107 cells/ml. Respective amounts of siRNA duplexes were transferred to a 4-mm cuvette (Peqlab Biotechnologie) and filled up to a final volume of 100 µl with Opti-MEM. One hundred microliters of cell suspension (containing 4 x 106 cells) was added and immediately pulsed in a Gene Pulser II apparatus (Bio-Rad). Pulse conditions were 300 V, 150 µF, and 100 ohms. Immediately after electroporation, the cells were transferred into an autologous medium supplemented with the previously indicated concentrations of GM-CSF and IL-4 or, in case of maturation, transferred to a medium containing the maturation mixture.

RNA isolation and reverse transcription

Cells were harvested and washed with PBS. Total RNA was isolated using the RNeasy mini kit and QIAshredder spin columns (Qiagen). Traces of genomic DNA were removed by DNase digestion with the RNase-free DNase set (Qiagen). Subsequently, 1 µg of each RNA was reverse transcribed into a single-stranded cDNA by using avian myeloblastosis virus reverse transcriptase as specified by the manufacturer (Promega).

Real-time PCR and relative quantification

Real time PCR using the LightCycler system (Roche Diagnostics) was performed in glass capillaries at a total volume of 20 µl in the presence of 2 µl of 10x reaction buffer (Taq polymerase, dNTPs, MgCl2, and SYBR Green; Roche Diagnostics), and 1 µl of cDNA. Oligonucleotide primers (11.25 pmol of each) were added. Real time PCR was performed with an initial denaturation step at 95°C for 15 min followed by 50 cycles of denaturation at 94°C for 20 s, annealing at 57°C for 60 s, and 30 s of elongation time at 72°C. At the end of each cycle, the fluorescence emitted by SYBR Green was measured. After completion of the cycling process, samples were subjected to a temperature ramp (from 65 to 95°C at 0.1°C/sec) with continuous fluorescence monitoring for melting curve analysis. The primers used were as follows: CD80 (sense, 5'-GGACATGAATATATGGCCCG-3'; antisense, 5'-CAACACACTCGTATGTGCCC-3'; as described previously in Ref. 4), CD83 (sense, 5'-CCTCCAGCTTCTGCTCCTGA-3'; antisense, 5'-TCGGAGCAAGCCACCTTCAC-3'), and CD86 (sense, 5'-TTGCAAACTCTCAAAACCAA-3'; antisense, 5'-GGAATGAACACTGTCAAATT-3'). As a reference primer, beta-glucuronidase (sense, 5'-CTCATTTGGAATTTTGCCGATT-3'; antisense, 5'-CCGAGTGAAGATCCCCTTTTTA-3'; as described previously in Ref. 34) was used. Relative quantification was performed by using LightCycler software according to the manufacturer’s instructions.

Western blot analysis

DCs (4 x 106) were lysed in 150 µl of lysis buffer (10% glycerol, 2 mM EDTA (pH 8.0), 0.5% Nonidet P-40, 137 mM NaCl, and 50 mM Tris-HCl (pH 8.0)), separated on 12% SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane. The following primary Abs were used for Western blot analysis: anti-CD83 mAb (clone 1G11; 1/500) (35) and anti-beta-actin mAb (clone AC-74, Sigma-Aldrich; 1/1000). After incubation with the appropriate secondary HRP-labeled Ab, detection was performed using an ECL Western blotting substrate (Pierce).

FACS analysis

The cell surface phenotype was analyzed by FACS. The following mAbs were used: CD40 (clone 5C3), CD80 (clone L307.4), CD83 (clone HB15e), CD86 (clone 2331(FUN-1)), B7-H1 (clone M1H1), B7-H2 (clone 2D3/B7-H2), and MHC-class II (clone G46-6(L243)) together with the respective isotype controls IgG1 (clone MOPC-21), IgG2a (clone G155-178), or IgG2b (clone 27-35) (all obtained from BD Biosciences and used according to the manufacturer’s instructions). For intracellular CD83 staining, a FITC-labeled CD83 (clone HB15e) and a FITC-labeled IgG1 (clone MOPC-21) isotype control (BD Biosciences) were used according to the manufacturer’s instructions. Simultaneous intracellular and extracellular staining was performed with the BD Cytofix/Cytoperm kit (BD Biosciences) as described by the manufacturer.

Allogeneic MLR

T cells (2 x 105 per well) and DCs were cocultivated for 4 days in 200 µl of RPMI 1640 supplemented with 5% human serum from a single AB donor in 96-well cell culture dishes. Then, cells were pulsed with [3H]thymidine (1µCi/well; Amersham Biosciences) for 24 h. The culture supernatants were harvested onto glass fiber filtermates using an IH-110 harvester (Inotech) and filters were counted in a 1450 microplate counter (Wallac).

Cytometric bead array (cytokine profiling)

Relative cytokine protein expression was determined using a cytometric bead array system according to manufacturer’s instructions (human inflammation kit and Th1/Th2 kit, BD Biosciences). Cytokine expression of T cells was determined by directly removing 50 µl of the supernatant from the MLR experiments as described above. Afterward the supernatant was added to the reaction mix containing Ab-coated microbeads and incubated at room temperature for 3 h. After intensive washing, the mean fluorescence intensity (MFI) for each cytokine was determined by FACS analyses. To calculate relative MFI (rMFI), the MFI of untreated DCs was set as 100% and electroporated cells were set in relationship to this population.

Statistical methods

If not indicated otherwise, results are shown as mean ± SD. To determine the significance of variance in the experiments, data were analyzed using Student’s t test. Significance was accepted if p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Identification of a CD83 targeted siRNA duplex that shows specific and efficient knockdown of CD83 mRNA

To knock down a cellular target gene specifically and efficiently, a functional siRNA duplex—directed against the mRNA of the respective protein—had to be identified (27, 28, 29, 30). To find a siRNA duplex that is able to reduce the level of CD83 mRNA and thereby reduces the total amount of the CD83 protein, five different duplexes were synthesized (for target sequences and the sequences of the duplexes see Table I).

iDCs were electroporated with 5.0 µg of the respective siRNA duplex and incubated for 4 h in DC medium. Afterward they were matured for 24 h and finally the total RNA was isolated, digested with DNase I, and reverse-transcribed into cDNA. RT-PCR for CD83 was performed using a LightCylcer system (Roche) with beta-glucuronidase mRNA as internal control. The normalized ratio of the CD83 mRNA compared with the beta-glucuronidase internal control was calculated using LightCycler software. Fig. 1A demonstrates the knockdown efficiency of the different CD83 duplexes. The normalized ratio of untreated cells was set as 100% (Fig. 1A, column 1) and compared with the normalized ratio of cells that were either electroporated without siRNA (Fig. 1A, column 2) or together with 5.0 µg of the respective siRNA duplex (Fig. 1A, columns 3–7). A significant reduction of the total amount of CD83 mRNA to 50–60% was only detectable in case of duplex no. 1 and duplex no.5 (p < 0.01, marked by asterisks; Fig. 1A, columns 3 and 7). Duplexes no. 2, no. 3, and no. 4 (columns 4–6) did not lead to a significant effect on CD83 mRNA. An influence of the electroporation procedure on the expression of CD83 mRNA could be excluded by a mock electroporation experiment in which no siRNA duplex was delivered into the cells at all (Fig. 1A, column 2).


Figure 1
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  		FIGURE 1. Identification of an efficient and specific siRNA duplex targeted against CD83. A, iDCs were electroporated with 5.0 µg of different CD83-siRNA duplexes and maturation was induced 4 h after electroporation. After 24 h of maturation time, total RNA was isolated and reverse transcribed and real-time PCRs were performed with CD83 mRNA as the target and beta-glucuronidase as an internal control. The normalized ratio of CD83 to beta-glucuronidase in untreated cells was set to 100% (column 1). iDCs electroporated without any siRNA demonstrated no significant down-regulation of the CD83 messenger (column 2). A significant effect (*, p < 0.01) on CD83 mRNA was detected for duplex no.1 and no.5 (columns 3 and 7). The specific mRNA level was reduced up to 50%. The data represent the mean ± SD of three independent experiments. Changes were considered as significant if p < 0.01. B, Additional real-time PCRs were performed to quantify the mRNA amounts of the typical DC surface markers CD80 and CD86. The normalized ratios of iDCs electroporated without any siRNA were set to 100% and compared with cells that were electroporated with 7.5 µg of duplex no.1. Only in the case of CD83 mRNA was a significant down-regulation observed (column 2). Neither CD80 (column 3) nor CD86 (column 4) were influenced by the siRNA duplex, demonstrating the specificity of the knockdown. The data represent the mean ± SD of three independent experiments. Changes were considered as significant if p < 0.01.

 
To prove whether a combination of duplexes no. 1 and no. 5 would increase the mRNA knockdown, we performed additional electroporation experiments. This time 5.0 µg of each duplex was electroporated simultaneously and subsequent processing was essentially performed as described above. However, we did not detect an increase in CD83 mRNA down-regulation (data not shown). This observation might be explained by a saturation of the RNAi machinery that is able to process only certain amounts of siRNA molecules (discussed, for example, in Ref. 36).

To prove the specificity of the siRNA-mediated gene silencing, additional real-time PCRs were performed for typical DC surface markers. iDCs were electroporated with 7.5 µg of duplex no.1 and treated as described above. Fig. 1B shows the relative expression levels of CD83 (column 2), CD80 (column 3), and CD86 (column 4) compared with DCs electroporated without siRNA that were set as 100% (column 1). Although in the case of CD83 mRNA a strong and significant (p < 0.01) down-regulation could be observed (Fig. 1B, column 2), the mRNA levels for CD80 (Fig. 1B, column 3) and CD86 (Fig. 1B, column 4) were not influenced at all (p = 0.59 in the case of CD80; p = 0.71 in the case of CD86). Therefore, we conclude that duplex no.1 represents a suitable duplex for siRNA-mediated silencing of CD83 in DCs (please note that from this point duplex no.1 is also referred to as "CD83 siRNA").

CD83 is stable over a long time and its expression is rapidly induced upon maturation

The efficiency of siRNA mediated gene knockdown is strictly dependent on the stability of the target protein. Although efficient silencing has been achieved on the mRNA level, cells might still contain large amounts of the respective protein because of its high stability. To evaluate the stability of CD83 on the surface of fully matured DCs, we used cycloheximide (CHX) to block the intracellular supply of newly synthesized proteins. CHX has been described as a powerful drug for inhibiting peptide synthesis (37).

Mature DCs were either incubated for 24, 48, or 72 h in the absence (Fig. 2A, open symbols) or presence (Fig. 2A, filled symbols) of 10 µg/ml CHX and analyzed by FACS staining (please note that we gated on living cells) for extracellular CD83 (panel A1), CD80 (panel A2), CD86 (panel A3), and HLA-DR (MHC-II; panel A4) expression afterward. Fig. 2A shows a time course of the cells that were positive for the respective surface marker. Although CD80, CD86, and HLA-DR are almost not affected by the CHX treatment (Fig. 2, panels A2–A4), CD83 surface expression is slightly influenced by the inhibitor (Fig. 2, panel A1). In this case, treatment with CHX led to a loss of surface-bound CD83 of ~10–20%. However, cells still showed high amounts of CD83 on their surface, indicating that CD83 is very stable in its membrane-bound form. To determine the effect of CHX treatment, additional FACS stainings for annexin V (Fig. 2, panel B3) and propidium iodide (PI; Fig. 2, panel B2) were performed. Furthermore, we provide data regarding the viability of the cells by using trypan blue staining (Fig. 2, panel B1). Therefore, the percentage of trypan blue negative cells was considered as viable cells. The number of living cells was reduced over time from ~95 to 20% (Fig. 2, panel B1, line with filled squares) and the CHX treatment increased the number of PI-positive cells from 3 to 20% (Fig. 2, panel B2, line with filled squares) and the number of annexin V-positive cells from 20% to 60% (Fig. 2, panel B3, line with filled squares). However, although the number of dying cells strongly increased due to the treatment with CHX, these cells still show high levels of CD83 surface expression (Fig. 2, panel A1, line with filled circles). These data proved the high stability of the CD83 molecule and underlined the necessity of using DCs that do not yet express large amounts of CD83 for knockout experiments. Thus, iDCs represent the most appropriate target for achieving an efficient knockdown of this protein.


Figure 2
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  		FIGURE 2. CD83 is stable over a long time and its expression is rapidly induced upon maturation. A, Mature DCs were incubated for 24, 48, or 72 h in the absence (open symbols) or presence (filled symbols) of 10 µg/ml CHX. The percentage of cells positive for CD83 (panel 1), CD80 (panel 2), CD86 (panel 3), and HLA-DR (panel 4) was determined by FACS (cells were gated on living cells) analyses at the indicated time points. Although CD80, CD86, and HLA-DR show almost constant surface expression regardless of whether cells were incubated with CHX or not, CD83 surface expression was slightly influenced by CHX treatment. After 24 h, ~98% of the untreated cells were CD83 positive. The treatment with CHX reduced the CD83 surface expression levels to ~80%. After 72 h, both untreated and CHX-treated cells showed equal expression of ~80%. These data represent the mean ± SD of three independent experiments with cells from different donors. However, as shown in panel 1 CD83 expression was only slightly altered, indicating the high stability of CD83. B, Cell viability was determined using trypan blue, PI, and annexin V staining. The number of living cells (i.e., the percentage of trypan blue-negative cells) was reduced over time. The CHX treatment also increased the number of PI-positive cells (panel 2, line with filled squares) and the number of annexin V-positive cells (panel 3, line with filled squares). The data represent the mean ± SD of three independent experiments with cells from different donors. C, Immature DCs were simultaneously analyzed for intracellular and extracellular CD83 expression in the absence or presence of maturation stimuli. Freshly prepared iDCs (topmost panel) do not express large amounts of CD83, as only 2% of the population was double positive for intracellular and extracellular CD83. The induction of maturation (right column) led to rapid expression of both intracellular and extracellular CD83: 89% of the cells were double positive after 6 h and 98% after 24 h. Although no maturation stimuli were present in the medium, iDCs accumulated intracellular CD83, probably due to the mechanical treatment/stress. After 24 h, 96% of the cells express intracellular CD83 (left column, lower panel, upper and lower right quadrants; representing fluorescence channel 1). The data are representative of three independent experiments with cells from different donors.

 
To address this point, we next tried to identify the population of DCs that bears the lowest amounts of both intracellular and extracellular CD83. Freshly prepared iDCs (day 5) were removed from the cell culture flasks by soft shaking and centrifugation. For simultaneous intracellular and extracellular staining, first the extracellular CD83 was stained with a PE-labeled Ab. After fixation and permeabilization, the intracellular CD83 was detected using a FITC-labeled Ab that recognized the same epitope as the first Ab. In Fig. 2C (topmost panel) the intracellular and extracellular levels of CD83 are shown. Only 2% of the cells are double positive for intracellular and extracellular CD83. This result clearly demonstrated that iDCs contain almost no CD83.

Next, we transferred the iDCs to either a DC medium without maturation mixture (Fig. 2C, left column) or a DC medium containing the maturation stimuli (Fig. 2C, right column). After 6 h of incubation the cells were removed and again stained for both intracellular and extracellular CD83. When no maturation stimuli were present, only 5% of the cells were double positive for intracellular and extracellular CD83 (Fig. 2C, left column, upper panel). In strong contrast, when the maturation mixture was present for 6 h 89% of the cells became positive for CD83 (Fig. 2C, right column, upper panel). A final determination of CD83 after 24 h revealed an interesting phenomenon: although maturation was not induced, 25% of the cells became double positive (Fig. 2C, left column, lower panel). Thus, it seems that the soft mechanical treatment of the iDCs during transfer from the culture flasks to the DC medium in 6-well plates led to an intracellular expression/accumulation of CD83 as indicated by 96% of the cells being positive in fluorescence channel 1 (Fig. 2C, left column, lower panel, upper right quadrant 25% and lower right quadrant 71%). In fact, already 6 h after transfer of the cells a slight accumulation of intracellular CD83 (10%) was detectable (Fig. 2C, left column, upper panel, upper right quadrant 5% and lower right quadrant 5%) However, this CD83 was not completely transported to the cell surface as indicated by the low percentage of cells positive for fluorescence 2 (Fig. 2C, left column, lower panel, upper left quadrant 0% and upper right quadrant 25%). In strong contrast, the presence of maturation stimuli led to 98% double-positive cells (Fig. 2C, right column, lower panel).

Taken together, these data showed that freshly prepared iDCs contained almost no CD83 and therefore represented the most favorable target for CD83 knockdown. Furthermore, we were able to show that physical treatment of the cells induced intracellular expression/accumulation of CD83 and should therefore be avoided or at least strongly reduced to obtain low expressing iDCs.

The achieved CD83 knockdown is efficient, specific, and long lasting

As the optimal time point for electroporation and a functional duplex was identified, we wanted to obtain an insight on the protein level, i.e., the total level of cellular CD83 as well as the CD83 surface expression. Therefore, we electroporated freshly prepared iDCs (tested by FACS for low levels of both intracellular and extracellular CD83; data not shown) with 7.5 µg of siRNA duplex no.1. After 4 h, maturation was induced by adding the appropriate stimuli and then the DCs were allowed to mature for 48 h.

After that time, DCs were harvested, lysed and total cellular protein extracts were separated on a 12% SDS-polyacrylamide gel. Proteins were transferred to a nylon membrane and the total amount of CD83 was detected using an anti-CD83 Ab (clone 1G11). Afterward, the membranes were stripped and reprobed with an anti-beta-actin Ab. Although untreated DCs (Fig. 3A, lane 1) and mock electroporated DCs (Fig. 3A, lane 2) showed almost equal levels of highly glycosylated CD83 (35), the electroporated cells of CD83 siRNA duplex no.1 (Fig. 3A, lane 3) showed dramatically reduced levels of CD83.


Figure 3
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  		FIGURE 3. CD83 knockdown is also specific and long lasting at the protein level. A, Western blot analyses. CD83 siRNA electroporated DCs showed dramatically reduced CD83 levels when compared with untreated and mock electroporated (EP) cells. After 48 h, the total cellular protein extract of electroporated DCs was separated on a 12% SDS-polyacrylamide gel. CD83 was detected using an anti-CD83 Ab (clone 1G11). Afterward, the membranes were stripped and reprobed with an anti-beta-actin Ab. Although untreated DCs (lane 1) and mock electroporated DCs (lane 2) showed almost equal expression levels of highly glycosylated CD83 (35 ), the CD83 siRNA duplex no.1 electroporated cells (lane 3) showed dramatically reduced levels of CD83. The data are representative of two independent experiments with cells from different donors. B–D, FACS analyses of the surface expression of CD83 (B), CD80 (C), and CD86 (D). Immature DCs were either left untreated (B–D, left columns), mock electroporated (EP) (B–D; middle column), or electroporated with 7.5 µg of CD83 siRNA duplex no.1 (B–D; right columns). Although the surface expression of CD80 and CD86 was not influenced at any condition, CD83 was efficiently knocked down from the cell’s surface (B, left column, untreated cells, 81–85%; middle column, mock-electroporated cells, 80–86%; right column, CD83 duplex no.1 electroporated cells, 20–35%). The silencing of CD83 had also a long lasting effect, because after 72 h only 29% of the cells still expressed CD83 on their surface (B, right column, lower panel). The data are representative for five independent experiments with cells from different donors. E, Influence of CD83 siRNA electroporation on other surface markers. Immature DCs were either left untreated (upper row), mock electroporated (EP) (middle row), or electroporated with 7.5 µg of CD83 siRNA duplex no.1 (bottom row). Forty-eight hours after electroporation, CD40 (left column), B7-H1 (second column from left), B7-H2 (third column from the left), and HLA-DR (MHC-II; right column) were not significantly influenced in their expression. These data are representative for three independent experiments with cells from different donors.

 
Cells from populations that were not treated at all (Fig. 3, B–D, left columns), cells that were mock electroporated (Fig. 3B, middle column), and cells that were electroporated with duplex no.1 (Fig. 3B, right column) were analyzed for surface expression of CD83 (Fig. 3B), CD80 (Fig. 3C), and CD86 (Fig. 3D) after 24, 48, and 72 h. No significant differences in surface expression of CD80 and CD86 were detected at any given condition. The cells always showed high expression of these very important (4, 38) costimulatory molecules, typical for mDCs. In strong contrast, populations that were siRNA electroporated before maturation (Fig. 3B, right column) showed dramatically reduced numbers of CD83-positive cells compared with untreated and mock electroporated cells. After 48 h (the time point at which DCs are usually considered mature), only 20% were still positive for CD83 (Fig. 3B, right column, middle panel), while 81% of the untreated (Fig. 3B, left column, middle panel) and 80% of the mock electroporated population (Fig. 3B, middle column, middle panel) were positive for CD83. It is noteworthy that even 72 h after the induction of maturation only 29% of the duplex no.1 electroporated cells were positive for CD83 (Fig. 3B, right column, lower panel). This result clearly demonstrated that the achieved knockdown was specific for the CD83 protein and also had an efficient long-lasting effect.

To exclude an influence of CD83 siRNA on other typical surface markers, DCs were either left untreated (Fig. 3E, upper row), mock-electroporated (Fig. 3E, middle row), or electroporated with 7.5 µg of CD83 siRNA (Fig. 3E, bottom row) and, after 4 h, maturation was allowed to take place for 48 h. As already shown for CD80 and CD86 (see Fig. 3, C and D), CD40 (Fig. 3E, left column), B7-H1 (Fig. 3E, second column from left), B7-H2 (Fig. 3E, third column from left), and HLA-DR (Fig. 3E, right column) were not significantly influenced in their surface expression. This data further enhanced our previous observation that duplex no.1 did not produce unwanted off-target effects.

CD83 siRNA electroporated mature DCs show reduced capacity to stimulate allogeneic T cells

To test whether CD83 siRNA electroporation influenced DC-mediated T cell proliferation, their stimulatory capacity was tested in an allogeneic MLR. iDCs were electroporated, incubated for 4 h in DC medium, and afterward matured for 24 h. The mDCs were then seeded together with T cells for 72 h, pulsed with [3H]thymidine, harvested 24 h later, and the filters were then counted.

Cells that were electroporated with CD83 siRNA (Fig. 4, open squares) showed dramatically reduced capacity to stimulate T cell proliferation when compared with cells that were mock electroporated (Fig. 4, filled squares) and cells that were left untreated (Fig. 4, filled inverted triangle). This observation provided evidence that membrane-bound CD83 had the function of an enhancer during DC mediated T cell proliferation and was critical for effective T cell stimulation.


Figure 4
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  		FIGURE 4. CD83 siRNA electroporated mature DCs show reduced capacity to stimulate allogenic T cells. Immature DCs were electroporated with 7.5 µg of CD83 siRNA duplex no.1, incubated for 4 h, and then matured for 24 h and finally seeded together with allogeneic T cells for 72 h. Cells were then pulsed with [3H]thymidine for 24 h and harvested and the filters were counted. Cells that were electroporated with the CD83 siRNA ({square}) showed a dramatically reduced capacity to induce T cell proliferation when compared with cells that were mock electroporated ({blacksquare}) or left untreated ({blacktriangledown}). These results are representative of three independent experiments with cells from different donors.

 
T cells cocultured with CD83 siRNA electroporated DCs show significant changes in their cytokine expression profiles

A functional consequence of CD83 knockdown in DCs—apart from the reduced capability to induce T cell proliferation as shown above by MLR—might be a change in the cytokine expression profile.

To investigate this possibility, cytokine bead array assays were performed to determine the cytokine secretion profile of T cells that were stimulated with siRNA electroporated mDCs. Therefore, experiments were performed essentially as described above for the MLR assays. After 72 h of cocultivation, supernatants were removed and cytokine expression was determined. The interaction of fully matured, monocyte-derived DCs with T cells normally induces the expression of several cytokines such as IFN-{gamma}, TNF, IL-2, IL-4, IL-5, and IL-10 (3, 39, 40).

