Fig. 2: T-ALL human cells arrested at the β-selection checkpoint display LIC potential.

a, CD3 versus TCRαβ expression of CD4⁺ CD8⁺ double-positive gated cells from peripheral blood samples of three individuals with T-ALL (T-ALL9, T-ALL17, T-ALL18) analyzed by flow cytometry at the time of diagnosis (patient), or after serial transplantation into consecutive NSG mice (1st and 2nd transfer). Human cells engrafting the bone marrow of transplanted mice were analyzed when mice presented advanced symptoms of disease (70–92% leukemic burden). Numbers indicate percentages of gated CD3^loTCRαβ^neg and TCRαβ⁺ cells. b, Mean ± s.e.m. percentages (when n = 3) of human CD3^loTCRαβ^neg cells from T-ALL9 and T-ALL17 human samples engrafting the bone marrow and spleen of serially transplanted mice in a. For T-ALL9 bone marrow and spleen values from the first transfer, n = 3 in both tissues; from the second transfer, n = 1 in both tissues. For T-ALL17 bone marrow and spleen values from the first transfer, n = 3 in both tissues; from the second transfer, n = 2 and n = 1, respectively. Percentages at the time of diagnosis (human, n = 1 each) are shown for comparison. c,f, Sorting strategy used to isolate CD3^loTCRαβ^neg and CD3⁺ TCRαβ⁺ T-ALL17 (c) and T-ALL43 (f) human cells recovered from spleen xenografts of NSG mice (left histograms). CD3 versus TCRαβ and cTCRβ expression of sorted populations is shown in the middle and right histograms, respectively. d,g, LIC potential of CD3⁺ TCRαβ⁺ and CD3^loTCRαβ^neg human T-ALL17 (d) and T-ALL43 (g) sorted populations shown in c and f and transplanted into NSG mice under limiting dilution conditions (from 10⁵ to 10 cells per mouse, 3–4 mice per dose). LIC frequency was calculated using ELDA software by recording numbers of leukemia-free mice in the peripheral blood by 29–32 weeks after transplant. e,h, Kaplan–Meier survival curves of mice transplanted in d and g with 10³ FACS-sorted CD3⁺ TCRαβ⁺ or CD3^loTCRαβ^neg human T-ALL cells were compared using log-rank Mantel–Cox test. *P = 0.0266 and *P = 0.0339, respectively. BM, bone marrow.

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