Efficient Induction of CD8 T-Associated Immune Protection by Vaccination with mRNA Transfected Dendritic Cells Shohreh Zarei, Jean-François Arrighi,

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Efficient Induction of CD8 T-Associated Immune Protection by Vaccination with mRNA Transfected Dendritic Cells Shohreh Zarei, Jean-François Arrighi, Gioele Ongaro, Thomas Calzascia, Olivier Haller, Christophe Frossard, Vincent Piguet, Paul R. Walker, Conrad Hauser Journal of Investigative Dermatology Volume 121, Issue 4, Pages 745-750 (October 2003) DOI: 10.1046/j.1523-1747.2003.12492.x Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Flow cytometric analysis of transgene expression in murine DC following EGFP mRNA transfection. Murine DC were electroporated with mRNA encoding EGFP and were analyzed after 24 h by flow cytometry. Viability was determined by 7-amino-actinomycin D (7-AAD) staining. These results are representative of five independent experiments. MHCII, MHC class II. Journal of Investigative Dermatology 2003 121, 745-750DOI: (10.1046/j.1523-1747.2003.12492.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Electroporation of DC does not interfere with their maturation process. After transfection of DC with EGFP encoding mRNA, cells were incubated with or without LPS. After 24 h, the expression of surface markers of transfected (EGFP+) and nontransfected DC from the same experiment were analyzed by FACScalibur. Isotype controls are shown by dotted lines, immature DC are presented with filled histograms, and LPS-treated cells are represented by bold lines. These results are representative of five independent experiments. GFP, green fluorescent protein. MHCII, MHC class II. Journal of Investigative Dermatology 2003 121, 745-750DOI: (10.1046/j.1523-1747.2003.12492.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Transfected DC are able to present the LCMV glycoprotein to transgenic CD8 T cells specific for the 33 to 41 epitope. Transgenic T cells (5×104 cells per well) were incubated with graded numbers of DC transfected with LCMV glycoprotein mRNA (▪), DC pulsed with 10–7 M glycoprotein 33 to 41 peptide (□), or 5×103 DC pulsed with different concentrations of glycoprotein 33 to 41 peptide (). *DC/transgenic CD8 T cell ratio 1 : 10. These results are representative of two independent experiments. gp, glycoprotein; tg, transgenic. Journal of Investigative Dermatology 2003 121, 745-750DOI: (10.1046/j.1523-1747.2003.12492.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Immunization with DC electroporated with LCMV mRNA leads to expansion of antigen-specific CD8+ T cells as determined by staining with MHC class I tetramers. Density plot of blood cells stained with glycoprotein 33 to 41 tetramer and CD8 from C57/BL6 mice immunized under the indicated conditions and then challenged with LCMV WE. Peripheral blood mononuclear cells from mice infected with LCMV WE but not challenged were stained with tetramer 13 d postinfection (200 PFU LCMV WE). The percentage of tetramer-positive cells among CD8+ T cells is indicated in each panel. Nonpulsed DC induced 0.29% tetramer-positive cells among CD8+ T cells. This experiment is representative of two independent experiments. gp, glycoprotein. Journal of Investigative Dermatology 2003 121, 745-750DOI: (10.1046/j.1523-1747.2003.12492.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Protection of mice immunized with DC transfected LCMV glycoprotein encoding mRNA. Mice were immunized twice in weekly intervals with the indicated numbers of DC. One week later, mice were challenged with 200 PFU LCMV (WE strain). Five days later, LCMV titers were determined in the spleen. *p=0.05; PFU per g, PFU per gram wet tissue. These results are representative of five independent experiments. gp, glycoprotein. Journal of Investigative Dermatology 2003 121, 745-750DOI: (10.1046/j.1523-1747.2003.12492.x) Copyright © 2003 The Society for Investigative Dermatology, Inc Terms and Conditions