EphA2 is the main EFNA4 receptor found in the CD31+ vascular endothelium of human LN. (A) Flow cytometric analyses of EFNA4-Fc binding by LN cells from patients with CLL (a representative experiment is shown, CLL LN no. 2). Cell suspensions (3 × 105/sample tube) from fresh CLL LN biopsies were preincubated with hFc before the addition of poly-His–tagged EFNA4-Fc homodimers (0.5 μg/106 cells), then followed by an anti His-FITC mAb. The percentage of EFNA4-Fc binding cells (black profiles) was analyzed within gated subpopulations according to CD5, CD19, and CD31 costainings (empty profiles: control anti-His–FITC stainings). (B) RT-PCR analyses of EphA mRNAs expression of enriched CD2− CD5− CD19− CD13− CD14− CD31+ LN cells from CLL patients or control subjects (≥ 95% purity, 2-5 × 105 cells per LN fragment). (C-D) Immunofluorescence analysis of EphA receptor expression on LN tissue sections. LN cryosections (8 μm thick) were immunostained for detection of EphA2, EphA3, EphA4, or EphA8 (Alexa Fluor 546, green), CD31 (Alexa Fluor 647, red), and CD19 (Alexa Fluor 488, blue). Objectives: 20× multi-immersion, 1.20 NA. (D) High magnification images (objective: 63× oil immersion, 1.40 NA) of CLL or normal LN showing expression of EphA2 (green) in the high endothelial venules (CD31+ structures, red; CLL LN no. 2 in panels C-D; Normal reactive LN in panel D). Fluorescence images were acquired with a confocal microscope (Leica TCS-SP2 AOBS).
