Notch1-dependent T-ALL pathogenesis parallels the induction of IL-7R expression. (A) Schematic diagram of mouse T-ALL generation. BM Lin⁻ c-kit⁺ HPCs from C57BL/6 (H2-Kb⁺) mice were transduced with a lentiviral vector encoding the active form of Notch1 plus GFP (ICN1⁺) and then transplanted into NSG (H2-Kd⁺) immunodeficient mice. Mice were euthanized and analyzed when advanced symptoms of disease were evident. (B) CD4, CD8, CD3, and TCRαβ expression analyzed by flow cytometry (right panels) on electronically gated (left panel) ICN1-transduced (ICN1⁺) or nontransduced (ICN1⁻) H2-Kb⁺ donor cells engrafting the BM of recipient mice at 2 weeks posttransplant. (C) Percentages of ICN1⁺ and ICN1⁻ H2-Kb⁺ donor cells engrafting the BM of mice transplanted as in (A) at the indicated days posttransplant. Percentages at the day of transplant (t=0) are shown for comparison. Mean ± standard error of the mean (SEM) of 3 to 7 mice per group are shown. (D) Kaplan-Meier survival curves of mice transplanted with BM HPCs transduced with ICN1 and GFP (ICN1) or GFP alone (Ctrl), as in (A). (E) Flow cytometry of IL-7R (right panels) and CD4/CD8 (left panels) expression displayed by conventional CD4⁺CD8⁺ DP cells from a normal C57BL/6 thymus (top row) or by moT-ALL1 and moT-ALL2 primary mouse leukemias (middle and bottom rows, respectively) generated in 2 independent mice transplanted as in (A). Shaded graphs show background staining with irrelevant isotype-matched antibodies. (F) Percentages of IL-7R–expressing cells within normal DP thymocytes from C57BL/6 mice (normal thymus) or ICN1⁺ DP moT-ALL2 cells engrafting the indicated organs of mice transplanted as in (A). Mean ± SEM values of ≥7 mice per group are shown. (G) IL-7R expression levels (right panel) displayed by primary T-ALL cells (moT-ALL3) generated as in (A) and electronically gated for high (ICN1^hi) or intermediate (ICN1^int) ICN1 expression (left panel). ICN1 expression was determined by GFP flow cytometry. **P < .01, ***P < .001.