In vitro priming with adenovirus/gp100 antigen-transduced dendritic cells reveals the epitope specificity of HLA-A*0201-restricted CD8+ T cells in patients with melanoma.
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.
Linette
G P
GP
Hematology-Oncology Unit and Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA. linette.gerald@mgh.harvard.edu
Shankara
S
S
Longerich
S
S
Yang
S
S
Doll
R
R
Nicolette
C
C
Preffer
F I
FI
Roberts
B L
BL
Haluska
F G
FG
D000260:Adenoviruses, Human / Q000235:genetics / Q000276:immunology*; D000951:Antigens, Neoplasm; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000276:immunology* / Q000378:metabolism; D029822:Enterovirus B, Human / Q000235:genetics; D018984:Epitopes, T-Lymphocyte / Q000235:genetics / Q000276:immunology*; D005456:Fluorescent Dyes / Q000378:metabolism; D005822:Genetic Vectors / Q000276:immunology; D015789:HLA-A2 Antigen / Q000235:genetics / Q000276:immunology*; D006207:Half-Life; D006801:Humans; D016056:Immunodominant Epitopes / Q000235:genetics / Q000276:immunology; D016130:Immunophenotyping; D058965:MART-1 Antigen; D008545:Melanoma / Q000235:genetics / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D009363:Neoplasm Proteins / Q000235:genetics / Q000276:immunology*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011991:Receptors, Virus / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014771:Virion / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics; D058951:gp100 Melanoma Antigen
D000951:Antigens, Neoplasm; D018984:Epitopes, T-Lymphocyte; D005456:Fluorescent Dyes; D015789:HLA-A2 Antigen; D016056:Immunodominant Epitopes; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D010446:Peptide Fragments; D011991:Receptors, Virus; D058951:gp100 Melanoma Antigen