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. 2018 Jun 4;215(6):1709–1727. doi: 10.1084/jem.20180147

Figure 2.

Figure 2.

Down-regulation, antibody targeting, or genetic deletion of IL1RAP inhibits AML pathogenesis in vivo. (A) Experimental scheme for B. THP-1 AML cells or HEL AML cells were transduced with lentiviral control or IL1RAP-directed shRNAs, isolated by FACS using the GFP marker, and 2.5 × 105 (THP-1) or 2.8 × 105 (HEL) transduced cells were transplanted in each NSG mouse. (B) Chimerism of shRNA-transduced human THP-1 cells or HEL cells in the BM of mice at termination. Each symbol represents an individual mouse. Mean ± SD is shown. P-values were calculated using unpaired two-tailed t tests. (C) Experimental scheme for D–E. THP-1 cells were transplanted into NSG mice. Mice were dosed three times per week with 10 mg/kg IL1RAP pAb or isotype control pAb for 6 wk. Blue arrowhead indicates one dose. (D) Chimerism of THP-1 cells in BM of treated mice 33 d (left) and 45 d (right) after transplant. Each symbol represents an individual mouse. Mean ± SD is shown. P-values were calculated using unpaired two-tailed t tests. tx, treatment. (E) Sectioned and H&E–stained femurs of transplanted mice showing THP-1 cell infiltration (white arrowheads) and residual normal mouse BM (black arrowheads). Four magnifications of femurs of multiple mice are shown. (F) Experimental scheme for G–H. MLL-AF9 transduced Il1rap−/− or wt BM LSK cells were expanded ex vivo and transplanted into primary (1.2 × 105 cells/mouse) then secondary recipients (103 cells/mouse). (G) Leukemic cell chimerism in the blood of MLL-AF9 secondary recipient mice 4 wk after transplant. P-value was calculated using an unpaired two-tailed t test. (H) Kaplan-Meier plot showing survival of secondary recipient mice transplanted with wt versus Il1rap−/− MLL-AF9 cells. Recipient mice of cells from each donor are shown as individual curves. P-value was calculated using log-rank (Mantel-Cox) test. (I) Flow cytometric analysis of PB populations in wt, homozygous FLT3-ITD knock-in mice (Il1rap+/+FLT3ITD/ITD), and Il1rap−/−FLT3ITD/ITD mice at 12 mo of age. P-values were calculated using unpaired two-tailed t tests. (J) Cell number per femur in wt, Il1rap+/+FLT3ITD/ITD, and Il1rap−/−FLT3ITD/ITD mice at 12 mo of age. P-values were calculated using unpaired two-tailed t tests. (K) BM cells isolated from wt, Il1rap+/+FLT3ITD/ITD, and Il1rap−/−FLT3ITD/ITD mice at 12 mo of age were plated in methylcellulose medium and scored for colony formation after 14 d. Each colony type is indicated by a different color: B/CFU-E, burst-forming unit erythroid or colony forming unit erythroid; CFU-M, macrophage; CFU-G, granulocyte; CFU-GM, granulocyte/macrophage; CFU-GEMM, granulocyte, erythroid, macrophage, and megakaryocyte. Data represent the mean ± SD of three independent experiments. P-values were calculated using unpaired two-tailed t tests comparing counts for each type of colony. Statistical significance bar color refers to the colony type indicated in the legend. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. n.s, not significant.