Chemokines produced by NLCs impact B cell motility. (A) Graph comparing the mean speed of B cells loaded on NLC monolayers in 14-day culture-conditioned medium (cdt) or fresh medium. B cells were left untreated or incubated with anti-CCR7 (10 μg/mL), anti-CXCR4 (30 μg/mL), and anti-CXCR5 antibodies (20 μg/mL) before loading. CD68⁺ NLCs were incubated or not with anti-CCL21 antibody (4 μg/mL) before addition of untreated B cells. Motility was analyzed using Trackmate in ImageJ software. Only displacement lengths >10 μm were included in the analysis (n = 12). (B) Representative images of interactions between CLL B cells (green) and CD68⁺ NLCs (red) after 4 hours of coculture in conditions identical to panel A and vigorous washing (objective ×20; scale bar represents 10 μm). The areas in the white dotted boxes in the upper panel are ×3.5 zoomed in the lower panels to show B/NLC interactions. Green arrows show noninteracting B cells. (C) Graph comparing the ratios of colocalized B cells/NLCs detected in the different conditions. Postacquisition data analysis of images in panel B was performed using ImageJ software. To quantify B cells in contact with NLCs, a threshold was applied to obtain a mask of NLCs, and the number of B cells present in this mask was evaluated using the “analyze particles” tool and represented in the graph as number of B cells per NLC. Statistical analysis was carried out by Student t-test (***P < .001; ****P < .0001; n = 12).