Figure 3.

Binding of 10E8_scFv-exos to HIV Env-expressing cells in vitro.(A) Confocal microscopic images show colocalization of the exosomes and the membrane protein Env fused with GFP on the target CHO cells. Significant colocalization of DiI-labeled exosomes (red) with the Env protein was observed. (B) Exosomes purified from HEK293T were labeled with DiI and incubated with Env⁺ and Env^- cells at 37 °C for 24 h. 10E8_scFv-exos showed significantly higher binding to Env⁺ cells compared with the naked exosomes. (C) Env⁺ cells specifically bound to 10E8_scFv-exos. The mixture of Env^- and Env⁺ cells were treated with different doses of 10E8_scFv-exos. Binding curve of 10E8_scFv-exos to the Env^- fraction and the Env⁺ fraction were measured by the counts of cells that absorbed DiI- labeled exosomes (Exos-DiI). Red line shows the binding curve of the Env^- cell fraction; Blue line shows the binding curve of the Env⁺ cell fraction. Number shows the ratio of Exos-DiI- positive cells in the Env⁺ fraction (Blue) or Env^- fraction (Red). As the dose of 10E8_scFv-exos was reduced, Env⁺ cells retained a high binding curve (Blue) but Env^- cells binding curve (Red) decreased. (D) Uptake of DiI-labeled 10E8_scFv-exos was compared between that in the Env^low fraction and Env^high fraction of the Env⁺ CHO cells. Binding curves are shown by the Exos-DiI counts in each cell fraction. Red line shows the binding curve of the Env^low cell fraction; Blue line shows the binding curve of the Env^high cell fraction. (E) 10E8_scFv-exos adsorption onto Env⁺ cells could be competitively inhibited by soluble 10E8 IgG. Blank and Exos were used as control.