High efficiency gene transfer is an efficient way of defining therapeutic targets: a functional genomics approach

Abstract

BACKGROUND—Dendritic cells are the most potent antigen presenting cells and key to many aspects of the immune function. Studying the intracellular signalling mechanism used by dendritic cells would provide an insight into the functioning of these cells and give clues to strategies for immunomodulation.
METHOD—Highly efficient adenoviral infection of dendritic cells for the delivery of transgenes was obtained. These viral vectors were used to introduce IκBα into dendritic cells for the inhibition of NF-κB. This was used to investigate the role of NF-κB in dendritic cell function.
RESULTS—By blocking the NF-κB function a potent inhibition of the expression of costimulating molecules by dendritic cells with the concomitant loss of T cell stimulating function was demonstrated.
CONCLUSION—The use of adenoviral vectors may be a useful way of studying the role of genes in dendritic cell function.



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Figure 1  .

(A) Mature dendritic cells were generated by five days' culture in 50 ng/ml granulocyte macrophage colony stimulating factor and 10 ng/ml interleukin 4 (IL4) and a further three days in monocyte conditioned medium. Then they were plated on a 96 well, flat bottom plate at a density of 2×10⁵ cells/well and left either uninfected with a multiplicity of infection ranging from 40 to 500 of an adenovirus without insert (Adv0) or an adenovirus overexpressing β-Gal expression. Fluorescein-di-(β-D)-galactopyranoside (Sigma) was used as a substrate of β-galactosidase and cell fluorescence was analysed by FACS analysis. In (B) and (C), 10×10⁶ cells per condition were left either uninfected or infected with Adv0 or AdvIκBα at a multiplicity of infection of 300. Two days after infection, cells were left unstimulated or stimulated with lipopolysaccharide (LPS; 50 ng/ml) for 60 minutes. Cytosolic and nuclear extracts were then prepared and examined for IκBα or p42/44MAPK (loading control) expression by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (B), or for NF-κB DNA binding activity by electrophoretic mobility shift assay (C).

Figure 2  .

Mature dendritic cells (DC) were left uninfected, infected with Adv0, or infected with AdvIκBα at a multiplicity of infection of 300. DC were then plated in graded doses for 10⁵ purified, allogeneic T cells in triplicate in a 96 well, round bottom microtitre plate on day 1 after adenovirus infection. Proliferation was determined on day six with either a 5-bromo-2'-deoxyuridine (BrdU) labelling and detection kit III (Boehringer Mannheim, East Sussex, UK) (A) or by a [³H]thymidine ([³H]TdR) uptake assay (B). Each point represents the mean (SEM) of either six (A) or three (B) separate experiments.

Figure 3  .

The effect of AdvIκBα infection on dendritic cell antigen presenting and costimulatory molecules as well as the costimulatory cytokine interleukin 12 (IL12) was studied. IκBα was overexpressed in mature DC for two days and then cells were collected and stained by FACS for HLA (A) or CD80/86 (B), or stimulated by soluble CD40L for 24 hours, and then IL12 production was assayed by enzyme linked immunosorbent assay (ELISA) (C).