Real-time polymerase chain reaction monitoring of epithelial cell adhesion molecule-induced T-cell stimulation in patients with lung cancer and healthy individuals using LightCycler technology.
The epithelial cell adhesion molecule is strongly expressed in carcinomas of diverse histologic origin and has attracted attention as a target for immunotherapy. We have previously demonstrated that the epithelial cell adhesion molecule-derived human leukocyte antigen-A*0201-restricted nonamer peptide ILYENNVIT(184-192) can be efficiently recognized by peptide-specific cytotoxic T lymphocytes (CTL). Using this peptide (Ep-2) we could readily expand precursor CTL and demonstrate their cytotoxicity by standard chromium-release assay. We now sought to evaluate the immune reactivity of Ep-2-specific CTL expanded from the peripheral blood mononuclear cells by real-time quantitative polymerase chain reaction assay (LightCycler system) measuring the change in interferon-gamma mRNA levels upon stimulation. Cytotoxic T lymphocyte response against the Ep-2 peptide in two patients with epithelial cell adhesion molecule-expressing lung cancer and three healthy donors was compared with the chromium-release assay. In vitro expanded epithelial cell adhesion molecule-specific CTL precursors that exhibited significantly elevated interferon-gamma mRNA levels also lysed Ep-2 peptide-pulsed target cells. Our study indicates that quantitative polymerase chain reaction may be a supplement to chromium-release assay for monitoring in vitro expanded CTL precursor reactivity.
Trojan
Andreas
A
Division of Oncology, Department of Internal Medicine, University Hospital Zurich, Rämistrasse 100, CH-8091 Zurich, Switzerland. andreas.trojan@dim.usz.ch
Urosevic
Mirjana
M
Dummer
Reinhard
R
Nestle
Frank O
FO
Stahel
Rolf A
RA
D000951:Antigens, Neoplasm / Q000276:immunology*; D016827:CD8 Antigens / Q000235:genetics; D015815:Cell Adhesion Molecules / Q000276:immunology*; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D000071858:Epithelial Cell Adhesion Molecule; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D008175:Lung Neoplasms / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
D000951:Antigens, Neoplasm; D016827:CD8 Antigens; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; D012333:RNA, Messenger; D007371:Interferon-gamma