title: Identification of T-cell epitopes by a novel mRNA PCR-based epitope chase technique.
abstract: The identification of specific viral and tumor antigen T-cell epitopes remains a challenge. Indeed, epitope mapping methods are generally costly and time-consuming. Thus, few techniques allow for efficient CD4+ T-lymphocyte epitope identification. Here, we introduce a novel polymerase chain reaction-based mRNA epitope identification method, called mPEC, to rapidly and precisely identify relevant T-cell epitopes recognized by CD8+ or CD4+ T lymphocytes. This method is based on the use of mRNA fragments synthesized from polymerase chain reaction-amplified cDNA with a choice of 3'end deletions. mRNA fragments are electroporated into autologous antigen-presenting cells to deduce an epitope's localization in a given protein antigen. Considering mRNA's sensitivity to degradation, we also inserted a defined epitope at the mRNA's 3'end to control for electroporated mRNA's integrity and its capacity to be translated. Using this method, we rapidly and successfully identified the specific epitope of 2 CD8+ and 1 CD4+ T-lymphocyte clones derived from influenza model antigens. Hence, mPEC could be used to identify new, in vivo-relevant T-cell epitopes for cancer immunotherapy and vaccination in general.
authors.affiliation: CRCHUM--Hôpital Notre-Dame, Université de Montréal, Institut du Cancer de Montréal, Montréal, Québec, Canada. [corrected]
authors.forename: Jean-Daniel
authors.initials: JD
authors.lastname: Doucet
authors.forename: Dominique
authors.initials: D
authors.lastname: Gauchat
authors.forename: Réjean
authors.initials: R
authors.lastname: Lapointe
mesh_terms: D001402:B-Lymphocytes / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D019013:CD40 Antigens / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002460:Cell Line; D018604:Epitope Mapping / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000235:genetics*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D019590:Nucleocapsid Proteins; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000276:immunology; D014758:Viral Core Proteins / Q000276:immunology; D014763:Viral Matrix Proteins / Q000276:immunology
chemical_list: D019013:CD40 Antigens; D018984:Epitopes, T-Lymphocyte; C488936:M1 protein, Influenza A virus; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D014763:Viral Matrix Proteins