title: Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA.
abstract: Assessment of antigen-specific T-cell responses has been greatly facilitated by development of ELISPOT and intracellular cytokine flow cytometry (CFC) assays. The use of autologous antigen presenting cells transfected with in vitro transcribed RNA as stimulators allows in principle quantification of antigen-specific T-cells independent of the knowledge of the epitopes. We describe here a cytokine secretion assay that enables simultaneous assessment of both antigen-specific CD4+ as well as CD8+ T-cells directly from clinical samples without the need for generation of dendritic cells. To this aim, bulk PBMCs were electroporated with RNA encoding the antigen fused to trafficking signal sequences derived from a MHC class I molecule and used as stimulators. With human cytomegalovirus (HCMV) phosphoprotein 65 (pp65) as antigen we show that for measuring ex vivo T-cell responses in ELISPOT and CFC such stimulators are superior or at least equivalent to a pool of overlapping peptides representing the entire pp65 sequence as well as to untagged pp65 encoding RNA. This approach avoids the time consuming generation of dendritic cells as immune stimulators and, in particular when used in the context of the CFC, is robust, broadly applicable and fast.
authors.affiliation: Division of Experimental and Translational Oncology, Department of Internal Medicine III, Johannes Gutenberg University, Obere Zahlbacherstr. 63, 55131, Mainz, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Kreiter
authors.forename: Thorsten
authors.initials: T
authors.lastname: Konrad
authors.forename: Martina
authors.initials: M
authors.lastname: Sester
authors.forename: Christoph
authors.initials: C
authors.lastname: Huber
authors.forename: Ozlem
authors.initials: O
authors.lastname: Türeci
authors.forename: Ugur
authors.initials: U
authors.lastname: Sahin
mesh_terms: D017951:Antigen Presentation; D000941:Antigens / Q000235:genetics / Q000276:immunology*; D001681:Biological Assay; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D016207:Cytokines / Q000378:metabolism; D018274:Electroporation; D018984:Epitopes, T-Lymphocyte / Q000032:analysis* / Q000276:immunology; D005434:Flow Cytometry; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000276:immunology; D006801:Humans; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014158:Transcription, Genetic; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000941:Antigens; D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D010750:Phosphoproteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa