OP9 overexpressing the murine NOTCH1 ligand DL1 drives bulk and T-ALL L-IC proliferation. (A) To determine whether T-ALL cells proliferate in response to NOTCH1 activating signals, 5.5 × 10⁵ primary 7021 T-ALL cells were added to coculture on OP9-DL1 or OP9-GFP. Cocultures were performed in triplicates. Cells were counted weekly with graph showing the mean cell counts plus or minus the standard error. Cocultured cells were analyzed by flow cytometry weekly to evaluate immunophenotype to confirm leukemia cell expansion. OP9 coculture showed progressive expansion of bulk leukemia cells in coculture with OP9-DL1 but not with OP9-GFP. (B) Sorted 7021 T-ALL cells were added to OP9-DL1 stroma. CD7⁺CD1a⁻ (1 × 10⁵), CD7⁺CD1a⁺ (4 × 10⁴), and CD7⁻ (4 × 10⁴) cells were cocultured, and cells were counted weekly. (C) To confirm that cells remaining after 28 days of OP9-DL1 coculture were T-ALL cells, flow cytometric analysis was performed. The figure shows the results for T-ALL 0549 cocultured with OP9-DL1 (left column) and OP9-GFP (right column). T-ALL 0549 is positive for CD7 and CD5 surface expression but negative for surface CD4 and CD8. Analysis of CD4/CD8 (upper panels) and CD7/CD5 expression (lower panels) on cocultured cells revealed that T-ALL 0549 CD7⁺CD5⁺CD4⁻CD8⁻ cells persisted after 28 days of OP9-DL1 coculture (left panels) whereas almost no CD7⁺CD5⁺CD4⁻CD8⁻ cells were found after 28 days of OP9-GFP coculture (right panels).