Flow cytometry assessment of cell-surface CAR expression in cultured malignant CAR T cells and assessment of basal activation of unstimulated malignant CAR T cells. (A) Viable cells in stimulated tumor-derived malignant CAR T-cell cultures (red) compared with blood-derived CAR T cells (black) at the end of 4-week culture. (B) Cultured tumor-derived CD3⁻, CD2^high, CD4^low malignant CAR T cells (red dots) showing high CAR expression compared with the mixed CD4⁺ and CD8⁺, CD3⁺ CAR T cells expanded from the peripheral blood (black dots) over 2 weeks. (C) Histograms showing phosphorylation levels of mediators of activation in unstimulated CD4⁺ T cells from untransduced donor PBMC (donor, top row), the CAR T-cell product (product, middle row), and directly isolated malignant CAR T cells (CAR T malignancy, bottom row). Histograms from samples stained with the phosphor-specific antibody (red lines) were compared with fluorescence minus one (FMO) samples (black lines) stained with surface antibodies only. (D) Heat map of relative phosphorylation status of CD3ζ, ZAP70, and AKT in different conditions corrected for autofluorescence using the resolution metric (R_D), where R_D = (MFI_stained − MFI_FMO)/(rSD_stained + rSD_FMO). MFI, median fluorescent intensity; rSD, robust standard deviation; SSC-A, side scatter area.