Specific blockade of IL-9 decreases ALK⁺ ALCL-cell proliferation and colony formation potential. (A) anti–IL-9–neutralizing antibody induces concentration-dependent decrease in ALK⁺ ALCL-cell proliferation as revealed by the [³H]-thymidine incorporation assay. The decrease in cell proliferation reached approximately 40% of the baseline level at a concentration of 80 μg/mL (P < .001). Similar effects were not detected when the anti–IL-9–neutralizing antibody was preincubated with rhIL-9 before treating Karpas 299 cells, and cell proliferation remained stable with a very slight decrease to 90% of its baseline level at 80 μg/mL concentration of the antibody. Similarly, changes in cell proliferation were not seen in the negative control cells HT29 after treatment with anti–IL-9–neutralizing antibody. When the Hodgkin lymphoma cells L1236 were used as a positive control, a significant and gradual decrease in cell proliferation was observed, which became 69% of the baseline level at a concentration of 80 μg/mL (P < .01). The results are shown as means ± SE of at least 3 consistent experiments. *Statistically significant compared with baseline cells (0) treated with IgG. (B) anti–IL-9–neutralizing antibody (80 μg/mL) induces marked decrease in colony formation of Karpas 299 cells in soft agar. The top panel shows the means ± SD of 3 consistent experiments. Compared to a control level of 60 ± 8 colonies/plate in control cells treated with IgG, cells treated with the anti–IL-9–neutralizing antibody developed only 24 ± 6 colonies/plate. The bottom panel shows examples of the cultured plates. The plate on the right side contains Karpas 299 cells treated with anti–IL-9–neutralizing antibody before being cultured for 2 weeks. The plate on the left side contains control cells treated with IgG under the same experimental conditions.