title: Generation of transgenic mice expressing EGFP protein fused to NP68 MHC class I epitope using lentivirus vectors.
abstract: Immune tolerance to self-antigens is a complex process that utilizes multiple mechanisms working in concert to maintain homeostasis and prevent autoimmunity. Considerable progress in deciphering the mechanisms controlling the activation or deletion of T cells has been made by using T cell receptor (TCR) transgenic mice. One such model is the F5 model in which CD8 T cells express a TCR specific for an epitope derived from the influenza NP68 protein. Our aim was to create transgenic mouse models expressing constitutively the NP68 epitope fused to enhanced green fluorescent protein (EGFP) in order to assess unambiguously the relative levels of NP68 epitope expressed by single cells. We used a lentiviral-based approach to generate two independent transgenic mouse strains expressing the fusion protein EGFP-NP68 under the control of CAG (CMV immediate early enhancer and the chicken β-actin promoter) or spleen focus-forming virus (SFFV) promoters. Analysis of the pattern of EGFP expression in the hematopoietic compartment showed that CAG and SFFV promoters are differentially regulated during T cell development. However, both promoters drove high EGFP-NP68 expression in dendritic cells (pDCs, CD8α(+) cDCs, and CD8α(-) cDCs) from spleen or generated in vitro following differentiation from bone-marrow progenitors. NP68 epitope was properly processed and successfully presented by dendritic cells (DCs) by direct presentation and cross-presentation to F5 CD8 T cells. The models presented here are valuable tools to investigate the priming of F5 CD8 T cells by different subsets of DCs.
authors.affiliation: INSERM, U851, Lyon, F-69007, France.
authors.forename: Martine
authors.initials: M
authors.lastname: Tomkowiak
authors.forename: Raffaella
authors.initials: R
authors.lastname: Ghittoni
authors.forename: Marie
authors.initials: M
authors.lastname: Teixeira
authors.forename: Bariza
authors.initials: B
authors.lastname: Blanquier
authors.forename: Judit
authors.initials: J
authors.lastname: Szécsi
authors.forename: Didier
authors.initials: D
authors.lastname: Nègre
authors.forename: Denise
authors.initials: D
authors.lastname: Aubert
authors.forename: Charles-Antoine
authors.initials: CA
authors.lastname: Coupet
authors.forename: Molly
authors.initials: M
authors.lastname: Brunner
authors.forename: Els
authors.initials: E
authors.lastname: Verhoeyen
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Thoumas
authors.forename: François-Loïc
authors.initials: FL
authors.lastname: Cosset
authors.forename: Yann
authors.initials: Y
authors.lastname: Leverrier
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Marvel
mesh_terms: D000818:Animals; D003713:Dendritic Cells / Q000378:metabolism; D018984:Epitopes, T-Lymphocyte / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D015395:Histocompatibility Antigens Class I / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes / Q000378:metabolism; D019076:Transgenes; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D018984:Epitopes, T-Lymphocyte; D015395:Histocompatibility Antigens Class I; D014764:Viral Proteins; D049452:Green Fluorescent Proteins