Figure 3
Figure 3. Immature MoDCs release part of macropinocytosed Ag under its native form and B cells capture them. (A) MoDCs were pulsed 1 hour with 1 mg/mL HRP and then washed and membrane stripped or not, either with trypsin or with acid wash. Enzymatic activity of HRP was detected in the supernatants at different time points. (B) HRP release capacity of 5 different donors was measured as in panel A during 1 hour and up to 6 hours. Data are mean ± SEM of 5 independent experiments. (C) HRP releasing capacity was measured for immature MoDCs and monocyte-derived macrophages and expressed as a ratio of HRP found inside and outside cells after 1-hour pulse and 4-hour chase. Data are mean ± SEM of 3 independent experiments. (D) DCs were pulsed for 1 hour with fluorescent F(ab)′2 and then treated or not with acid wash and analyzed by flow cytometry. Data are mean ± SEM of 4 independent experiments. (E) DCs were pulsed for 1 hour with fluorescent F(ab)′2 or not (NS) and then chased for 4 hours, and the F(ab)′2 concentration in supernatants was measured by ELISA. (F) Primary B cells were incubated for 4 hours with increasing concentrations of fluorescent F(ab)′2 and then analyzed by flow cytometry. Results are expressed both in mean fluorescence intensities and in percentage of positive B cells. (G) MoDCs were unpulsed (NS) or pulsed with 50 μg/mL of Cy2-F(ab)′2 for 1 hour and then fixed with PFA (+PFA) or not (−PFA) and chased for 4 hours at 37°C. Supernatants were collected and applied to autologous primary B cells for 4 or 18 hours. As control (Ctrl+), B cells were directly incubated with 1 μg/mL of Cy2-F(ab)′2 or not (Ctrl−) for 4 hours. B cells were analyzed by flow cytometry, and results are shown as a histogram. (H) MoDCs were treated as in panel G, and results are expressed as percentage of Cy2-F(ab)′2-positive B cells compared with B cells incubated directly for 4 hours with 1 μg/mL of Cy2-F(ab)′2. Data are mean ± SEM of 4 independent experiments. (I) B cells were cocultured for 4 hours or 18 hours with DCs nonstimulated (NS) or pulsed with Cy2-F(ab)′2+ and fixed (+PFA) or not (−PFA) before B-cell addition. Quantifications were made with the ImageStream technology compiled with results obtained by flow cytometry. Results are expressed in percentages of CD19+/F(ab)′2+ cells compared with control B cells incubated directly for 4 hours with 1 μg/mL of Cy2-F(ab)′2. Data are mean ± SEM of 3 independent experiments. (J) DCs were pulsed with Cy2-F(ab)′2 or not (NS) for 1 hour and then cocultured with B cells for 4 hours. Wide field fluorescent microscopy Z-stack images show B cells identified by CD19 (red) and positive for Cy2-F(ab)′2 (green). Bar represents 5 μm.

Immature MoDCs release part of macropinocytosed Ag under its native form and B cells capture them. (A) MoDCs were pulsed 1 hour with 1 mg/mL HRP and then washed and membrane stripped or not, either with trypsin or with acid wash. Enzymatic activity of HRP was detected in the supernatants at different time points. (B) HRP release capacity of 5 different donors was measured as in panel A during 1 hour and up to 6 hours. Data are mean ± SEM of 5 independent experiments. (C) HRP releasing capacity was measured for immature MoDCs and monocyte-derived macrophages and expressed as a ratio of HRP found inside and outside cells after 1-hour pulse and 4-hour chase. Data are mean ± SEM of 3 independent experiments. (D) DCs were pulsed for 1 hour with fluorescent F(ab)′2 and then treated or not with acid wash and analyzed by flow cytometry. Data are mean ± SEM of 4 independent experiments. (E) DCs were pulsed for 1 hour with fluorescent F(ab)′2 or not (NS) and then chased for 4 hours, and the F(ab)′2 concentration in supernatants was measured by ELISA. (F) Primary B cells were incubated for 4 hours with increasing concentrations of fluorescent F(ab)′2 and then analyzed by flow cytometry. Results are expressed both in mean fluorescence intensities and in percentage of positive B cells. (G) MoDCs were unpulsed (NS) or pulsed with 50 μg/mL of Cy2-F(ab)′2 for 1 hour and then fixed with PFA (+PFA) or not (−PFA) and chased for 4 hours at 37°C. Supernatants were collected and applied to autologous primary B cells for 4 or 18 hours. As control (Ctrl+), B cells were directly incubated with 1 μg/mL of Cy2-F(ab)′2 or not (Ctrl−) for 4 hours. B cells were analyzed by flow cytometry, and results are shown as a histogram. (H) MoDCs were treated as in panel G, and results are expressed as percentage of Cy2-F(ab)′2-positive B cells compared with B cells incubated directly for 4 hours with 1 μg/mL of Cy2-F(ab)′2. Data are mean ± SEM of 4 independent experiments. (I) B cells were cocultured for 4 hours or 18 hours with DCs nonstimulated (NS) or pulsed with Cy2-F(ab)′2+ and fixed (+PFA) or not (−PFA) before B-cell addition. Quantifications were made with the ImageStream technology compiled with results obtained by flow cytometry. Results are expressed in percentages of CD19+/F(ab)′2+ cells compared with control B cells incubated directly for 4 hours with 1 μg/mL of Cy2-F(ab)′2. Data are mean ± SEM of 3 independent experiments. (J) DCs were pulsed with Cy2-F(ab)′2 or not (NS) for 1 hour and then cocultured with B cells for 4 hours. Wide field fluorescent microscopy Z-stack images show B cells identified by CD19 (red) and positive for Cy2-F(ab)′2 (green). Bar represents 5 μm.

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