Anti-adenoviral immune responses: a novel adenoviral reporter system to characterize IFNγ secretion from T cells onto virally infected cells in the brain in vivo. Part I: vector construction and in vitro characterization.

PUNTEL; BARRET; YANG; KROEGER; CASTRO; LOWENSTEIN

Boston, Massachisets, USA

Congreso; American Society of Gene Therapy; 2008

Anti-adenoviral immune responses: a novel adenoviral reporter system to characterize IFNγ secretion from T cells onto virally infected cells in the brain in vivo. Part I: vector construction and in vitro characterization.Puntel M., Barret R., Jieping Yang, Kroeger K.M., Castro M. G., Lowenstein P.R.Board of Governors’ Gene Therapeutics Research Institute, Cedars-Sinai Medical Center and Department of Medicine and Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, 90048. Correspondence: lowensteinp@cshs.orgImmunological synapses could be a special structure that allows for close communication between the antigen presenting cell and the anti-viral immune cell. We have already described the polarization of interferon gamma (IFNγ) in the CD8Tcell opposite to the infected astrocyte. A novel approach to study the immunological synapse and the fate of the infected astrocyte after immunization is to clarify the role of chemokines that the expressed by the CD8Tcell. Few Interferon Gamma Sensitive Element (GAS) elements have been described that confer IFNγ inducible expression of genes (i.e. MHCII, CD40, ). Here we describe the construction of novel Adenoviral bi-cistronic vectors encoding for the expression of different transgenes (i.e. Cre recombinase, Enhanced Green Fluorescent Protein, and Firefly Luciferase) driven by a GAS promoter; and a second cassette for EGFP under the human CMV promoter that confers constitutive expression of EGFP, or Thymidine kinase allowing for infected cell identification.We tested the inducible transgene expression under the presence of recombinant IFNgamma in vitro. For this, we infected different glioma cell lines and found that recombinant IFNγ did turn on the expression of luciferase our marker transgene and further we found Cre recombinase in a population of Ad infected cells (EGFP positive cells). Further, we describeWe conclude that IFNg regulated transgene expression constitutes a valuable tool for the study of IFNgamma targeting on the APC, as a downstream consequence of the close opposition and the formation of the IS structures between a CD8T cell and the infected cells in the brain of immune mice.