Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation
Overexpression of mHSF1 does not impair the phenotype and function of immature and mature DCs. Immature DCs were transduced with Ad5Luc1 or Ad5mHSF1 at 500 TCID50/cell or Mock-treated and were either left immature (“iDC”) or were matured (“mDC”) with a maturation cytokine cocktail (IL-1β, IL-6, TNF-α, and PGE2). (a) Western Blot analyses of cell lysates harvested 24 h after transduction on HSF1, mHSF1, Hsp40, Hsp70A, Hsp70B′, Hsp90, Grp94, and beta-actin protein expression. One representative experiment out of three is shown. (b, c) Flow cytometric analyses of iDCs and mDCs on CD25, CD80, CD83, CD86, HLA-ABC, and HLA-DR (b) as well as PI (c) 24 h and 48 h after transduction. Data are mean ± SEM of four independent experiments with DCs derived from different donors. (d) Cell culture supernatants derived from experiments shown in (b and c) were analyzed by cytometric bead array for the content of IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α. The red line in (d) indicates background levels of respective cytokines derived from the cytokines present in the maturation cocktail. Data are mean ± SEM of three independent experiments. (e) Twenty-four hours after adenoviral transduction accompanied by maturation of DCs, cells were either transfected with wild-type MelanA RNA (left panel) or loaded with MelanA analogue peptide (right panel). Antigen-loaded DCs were cocultured with MACS-sorted autologous CD8+ T cells for seven days. Induction of antigen-specific CTLs was determined by HLA-A2-MelanA/iTag MHC class I-tetramer and anti-CD8-PC7 staining using a FC500 cytofluorometer. Data are mean ± SEM of three independent experiments with different donors. (b–e) , , , and n.s.: not significant (); bars without annotation are not significant (), one-way, (e) or two-way ANOVA (b–d) with Bonferroni’s Multiple Comparison post hoc test.
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