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.