Fig. 5 shows the rMFI (see Material and Methods for details) of IFN-{gamma} (Fig. 5A), IL-1beta (Fig. 5B), IL-2 (Fig. 5C), IL-4 (Fig. 5D), IL-5 (Fig. 5E), IL-6 (Fig. 5F), IL-10 (Fig. 5G), and TNF-{alpha} (Fig. 5H). The MFI of untreated cells was set as 100%. A significant lower expression of each cytokine compared with mock electroporated cells was detectable for CD83 siRNA duplex no.1 electroporated cells (Fig. 5, right column in each graph). In strong contrast, the cytokine expression profile of the mock-electroporated cells (Fig. 5, middle column in each graph) was almost equal when compared with those of the untreated cells (Fig. 5, left column in each graph). The most significant cases of down-regulation were detected for IL-6 (Fig. 5F) and TNF-{alpha} (Fig. 5H). The expression of IL-12p70 was not detected. This fits to observations from another study, demonstrating that IL-12p70 is only expressed when DCs were matured together with CD40L (41).


Figure 5
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  		FIGURE 5. Coculture of T cells and CD83 siRNA electroporated DCs leads to strongly reduced cytokine expression. Shown are rMFI values of the following cytokines: IFN-{gamma} (A), IL-1beta (B), IL-2 (C), IL-4 (D), IL-5 (E), IL-6 (F), IL-10 (G), and TNF-{alpha} (H). A cytokine expression profile of T cells that were cocultivated with DCs is shown in each panel. Untreated (left column in each graph), mock-electroporated (middle column in each graph), and CD83 siRNA treated DCs (right column in each graph) were incubated with T cells essentially as described above for the MLR assays. After 72 h of cocultivation supernatants were removed and cytokine expression levels were determined by a cytokine bead array assay. A significant lower expression of each cytokine was detectable in the presence of CD83 siRNA duplex no.1 electroporated cells, while mock electroporated cells (middle column in each graph) as well as untreated cells (left column in each graph) induced similar expression levels. The data represents the mean ± SD of three independent experiments with cells from different donors. The p values were calculated using Student’s t test.

 
These data clearly demonstrate that the CD83 knockdown had a dramatic influence on the cytokine expression profiles of cocultured T cells. This strongly supported our observation from the MLR experiments and supported the hypothesis that CD83 enhanced the stimulation and acted as a costimulatory molecule.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Maturation is the critical step for DCs to become potent APCs and to be able to activate naive T cells. During this maturation process, specific surface molecules such as CD83 or MHC class II are strongly up-regulated together with costimulatory molecules such as CD80 and CD86. The inhibition of an entire DC maturation leads to tolerance induction and suppression of unwanted immune responses (1, 42). Several studies describe phenomena that interfere with the maturation of DCs. When the specific CRM-1 nucleocytoplasmic mRNA export pathway is blocked by a low m.w. inhibitor (GC7), iDCs do not achieve their mature state because CD83 is not expressed on the cell surface. This is due to an accumulation of the CD83 mRNA inside the nucleus and leads in consequence to a reduction of the ability of DCs to induce allogenic T cell proliferation (18). A similar observation on DCs in vitro and in vivo has been described by Zinser et al. (43). The addition of CNI-1493 (also an inhibitor of the CRM1 export pathway; Ref. 44) reduces the expression of CD83 during maturation and DC-mediated T cell stimulation (43). It additionally reduces the clinical symptoms of an experimental autoimmune encephalomyelitis model when applied in an early therapeutic setting (43).

Further evidence of the importance of CD83 during T cell stimulation came from observations with viruses of the Herpesviridae family: after infection with HSV-1 CD83 is specifically down-regulated from cell surface and, in consequence, the mDCs are no longer able to induce proper T cell responses (14). The infection of mDCs with the human CMV leads to the shedding of CD83 from the surface, although the intracellular expression of the protein remained constant. The resulting sCD83 accumulated in the supernatant of the cells and was also able to interfere with the T cell-activating properties of DCs (15). However, all of the influences on CD83 surface expression mentioned above might not be completely free of other side effects. When blocking the intracellular mRNA transport by GC7 or CNI-1493, it cannot be ruled out that these drugs may also affect the metabolism of additional mRNAs, leading to impairment with the T cell stimulatory capacity. Viruses (esp. HSV-1) have been shown to act on several stages to evade the immune system. HSV-1 for example is able to block the maturation of DCs (13) and prevents mature DCs from migrating to the areas of T cell activation, i.e., into the secondary lymphoid organs (45).

In this study, we describe for the first time an efficient method for preventing the proper up-regulation of CD83 by the use of siRNA electroporation into iDCs without interfering with other molecules. In a recent article we showed that the delivery of siRNA by electroporation does not influence the biology of DCs and is therefore a suitable method for modulating DCs without the use of chemical transfection reagents (31). Specific knockdown of a gene without directly influencing any other mRNA is a typical and outstanding attribute of siRNA and has been intensively investigated in detail by numerous researchers (for examples, see Refs28 , 46 , and 47). However, in this respect some interesting observations have to be considered: Cella and colleagues presented results showing that iDCs could be activated by double-stranded RNA (48). Furthermore, recently two other groups provided evidence that under certain circumstances siRNAs are able to initiate DC maturation (49, 50). This is not a general effect of siRNA but is dependent on the sequence of the duplex and has to be considered as an additional biological activity of siRNA. For this reason, they termed this immunostimulatory function immunostimulatory siRNA (isRNA) (49). During our evaluation of potential siRNA duplexes, we found an increase in CD83 mRNA levels after application of duplex no.4 (Fig. 1A). This observation might be explained by RNA stabilization effects that are initiated after the activation of immune cells by an isRNA/TLR interaction and counteract the intended knockdown (discussed in Ref. 51). However, we were not able to detect any (stimulatory) effects either on iDCs that were incubated with large amounts of duplex no.4 or on iDCs that were matured in the presence of this siRNA (data not shown). Although target recognition of siRNA is usually highly specific (52), under certain circumstances siRNA may target unrelated genes with only partial sequence complementarity (off-target effects) (53). Hence, it is important to carefully evaluate the possibility of unwanted side effects to perform reliable gene knockdown experiments. In this respect we performed additional experiments to identify possible effects on essential surface molecules (see Figs. 1B and 3, B–E). However, apart from the desired CD83 down-modulation, we did not observe any negative side effects.

