Technical Reference #3089

3089.

Transport of Streptococcus pneumoniae Capsular Polysaccharide in MHC Class II Tubules Tom Li Stephen; Mario Fabri; Laura Groneck; Till A. Ro¨hn; Helena Hafke; Nirmal Robinson; Jens Rietdorf ; David Schrama; Ju¨ rgen C. Becker; Georg Plum; Martin Kro¨nke; Harald Kropshofer; Wiltrud M. Kalka-Moll, University of Cologne, PLoS Pathogens, 3(3089), (2007)
Link To Paper

Abstract:
Bacterial capsular polysaccharides are virulence factors and are considered T cell–independent antigens. However the capsular polysaccharide Sp1 from Streptococcus pneumoniae serotype 1 has been shown to activate CD4þ T cells in a major histocompatibility complex (MHC) class II–dependent manner. The mechanism of carbohydrate presentation to CD4þ T cells is unknown. We show in live murine dendritic cells (DCs) that Sp1 translocates from lysosomal compartments to the plasma membrane in MHCII-positive tubules. Sp1 cell surface presentation results in reduction of self-peptide presentation without alteration of the MHCII self peptide repertoire. In DM-deficient mice retrograde transport of Sp1/MHCII complexes resulting in T cell–dependent immune responses to the polysaccharide in vitro and in vivo is significantly reduced. The results demonstrate the capacity of a bacterial capsular polysaccharide antigen to use DC tubules as a vehicle for its transport as an MHCII/saccharide complex to the cell surface for the induction of T cell activation. Furthermore retrograde transport requires the functional role of DM in self peptide–carbohydrate exchange. These observations open new opportunities for the design of vaccines against microbial encapsulated pathogens.

Materials & Methods:
Live fluorescent microscopy. To investigate intracellular trafficking of Sp1 live cell imaging was performed. Cells were plated for 30 min on poly-D-lysine–pre-coated number 1.5 coverslips attached to 35-mm dishes (MatTek http://www.mattek.com) and fresh medium was added. To study mechanisms of internalization of Sp1 cells were incubated with competitors or chemical inhibitors for 30 min at 4 8C or 37 8C before Sp1 treatment. To monitor Sp1 APCs were loaded with markers for cellular compartments before or at the same time point of Sp1 addition. Cells were washed before and after Sp1 treatment three times in ice-cold medium. Inverted fluorescent microscopy was performed on an Olympus IX81 microscope (http:// www.olympus-europe.com/microscopes/index.htm). Temperature control at 37 8C was achieved with a heating dish. Acquisition was performed using AnalySIS Imaging System software (Olympus http:// www.olympus.de). Confocal microscopy was done on a PerkinElmer UltraView LCI spinning disc system (http://las.perkinelmer.com) equipped with a suitable multi-band beamsplitter and a MellesGriot Omnichrome 643-RYB-A02 ArKr gas laser (http://www.mellesgriot. com) providing 488-nm and 568-nm lines for excitation. A Nikon Plan Fluor 3100 1.3NA oil immersion objective (http://www.nikon. com) and 525/50 and 607/45 emitter filters were used for GFP FITC Alexa 488 and Alexa 594 and Texas Red stains respectively.

Microscopic Technique
Live fluorescent microscopy

Cell Type(s)
dendritic cells