MD for the prediction of IL-3 mutants with low binding affinity to CD123. (A-B), WT and Mut4 interface interaction between IL-3 and IL-3Ra. HB is represented in yellow; salt bridge is represented in magenta; distance in Å for each interaction are also reported in magenta color. (C) ΔG_binding energies vs simulation time in WT and Mut4 systems. (D) Scatter plot of ΔG_binding energies of all analyzed frames in WT and Mut4 systems. (E) Time-course analysis of apoptosis measured by caspase-3 activation on target TIME cells cocultured for 8 hours with NT, IL-3z WT CAR-, and IL-3z mutant CAR–engineered CIK cells (E:T ratio of 5:1) using Operetta CLS. Custom-made MatLab-based software provided the image analyses for extraction of the populated area of the different dyes; n = 3 wells. (F) Live-cell imaging of the dynamic processes over time and space. First row represents the frame took at time 0, whereas the second row reports the frame taken after 8 hours.