TLR4 ligand exposure triggers extensive B cell interactions in the LN follicle. (A) B cell clusters. MIP from TP-LSM of TLR4-stimulated B cell clusters (red circles) 1 d after transfer. Two separate regions (top and bottom) are shown. Bar, 20 µm. (B) Tracking clustered B cells. MIP and tracks of TLR4-stimulated B cell (green) 48 h after transfer. White circles indicate highly interactive cells and the time colored tracks (red, blue, and yellow) show movement of the TLR4-stimulated B cells over 20 min. Arrows point to several representative tracks. Bar, 20 µm. (C) Quantification of cell–cell interactions. The durations in which LPS-activated B cells contacted other activated B cells or nonstimulated B cells contacted other nonstimulated B cells are shown (left). Each cell was tracked for 15 min and the number of interactions is shown (right). Black bars indicate mean values (*, P < 0.05). (D) B cell–B cell interactions (arrows). MIPs of intravital TP-LSM of TLR4-stimulated B cells 72 h after transfer in the iLN. Image spaces are 130 × 115 × 33 µm (first and second) and 130 × 130 × 33 µm (third and fourth). Individual cells were tracked every 30 s for 30 min and their previous 20 positions are indicated by a dragon tail. Three frames from two sets of imaging data showing colliding LPS-stimulated B cells at various time points during the imaging. Track 1 is further analyzed in F. Bar, 20 µm. (E) Tracking interacting B cells. Tracks of TLR4-stimulated B cells 72 h after transfer. Imaging space is 240 × 190 × 33 µm. Individual tracks were sorted by duration and the top 18 of 60 tracks were selected. Interactions with other TLR4-stimulated B cells (both selected and nonselected) in the imaging space are indicated with arrows. Track 2 is further analyzed in F. (F) Velocity profiles. Instantaneous velocities of two tracks from TLR4-stimulated B cells are shown. Cell–cell interactions are indicated by shading. The tracks are from D and E. Imaging data shown is from the analysis of three recipient mice.