title: Evaluation of Cross-presentation in Bone Marrow-derived Dendritic Cellsin vitroand Splenic Dendritic Cellsex vivoUsing Antigen-coated Beads.
abstract: Antigen presentation by MHC class I molecules, also referred to as cross-presentation, elicits cytotoxic immune responses. In particular, dendritic cells (DC) are the most proficient cross-presenting cells, since they have developed unique means to control phagocytic and degradative pathways. This protocol allows the evaluation of antigen cross-presentation bothin vitro(by using bone marrow-derived DC) andex vivo(by purifying CD8+DC from spleen after incorporation of particulate antigen) using ovalbumin (OVA)-coupled particles. Cross-presentation efficiency is measured by three different readouts: the B3Z hybridoma T cell line (Karttunenet al.,1992) and stimulation of antigen-specific CD8+T cells (OT-I) (Kurtset al.,1996), either analyzing OT-I activation by CD69 expression or OT-I proliferation after labeling them with carboxyfluorescein succinimidyl ester (CFSE). By using this approach, we could show recently that DCs are able to increase cross-presentation efficiency transiently upon engagement of TLR4 (Alloattiet al.,2015).
authors.affiliation: Institute Curie, PSL Research University, INSERM U932, Paris, France.
authors.forename: Andrés
authors.initials: A
authors.lastname: Alloatti
authors.affiliation: Institute Curie, PSL Research University, INSERM U932, Paris, France; Department of Virology, University of Buenos Aires, Buenos Aires, Argentina.
authors.forename: Fiorella
authors.initials: F
authors.lastname: Kotsias
authors.affiliation: Institute Curie, PSL Research University, INSERM U932, Paris, France; VIB Inflammation Research Center, Unit of Molecular Signal Transduction in Inflammation, Ghent, Belgium; Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
authors.forename: Eik
authors.initials: E
authors.lastname: Hoffmann
authors.affiliation: Institute Curie, PSL Research University, INSERM U932, Paris, France.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Amigorena