IL-21 enhances NK-mediated ADCC of rituximab-coated CLL cells. (A) IL-21 mediates tyrosine phosphorylation of STAT1 and STAT5 in NK cells. NK cells (CLL patient derived) were stimulated with media, IL-21 (10 ng/mL), IL-2 (10 U), or IFN-α (10⁴ U/mL). Cells were permeabilized and stained for p-STAT1 and p-STAT5 with appropriate controls as described in “Assessment of STAT and STAT3 signaling by flow cytometry” and analyzed by flow cytometry. Shown is a representative histogram of 3 independent experiments. (B) IL-21 enhances autologous NK cell–mediated ADCC of rituximab-coated CLL cells. ⁵¹Cr-labeled freshly isolated B-CLL cells were incubated with alemtuzumab, rituximab, or isotype control trastuzumab. CLL patient–derived NK cells treated with media alone (M) or 100 ng/mL of IL-21 (E) were incubated with autologous B-CLL cells at 37°C for 18 hours. Percentages of relative cytotoxicity were measured after 4 hours as described in “Antibody-dependent cellular cytotoxicity assay.” Data shown here are summary of 3 patient samples, and error bars represent SD between patients. IL-21 plus rituximab significantly enhances ADCC compared with rituximab alone (P < .001 at all E:T ratios). (C) IL-21 enhances allogeneic NK cell–mediated ADCC of rituximab-coated CLL cells. Freshly isolated CD19⁺ B-CLL cells were incubated with alemtuzumab, rituximab, or isotype control trastuzumab. Donor CLL patient–derived NK cells were treated with media alone or 100 ng/mL of IL-21 (18 hours) and incubated with B-CLL cells at 37°C. Percentages of relative cytotoxicity was measured after 4 hours as described above. Data shown here are a summary of 3 patient samples, and error bars represent SD between patients (n = 3). IL-21 plus rituximab significantly enhances ADCC compared with rituximab alone (P = .04 at an E:T ratio of 25:1).