Figure 4
Figure 4. Extracellular release of macropinocytosed Ag occurs from late endosomes. (A) DCs were incubated with Cy2-F(ab)′2 for 1 hour and then either fixed before B-cell addition (PFA) or cocultured with B cells for 4 hours in the presence of 25 μg/mL monensin (Mo) or not (NT). MoDC images were obtained with the ImageStream multispectral imaging technology. (B) DCs were treated as in panel A. Quantifications show the percentage of cell-associated F(ab)′2 in MoDCs after each treatment, compared with cells fixed after 1-hour pulse. Data are mean ± SEM of 3 independent experiments. (C) DCs were incubated with HRP for 1 hour and then treated or not (NT) with monensin (Mo) during 4 and 6 hours of chase. Results are expressed as fold increase of extracellular HRP measured in monensin-treated cells compared with nontreated cells (NT) at each time point. (D) DCs were incubated with Cy2-F(ab)′2 for 1 hour and then treated or not (NT) with monensin (Mo) during 4 hours of chase in the presence of B cells (contact). Supernatants were collected and applied to B cells (Spnt) for 4 hours. Cell-associated Cy2-F(ab)′2 was measured by flow cytometry gated on CD19+ B cells. Fold increase of efficiency of Ag transfer from DCs to B cells, from supernatants, or in cocultures in the presence of monensin were calculated compared with control (NT) conditions. Graph is representative of 2 independent experiments. (E) MoDCs were nucleofected to express GFP alone or dominant-negative form of Rab27a (Rab27aTN-GFP) or b (Rab27bTN-GFP) fused to GFP. After 6 hours, cells were pulsed with 50 μg/mL of Cy5-F(ab)′2 for 1 hour, then chased for 4 hours at 37°C, and fixed and analyzed by wide field fluorescent microscopy. (F) MoDCs were nucleofected to express dominant-negative forms of Rab27a (Rab27aTN-GFP and Rab27aNI-GFP) or Rab27b (Rab27bTN-GFP and Rab27bNI-GFP) fused to GFP and then analyzed by flow cytometry to measure the quantity of intracellular Cy5-F(ab)′2+ in GFP+ and GFP− cells (Neg). Results are expressed as ratio compared with Ag measured in GFP− DCs. Data are mean ± SEM of 3 independent experiments.

Extracellular release of macropinocytosed Ag occurs from late endosomes. (A) DCs were incubated with Cy2-F(ab)′2 for 1 hour and then either fixed before B-cell addition (PFA) or cocultured with B cells for 4 hours in the presence of 25 μg/mL monensin (Mo) or not (NT). MoDC images were obtained with the ImageStream multispectral imaging technology. (B) DCs were treated as in panel A. Quantifications show the percentage of cell-associated F(ab)′2 in MoDCs after each treatment, compared with cells fixed after 1-hour pulse. Data are mean ± SEM of 3 independent experiments. (C) DCs were incubated with HRP for 1 hour and then treated or not (NT) with monensin (Mo) during 4 and 6 hours of chase. Results are expressed as fold increase of extracellular HRP measured in monensin-treated cells compared with nontreated cells (NT) at each time point. (D) DCs were incubated with Cy2-F(ab)′2 for 1 hour and then treated or not (NT) with monensin (Mo) during 4 hours of chase in the presence of B cells (contact). Supernatants were collected and applied to B cells (Spnt) for 4 hours. Cell-associated Cy2-F(ab)′2 was measured by flow cytometry gated on CD19+ B cells. Fold increase of efficiency of Ag transfer from DCs to B cells, from supernatants, or in cocultures in the presence of monensin were calculated compared with control (NT) conditions. Graph is representative of 2 independent experiments. (E) MoDCs were nucleofected to express GFP alone or dominant-negative form of Rab27a (Rab27aTN-GFP) or b (Rab27bTN-GFP) fused to GFP. After 6 hours, cells were pulsed with 50 μg/mL of Cy5-F(ab)′2 for 1 hour, then chased for 4 hours at 37°C, and fixed and analyzed by wide field fluorescent microscopy. (F) MoDCs were nucleofected to express dominant-negative forms of Rab27a (Rab27aTN-GFP and Rab27aNI-GFP) or Rab27b (Rab27bTN-GFP and Rab27bNI-GFP) fused to GFP and then analyzed by flow cytometry to measure the quantity of intracellular Cy5-F(ab)′2+ in GFP+ and GFP− cells (Neg). Results are expressed as ratio compared with Ag measured in GFP− DCs. Data are mean ± SEM of 3 independent experiments.

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