IL-21R, CD132, and IL-21 expression on HL cells. Flow cytometric analysis for IL-21R and γc chain (CD132) expression on (A) primary HL tissue and (B) HL cell lines, L1236, L591, and L428. For IL-21R expression on primary HL cells we gated on the CD30⁺CD15⁺ cells and γc chain expression is gated on CD15⁺ cells. Gray histogram is isotype control. Representative of 5 different donors. (C) RT-PCR for IL-21 and actin expression on HL-infiltrating sorted T cells (n = 2). (D) Immunoblot analysis for IL-21 and actin as a loading control was performed on 3 HL cell lines (n = 3). (E) RT-PCR for IL-21 and actin on cDNA generated from laser capture-isolated purified HL cells from 10 different donors (nos. 1-10). (F-H) Multinucleated HL cells stained positive for IL-21 protein by immunohistochemistry (brown staining). Stainings from 3 different representative patient tumor samples (of 6 patients tested) are shown. Immunohistochemistry for IL-21 was performed on paraffin-embedded tumor sections from HL patients using a rabbit polyclonal anti–IL-21 Ab (0.5 μg/ml; eBioscience). Functionality of the anti–IL-21 Ab was established on paraffin-embedded tonsil tissue as described.³³  IL-21 staining was developed using the CSA-II kit (Dako Cytomation) with DAB detection; tissue was counterstained with hematoxylin. Slides were visualized on an Olympus BX51 light microscope (Olympus, Zoeterwoude, The Netherlands) using a UPlan/Apo 40×/0.85 objective and Olympus DP70 camera. Images were captured with Olympus DP Controller software version 1.2.1.108 and were processed with Adobe Photoshop 7.0. (Adobe Systems, San Jose, CA).