CXCR4 is functional, and its surface expression segregates with B-cell phenotype in hu/SCID tumor cells. (A) Effect of CXCL12 on migration of hu/SCID lymphoma cells. Lymphoma cells were added to the chemotaxis chamber in the presence (+) or absence (–) of the following reagents: 100 ng/mL CXCL12 (SDF-1α), neutralizing anti-CXCL12 Abs (1:100), and TC14012 (1 and 5 μM). Migrated cells were recovered from the lower chamber after 2 hours at 37°C and counted. Results are the mean of 2 separate experiments executed in triplicate (± SD). The single asterisk denotes a significant difference compared with untreated cells (P < .05); 2 asterisks denote a significant inhibition by TC14012 or anti-CXCL12 Abs of the chemotactic properties of CXCL12-stimulated cells (P < .05). (B) Tumor cells were stained with the FITC-conjugated anti-CD23 mAb and the PE-conjugated anti-CXCR4 mAb before being analyzed by cytofluorimetry. The fluorograms represent the expression pattern of CD23 in a representative hu/SCID tumor sample (top) and LCL cells (bottom). (C) The expression of surface and intracellular CXCR4 (upper and lower panels, respectively) in the CD23^low (left diagrams) and CD23^int (right diagrams) tumor cell subsets is shown. A representative experiment of 3 consecutive experiments is shown. (D) Effect of CXCL12 on actin polymerization in the 2 hu/SCID lymphoma cell subsets. Lymphoma cells were labeled with PE-conjugated anti-CD23 Ab and tested by flow cytometry for CXCL12-induced cytoskeleton rearrangement. Results (mean ± SD from 2 experiments) show the kinetics of actin polymerization following addition of different concentrations of CXCL12; 100% corresponds to the baseline level.