In Vitro Priming with Adenovirus/gp100 Antigen-Transduced Dendritic Cells Reveals the Epitope Specificity of HLA-A0201-Restricted CD8+ T Cells in Patients with Melanoma

• Published in: The Journal of immunology (1950) 2000-03, Vol.164 (6), p.3402-3412
• Main Authors: Linette, Gerald P, Shankara, Srinivas, Longerich, Simonne, Yang, Sixun, Doll, Rhonda, Nicolette, Charles, Preffer, Frederic I, Roberts, Bruce L, Haluska, Frank G
• Format: Article
• Language: English
• Subjects: Adenoviruses, Human - genetics; Adenoviruses, Human - immunology; AIDS/HIV; Antigens, Neoplasm; CD8-Positive T-Lymphocytes - immunology; Cells, Cultured; Dendritic Cells - immunology; Dendritic Cells - metabolism; Enterovirus B, Human - genetics; Epitopes, T-Lymphocyte - genetics; Epitopes, T-Lymphocyte - immunology; Fluorescent Dyes - metabolism; Genetic Vectors - immunology; gp100 Melanoma Antigen; Half-Life; HLA-A2 Antigen - genetics; HLA-A2 Antigen - immunology; Humans; Immunodominant Epitopes - genetics; Immunodominant Epitopes - immunology; Immunophenotyping; MART-1 Antigen; Melanoma - genetics; Melanoma - immunology; Membrane Glycoproteins - genetics; Membrane Glycoproteins - immunology; Neoplasm Proteins - genetics; Neoplasm Proteins - immunology; Peptide Fragments - genetics; Peptide Fragments - metabolism; Receptors, Virus - genetics; T-Lymphocytes, Cytotoxic - immunology; Virion - genetics; Virion - metabolism; Virus Replication - genetics
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.164.6.3402

Replication-deficient recombinant adenovirus (Ad) encoding human gp100 or MART-1 melanoma Ag was used to transduce human dendritic cells (DC) ex vivo as a model system for cancer vaccine therapy. A second generation E1/E4 region deleted Ad which harbors the CMV immediate-early promoter/enhancer and a unique E4-ORF6/pIX chimeric gene was employed as the backbone vector. We demonstrate that human monocyte-derived DC are permissive to Ad infection at multiplicity of infection between 100 and 500 and occurs independent of the coxsackie Ad receptor. Fluorescent-labeled Ad was used to assess the kinetics and distribution of viral vector within DC. Ad-transduced DC show peak transgene expression at 24-48 h and expression remains detectable for at least 7 days. DC transduced with replication-deficient Ad do not exhibit any unusual phenotypic characteristics or cytopathic effects. DC transduced with Ad2/gp100v2 can elicit tumor-specific CTL in vitro from patients bearing gp100+ metastatic melanoma. Using a panel of gp100-derived synthetic peptides, we show that Ad2/gp100v2-transduced DC elicit Ag-specific CTL that recognize only the G209 and G280 epitopes, both of which display relatively short half-lives ( approximately 7-8 h) on the surface of HLA-A*0201+ cells. Thus, patients with metastatic melanoma are not tolerant to gp100 Ag based on the detection of CD8+ T cells specific for multiple HLA-A*0201-restricted, gp100-derived epitopes.