Fig 2: CAR TN/SCM display an indolent effector signature in vitro.(A) Schematic representation of CAR T cell manufacturing. Briefly, double-positive CD62L+CD45RA+ TN/SCM cells were isolated by FACS and bulk unselected T cells (TBULK) were employed as control. TN/SCM and TBULK were activated with TransAct (anti-CD3/anti-CD28 [aCD3/28]), transduced with a lentiviral vector (LV) encoding a CD19.28z CAR, and expanded in culture with IL-7 and IL-15. (B) CD19.28z CAR expression (mean fluorescence intensity, MFI; n = 5), (C) TSCM enrichment (n = 16), (D) CD4+/CD8+ ratio (n = 20), and (E) HLA-DR expression (percentage of positive cells, n = 18) at the end of CAR T cell manufacturing. (F) Fold expansion at different days of culture (n = 12). (G) Degranulation assay performed by coculturing CAR TN/SCM, CAR TBULK, and Mock control with CD19+ targets for 24 hours (n = 14 donors challenged against NALM-6, BV173, and ALL-CM CD19+ target cell lines). (H) Killing activity expressed as elimination index (see Methods) and measured by coculturing CAR T cells with CD19+ tumor cells for 4 days at different effector/target (E:T) ratios (n = 15 for CAR TBULK, n = 14 for CAR TN/SCM against NALM-6 cell line; n = 7 for CAR TBULK, n = 6 for CAR TN/SCM against BV173 cell line; n = 8 for CAR TN/SCM, n = 9 for CAR TBULK against ALL-CM cell line). (I) Cytokine (CTK) production after 24-hour coculture of T cells with CD19+ tumor cells at a 1:10 E:T ratio (n = 5 donors challenged against NALM-6, BV173, and ALL-CM cell lines). (J) T cell proliferation after 4-day coculture with CD19+ tumor cells, measured by intracellular staining of Ki-67 (n = 15 donors challenged against NALM-6, BV173, and ALL-CM cell lines). Data are represented as mean ± SEM or mean ± SEM together with overlapping scattered values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by paired t test (B–E, G, and L) or 2-way ANOVA (F, H, and I).