Figure 5: The ratio of SYK and ZAP70 expression sets thresholds for negative B-cell selection.
(a) Syk+/+ or SykZap70/Zap70 BCR-ABL1-driven B-ALL cells were transduced with GFP-tagged EV or LMP2A and relative changes of transduced (GFP+) cells were monitored by flow cytometry. Data are shown as mean ± standard error of mean (SEM) of three independent experiments. Statistical significance for day 5 was calculated using an unpaired Student’s t-test (*** p<0.001). (b) B-ALL cells with Zap70 overexpression were transduced with GFP-tagged EV or LMP2A and relative changes of transduced (GFP+) cells were monitored by flow cytometry. Data are shown as mean ± SEM of three independent experiments. Statistical significance for day 7 was calculated using an unpaired Student’s t-test (**p<0.01). (c) B-ALL cells carrying a fluorescent calcium reporter (GCaMP6s) were engineered to express a doxycycline-inducible LMP2A, or EV control, in the presence or absence of Zap70. Calcium oscillations were measured in these following addition of doxycyline (1μg/mL) to induce expression of LMP2A. Images were taken of each well at 40 second intervals for one hour in a 37°C chamber. 40 individual cells analyzed per condition are represented. Statistical significance of changes in frequency or amplitude of calcium oscillations was calculated using a Mann-Whitney test. (d) B-ALL cells pre-treated with control (DMSO), AKT inhibitor, AZD5363 (3 μM) or BTK inhibitor, ibrutinib (2.5 μM) for 16 hours were transduced with GFP-tagged EV or LMP2A and relative changes in percentages of transduced (GFP) cells were monitored by flow cytometry. Data are shown as mean ± SEM of two independent experiments. Statistical significance for day 5 was calculated using an unpaired Student’s t-test (**p<0.01).