Figure 2.
IL-7R signaling is essential for LIC function and progression of human T-ALL in vivo. (A) LIC potential of IL-7Rhi (blue) and IL-7R−/lo (red) primary human T-ALL8 cells (from T-ALL patient 8) that were sorted by fluorescence-activated cell sorting based on IL-7R expression levels (high or negative-to-low, respectively), as shown in supplemental Figure 2, and transplanted into NSG mice under limiting dilution conditions (from 80 to 8 × 104 cells per mouse, ≥4 mice per dose). LIC frequency was calculated using ELDA software by recording numbers of leukemia-free mice in the PB at 5 and 7 weeks posttransplant (p.t.). (B) Kaplan-Meier curves of leukemia-free mice transplanted in (A) with 80 FACS-sorted IL-7Rhi or IL-7R−/lo T-ALL8 cells. Flow cytometry analysis of the efficiency of transduction of human primary T-ALL5 (from T-ALL patient 5) (C) and T-ALL8 (E) cells with lentiviral vectors encoding an shRNA against IL-7R (shIL7R) or an shsc, together with GFP. Numbers indicate the percentages of transduced (GFP+) cells. Relative numbers of transduced (GFP+) T-ALL5 (D) or T-ALL8 (F) cells engrafting the indicated organs of NSG mice euthanized when they presented advanced symptoms of disease (7-8 weeks posttransplant) and analyzed by flow cytometry. Data are percentages (mean ± standard error of the mean [SEM]) of GFP+ engrafted cells normalized to percentages of GFP+ injected cells (C,E) (n = 5). (G) Flow cytometry of IL-7R surface expression on primary human T-ALL5 cells upon transduction with shIL7R or shsc (GFP+) and culture for 3 days onto OP9-GFP cells plus rhIL-7 (left panels). Line graphs show percentages of IL-7R+ cells on electronically gated GFP+ transduced cells from a representative experiment (right panels). Shaded graphs show background staining with irrelevant isotype-matched antibodies. (H) Surface IL-7R expression levels on primary T-ALL5 cells transduced and cultured as in (G). Data are mean ± SEM of mean fluorescence intensity (MFI) values of GFP+ transduced cells (n = 3). (I) Percentages of T-ALL5 cycling cells transduced and cultured as in (G). Data are percentages (mean ± SEM) of GFP+ transduced cells in the S+G2/M phases of the cell cycle, as determined by Hoestch labeling and flow cytometry (n = 3). *P < .05, **P < .01.

IL-7R signaling is essential for LIC function and progression of human T-ALL in vivo. (A) LIC potential of IL-7Rhi (blue) and IL-7R−/lo (red) primary human T-ALL8 cells (from T-ALL patient 8) that were sorted by fluorescence-activated cell sorting based on IL-7R expression levels (high or negative-to-low, respectively), as shown in supplemental Figure 2, and transplanted into NSG mice under limiting dilution conditions (from 80 to 8 × 104 cells per mouse, ≥4 mice per dose). LIC frequency was calculated using ELDA software by recording numbers of leukemia-free mice in the PB at 5 and 7 weeks posttransplant (p.t.). (B) Kaplan-Meier curves of leukemia-free mice transplanted in (A) with 80 FACS-sorted IL-7Rhi or IL-7R−/lo T-ALL8 cells. Flow cytometry analysis of the efficiency of transduction of human primary T-ALL5 (from T-ALL patient 5) (C) and T-ALL8 (E) cells with lentiviral vectors encoding an shRNA against IL-7R (shIL7R) or an shsc, together with GFP. Numbers indicate the percentages of transduced (GFP+) cells. Relative numbers of transduced (GFP+) T-ALL5 (D) or T-ALL8 (F) cells engrafting the indicated organs of NSG mice euthanized when they presented advanced symptoms of disease (7-8 weeks posttransplant) and analyzed by flow cytometry. Data are percentages (mean ± standard error of the mean [SEM]) of GFP+ engrafted cells normalized to percentages of GFP+ injected cells (C,E) (n = 5). (G) Flow cytometry of IL-7R surface expression on primary human T-ALL5 cells upon transduction with shIL7R or shsc (GFP+) and culture for 3 days onto OP9-GFP cells plus rhIL-7 (left panels). Line graphs show percentages of IL-7R+ cells on electronically gated GFP+ transduced cells from a representative experiment (right panels). Shaded graphs show background staining with irrelevant isotype-matched antibodies. (H) Surface IL-7R expression levels on primary T-ALL5 cells transduced and cultured as in (G). Data are mean ± SEM of mean fluorescence intensity (MFI) values of GFP+ transduced cells (n = 3). (I) Percentages of T-ALL5 cycling cells transduced and cultured as in (G). Data are percentages (mean ± SEM) of GFP+ transduced cells in the S+G2/M phases of the cell cycle, as determined by Hoestch labeling and flow cytometry (n = 3). *P < .05, **P < .01.

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