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. 2022 Feb 14;10(4):498–511. doi: 10.1158/2326-6066.CIR-21-0853

Figure 2.

Figure 2. Leukemia escape from immune pressure. A and B, NALM6 cells were cocultured with NT-T, CAR-T19, or STAb-T19 cells at the indicated E:T ratios, and the relative percentage of CD3+CD19−, CD3−CD19+, and CD3−CD19− cells were measured by FACS. A, Results are shown as the mean of three independent experiments. B, Representative FACS dot plots of the CAR-T19 1:8 E:T ratio sample. Gray, nonviable (NV), in which number of cells in the culture was <500. C and D, NALM6 coculture as in A–B, 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). One representative experiment out of two is shown. Dot plots (D) showing the cell populations cocultured at a 1:16 E:T ratio. E, The percentages of CD3+CD19−, CD3−CD19+, and CD3−CD19− and CD3+CD19+ NALM6 or SEM cell lines after 2 hours of coculture with A-T cells at a 2:1 E:T ratio. The results are means of 3 ± SD similar experiments. F and G, The percentages of CD3+CD19−, CD3−CD19+, CD3−CD19−, and CD3+CD19+ NALM6 cells after coculture 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. H, Representative dot plots showing the downmodulation of 19-CAR in A3B1CAR-T19 cells after 2 hours of coculture with NALM6 cells. One representative experiment out of three independent experiments is shown. I, The percentage of CD3+CD19−, CD3−CD19+, CD3−CD19−, and CD3+CD19+ primary human B-ALL cells from two different patients (B-ALL1 and B-ALL2, >90% of CD19+ B-ALL blasts) after coculture with primary A-T cells at a 2:1 E:T ratio. The results are means ± SD of 3 similar experiments. J, The number of alive (7AAD−) target B-ALL1 and B-ALL2 cells determined after 24- and 48-hour coculture with primary NT-T, CAR-T19, or STAb-T19 cells at 1:2 and 1:1 E:T ratios. Results are shown as mean ± SD from 3 experiments. Significance was calculated by an unpaired Student t test.

Leukemia escape from immune pressure. A and B, NALM6 cells were cocultured with NT-T, CAR-T19, or STAb-T19 cells at the indicated E:T ratios, and the relative percentage of CD3+CD19−, CD3−CD19+, and CD3−CD19− cells were measured by FACS. A, Results are shown as the mean of three independent experiments. B, Representative FACS dot plots of the CAR-T19 1:8 E:T ratio sample. Gray, nonviable (NV), in which number of cells in the culture was <500. C and D, NALM6 coculture as in A–B, 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). One representative experiment out of two is shown. Dot plots (D) showing the cell populations cocultured at a 1:16 E:T ratio. E, The percentages of CD3+CD19−, CD3−CD19+, and CD3−CD19− and CD3+CD19+ NALM6 or SEM cell lines after 2 hours of coculture with A-T cells at a 2:1 E:T ratio. The results are means of 3 ± SD similar experiments. F and G, The percentages of CD3+CD19−, CD3−CD19+, CD3−CD19−, and CD3+CD19+ NALM6 cells after coculture 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. H, Representative dot plots showing the downmodulation of 19-CAR in A3B1CAR-T19 cells after 2 hours of coculture with NALM6 cells. One representative experiment out of three independent experiments is shown. I, The percentage of CD3+CD19−, CD3−CD19+, CD3−CD19−, and CD3+CD19+ primary human B-ALL cells from two different patients (B-ALL1 and B-ALL2, >90% of CD19+ B-ALL blasts) after coculture with primary A-T cells at a 2:1 E:T ratio. The results are means ± SD of 3 similar experiments. J, The number of alive (7AAD−) target B-ALL1 and B-ALL2 cells determined after 24- and 48-hour coculture with primary NT-T, CAR-T19, or STAb-T19 cells at 1:2 and 1:1 E:T ratios. Results are shown as mean ± SD from 3 experiments. Significance was calculated by an unpaired Student t test.