Figure 7. Soluble human ephrinA4 in HEV of lymphadenopathies inversely correlates with apoptotic cells near HEV dramatically enhancing the number and viability of human CLL cells recovered from lymph node of adoptively transferred mice.

A-B. Seven μm thick sections from frozen CLL lymphadenopathies (LA n.1, 2 and 3 corresponding to CLL patients n. 3, 18 and 28, respectively) were fixed in acetone and sequentially incubated with A) TUNEL (white) and antibodies against PNAd (red) or B) antibodies against PNAd (magenta), ephrinA4 (green), EphA2 (red). In all cases nuclei were counterstained with Hoechst (blue). Fluorescent images were taken in a laser confocal microscope (TCS SP-2 AOBS, Leica). A) Representative low magnification images (20x) of areas near the capsule (“Cortical” region, left panel) or deeper (>200um far from capsule, “Inner” regions; right panel). ii) Frequency of apoptotic nuclei around (≤ 20 μm) or separated from HEV in cortical and inner LA regions (*p<0.05; **p<0.01; ***p<0.001). B) i) HEV (PNAd+, magenta; dotted line) showed strong ephrinA4 staining co-localizing with EphA2. (63x). ii) Regions of interest (ROI) were traced around HEV according to EphA2 staining (left). Intensity of ephrinA4 staining within ROIs was measured (pseudocolor scale inset). iii) HEVs containing ≥ 10% pixels above 200 gray-scales were considered as positive. Whisper-box plots of > 20 measured fields from each LA section. Unpaired two-tailed Student's t-test significance values were *p<0.01; **p<0.005; ***p<0.001. C. Human CLL cells were stained with CFSE fluorescent tracer and adoptively transferred into germ-free young Balb/c mice (2×10⁷ per mice) by intravenous injection through the tail vein along with increasing concentrations of human soluble ephrinA4 purified from the sera of patients. Mice were sacrificed 24 hours later and cell suspensions from popliteal lymph nodes stained with an anti-mouse CD45 antibody, AnnexinV-PE and 7AAD for flow cytometry analysis. Human CLL cells were gated according to CFSE positivity and negativity for anti-mouse CD45 (Suppl. Figure S1). Data (mean±SD) were compared to mice receiving human CLL cells in PBS without ephrinA4. Five animals were used per sample and experimental condition. Paired two-tailed Student's T test significance values were *p<0.05; **p<0.01; ***p<0.001.