Humanized CAR T cells exhibited antigen-specific effector function. (A) The growth curve for Mock, FMC63, VH4vκ1, and 4D5 is shown as fold change normalized to day 0. (B) Surface expression of CD19 antigen, costimulatory molecules CD80, CD86, and inhibitory ligand PD-L1 on tumor lines KG1a, Nalm6, and Raji. Tn/mem cells expressing the specified CAR constructs were analyzed for their ability to (C) degranulate (107a), (D) produce IFN-γ and in response to KG1a, CD19⁺ Raji, and CD19⁺ Nalm6 by flow cytometry. Data are shown as mean ± SEM for CAR⁺ CD107a⁺ (n = 4) and CAR⁺ IFN-γ (n = 3). Tn/mem cells expressing the specified CAR constructs were challenged with CD19⁺ green fluorescent protein-positive (GFP⁺) Raji cells at an E:T ratio of 2:1 on day 0. CAR T cells were subsequently rechallenged with the same number of Raji cells every 3 days. (E) The remaining Raji tumor is presented as the number of GFP⁺ events on days 3, 6, 9, and 12 in the control mock group and the 3 CAR groups. (day 6, 9, and 12: P < .01 Mock vs FMC63, VH4vκ1, and 4D5; day 9: P < .05 FMC63 vs VH4vκ1 and P < .01 VH4vκ1 vs 4D5; day 12: P < .01 VH4vκ1 vs 4D5). (F) Proliferation of CAR T cells is presented as the number of EGFR⁺ events on days 3, 6, 9, and 12 (day 9: P < .05 FMC63 vs 4D5 and VH4vκ1 vs 4D5). (G) Exhaustion markers on EGFR⁺ cells such as PD1, LAG3, and TIM3 are analyzed on day 12 (ns). For panels E-G, data are presented as mean ± SEM (n = 3).