EphA2-EFNA4 interactions can take place in the adhesion between HUVECs and B cells during TEM. (A) Flow cytometry analysis of EFNA4-Fc binding (top histogram, gray) or EphA2 expression (bottom histogram, gray) of TNF-α–activated HUVECs (>95% CD31, CD54 double positive, dot-plot; black histogram: control secondary PE-Abs; white: unstained cells). (B) CLL cells or normal B cells were cocultured for 2 hours with TNF-α–activated HUVEC monolayers, the nonadhered cells were washed out, and slides were fixed and immunostained for EphA2 (Alexa Fluor 546, red, bottom) and ICAM-1 (Alexa Fluor 488, green, top; blue, Hoechst 33 342 counterstained cell nuclei; confocal microscope: Leica TCS-SP2 AOBS; objectives: 20× oil-immersion, 1.20 NA). Note the high number of normal B cells transmigrated (dark appearance) compared with cultures containing CLL cells in the phase-contrast images. (C) Flow cytometric analyses of EphA2-Fc binding by CLL cells directly incubated with EphA2-Fc homodimers (gray, top) or after preincubation with 2 different doses of an anti-EFNA4 polyclonal Ab (0.10 or 0.20 μg/10⁶ cells, middle and bottom, respectively; white, control anti-His FITC; black, control unstained cells). A representative experiment is shown (CLL no. 1). Values are mean fluorescence intensity (MFI; mean ± SEM) from triplicates of the same sample. (D) TNF-α–activated HUVEC monolayers were cultured for 60 minutes with fluorescent preclustered EFNA4-Fc complexes (red). After cells fixation, slides were immunostained with anti-EphA2 or -EphA4 Abs (Alexa Fluor 488, green). Colocalization plots (bottom) show that EFNA4-Fc preferentially binds to EphA2 (ROI indicates region of interest). Leica TCS-SP2 AOBS confocal microscope; objective: 63× oil immersion, 1.40 NA.