Optimized T-cell manufacturing augments CAR expression and T-cell phenotype. NHP CAR T-cell products manufactured with PBMCs from 3 uninfected animals were compared in vitro using 1 of 2 manufacturing schemes: traditional isolation of total CD3⁺ cells, bead-based stimulation, and culture with IL-2 (“Old”) or an updated scheme including separate isolation of CD4⁺ and CD8⁺ cells, cell-based stimulation, and no IL-2 (”New”). (A) CAR expression in total CD3⁺ (left), CD3⁺CD8^– (middle), or CD3⁺CD8⁺ subsets (right). (B) CD4:CD8 ratio. (C) T-cell memory:effector ratio, determined via flow-based staining with antibodies for CD95 and CD28. (D) T-cell memory subset distribution, determined via flow-based staining with antibodies for CCR7 and CD45RA. Samples in panels B-D were collected on manufacturing day 8. Statistical significance was determined by using the Holm-Šídák method, with α = 0.05. DP, double positive; DN, double negative; NS, not significant; T_CM, T central memory; Tdx, CAR-transduced; T_EM, T effector memory; T_EMRA, T effector memory RA.