Transfer of Nef protein from Jurkat T cells into hepatocytes.

<p>(A) Formation of conduits in nef.GFP-expressing Jurkat T cells. gfp- or nef.GFP-expressing Jurkat T cells were adhered to the poly-lysine coated cover slip, and cell morphology was investigated by confocal microscopy. Arrows indicate conduits. (B) Extension of conduits from nef.GFP-expressing Jurkat cells to hepatocytes. RLuc replicon cells grown onto a poly-lysine coated cover slip were stained with SP-DilC18(3), and the stained cells (red) were co-cultured with nef.GFP-expressing Jurkat cells (green) for 24 hr in complete DMEM media. Extension of conduits from Jurkat cells to RLuc cells (arrows) was then analyzed by confocal microscopy. (C) Quantification of Nef transferred from Jurkat to RLuc. (Co-cultivation) RLuc (1×10⁶ cells) stained with SP-DilC18(3) was co-cultivated with the same number of gfp- (closed bar) or nef.GFP (open bar)-expressing Jurkat cells for 24 hr in a 6 well plate, as described above. After Jurkat cells were removed by gentle wash with PBS, RLuc cells were detached from the plate by trypsinization, washed twice, and subjected to flow cytomery to quantify the transfer of GFP to the target RLuc cells. (Exosomes) The same number of RLuc cells was cultured with exosomes in the culture supernatants of gfp- or nef.GFP-expressing Jurkat cells, and the transfer of Nef in the exosomes to the hepatocytes was quantified in a similar manner, as described. Bar graph indicates % presentation of green positive cells to total red positive cells (RLuc cells).