Ibrutinib impacts B cells/NLC interaction. (A) Graph shows the CCL21mRNA MFI values detected in NLC cells exposed or not to ibrutinib 24 hours before analysis (P = ns; n = 16) or in NLCs derived from patients treated with ibrutinib (n = 4). (B) Image galleries of single macrophages or aggregates (as defined in Figure 3A) exposed or not to ibrutinib for 24 hours (upper panels) or derived from patients treated with ibrutinib (lower panels) showing CD19 (purple), CCL21 (green), CD163 (yellow), CCL21 mRNA (red), and CD68 (cyan) fluorescent signals and Brightfield (BF) in permeabilized conditions and in presence of BFA (a) or nonpermeabilized conditions and no BFA treatment (b). (C) Graph comparing the ratios of colocalized B cells/NLCs after 4 hours of coculture exposed (+) or not (−) to ibrutinib for 24 hours (n = 6) or in cocultures derived from patients treated long-term with ibrutinib (n = 3). Statistical analysis was carried out by Student t-test (***P < .001; ****P < .0001) comparing untreated and treated samples. (D) Representative images of interactions between CLL B cells (green) and CD68⁺ NLCs (red) after 4 hours of coculture priorly treated or not with ibrutinib (24 hours) or in cocultures derived from patients treated with ibrutinib (objective ×10; scale bar represents 10 μm). The areas in the white dotted boxes in the upper panel are ×2.5 zoomed in the lower panels to show B cell/NLC interactions.