Transfection of human monocyte-derived dendritic cells with native tumor DNA induces antigen-specific T-Cell responses in vitro.

Abstract

Objective: Nucleofection of genomic tumor (Tu) DNA into human monocyte-derived dendritic cells (hMoDC) was evaluated for use in producing anti-tumor vaccines able to induce effective T-cell specific immune responses.Methods: Cultured hMoDC obtained from HLA-A2+ normal donors were nucleofected with genomic DNA extracted from an HLA-A2+gp100+ Mel 526 cell line and 3' end-labeled with biotinylated TdT nucleotides or from a genetically-modified Mel 526 expressing enhanced green fluorescent protein (EGFP). An Amaxa Nucleofector™ system was used for electroporation. Nucleofected hMoDC were matured in the presence of cytokines and examined in ELISPOT assays for the ability to present the gp100 [209][210][211][212][213][214][215][216][217] epitope to epitope-specific T cells or to prime autologous naïve T cells in culture.Results: The nucleofected hMoDC presented gp100 protein to HLA-A2+gp100-specific T cells as observed in IFN-g ELISPOT assays. Spot formation was inhibited by anti-HLA class I and HLA-A2 but not anti-HLA class II antibodies (Abs). Tu DNA-nucleofected hMoDC also primed naïve autologous peripheral blood T cells in culture to develop into Tu-reactive effector cells (CTL). These CTL recognized Tu cells which had donated genomic DNA, and these responses were MHC class I-and class II-restricted. The CTL recognized shared Tu antigens encoded in Tu-derived DNA.Conclusion: Nucleofection of hMoDC with genomic Tu-derived DNA is a useful strategy for Tu vaccine production: it is feasible, does not require Tu epitope isolation, can be used when few Tu cells are available, and avoids Tu-induced DC suppression.

Immunobiology of head and neck cancer

“…The expectation is that genes encoding weakly immunogenic TAA will be expressed in a highly immunogenic form by DC, which can integrate tumor DNA within its own DNA, express the transferred genes and present the endogenous epitopes to immune cells. The pre-clinical experiments with DNA derived from HLA-A2+ tumor and transferred to human semi-allogeneic HLA-A2+ immature DC show that these DC can prime autologous T cells, leading to expansion to effector T cells capable of recognizing and responding specifically to the tumor donating the DNA [37]. This approach is applicable to DNA obtained from very small biopsy specimens, as only minimal amounts of DNA are needed for its transfer to DC.…”

High-Level Antigen Expression and Sustained Antigen Presentation in Dendritic Cells Nucleofected with Wild-Type Viral mRNA but Not DNA

“…Non-viral-based approaches for delivering genes to DC for cancer immunotherapy have distinct advantages over recombinant virus-based strategies, in particular because DNA or RNA can be amplified directly from a tumor and serve as a source of polyvalent patient-specific antigens (2,19,42). This strategy is being employed in the design of therapeutic DCbased vaccines for human immunodeficiency virus (HIV)-infected individuals as well, as viral mRNA expressing patientderived sequences or sequences from SIV-infected monkeys can readily be introduced into monocyte-derived DC for stimulation of autologous T cells (33,48).…”

“…An attractive alternative to vector-mediated delivery of antigens into DC is nonviral gene transfer based on DNA or mRNA. DNA transfection of DC has been used successfully in some studies but is limited by poor expression levels and toxicity (2,29,41,45,49), although methods to enhance expression based on DC maturation have been proposed (28). mRNA transfection of DC is being increasingly utilized for cancer immunotherapy and in vitro stimulation of virus-specific T cells, but approaches to deliver mRNA into primary DC cultures have been limited (16,18,25,39,40,43,48,50).…”

Head and neck cancer immunotherapy: Clinical evaluation

“…These tumor DNA-transfected dendritic cells are reinfused intranodally in advanced or recurrent resectable disease (excluding laryngeal cancer) [19].…”