Figure 3.
Functional characterization of STAT5B1∗6-expressing Lmo2Tg pre-LSCs. (A) Scheme for serial transplantation into primary (I), secondary (II), tertiary (III), and quaternary (IV) recipients, using CD2-rtTA3-STAT5B1∗6; Lmo2Tg thymocytes. (B) Proportion of donor-derived (Cd45.2+) cells enumerated in the thymus of primary recipient mice. Mean ± SEM, Student t test, ∗∗∗P < .001. (C) Immunophenotype of donor-derived cells in the thymus of primary recipients. DN, DP, and SP represent the CD4−CD8− double-negative, CD4+CD8+ DP, and CD4+CD8− single-positive with CD4−CD8+ single-positive populations, respectively. Mean ± SEM, Student t test (N = 6), ∗∗∗P < .001. (D) Fold expansion of donor-derived CD2-rtTA3-STAT5B1∗6; Lmo2Tg DN3a cells in the thymus of serially injected recipients maintained (+DOX) or not (untreated) on DOX-enriched feed. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗∗P < .001 compared with mice maintained on standard diet. (E-F) Cell-cycle (E) and apoptosis (F) analysis in DN3a thymocytes from CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice after 7 days administration of DOX. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗∗P < .001 compared with mice maintained on standard diet. (G) Competitive assay of LSCs assessed by flow cytometry 4 weeks after transplantation of equal numbers of CD2-rtTA3-STAT5B1∗6; Lmo2Tg, and YFP; Lmo2Tg (yellow) thymocytes into sublethally irradiated recipients. Proportion of donor-derived CD2-rtTA3-STAT5B1∗6; Lmo2Tg (gray or red) and YFP; Lmo2Tg (yellow) DN3a thymocytes from recipients maintained (+DOX) or not maintained on doxycycline-enriched feed. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗P < .01 compared with YFP; Lmo2Tg cells. (H) Survival of DN3a thymocytes from CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice treated with induction-like therapy for T-ALL (VXL43) after 7 days administration of DOX. Numbers of DN3a thymocytes in mice treated with vehicle (supplemental Figure 3B) was used as reference and defined as 100%. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. (I) Fold expansion of donor-derived DN3a cells enumerated in the thymus of recipients injected with CD2-rtTA3-STAT5B1∗6; Lmo2Tg thymocytes from panel H. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05.

Functional characterization of STAT5B1∗6-expressing Lmo2Tg pre-LSCs. (A) Scheme for serial transplantation into primary (I), secondary (II), tertiary (III), and quaternary (IV) recipients, using CD2-rtTA3-STAT5B1∗6; Lmo2Tg thymocytes. (B) Proportion of donor-derived (Cd45.2+) cells enumerated in the thymus of primary recipient mice. Mean ± SEM, Student t test, ∗∗∗P < .001. (C) Immunophenotype of donor-derived cells in the thymus of primary recipients. DN, DP, and SP represent the CD4−CD8− double-negative, CD4+CD8+ DP, and CD4+CD8− single-positive with CD4−CD8+ single-positive populations, respectively. Mean ± SEM, Student t test (N = 6), ∗∗∗P < .001. (D) Fold expansion of donor-derived CD2-rtTA3-STAT5B1∗6; Lmo2Tg DN3a cells in the thymus of serially injected recipients maintained (+DOX) or not (untreated) on DOX-enriched feed. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗∗P < .001 compared with mice maintained on standard diet. (E-F) Cell-cycle (E) and apoptosis (F) analysis in DN3a thymocytes from CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice after 7 days administration of DOX. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗∗P < .001 compared with mice maintained on standard diet. (G) Competitive assay of LSCs assessed by flow cytometry 4 weeks after transplantation of equal numbers of CD2-rtTA3-STAT5B1∗6; Lmo2Tg, and YFP; Lmo2Tg (yellow) thymocytes into sublethally irradiated recipients. Proportion of donor-derived CD2-rtTA3-STAT5B1∗6; Lmo2Tg (gray or red) and YFP; Lmo2Tg (yellow) DN3a thymocytes from recipients maintained (+DOX) or not maintained on doxycycline-enriched feed. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗P < .01 compared with YFP; Lmo2Tg cells. (H) Survival of DN3a thymocytes from CD2-rtTA3-STAT5B1∗6; Lmo2Tg mice treated with induction-like therapy for T-ALL (VXL43) after 7 days administration of DOX. Numbers of DN3a thymocytes in mice treated with vehicle (supplemental Figure 3B) was used as reference and defined as 100%. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001. (I) Fold expansion of donor-derived DN3a cells enumerated in the thymus of recipients injected with CD2-rtTA3-STAT5B1∗6; Lmo2Tg thymocytes from panel H. Mean ± SEM, 2-way ANOVA with Tukey correction test; ∗P < .05.

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