title: Generation of peptide-specific CD8+ T cells by phytohemagglutinin-stimulated antigen-mRNA-transduced CD4+ T cells.
abstract: Functional analysis of antigen-specific CD8(+) T cells is important for understanding the immune response in various immunological disorders. To analyze CD8(+) T cell responses to a variety of antigens with no readily defined peptides available, we developed a system using CD4(+) phytohemagglutinin (PHA) blasts transduced with mRNA for antigen molecules. CD4(+) PHA blasts express MHC class I and II, and also CD80 and CD86 and are thus expected to serve as potent antigen presenting cells. EGFP mRNA could be transduced into and the protein expressed by more than 90% of either LCL or CD4(+) PHA blasts. Its expression stably persisted for more than 2 weeks after transduction. In experiments with HLA-A*2402 restricted CD8(+) CTL clones for either EBNA3A or a cancer-testis antigen, SAGE, mRNA-transduced lymphoid cells were appropriate target cells in ELISPOT assays or (51)Cr releasing assays. Finally, using CD4(+) PHA blasts transduced with mRNA of a cancer-testis antigen MAGE-A4, we successfully generated specific CTL clones that recognized a novel HLA-B*4002 restricted epitope, MAGE-A4(223-231). Messenger RNA-transduced CD4(+) PHA blasts are thus useful antigen presenting cells for analysis of CD8(+) T cell responses and induction of specific T cells for potential immunotherapy.
authors.affiliation: Second Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Naota
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Miyahara
authors.forename: Satoshi
authors.initials: S
authors.lastname: Okumura
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Kuzushima
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Akatsuka
authors.forename: Atsunori
authors.initials: A
authors.lastname: Hiasa
authors.forename: Shigehisa
authors.initials: S
authors.lastname: Kitano
authors.forename: Toshitada
authors.initials: T
authors.lastname: Takahashi
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yuta
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Majima
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Shiku
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D000941:Antigens / Q000737:chemistry; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002460:Cell Line; D018274:Electroporation; D000939:Epitopes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D015234:HLA-A Antigens / Q000276:immunology; D059871:HLA-A24 Antigen; D051379:Mice; D009363:Neoplasm Proteins / Q000737:chemistry / Q000276:immunology; D010455:Peptides / Q000737:chemistry*; D010835:Phytohemagglutinins / Q000494:pharmacology*; D012333:RNA, Messenger / Q000737:chemistry*; D014161:Transduction, Genetic
chemical_list: D000941:Antigens; D000951:Antigens, Neoplasm; D000939:Epitopes; D015234:HLA-A Antigens; C438506:HLA-A*24:02 antigen; D059871:HLA-A24 Antigen; C095204:MAGEA4 protein, human; D009363:Neoplasm Proteins; D010455:Peptides; D010835:Phytohemagglutinins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins