title: Small interfering RNA-mediated knockdown of notch ligands in primary CD4+ T cells and dendritic cells enhances cytokine production.
abstract: The key interaction in the adaptive immune system's response to pathogenic challenge occurs at the interface between APCs and T cells. Families of costimulatory and coinhibitory molecules function in association with the cytokine microenvironment to orchestrate appropriate T cell activation programs. Recent data have demonstrated that the Notch receptor and its ligands also function at the APC:T interface. In this study, we describe synthetic small interfering RNA (siRNA) sequences targeting the human Notch ligands Delta1, Jagged1 and Jagged2. Transfection of these siRNAs into human primary CD4(+) T cells and monocyte-derived dendritic cells leads to knockdown of endogenous Notch ligand message. Knockdown of any one of these three Notch ligands in dendritic cells enhanced IFN-gamma production from allogeneic CD4(+) T cells in MLR. In contrast, Delta1 knockdown in CD4(+) T cells selectively enhanced production of IFN-gamma, IL-2, and IL-5 in response to polyclonal stimulation, while Jagged1 or Jagged2 knockdown had no effect. Strikingly, blockade of Notch cleavage with a gamma secretase inhibitor failed to affect cytokine production in this system, implying that Delta1 can influence cytokine production via a Notch cleavage-independent mechanism. These data show for the first time that the Notch pathway can be targeted by siRNA, and that its antagonism may be a unique therapeutic opportunity for immune enhancement.
authors.affiliation: Lorantis Ltd., Cambridge, United Kingdom.
authors.forename: Yvette
authors.initials: Y
authors.lastname: Stallwood
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Briend
authors.forename: Katrina M
authors.initials: KM
authors.lastname: Ray
authors.forename: George A
authors.initials: GA
authors.lastname: Ward
authors.forename: Beverley J
authors.initials: BJ
authors.lastname: Smith
authors.forename: Emma
authors.initials: E
authors.lastname: Nye
authors.forename: Brian R
authors.initials: BR
authors.lastname: Champion
authors.forename: Grahame J
authors.initials: GJ
authors.lastname: McKenzie
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism*; D016466:CHO Cells; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D002478:Cells, Cultured; D006224:Cricetinae; D016207:Cytokines / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D003713:Dendritic Cells / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D000072100:Jagged-1 Protein; D000072101:Jagged-2 Protein; D008024:Ligands; D007959:Lymphocyte Culture Test, Mixed; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics*; D051880:Receptors, Notch / Q000037:antagonists & inhibitors* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D014162:Transfection; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology
chemical_list: D002135:Calcium-Binding Proteins; D016207:Cytokines; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; C490504:JAG2 protein, human; D000072100:Jagged-1 Protein; D000072101:Jagged-2 Protein; D008024:Ligands; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D051880:Receptors, Notch; D011994:Recombinant Proteins; D000072099:Serrate-Jagged Proteins; C064045:delta protein