Technical Reference #2059

2059.

Quantum Dots for Tracking Dendritic Cells and Priming an Immune Response In Vitro and In Vivo Debasish Sen; Thomas J. Deerinck; Mark H. Ellisman; Ian Parker; Michael D. Cahalan, University of California Irvine, PloS ONE, 3(2059), (2008)
Link To Paper

Abstract:
Dendritic cells (DCs) play a key role in initiating adaptive immune response by presenting antigen to T cells in lymphoid organs. Here we investigate the potential of quantum dots (QDs) as fluorescent nanoparticles for in vitro and in vivo imaging of DCs and as a particle-based antigen-delivery system to enhance DC-mediated immune responses. We used confocal two-photon and electron microscopies to visualize QD uptake into DCs and compared CD69 expression T cell proliferation and IFN-c production by DO11.10 and OT-II T cells in vivo in response to free antigen or antigen-conjugated to QDs. CD11c+ DCs avidly and preferentially endocytosed QDs initially into small vesicles near the plasma membrane by an actin-dependent mechanism. Within 10 min DCs contained vesicles of varying size motion and brightness distributed throughout the cytoplasm. At later times endocytosed QDs were compartmentalized inside lysosomes. LPS-induced maturation of DCs reduced the rate of endocytosis and the proportion of cells taking up QDs. Following subcutaneous injection of QDs in an adjuvant depot DCs that had endocytosed QDs were visualized up to 400 mm deep within draining lymph nodes. When antigen-conjugated QDs were used T cells formed stable clusters in contact with DCs. Antigenconjugated QDs induced CD69 expression T cell proliferation and IFN-c production in vivo with greater efficiency than equivalent amounts of free antigen. These results establish QDs as a versatile platform for immunoimaging of dendritic cells and as an efficient nanoparticle-based antigen delivery system for priming an immune response.

Materials & Methods:
Harvested DCs were cultured overnight in glass-bottom culture dishes (MattekTM) incubated with 2 nM QD-containing medium for varying times at 37uC and processed as described [23]. Briefly DCs were fixed with 2% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M sodium cacodylate (Ted PellaTM) buffer (pH 7.3) for 20 min washed with 0.1% cacodylate buffer and postfixed with 1% osmium tetroxide (Electron Microscopy Sciences) solution for 30 min. Subsequently the DCs were counterstained with 4% uranyl acetate (Electron Microscopy Sciences) for 30 min washed with distilled water dehydrated in 100% ethanol and embedded in DurcupanTM ACM resin (FlukaTM).

Microscopic Technique
Electron Microscopy

Cell Type(s)
DC cells