Fig. 4: Pre-TCR signaling is required for LIC activity and tumor progression of human T-ALL.

a, Schematic of the experimental design for abrogation of pre-TCR function in T-ALL human cells. T-ALL (T-ALL5 and T-ALL9) or B-ALL (B-ALL9) cells recovered from bone marrow xenografts of NSG mice were transduced with a lentiviral vector encoding an shRNA against CMS (shCMS) along with GFP as the cell tracer, or a control scramble RNA (shSC) and GFP, before infusion into sublethally irradiated NSG mice. b,e,h, Transduction efficiencies of T-ALL5 (b) T-ALL9 (e) and B-ALL9 (h) analyzed by flow cytometry. Numbers indicate percentages of transduced (GFP⁺) cells before transplantation. c,f,i, Percentages ± s.e.m. of CD45⁺ GFP⁺ T-ALL5 (c), T-ALL9 (f) and B-ALL9 (i) cells infiltrating the peripheral blood (PB) of transplanted mice. For shSC-transduced and shCMS-transduced T-ALL5 cells, n = 6 and n = 8 mice, respectively, from two independent experiments, at both 8 and 10 weeks after transplant (***P = 0.0007 for both times, c); for shSC-transduced and shCMS-transduced T-ALL9 cells, n = 4 and n = 3 mice, respectively, from one experiment, at both 7 and 8 weeks after transplant (*P = 0.0326 and **P = 0.0083, respectively, f); for shSC-transduced and shCMS-transduced B-ALL9 cells, n = 4 and n = 3 mice, respectively, from one experiment, at 5 weeks after transplant (i). d,g,j, Relative numbers ± s.e.m. of T-ALL5 (d), T-ALL9 (g) and B-ALL9 (j) cells transduced with shCMS or shSC, engrafting the indicated organs of NSG mice euthanized when they presented advanced symptoms of disease (13, 11 and 7 weeks after transplant for T-ALL5, T-ALL9 and B-ALL9, respectively). Data are shown as the mean ± s.e.m. percentages of CD45⁺ GFP⁺ engrafted cells normalized to percentages of GFP⁺ injected cells (b, e and h). For shSC-transduced and shCMS-transduced T-ALL5 cells (d), n = 4 thymus samples from one experiment, **P = 0.0075; n = 6 and n = 8, respectively, bone marrow samples from two independent experiments, **P = 0.0032; n = 2 and n = 4, respectively, brain samples, from one experiment; n = 6 and n = 8, respectively, spleen and liver samples from two independent experiments, ***P = 0.0002 for both samples. For shSC-transduced and shCMS-transduced T-ALL9 cells (g), n = 4 thymus samples from one experiment, ***P = 0.0002; n = 4 bone marrow samples, from one experiment, **P = 0.0015; n = 4 spleen and liver samples from one experiment, ****P < 0.0001, and n = 4 LN samples from one experiment, **P = 0.0056. For shSC-transduced and shCMS-transduced B-ALL9 cells (j), n = 4 and n = 3, respectively, bone marrow and liver samples from one experiment. Data were analyzed by a two-tailed unpaired t-test with Mann–Whitney correction (c, f and i) and two-tailed unpaired multiple t-test (d, g and j). LN, lymph node.

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