CLL cells consistently express CXCR4 in the peripheral blood and lymphoid organs, whereas CXCR7 expression is significantly lower, (A) Healthy tonsil and CLL spleen sections were stained with antibodies against CXCR4 or CXCR7. In normal tonsil, CXCR4 is widely expressed but with noticeably stronger expression in follicles. In contrast, expression of CXCR7 is weak, and it is expressed primarily by vascular endothelial cells. In organs infiltrated by CLL (shown in spleen), expression of CXCR4 and CXCR7 is observed across the entire sample. CXCR4 staining in proliferation zones is weaker than in other regions of the sample but is still observed. CXCR7 expression is detected, but it is far weaker than CXCR4, with no strong differences observed between proliferation centers and the surrounding tissue. The insets in the upper panels are shown at increased magnification in the lower panels. HRP/DAB technique was used with hematoxylin counterstain. Scale bars, 500 µm. (B) Immunoblot showing relative protein expression levels of CXCR4 and CXCR7 in primary CLL cells, with total ERK1/2 presented as a protein expression control. Densitometric quantification of the blots is shown with adjustment relative to the protein expression control on each blot. Percentage errors were calculated as the average variation between equivalent blots across several experiments. (C) Immunocytofluorescence of CXCR4 assessed by flow cytometry using a phycoerythrin-conjugated CXCR4 antibody for a representative case (shaded graph), with the isotype-control peak shown as a dotted line. (D) Median CXCR4 staining of CLL PBMCs from 10 cases was assessed with flow cytometry using a phycoerythrin-conjugated CXCR4 antibody (●) compared with isotype control (⃝). The median and range of each distribution are represented by horizontal lines. Strong CXCR4 expression is consistently observed, although a significant variation in median staining level was also seen.