Activation of multiple CD4 and CD8 effector functions by CD123-specific CARs following coculture with primary AML samples. Pair-matched CAR-engineered T cells were cocultured for 6 hours with 3 different primary AML patient samples (AML 179, 373, and 605) and analyzed for surface CD107a expression and intracellular IFN-γ or TNF-α production. (A, bar graphs) Percentage of DAPI⁻CD3⁺CD8⁺ EGFRt⁺ cells expressing CD107a. Data represent mean values + SD. (A, pie charts) The fractions of CD3⁺CD8⁺EGFRt⁺ cells undergoing degranulation and producing IFN-γ and/or TNF-α are plotted in the pie charts. Percentages in each subset are indicated. (B) DAPI⁻CD3⁺CD4⁺EGFRt⁺ population data from the same experiment as described in panel A. (C) Pair-matched CFSE-labeled CD19- or CD123-specific T cells were cocultured with the indicated stimulator cells for 72 hours at an E:T of 2:1 and analyzed by flow cytometry for CFSE dilution in the DAPI⁻CD3⁺EGFRt⁺ population. LCL and K562 cell lines served as positive and negative controls, respectively. Pre-B ALL 802 is a primary patient sample double positive for CD19 and CD123. Quadrant placement is based on unstimulated T cells.