CD83 Knockdown in Monocyte-Derived Dendritic Cells by Small Interfering RNA Leads to a Diminished T Cell Stimulation

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.

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-β-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.

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 (□) showed a dramatically reduced capacity to induce T cell proliferation when compared with cells that were mock electroporated (▪) or left untreated (▾). These results are representative of three independent experiments with cells from different donors.