Fig 2: Spleen is required for extrathymic CD4+CD8+ cell development in DLL4-driven T-ALL. A) Three-week-old Tg8 mice underwent thymectomy (thyX) or sham surgery (mock) and blood cells were collected and stained for CD3, CD4 and CD8 every week after surgery. Cells were gated on CD3 expression, and analyzed for CD4 and CD8 expression. The left panel shows a representative analysis at week 3 after surgery. The right panel shows the CD4+CD8+/CD4+ (DP/4SP) ratio increase after surgery (n = 6 mice per treatment). B) Three-week-old pre-tumoral Tg8 mice underwent splenectomy (splX) or sham surgery (mock). One month after surgery, mice were sacrificed and CD4+CD8+ cell accumulation in mesenteric lymph nodes (mLN), bone marrow (BM), liver, and spleen of Tg8 mice determined by flow cytometry (data are representative of n=4 mice for each indicated time point). Cells were positively gated using CD3 and analyzed for the expression of CD4 and CD8. Data are representative of n = 4 mice per treatment. C) Survival curves of mice that underwent splenectomy (splX) or sham surgery (sham) at 3 weeks of age. n = 12 mice per treatment coming from 2 different experiments with 5 and 7 mice per group, respectively. In the first experiment, splenectomized mice were killed when the last sham-operated mouse died of T-ALL and they appear as censored (with a tick mark). In the second experiment, splenectomized mice were kept alive for another month after the last sham-operated mouse died, and also appear as censored (with a tick mark). Kaplan-Meier survival curves were analyzed with the Gehan-Breslow-Wilcoxon test (p = 0.000012). **** p < 0.0001.

Fig 3: Therapeutic effect of demcizumab in mice harboring DLL4-expressing T-ALL cells. A) PDTALL13 cells were injected IV in NRG mice that were then treated with vehicle (IgG, n = 8), DBZ (n = 8), murine anti-DLL4 (n = 8), or human anti-DLL4 antibodies (demcizumab, n = 8). The Y-axis shows the percentage of alive animals and the X-axis the days after treatment. Data were compared with the Gehan-Breslow-Wilcoxon test: vehicle vs DBZ (p = 0.02), vehicle vs demcizumab (p = 0.006), murine anti-DLL4 antibody vs DBZ (p = 0.0003), and murine anti-DLL4 antibody vs demcizumab (p = 0. 0002). The other comparisons were not significant. Asterisks in the figure are for the comparison with the vehicle group. B) Protein expression of the indicated proteins by western blotting of whole cell extracts from spleens of NRG mice treated as in (A). C) Formalin fixed-paraffin embedded spleen sections from NRG mice treated as in (A) were stained with anti-HES1 antibodies, DAPI and secondary antibodies conjugated with fluorescent dyes. Four random photographs at 20X magnification were taken for each condition and the number of HES1high cells (left) and HES1low cells (right) is presented below. Statistical analyses (treatment vs control) were performed by one-way ANOVA for each PDTALL line. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.