Further, we were able to show by simultaneous intracellular and extracellular FACS staining that freshly prepared immature DCs are the most suitable targets for the application of siRNA molecules directed against CD83 because they do not express large amounts of CD83 (see Fig. 2C). This observation is in contrast to data reported by Klein et al (54). In their experiments they showed by Western blot analyses that iDCs already contain a pool of preformed CD83 and speculated that upon maturation the CD83 is transported from this intracellular reservoir to the surface, where it is expressed fully glycosylated as the membrane-bound form. However, from our data we conclude that probably mechanical stress during the treatment of the immature DCs is most likely the reason for intracellular accumulation.

Little is known about the exact function of human CD83; whether a corresponding ligand for this molecule exists on DCs and/or T cells is still controversial (16, 17). Nevertheless, CD83 is always put into the spotlight when trying to identify factors that induce or promote the generation of T cell immunity; recently Hirano et al. reported that a CD83 binding molecule is induced in both CD4+ and CD8+ human T cells by CD28-mediated costimulation (55). Furthermore, they were able to show that CD83 delivers a signal that enhances the in vitro generation of CTLs (55). Data supporting the observation made by Fujimoto et al. (26) came from Garcia-Martinez et al. who found that CD4+ T cell development is substantially reduced in mice that bear a mutated form of the CD83 gene (56).

The aim of our study was to gain further information of the function of surface-associated CD83 during the activation of T cell proliferation. A population of mature DCs with a CD83 knockdown showed a significant reduction in the ability to prime naive allogenic T cells (see Fig. 4). It is noteworthy that our observation fits perfectly to data achieved by other studies. Zinser et al. (43) as well as Kruse et al. (18) were able to block CD83 surface expression and, in consequence, found a reduced capability of DCs to activate T cells that correlated very well with our results. However, in contrast to these studies we were able to reduce specifically CD83 surface expression without interfering with other molecules.

Dudziak et al. (22) recently reported that during the development of DCs different CD83 proteins are produced by alternative splicing. The longest form of these transcripts codes for the membrane-bound form, whereas the smaller transcripts are splice variants of full-length CD83 coding for putative sCD83 proteins (22). This might be a first hint that sCD83 is not necessarily produced exclusively by the shedding of mCD83 (12). However, they also found that the stimulation of PBMCs leads to a strong up-regulation of the full-length CD83 transcript and to a strong down-regulation of two of the three smaller transcripts. In iDCs almost none of the smaller transcripts were found. Hence, we are convinced that the membrane-bound form represents the optimal target for the further investigation of CD83 function during T cell stimulation.

Under physiological conditions the interaction of fully matured DCs and naive T cells leads to the establishment of a so-called immunological synapse (57, 58, 59). During this cell-cell contact, among other changes, specific signals induce the production of cytokines by T cells (39, 40, 60). In our studies we were able to show that the interaction of T cells with CD83-specific siRNA electroporated DCs led to strongly reduced cytokine production compared with mock electroporated DCs (Fig. 5).

Taken together, our data provide evidence that the membrane-bound form of CD83 acts as an enhancer/costimulator during the DC:T cell interaction.


   Acknowledgment
 
We thank Dr. Kerstin Zander for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the "ELAN-Fonds" Grant 04.08.08.2 of the University Hospital Erlangen and by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 643, Grant A4. A.A.T. was supported by Research Training Grant GK592 from the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Alexander T. Prechtel, Department of Dermatology, University Hospital Erlangen, Hartmannstrasse 14, Erlangen, Germany. E-mail address: alexander.prechtel{at}derma.imed.uni-erlangen.de Back

3 Abbreviations used in this paper: mDC, mature dendritic cell; DC, monocyte-derived dendritic cell; iDC, immature DC; sCD83, soluble CD83; mCD83, membrane-bound CD83; CHX, cycloheximide; eIF, eukaryotic initiation factor; MFI, mean fluorescence intensity; rMFI, relative MFI; PI, propidium iodide; RNAi, RNA interference; isRNA, immunostimulatory RNA; siRNA, small interfering RNA. Back

