Signaling through IL-21R activates STAT3 and STAT5. (A) Tyrosine phosphorylation of STAT3 and STAT5 upon IL-21 treatment. Immunoblot analysis for tyrosine-phosphorylated STAT3 (pSTAT3) and pSTAT5 was performed on the HL cell lines L1236, L591, and L428. As loading controls, blotting for total STAT3 and STAT5 was performed (n = 3). (B) Proliferation assay was performed on L591 (□) and L1236 (■) with different concentrations of the IL-21R-Fc protein to block IL-21 signaling. Proliferation was measured at 72 hours by [³H]-thymidine incorporation. The y-axis shows the proliferation of the treated culture as a percentage of the untreated control cells, calculated as the mean of triplicate samples (n = 3). (C) Flow cytometric analysis of pSTAT5 expression in cells from primary HL tissue. The pSTAT5 (bold line) histogram was obtained after gating on CD30⁺CD15⁺ cells, and the expression level was compared with an isotype control antibody gated on the CD30⁺CD15⁺ cells (thin line). Isotype control histogram gave similar results as when gated on the CD30⁻CD15⁻ cells. Representative example of 5 different patients is shown.