CD83+ human dendritic cells transfected with tumor peptide cDNA by electroporation induce specific T-cell responses: A potential tool for gene immunotherapy

Abstract

Dendritic cells (DC) are the most potent immunostimulatory cells, with the capacity to induce primary T-cell responses. Functional autologous DC can be generated from fetal calf serum-free peripheral blood mononuclear cells in the presence of interleukin-4 and granulocyte-macrophage colony-stimulating factor and are stimulated with a defined cytokine cocktail for terminal maturation. We were able to establish a nonviral transfection protocol for these DC by electroporation. Using enhanced green fluorescent protein as a reporter gene, we achieved transfection efficiencies of up to 10%. FACScan analyses revealed a stable phenotype, and the expression of major histocompatibility complex class II and CD83 was not affected by the transfection conditions used. Like their untransfected counterparts, DC that were functionally transfected with green fluorescent protein were potent inducers of allogeneic T cells. To assess whether cDNAs transfected into DC are functionally expressed, human tyrosinase cDNA was transfected into DC. Tyrosinase-transfected DC, but not controls, resulted in antigen-specific tumor necrosis factor-␣ release of the tyrosinase-specific cytolytic T-cell clone IVSB. Taken together, the data show that genuine (CD83 ϩ ) mature DC can be transfected using a nonviral method, and that the DC retain their functionality. These DC are ideal candidates for immunotherapy (e.g., cancer therapy). Cancer Gene Therapy (2000) D endritic cells (DC) are the most potent antigen (Ag)-presenting cells (APCs) of the immune system, with the capacity to activate resting naive T cells most efficiently and to initiate primary immune responses. Due to their functional properties, DC are ideal adjuvants for immunotherapeutic strategies using DC pulsed with tumor-associated Ags (TAA) for vaccination. 1,2 Recently several TAA have been characterized, and they are expressed by the tumor cells. TAA peptides in association with major histocompatibility complex (MHC) are presented on the tumor cell surface, are recognized by Ag-specific T cells, and provide a potential target for immunotherapeutic vaccination approaches. [3][4][5] In various murine studies, DC pulsed with TAA peptide can induce Ag-specific antitumoral responses in vivo. 6,7 There are even promising reports about clinical trials in which patients with B-cell lymphoma were successfully vaccinated using autologous Ag-pulsed DC. 8 Recently, a clinical pilot study was published that reports on the successful vaccination of melanoma patients with peptide-or tumor lysate-pulsed DC. 9 The use of genetically engineered DC as tumor vaccines combines the advantages of immunotherapy with those of gene therapy. The application of peptide-pulsed DC is limited by the human histocompatibility leukocyte Ag (HLA) restriction of characterized TAA epitopes. 3 Using recombinant techniques, the full-length protein Ag is expressed in the genetically engineered APCs and is accessible to the cellular Ag-processing machinery. Therefore, the repertoire of the epitopes presented in context w...

Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications

“…Thus, hematopoietic cells can now be efficiently transduced and the main cause of mortality, apoptosis, can be highly reduced by using caspase inhibitors [29]. Similarly, dendritic cells have been transfected thanks to electroporation and retain a stable phenotype as their functionality [30]. In addition, optimized electroporation conditions have been established for gene transfer into embryonic and postnatal neural cells, which allow preferential transfection of subsets of neural cells, depending on voltage and capacitance conditions [31].…”

Augmentation of Antiproliferative and Antitumor Effect on Human Cancer Cells in Combined Use of Electroporation with a Plant Toxin, Saporin

“…Electroporation (EP) has been widely utilized in the field of cancer therapy, such as gene therapy [11][12][13][14] or chemotherapy. 6-10 Bleomycin, 7-10 cisplatin 6,15,16 and actinomycin D 17 have been reported to augment cytotoxic effect of EP.…”

Evaluation of immune response according to the metastatic status in the regional lymph nodes in patients with gastric carcinoma

“…CD1a, a cell surface glycoprotein that is structurally related to the MHC molecules and mediates an MHCindependent antigen presentation pathway, is a highly specific and sensitive marker of immature DCs and Langerhans cells, a population of DCs found in the skin (7,8). CD83 is a glycoprotein member of the immunoglobulin superfamily that is strongly up-regulated during DC maturation, thus making it a robust marker for mature DCs (9)(10)(11)(12). Activation of naïve T-cell by mature DCs requires two signals.…”