Dendritic Cell Function After Gene Transfer with Adenovirus-calcium Phosphate Co-precipitates

Abstract

Dendritic cells (DCs) are essential for initiating and directing antigen-specific T-cell responses. Genetic modification of DC is under study for cancer immunotherapy, vaccine development, and antigen-targeted immunosuppression. Adenovirus (Ad) type 5 (Ad5)-mediated gene transfer to mouse bone marrow DCs and human monocyte-derived DCs is inefficient because neither express the cognate high-affinity Ads receptor. We show that co-precipitating adenoviral vectors with calcium phosphate (CaPi) increased gene expression (2000-fold) and transduction efficiency (50-fold) in mouse DC, primarily owing to receptor-independent viral uptake. Moreover, Ad5:CaPi-treated DCs were activated to express the maturation surface molecules CD40 and CD86, and to secrete proinflammatory cytokines tumor necrosis factor-alpha and interleukin 6. However, neither DC transduction nor maturation was dependent on viral protein interactions with cell surface integrin. Ad5:CaPi also transduced human DC more efficiently than Ad5 alone, similar to a genetically modified vector (Ad5f35) targeted to the CD46 receptor. As such, this approach combines the efficiency of adenoviral-mediated endosomal escape and nuclear trafficking with the receptor independence of nonviral gene delivery. Importantly, CaPi co-precipitation could be used to functionally modify DC to activate and expand cytomegalovirus-specific memory cytotoxic T lymphocytes. This study identifies a simple technique to improve the efficacy of current Ad5 gene transfer, in support of clinical adoptive immunotherapy.

Dendritic Cell-Based Cancer Gene Therapy

“…On the other hand, their immunogenicity may also function as a trigger for enhanced DC maturation and may provide the necessary danger signals in the vaccine, resulting in more potent antitumor immunity (Dietz et al, 2001;Timares et al, 2004;Perreau et al, 2007;Seiler et al, 2007). In preclinical murine models, adenovirally transduced DCs were shown to be more potent than their peptide-loaded counterparts for generating antitumor immunity (Nakamura et al, 2005;Oh et al, 2006).…”

Cytokine-Conditioned Dendritic Cells Induce Humoral Tolerance to Protein Therapy in Mice

“…DC precursors were harvested from femurs and tibias, and cultured as described (Seiler et al, 2007). DCs were cultured in complete medium (RPMI 1640 with 10% fetal bovine serum, 2 mM l-glutamine, 50 lM 2-mercaptoethanol, penicillin [100 U/ml], and streptomycin [100 lg/ml]) supplemented with mouse granulocyte-macrophage colonystimulating factor (GM-CSF, 20 ng/ml; ProSpec, East Brunswick, NJ) and mouse IL-4 (10 ng/ml; ProSpec) for DCs alone; human TGF-b 1 (hTGF-b 1 , 10 ng/ml; eBioscience, San Diego, CA) for DCs + TGF-b; human IL-10 (hIL-10, 10 ng/ml; eBioscience) for DCs + IL-10; or TGF-b 1 and IL-10 for DCs + TGF-b + IL-10, for 6 days with medium change on every alternate day of culture.…”

Induction of T cells suppression by dendritic cells transfected with VSIG4 recombinant adenovirus

“…In vivo transfer of Agloaded DCs with a tolerogenic character is regarded as a promising therapeutic means to repress the immune response [12,13]. Transduction with recombinant, replication-defective adenoviral (Ad) vectors encoding transgenes has been shown to be an efficient method for gene transfer into DCs [14][15][16].…”