Figure 1.

CD40L and gp350 are incorporated into EVs and confer B-cell tropism.

HEK293 cells were transfected with CD40L and gp350 expression plasmids and EVs were isolated from conditioned cell culture supernatant by serial (ultra-) centrifugation and density gradient centrifugation. (a) The fractions of a bottom-up iodixanol gradient were analysed by Western blotting. EVs carrying CD40L and gp350 and the EV-enriched proteins CD63, TSG101 and Alix were mainly detected in fractions two and three. CL: cell lysate. (b) Electron microscopy analysis of EV preparations. For immune electron microscopy antibodies against CD63, CD40L, gp350 or an isotype control were used and detected by a gold-conjugated secondary antibody. (c) Confocal microscopy of CLL cells incubated for 24 h with CD40L+/gp350+ EVs labelled with the fluorescent dye PKH26. Cell nuclei were counterstained with DAPI (40× magnification). (d) EVs from untransfected HEK293 cells or cells expressing gp350 were incubated with PBMCs from a healthy donor or primary CLL cells. After 48 h, cells were washed, stained with CD19- and gp350-specific antibodies and analysed by flow cytometry.