CXCR5 receptor eudocytosis in CLL cells. CXCL13 induces CXCR5 receptor endocytosis in a time-dependent (A) and dose-dependent (B) fashion, and coculture with NLCs induces CXCR5 down-regulation on CLL B cells (C). (A) CXCR5 expression was determined before and after incubation of CLL cells with 1 μg/mL CXCL13 at various time points; the different time points are displayed on the horizontal axis. The vertical axis displays the mean (± STDV, n = 5) relative CXCR5 expression on the CLL cells compared with the CXCR5 expression before addition of CXCL13. (B) CLL cells were exposed to increasing concentrations of CXCL13, as displayed on the horizontal axis. Then, CLL cells were stained with anti-CXCR5 antibodies, and surface CXCR5 expression was detected by flow cytometry. Increasing concentrations of CXCL13 induced down-regulation of surface CXCR5 that was significantly lower than CXCR5 expression of controls that were not exposed to CXCL13 at higher concentrations, as indicated by the asterisks. Displayed are the mean (± SEM) relative CXCR5 expression levels from 6 different patients compared with the CXCR5 levels of the untreated controls, as indicated on the horizontal axis. (C) CXCR5 surface expression was determined at initiation of NLC cocultures (n = 13), and subsequently aliquots of CLL cells were removed from NLC cocultures to determine whether this coculture system affects CXCR5 levels. After 14 days (n = 9) and 35 days (n = 8), CLL cells displayed significantly lower CXCR5 MFIRs than the respective CLL samples at the initiation of the cultures, suggesting CXCR5 endocytosis by CXCL13 that is secreted by NLCs.