CXCR5 determines localization and migration speed in B-cell follicles and T-cell area. (A) PLN homing and egress of WT and CXCR5^−/− B cells. Each dot represents the ratio of WT and CXCR5^−/− B cells from pooled PLNs of 1 recipient mouse before and 12 hours after Mel-14 treatment. (B) Three-dimensional reconstruction of a part of the selective plane illumination microscopy–scanned PLN shown in supplemental Figure 1. Asterisks depict accumulation of WT B cells in areas lacking HEV-representing B-cell follicles, arrows highlight the close association of CXCR5^−/− B cells with HEV. Grid length, 123 µm. (C) Euclidian distance analysis of WT and CXCR5^−/− B cells to the nearest HEV. CXCR5^−/− B cells are, on average, closer to HEV located in the T-cell area (31 µm vs 61 µm), similar to adoptively transferred T cells (39 µm; not shown). (D) Schematic outline of the 2-P microscopy experiments with anatomical landmarking. WT B cells (blue) and MECA-79-Alexa633 were used to delineate the B-cell follicle and HEV of the T-cell area, respectively. Low numbers of WT and CXCR5^−/− B cells were transferred for cell tracking. (E) Example of 2-P microscopy image (top left panel) and microenvironmental classification (top right panel). Individual WT (bottom left panel) and CXCR5^−/− (bottom right panel) B-cell tracks are depicted. Scale bar, 40 µm. (F) Percentage of microenvironmental track distribution as determined by 2-P microscopy. (G) Track speeds of WT and CXCR5^−/− B cells in T-cell area and B-cell follicle. Each dot represents an individual track. The red bar represents the mean. (H) Turning angle distribution of WT and CXCR5^−/− B cells in T-cell area and B-cell follicle. (I) Motility coefficients of WT and CXCR5^−/− B cells in T-cell area and B-cell follicles. Data in F through I are pooled from 7 mice, 12 image sequences, and 697 WT and 591 CXCR5^−/− B-cell tracks. Fo, B-cell follicle; H, HEV; T, T-cell area. **P < .01, ***P < .001.