CD73 is expressed by proliferating CLL cells and is enriched in perivascular areas. (A) Triple staining of a representative CLL LN section with anti-CD23 (green), anti-CD2 (red), and anti–Ki-67 (blue). CLL proliferation centers are defined as areas enriched in Ki-67⁺ (blue) CD23⁺ (green) CLL cells and in CD2⁺ T lymphocytes (red). Original magnification ×20 for image on the left and ×63 for that on the right. Scale bar represents 75 μm. (B) Cumulative analysis of CD2 pixel intensity (arbitrary units, a.u.) indicates that expression is significantly higher in Ki-67⁺ than in Ki-67⁻ areas. (C) Triple staining for CD23 (green), CD73 (red), and Ki-67 (blue). Original magnification ×63; zoom factor of 3 for image on the right. Scale bar represents 25 μm. (D) Cumulative data of CD73 pixel intensity (a.u.) from Ki-67⁺ and Ki-67⁻ areas. (E) Triple staining (left) for CD23 (green), CD73 (red), and CD3 (white) and double staining (right) for CD73 (red) and CD3 (white). Original magnification ×63; zoom factor of 3 for image on the right. Scale bar represents 25 μm. (F) Comparative analysis of CD73 pixel intensity (a.u.) in T cell–rich vs T cell–poor areas from different LN sections. (G) Triple staining for CD23 (green), CD73 (red), and CD31 (white). Original magnification ×63; zoom factor of 3 for image on the right. Scale bar represents 25 μm. (H) Cumulative analysis of CD73 pixel intensity confirms significantly higher intensity in perivascular versus nonperivascular areas. For cumulative analysis, 4 randomly chosen fields from 4 different samples were counted. All samples were analyzed using a TCS SP5 laser scanning confocal microscope (Leica Microsystems) with a 20×/0.5 and an oil immersion 63×/1.4 objective lenses, images were acquired with LAS AF Version Lite 2.4 software and processed with Photoshop (Adobe Systems). Pixel intensities were calculated with ImageJ software (freely downloadable at http://rsbweb.nih.gov/ij/), and statistical analysis was performed using Student t test.