Received for publication March 14, 2006. Accepted for publication February 9, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245-252. [Medline]
  2. Dustin, M. L., J. A. Cooper. 2000. The immunological synapse and the actin cytoskeleton: molecular hardware for T cell signaling. Nat. Immunol. 1: 23-29. [Medline]
  3. Lanzavecchia, A., F. Sallusto. 2001. Regulation of T cell immunity by dendritic cells. Cell 106: 263-266. [Medline]
  4. Dilioglou, S., J. M. Cruse, R. E. Lewis. 2003. Function of CD80 and CD86 on monocyte- and stem cell-derived dendritic cells. Exp. Mol. Pathol. 75: 217-227. [Medline]
  5. Pardoll, D. M.. 2002. Spinning molecular immunology into successful immunotherapy. Nat Rev. Immunol. 2: 227-238. [Medline]
  6. Cao, W., S. H. Lee, J. Lu. 2005. CD83 is preformed inside monocytes, macrophages and dendritic cells, but it is only stably expressed on activated dendritic cells. Biochem. J. 385: 85-93. [Medline]
  7. Wolenski, M., S. O. Cramer, S. Ehrlich, C. Steeg, B. Fleischer, A. von Bonin. 2003. Enhanced activation of CD83-positive T cells. Scand. J. Immunol. 58: 306-311. [Medline]
  8. Zhou, L. J., R. Schwarting, H. M. Smith, T. F. Tedder. 1992. A novel cell-surface molecule expressed by human interdigitating reticulum cells, Langerhans cells, and activated lymphocytes is a new member of the Ig superfamily. J. Immunol. 149: 735-742. [Abstract]
  9. Zhou, L. J., T. F. Tedder. 1995. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily. J. Immunol. 154: 3821-3835. [Abstract]
  10. Zhou, L. J., T. F. Tedder. 1996. CD14+ blood monocytes can differentiate into functionally mature CD83+ dendritic cells. Proc. Natl. Acad. Sci. USA 93: 2588-2592. [Abstract/Free Full Text]
  11. Hock, B. D., M. Kato, J. L. McKenzie, D. N. J. Hart. 2001. A soluble form of CD83 is released from activated dendritic cells and B lymphocytes, and is detectable in normal human sera. Int. Immunol. 13: 959-967. [Abstract/Free Full Text]
  12. Hock, B. D., L. F. Haring, A. Steinkasserer, K. G. Taylor, W. N. Patton, J. L. McKenzie. 2004. The soluble form of CD83 is present at elevated levels in a number of hematological malignancies. Leuk. Res. 28: 237-241. [Medline]
  13. Salio, M., M. Cella, M. Suter, A. Lanzavecchia. 1999. Inhibition of dendritic cell maturation by herpes simplex virus. Eur. J. Immunol. 29: 3245-3253. [Medline]
  14. Kruse, M., O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G. Schuler, J. Hauber, A. Steinkasserer. 2000. Mature dendritic cells infected with herpes simplex virus type 1 exhibit inhibited T-cell stimulatory capacity. J. Virol. 74: 7127-7136. [Abstract/Free Full Text]
  15. Senechal, B., A. M. Boruchov, J. L. Reagan, D. N. Hart, J. W. Young. 2004. Infection of mature monocyte-derived dendritic cells with human cytomegalovirus inhibits stimulation of T-cell proliferation via the release of soluble CD83. Blood 103: 4207-4215. [Abstract/Free Full Text]
  16. Lechmann, M., D. J. Krooshoop, D. Dudziak, E. Kremmer, C. Kuhnt, C. G. Figdor, G. Schuler, A. Steinkasserer. 2001. The extracellular domain of CD83 inhibits dendritic cell-mediated T cell stimulation and binds to a ligand on dendritic cells. J. Exp. Med. 194: 1813-1821. [Abstract/Free Full Text]
  17. Scholler, N., M. Hayden-Ledbetter, A. Dahlin, I. Hellstrom, K. E. Hellstrom, J. A. Ledbetter. 2002. Cutting edge: CD83 regulates the development of cellular immunity. J. Immunol. 168: 2599-2602. [Abstract/Free Full Text]
  18. Kruse, M., O. Rosorius, F. Kratzer, D. Bevec, C. Kuhnt, A. Steinkasserer, G. Schuler, J. Hauber. 2000. Inhibition of CD83 cell surface expression during dendritic cell maturation by interference with nuclear export of CD83 mRNA. J. Exp. Med. 191: 1581-1590. [Abstract/Free Full Text]
  19. Bevec, D., J. Hauber. 1997. Eukaryotic initiation factor 5A activity and HIV-1 Rev function. Biol. Signals 6: 124-133. [Medline]
  20. Park, M. H., E. C. Wolff, J. E. Folk. 1993. Hypusine: its post-translational formation in eukaryotic initiation factor 5A and its potential role in cellular regulation. Biofactors 4: 95-104. [Medline]
  21. Zinser, E., M. Lechmann, A. Golka, M. B. Lutz, A. Steinkasserer. 2004. Prevention and treatment of experimental autoimmune encephalomyelitis by soluble CD83. J. Exp. Med. 200: 345-351. [Abstract/Free Full Text]
  22. Dudziak, D., F. Nimmerjahn, G. W. Bornkamm, G. Laux. 2005. Alternative splicing generates putative soluble CD83 proteins that inhibit T cell proliferation. J. Immunol. 174: 6672-6676. [Abstract/Free Full Text]
  23. Hock, B. D., J. L. O’Donnell, K. Taylor, A. Steinkasserer, J. L. McKenzie, A. G. Rothwell, K. L. Summers. 2006. Levels of the soluble forms of CD80, CD86, and CD83 are elevated in the synovial fluid of rheumatoid arthritis patients. Tissue Antigens 67: 57-60. [Medline]
  24. Scholler, N., M. Hayden-Ledbetter, K. E. Hellstrom, I. Hellstrom, J. A. Ledbetter. 2001. CD83 is a sialic acid-binding Ig-like lectin (Siglec) adhesion receptor that binds monocytes and a subset of activated CD8+ T cells. J. Immunol. 166: 3865-3872. [Abstract/Free Full Text]
  25. Yang, S., Y. Yang, J. Raycraft, H. Zhang, S. Kanan, Y. Guo, Z. Ronai, I. Hellstrom, K. E. Hellstrom. 2004. Melanoma cells transfected to express CD83 induce antitumor immunity that can be increased by also engaging CD137. Proc. Natl. Acad. Sci. USA 101: 4990-4995. [Abstract/Free Full Text]
  26. Fujimoto, Y., L. Tu, A. S. Miller, C. Bock, M. Fujimoto, C. Doyle, D. A. Steeber, T. F. Tedder. 2002. CD83 expression influences CD4+ T cell development in the thymus. Cell 108: 755-767. [Medline]
  27. Tuschl, T., A. Borkhardt. 2002. Small interfering RNAs: a revolutionary tool for the analysis of gene function and gene therapy. Mol. Interv. 2: 158-167. [Abstract/Free Full Text]
  28. Tuschl, T.. 2001. RNA interference and small interfering RNAs. Chembiochem 2: 239-245. [Medline]
  29. Elbashir, S. M., J. Harborth, W. Lendeckel, A. Yalcin, K. Weber, T. Tuschl. 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494-498. [Medline]
