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Department of Dermatology, University Hospital Erlangen, Erlangen, Germany
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.
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.
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
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.
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