CAR.CD33, and ΔCD19 sel. iC9-CAR.CD33 ATCs displayed effectors function after stimulation with the CD33⁺ acute myeloid leukemia cell line, MV4-11.

<p>(a) Overnight co-culture experiments using non transduced (NT), CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 activated T-cells (ATCs) against the MV 4-11eGFP⁺ CD33⁺ cell line, followed by evaluation of enhanced green fluorescent protein (eGFP)/CD33⁺ cell killing by flow cytometry, as compared with co-culture employing NT ATCs as effectors; (mean±SEM of 3 experiments using ATCs from 3 healthy donors) <i>(left)</i>. Annexin V and 7-AAD expression from one representative co-culture experiment <i>(right)</i>. (b) Luciferase-based cytotoxicity assay using NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs after overnight incubation with the CD33⁺ MV4-11 cell line engineered to express the eGFP-firefly-luciferase (eGFP-ffLuc) construct. Effector:target (E:T) ratios ranged from 10:1 to 0.6:1; (N: 3–7 experiments with ATCs generated from 5 healthy donors). (c) Evaluation of CD107a degranulation marker expression on CAR.CD33 or ΔCD19 sel. iC9-CAR.CD33 ATCs (as compared with NT ATCs) after 4 hours of stimulation with MV4-11 CD33⁺ targets (E:T = 4:1); (N: mean±SEM of 4–6 experiments with ATCs from 4 healthy donors). (d) CFSE-based proliferation assay stimulating NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs with irradiated (40 Gy) MV4-11 CD33⁺ cell line <i>(gray)</i>, or no target cells <i>(blank)</i>, and cultured for additional 5 days (E:T = 1:1). Data from one representative experiment is shown. (e) Cytokines production on supernatant collected after overnight incubation of NT, CAR.CD33, or ΔCD19 sel. iC9-CAR.CD33 ATCs with the MV4-11 CD33⁺ cell line (E:T = 4:1); (mean±SEM of 3–9 independent experiments from 3–4 different healthy donors). SEM: standard error of the mean; NS: not statistically significant.