Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) ICN1-transduced human ETPs were treated with either blocking anti-CD44 mAb (515; IgG1) or control IgG1 before transplantation into RAG-2–/– × γc–/– mice. Absolute ICN1+ cell numbers infiltrating the BM of 5 mice/group were analyzed at 3 weeks after transplant. (B) Schematic representation of experiment design (left): RAG-2–/– × γc–/– mice transplanted with ICN1-transduced ETPs received 3 weekly i.p. injections of either blocking anti-CD44 (HP2/9; IgG1) mAb or control IgG1, starting at day 5 after transplant. (Right) Absolute numbers of human ICN1+ cells infiltrating the BM of 4 mice/group analyzed at day 15 after transplant. (C) Human cell numbers infiltrating the BM of RAG-2–/– × γc–/– mice (5/group) after 3 weeks of transplant with ETPs transduced with either ICN1 or CD44. Data in A–C were normalized to 105 transduced input cells (n = 3). (D) Mean percentages ± SEM of human cells infiltrating the BM of RAG-2–/– × γc–/– mice (4/group) transplanted with ETPs cotransduced with ICN1 along with either a CD44-encoding or a control retrovirus (n = 4). (E) Cells obtained from the BM of a T-ALL patient (T-ALL1) were pretreated with either blocking anti-CD44 mAb 515 or control IgG1 and then transplanted into RAG-2–/– × γc–/– mice. Relative T-ALL1 cell numbers infiltrating the BM, PB, spleen, and thymus of 4 mice/group were analyzed at the indicated times after transplant (n = 3). *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, Transplantation Assay, Transduction

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) RAG-2–/– × γc–/– mice transplanted with primary human T-ALL1 or T-ALL2 cells (Supplemental Figure 4) received 3 weekly i.p. injections of either blocking anti-CD44 mAb (HP2/9) or control IgG1 during 4 weeks, starting at 1 week (B/C) or 5 weeks (D/E/F) after transplant. When indicated, T-ALL cells recovered from the BM of transplanted mice at the end of treatment were transplanted into secondary hosts. (B) Percentages of T-ALL1 and T-ALL2 cells infiltrating the BM, PB, and spleen of mice treated from weeks 1 to 5 after transplant as shown in A. Mean values from 3 independent experiments with a total of 13 to 18 mice for T-ALL and 3 to 8 mice for T-ALL2 are shown. (C) Image of representative spleens obtained at the end of treatment from mice shown in B. (D) Thorough analysis of anti-CD44 in vivo treatment showing percentages of human T-ALL2 cells infiltrating the BM (left) and PB (right) of 5 mice/group treated from weeks 5 to 9 after transplant as shown in A. Empty symbols represent cell percentages before the onset of Ab treatment. Boxes identify individual donors for secondary transplantations shown in F. (E) Kaplan-Meier survival curve of mice treated with anti-CD44 mAb (HP2/9) or control IgG1 in D. (F) Kaplan-Meier survival curve of secondary recipients transplanted with T-ALL2 cells (33 cells/mouse) obtained from the BM of individual donors represented as boxed in D. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, In Vivo

Journal: The Journal of Clinical Investigation

Article Title: The NOTCH1/CD44 axis drives pathogenesis in a T cell acute lymphoblastic leukemia model

doi: 10.1172/JCI92981

Figure Lengend Snippet: (A) NSG mice transplanted with primary T-ALL2 cells received 3 weekly i.p. injections of either blocking anti-CD44 mAb (HP2/9) or control IgG1 during 7 weeks, starting at 1 week after transplant. (B) Image of representative spleens obtained at the end of treatment from mice shown in A. (C) Percentages of T-ALL2 cells infiltrating the BM, PB, and spleen of 5 to 10 mice treated as shown in A. (D) Representative FACS analysis showing persistence of anti-CD44 HP2/9 mAb on the surface of T-ALL cells recovered from the BM of NSG mice represented in A at the end of treatment with either anti-CD44 mAb or IgG1. Bound HP2/9 mAb was detected by reactivity with PE-labeled anti-mouse IgG1 (n = 3). (E) FACS analysis showing levels of HP2/9 anti-CD44 mAb persisting on the surface of T-ALL2 cells that infiltrate the BM of anti-CD44–treated mice shown in A at the indicated weeks after transplant. Shown are MFI values from a total of 7 mice, determined as in D. (F) BM and PB infiltration of T-ALL1 cells transduced with a lentiviral vector encoding GFP and either CD44-specific shRNA (28% transduction efficiency) or a scramble control shRNA (36%). Data show percentages of GFP+ cells within infiltrating CD45+ T-ALL1 cells of 5 NSG mice/group at 11 weeks after transplant. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: In vivo persistence of HP2/9 mAb on the surface of T-ALL cells was analyzed by FACS using a PE-labeled goat anti-mouse IgG1 Ab (Southern Biotech).

Techniques: Blocking Assay, Labeling, Transduction, Plasmid Preparation, shRNA