title: Identification of immune dominant cytomegalovirus epitopes using quantitative real-time polymerase chain reactions to measure interferon-gamma production by peptide-stimulated peripheral blood mononuclear cells.
abstract: The identification of HLA restricted immune dominant cytotoxic T cell (CTL) epitopes limits immune therapy. Cytomegalovirus (CMV) disease remains a significant cause of morbidity after allogeneic stem cell transplantation. Adoptive immune therapy using CTLs stimulated with immune dominant CMV pp65 peptides may be effective in preventing CMV disease, but immune dominant CMV peptides have been identified for only a few HLA class I molecules. The purpose of this study was to use a novel molecular system to establish a rapid and precise method to identify new HLA-restricted CMV epitopes. Cytomegalovirus pp65 peptides expected to bind to the HLA-24 molecule were identified with a computer algorithm. Five candidate peptides were screened by direct ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) from CMV-seropositive HLA-A*2402 individuals, and quantitative real time PCR (qRT-PCR) was used to evaluate CTL responses by measuring interferon-gamma (IFN-gamma) transcripts. One of the five candidate peptides, pp65341-350 (QYDPVAALFF), induced significant quantities of IFN-gamma mRNA production after 3 hours. PBMCs from CMV-seropositive HLA-A*2402 individuals sensitized in vitro with pp65341-350 also recognized CMV-infected targets. In conclusion, the measurement of IFN-gamma mRNA by qRT-PCR can be used to detect CTL responses 3 hours after peptide stimulation of a small quantity of PBMCs. This method has an advantage over other methods used to identify immune dominant epitopes in that it does not require in vitro expansion of CTLs with cytokines or virally infected targets. As a result, this method measures naturally induced immune reactions.
authors.affiliation: Department of Transfusion Medicine, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Provenzano
authors.forename: Simone
authors.initials: S
authors.lastname: Mocellin
authors.forename: Maria
authors.initials: M
authors.lastname: Bettinotti
authors.forename: Jeanne
authors.initials: J
authors.lastname: Preuss
authors.forename: Vladia
authors.initials: V
authors.lastname: Monsurrò
authors.forename: Francesco M
authors.initials: FM
authors.lastname: Marincola
authors.forename: David
authors.initials: D
authors.lastname: Stroncek
mesh_terms: D000956:Antigens, Viral / Q000276:immunology*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000276:immunology*; D018604:Epitope Mapping / Q000379:methods; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D015234:HLA-A Antigens / Q000276:immunology*; D059871:HLA-A24 Antigen; D006801:Humans; D016056:Immunodominant Epitopes / Q000276:immunology*; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology / Q000821:virology; D010455:Peptides / Q000276:immunology; D010750:Phosphoproteins / Q000138:chemical synthesis / Q000276:immunology*; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000096:biosynthesis; D014763:Viral Matrix Proteins / Q000138:chemical synthesis / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D018984:Epitopes, T-Lymphocyte; D015234:HLA-A Antigens; D059871:HLA-A24 Antigen; D016056:Immunodominant Epitopes; D010455:Peptides; D010750:Phosphoproteins; D012333:RNA, Messenger; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa; D007371:Interferon-gamma