title: Stable silencing of the glucocorticoid receptor in myelin-specific T effector cells by retroviral delivery of shRNA: insight into neuroinflammatory disease.
abstract: Autoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factors remain partially elusive due to the lack of appropriate methods to achieve gene inactivation. Here we describe a protocol for stable gene silencing in differentiated rat T cells by retroviral transfer of small hairpin RNAs. Through the combination of an expression cassette containing the green fluorescent protein with a puromycin selection cassette this allows for the generation of pure knockdown cell lines suitable for tracking in animals. Exemplified for the glucocorticoid receptor, we demonstrate that gene silencing renders T effector cells unresponsive to ligand-induced apoptosis and gene regulation without affecting their ability to induce EAE in rats. Interestingly, glucocorticoid administration remains effective in the treatment of EAE despite strongly diminished glucocorticoid receptor expression in antigen-specific T cells. This highlights an important role of other cell types and bystander T cells as targets of glucocorticoid therapy. Collectively, our approach provides a simple tool for stable and efficient gene silencing in T effector cells, which should help to better understand brain autoimmune pathophysiology.
authors.affiliation: Department of Cellular and Molecular Immunology, University of Göttingen Medical School, Göttingen, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Tischner
authors.forename: Jens
authors.initials: J
authors.lastname: van den Brandt
authors.forename: Andreas
authors.initials: A
authors.lastname: Weishaupt
authors.forename: Fred
authors.initials: F
authors.lastname: Lühder
authors.forename: Marco J
authors.initials: MJ
authors.lastname: Herold
authors.forename: Holger M
authors.initials: HM
authors.lastname: Reichardt
mesh_terms: D019264:Adoptive Transfer; D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000637:transplantation*; D004681:Encephalomyelitis, Autoimmune, Experimental / Q000188:drug therapy / Q000276:immunology*; D020868:Gene Silencing; D005938:Glucocorticoids / Q000494:pharmacology / Q000627:therapeutic use*; D034622:RNA Interference / Q000276:immunology; D051381:Rats; D011917:Rats, Inbred Lew; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D012190:Retroviridae
chemical_list: D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid