Transcriptional Targeting of Mature Dendritic Cells with Adenoviral Vectors via a Modular Promoter System for Antigen Expression and Functional Manipulation

Figure 5

The “modular promoter (MP)” system enables highly efficient and specific expression of therapeutic transgenes in mature DCs. (a) Schematic outline of the dual vector “modular promoter” system. Ad5E/P-510/mHSF1 drives expression of mHSF1 under the control of the cell type- and maturation-specific human CD83 promoter, which in turn induces specific induction of therapeutic transgenes MelanA and Il-12 by binding to the Hsp70B′_−29/−242 HRE encoded by Ad5MP2. (b, c) XS52 cells were cotransfected with a total amount of 2 μg of plasmid vectors pP-510/mHSF1, pEs/P-510/mHSF1, or pEas/P-510/mHSF1 in combination with plasmid vectors encoding the HRE alone (“pHsp70B′_−29/−242”) or the modular promoter system (“pMP2”). As a control, the empty pGL3-Basic vector (“Basic”) was used. (d, e) Human immature DCs were cotransduced with Ad5MP2 or Ad5MelA in combination with Ad5Luc1, Ad5mHSF1, Ad5P-510/mHSF1, Ad5Es/P-510/mHSF1, or Ad5Eas/P-510/mHSF1. Cells were either left immature or were matured by adding 0.1 ng/mL LPS. (b–e) Twenty hours after transfection, cell lysates were analyzed by Western Blot for expression of MelanA and beta-actin (b, d) and cell culture supernatants by ELISA for content of IL-12p40 and IL-12p70 (c, e). Either one representative (b, d) or data mean ± SEM from three independent experiments (c, e) is shown. ; ; bars without annotation are not significant (), one-way ANOVA (c) or two-way ANOVA (e) with Bonferroni’s Multiple Comparison post hoc test.

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