  30. Elbashir, S. M., W. Lendeckel, T. Tuschl. 2001. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15: 188-200. [Abstract/Free Full Text]
  31. Prechtel, A. T., N. M. Turza, A. A. Theodoridis, M. Kummer, A. Steinkasserer. 2006. Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation. J. Immunol. Methods 311: 139-152. [Medline]
  32. Liu, G., H. Ng, Y. Akasaki, X. Yuan, M. Ehtesham, D. Yin, K. L. Black, J. S. Yu. 2004. Small interference RNA modulation of IL-10 in human monocyte-derived dendritic cells enhances the Th1 response. Eur. J. Immunol. 34: 1680-1687. [Medline]
  33. Hill, J. A., T. E. Ichim, K. P. Kusznieruk, M. Li, X. Huang, X. Yan, R. Zhong, E. Cairns, D. A. Bell, W. P. Min. 2003. Immune modulation by silencing IL-12 production in dendritic cells using small interfering RNA. J. Immunol. 171: 691-696. [Abstract/Free Full Text]
  34. Lossos, I. S., D. K. Czerwinski, M. A. Wechser, R. Levy. 2003. Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. Leukemia 17: 789-795. [Medline]
  35. Lechmann, M., E. Kremmer, H. Sticht, A. Steinkasserer. 2002. Overexpression, purification, and biochemical characterization of the extracellular human CD83 domain and generation of monoclonal antibodies. Protein Expression Purif. 24: 445-452. [Medline]
  36. McManus, M. T., B. B. Haines, C. P. Dillon, C. E. Whitehurst, P. L. van, J. Chen, P. A. Sharp. 2002. Small interfering RNA-mediated gene silencing in T lymphocytes. J. Immunol. 169: 5754-5760. [Abstract/Free Full Text]
  37. Obrig, T. G., W. J. Culp, W. L. McKeehan, B. Hardesty. 1971. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J. Biol. Chem. 246: 174-181. [Abstract/Free Full Text]
  38. Gu, X., J. Xiang, Y. Yao, Z. Chen. 2006. Effects of RNA interference on CD80 and CD86 expression in bone marrow-derived murine dendritic cells. Scand. J. Immunol. 64: 588-594. [Medline]
  39. Kapsenberg, M. L.. 2003. Dendritic-cell control of pathogen-driven T-cell polarization. Nat. Rev. Immunol. 3: 984-993. [Medline]
  40. Langenkamp, A., M. Messi, A. Lanzavecchia, F. Sallusto. 2000. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat. Immunol. 1: 311-316. [Medline]
  41. Ebner, S., G. Ratzinger, B. Krosbacher, M. Schmuth, A. Weiss, D. Reider, R. A. Kroczek, M. Herold, C. Heufler, P. Fritsch, N. Romani. 2001. Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation, and is further enhanced by IL-4. J. Immunol. 166: 633-641. [Abstract/Free Full Text]
  42. Lutz, M. B., G. Schuler. 2002. Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity?. Trends Immunol. 23: 445-449. [Medline]
  43. Zinser, E., N. Turza, A. Steinkasserer. 2004. CNI-1493 mediated suppression of dendritic cell activation in vitro and in vivo. Immunobiology 209: 89-97. [Medline]
  44. Hauber, I., D. Bevec, J. Heukeshoven, F. Kratzer, F. Horn, A. Choidas, T. Harrer, J. Hauber. 2005. Identification of cellular deoxyhypusine synthase as a novel target for antiretroviral therapy. J. Clin. Invest. 115: 76-85. [Medline]
  45. Prechtel, A. T., N. M. Turza, D. J. Kobelt, J. I. Eisemann, R. S. Coffin, Y. McGrath, C. Hacker, X. Ju, M. Zenke, A. Steinkasserer. 2005. Infection of mature dendritic cells with herpes simplex virus type 1 dramatically reduces lymphoid chemokine-mediated migration. J. Gen. Virol. 86: 1645-1657. [Abstract/Free Full Text]
  46. Bantounas, I., L. A. Phylactou, J. B. Uney. 2004. RNA interference and the use of small interfering RNA to study gene function in mammalian systems. J. Mol. Endocrinol. 33: 545-557. [Abstract/Free Full Text]
  47. Hannon, G. J.. 2002. RNA interference. Nature 418: 244-251. [Medline]
  48. Cella, M., M. Salio, Y. Sakakibara, H. Langen, I. Julkunen, A. Lanzavecchia. 1999. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J. Exp. Med. 189: 821-829. [Abstract/Free Full Text]
  49. Hornung, V., M. Guenthner-Biller, C. Bourquin, A. Ablasser, M. Schlee, S. Uematsu, A. Noronha, M. Manoharan, S. Akira, A. de Fougerolles, et al 2005. Sequence-specific potent induction of IFN-{alpha} by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat. Med. 11: 263-270. [Medline]
  50. Sioud, M.. 2005. Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J. Mol. Biol. 348: 1079-1090. [Medline]
  51. Schlee, M., V. Hornung, G. Hartmann. 2006. siRNA and isRNA: two edges of one sword. Mol. Ther. 14: 463-470. [Medline]
  52. Cullen, B. R.. 2006. Enhancing and confirming the specificity of RNAi experiments. Nat. Methods 3: 677-681. [Medline]
  53. Pei, Y., T. Tuschl. 2006. On the art of identifying effective and specific siRNAs. Nat. Methods 3: 670-676. [Medline]
  54. Klein, E., S. Koch, B. Borm, J. Neumann, V. Herzog, N. Koch, T. Bieber. 2005. CD83 localization in a recycling compartment of immature human monocyte-derived dendritic cells. Int. Immunol. 17: 477-487. [Abstract/Free Full Text]
  55. Hirano, N., M. O. Butler, Z. Xia, S. Ansen, M. S. von Bergwelt-Baildon, D. Neuberg, G. J. Freeman, L. M. Nadler. 2006. Engagement of CD83 ligand induces prolonged expansion of CD8+ T cells and preferential enrichment for antigen specificity. Blood 107: 1528-1536. [Abstract/Free Full Text]
  56. Garcia-Martinez, L. F., M. W. Appleby, K. Staehling-Hampton, D. M. Andrews, Y. Chen, M. McEuen, P. Tang, R. L. Rhinehart, S. Proll, B. Paeper, et al 2004. A novel mutation in CD83 results in the development of a unique population of CD4+ T cells. J. Immunol. 173: 2995-3001. [Abstract/Free Full Text]
  57. Saito, T., T. Yokosuka. 2006. Immunological synapse and microclusters: the site for recognition and activation of T cells. Curr. Opin. Immunol. 18: 305-313. [Medline]
  58. Davis, D. M., M. L. Dustin. 2004. What is the importance of the immunological synapse?. Trends Immunol. 25: 323-327. [Medline]
  59. Lanzavecchia, A., F. Sallusto. 2001. Antigen decoding by T lymphocytes: from synapses to fate determination. Nat. Immunol. 2: 487-492. [Medline]
  60. Sallusto, F., A. Lanzavecchia. 2002. The instructive role of dendritic cells on T-cell responses. Arthritis Res. 4: S127-S132. [Medline]



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