Anti-CCR9 CAR has potent cytotoxicity against T-ALL cell lines in vitro. (A) Structure and vector design of anti-CCR9 and control anti-CD19 CAR used in the study, using “Campana” architecture with RQR8 marker/sort-suicide gene. (B) Expression of anti-CCR9 CAR on the surface of transduced T cells, detected by anti-murine Fab. (C) Fold expansion of NT, CAR19, or CARCCR9 cells 5 days after transduction. (D) Antigen density of CD19 and CCR9 on cell lines used in the study. (E) Cytotoxicity of CAR19 vs CARCCR9 against primary T-ALL cell lines, data normalized to NT condition, 48-hour coculture, data shown at 1:8 E:T ratio. (F) Secretion of interferon-γ (IFN-γ; top) and interleukin-2 (IL-2) (bottom) in 48-hour co-culture, 1:8 E:T ratio as in panel E. (G) Example flow plot of CFSE dilution on T cells after 7-day incubation with irradiated MOLT-4 cells at 1:2 ratio. (H) Quantification of T cell CFSE dilution, 3 donors. (I) Fold expansion of T cells after 7-day co-culture with irradiated SupT1 cells at 1:2 ratio, 3 donors. *P < .05, **P < .01, ***P < .001, ****P < .0001. ns, not significant. EFF, effectors.