Application of the optimized protocol to CD19 CAR T-cell products. BAFF-R and CD19 CARs were expressed in the same lentivirus backbone (4-1BB) and respective CAR T cells were generated from a single healthy donor. Bulk CAR T cells were stimulated with Nalm-6 (BAFF-R⁺ and CD19⁺) target cells or their respective control Nalm-6 BAFF-R⁻ or Nalm-6 CD19⁻ target cells at an E:T ratio of 1:2 for 5 hours. Bulk stimulated CD4⁺ and CD8⁺ T cells were then enriched, analyzed by FACS for EGFRt⁺ CAR T cells, or were loaded onto microchips for secretomic evaluation. The proportions of (A) EGFRt⁺ BAFF-R and (C) CD19 CAR T cells. Polyfunctionality is expressed as the percentage of single cells that released >2 cytokines and as PSI (B and D). S/E, stimulatory-to-effector cytokine ratios.