GÉTICA 2021

55 [ F I T C á n c e r - 7 ] Figure 2. Leukemia escape from immune pressure. (a,b) NALM6 cells were co-cultured with NT-T, CAR-T19 or STAb-T19 cells at the indicated E:T ratios, and the expression of CD3, CD2, CD19, and CD10 was analyzed at the indicated days. In those cases where 100 % of CD3+CD19- or CD3-CD19+ cells were reached the assay was discontinued. (a) Graphs showing the change over time in relative percentages of CD3+CD19-, CD3-CD19+ and CD3-CD19- cells. The results are shown as the mean of three independent experiments. (b) Representative FACS dot plots showing the different cell populations in CAR-T19/ NALM6 co-cultures performed at a 1:8 E:T ratio at different time points. (c,d) NALM6 cells were co-cultured with CAR-T19 bearing the anti-CD19 FMC63 scFv (FMC63CAR-T19), STAb-T19 or with NT-T cells in the presence of 100 ng/ml blinatumomab (BLI) at the indicated E:T ratios, and the expression of CD3, CD19 and CD10 was analyzed at different time points. (c) Graphs showing the change over time in relative percentages of CD3+CD19-, CD3-CD19+ and CD3-CD19- cells; in grey (not valuable, NV), conditions in which number of cells in the culture was < 500; one representative experiment is shown. (d) Dot plots showing the different cell populations in CAR-T19/NALM6 co-cultures performed at a 1:16 E:T ratio at different time points. (e) NALM6 or SEM cell lines were co-cultured with A-T cells at a 2:1 E:T ratio and the expression of CD3, CD19 and CD10 was analyzed after 2 hours; the percentages of CD3+CD19-, CD3- CD19+ and CD3-CD19- and CD3+CD19+ are shown. The results are means of 3 ± SD similar experiments. (f,g) NALM6 cells were co-cultured for 2 hours with NT-T cells, CAR-T19 cells bearing the anti-CD19 FMC63 scFv (FMC63CAR-T19) or the anti-CD19 A3B1 (A3B1CAR-T19), or STAb-T19 cells. (f) The percentages of CD3+CD19-, CD3-CD19+, CD3-CD19- and CD3+CD19+ cells were analyzed. (g) Dot plots showing the expression of CD3, CD19 and CD10 at time 0 and af- ter 2 hours of culture. One representative experiment is shown. (h) Representative dot plots showing the down-modulation of 19-BBζ CAR in A3B1-CAR-T19 cells after 2 hours of co-culture with NALM6 cells. One representative experiment out of three independent experiments is shown. (i) Human primary B-ALL cells from two different patients (B-ALL1 and B-ALL2, > 90 % of CD19+ B-ALL blasts) were co-cultured for 2 hours with primary A-T cells at a 2:1 E:T ratio and the percentage of CD3+CD19-, CD3-CD19+, CD3-CD19- and CD3+CD19+ cells analyzed. The results are means ± SD of 3 similar experiments. (j) Human primary B-ALL1 and B-ALL2 cells were co-cultured with primary NT-T, CAR-T19 or STAb-T19 cells at 1:2 and 1:1 E:T ratios, and the number of alive target cells (7AAD-) determined after 24 and 48 hours. Results are shown as mean ± SD from 3 experiments. Significance was calculated by an unpaired Student t test.