M2 macrophages trigger DC-SIGN–dependent BCR activation in FL B cells. (A) Purified CD14⁺ monocytes were differentiated into M1 vs M2 macrophages before staining with SytoxBlue, mouse IgG1 anti-CD68 mAb, and mouse IgG2b anti-DC-SIGN mAb. When indicated, IL-4 was omitted from terminal M2 differentiation. (B) Purified Hi-B FL and Lo-B FL B cells were cultured with M2 macrophages for 1 hour before fixation, and staining of macrophages with mouse IgG2b anti-DC-SIGN followed by A488-donkey anti-mouse IgG2b, and B cells with A549-goat anti-human IgM. Nuclei were counterstained with SytoxBlue. (C) Quantification of adherent FL B cells per 100 macrophages in the presence of anti-DC-SIGN blocking mAb or mouse IgG2b isotype control (n = 6 for Hi-B FL and n = 3 for Lo-B FL). ***P < .001. Scale bar, 15 µM. (D) Purified Hi-B FL B cells were cocultured for 24 hours with M2 macrophages in the presence of anti-DC-SIGN blocking mAb or isotype control. The percentages of CD19⁺Topro-3⁻ viable B cells were then evaluated and compared with that obtained in the presence of isotype control. Three different experiments were shown.