CCL6 and CCL9 are necessary for ICSBP-dependent protection against BCR-ABL–induced leukemia. (A) RT-PCR showing down-regulation by indicated lentiviral shRNAs of CCL6 and CCL9 expression in BaF3 cells that overexpress ICSBP and BCR-ABL. Two effective shRNAs were used for each chemokine; the control shRNA targeted luciferase. (B) Reduced expression of CCL6 or CCL9 prevents the antileukemic survival response normally mediated by ICSBP. Survival curves obtained after tail vein injection of 6- to 9-week-old Balb/c mice with: (left panel) 10⁶ BaF3 cells expressing BCR-ABL alone (×) or together with ICSBP (△) (experiment was repeated 4 times and n = 20 to 30 mice per cell line); (middle panel) 10⁶ BaF3 cells expressing ICSBP and BCR-ABL and a control shRNA (shControl, ◇) or either of the 2 shRNAs targeting CCL6 (♦) (experiments were repeated twice with n = 20 mice per condition); (right panel) 10⁶ BaF3 cells expressing ICSBP and BCR-ABL and a control shRNA (shControl, △) or either of the 2 shRNAs targeting CCL9 (♦) (experiments were repeated twice with n = 20 mice per condition). (C) In panel B, transformed cells were marked with GFP. Percentages of GFP⁺ blast cells in peripheral blood or spleen suspensions from mice injected with the indicated cell types are shown. Experiment was repeated 2 to 3 times with n = 3 mice per condition. (D) Representative images and weights of spleens harvested from a Balb/c control mouse, and from mice injected with BaF3 cells expressing the indicated genes. (E) Representative images of smears of peripheral blood cells from a Balb/c control mouse, and from mice injected with BaF3 cells expressing the indicated genes.