Murine B cells capture native Ag from pulsed DCs ex vivo. (A) CD11c⁺ purified DCs from spleen and lymph nodes were unpulsed (NS) or pulsed with 50 μg/mL of Cy5-F(ab)′₂ anti–mouse IgG for 1 hour, and then fixed with PFA (+PFA) or not (NT) and chased for 4 hours at 37°C in the presence of LPS or Monensin (Mo). (B) Supernatants were collected and applied to negatively purified splenic and lymph nodes B cells for 18 hours. Cells were stained with anti–CD19 antibodies and analyzed by using ImageStream technology and FACSCalibur flow cytometry. (C) Results are expressed as number of F(ab)′₂-positive B cells, and the graph represents 3 independent experiments. (D) B cells negatively purified from spleen and lymph nodes were incubated with anti–CD19-PE and QDot605-F(ab)′₂ anti–mouse IgG antibodies to show that 81.1% of the CD19-positive B cells were labeled by the anti–mouse IgG antibodies. (E) Model of Ag capture and regurgitation by DCs to target B cells. Immature DCs efficiently capture Ag by macropinocytosis. Part of the Ag is then processed and presented to activate T cells. Part of the Ag remains undegraded and is regurgitated in the extracellular medium from late endocytic compartments under the control of Rab27. On release, Ag is captured by B cells via their BCR, and this transfer is enhanced by the CXCL13 chemokine. In vivo, Ag-loaded DCs found in the lymph node were CD103⁺.