Evaluation of an mRNA lipofection procedure for human dendritic cells and induction of cytotoxic T lymphocytes against enhanced green fluorescence protein.
We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 microl/ml, and the mRNA concentration was 6 microg/ml. Under these conditions, ELISPOT and (51)Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-gamma-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs.
Department of Clinical Laboratory Science, Yamaguchi University School of Medicine, Ube, Japan.
Kozue
K
Okano
Mikiko
M
Fukui
Yutaka
Y
Suehiro
Yuichiro
Y
Hamanaka
Kohzoh
K
Imai
Yuji
Y
Hinoda
D002478:Cells, Cultured; D003713:Dendritic Cells / Q000276:immunology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D008081:Liposomes; D008164:Luminescent Proteins / Q000235:genetics* / Q000276:immunology*; D008213:Lymphocyte Activation; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014162:Transfection / Q000379:methods*
D008081:Liposomes; D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins