Fig. 2

Analysis of relative cell number increase using the trypan blue exclusion assay (N = 3) over 4 days in the two Irak1 KO clones shows no difference compared with the mock control (MC) BBC2 cells (A, left). Analysis of cell viability, relative to that seen in the MC cells, after 4 days (N = 3), using the CellTiter Glow assay (A, right), shows no difference between the KO and MC cells. Flow cytometric analysis (B, left) shows no difference in the distribution of cells in the S/G2/M phases of the cell cycle when the two KO clones were compared individually with the MC cells (N = 3). There is also no difference in levels of apoptosis as determined by annexin V staining (B, right). Analysis of BALB/c mice xenografted with either 10,000 (C, left) or 100,000 (C, center) cells (N = 6) from KO clones #5 and #7 and MC cells that show survival times of 14–17 days in mice xenografted with the MC BBC2 cells. In contrast, mice xenografted with the KO clones did not develop leukemia during the observation period up to 120 days regardless of inoculated tumor cell burden. When 10,000 cells from the MC and KO lines were engrafted into immunocompromized NSG mice (C, right) however, all mice (N = 5) developed disease and died within 13–19 days. The survival data shown in (C) was reflected in a significant reduction in spleen size and the proportion of GFP+ cells in the spleens from mice xenografted with the KO clones when compared individually with mice xenografted with the MC cells at autopsy (D). Spleen size in the NSG mice (E) was enlarged in all cohorts and levels of GFP+ cells were equally high. Treatment of mice (N = 5) with an anti-CD4 or anti-CD8 antibody shows a significant reduction in CD4+ or CD8+ cells, respectively (F and G) when compared individually with the isotype control. Conbination treatment with anti-CD4/CD8 antibodies led to significant loss of both cell types as shown in F and G. When IRAK KO #7 cells are xenografted into BALB/c mice treated with the various antibodies to deplete T-cells, while the isotype treated mice did not develop leulemia, mice experiencing depletion of CD4 or CD8 or both types of cells showed robust development of leukemia when each is compared with the isotype control (H, left). The development of leukemia was paralleled with increased spleen and liver weight (H, right). ns = not significant, * p < 0.05, ** p < 0.01, **** p < 0.0001