CD1a CARTs specifically target and eliminate CD1a⁺ T-ALL cell lines in vitro. (A) Scheme of the CD1aCAR construct used. (B) CAR detection in 293T cells using an anti-scFv mAb and GFP. (C) Representative CAR transduction and detection in CD4⁺ and CD8⁺ T cells (n = 6). (D) Proper T-cell activation (n = 3). (E) Robust expansion of activated T cells transduced with either MOCK or CD1a CAR reveals no signs of fratricide (n = 4). (F) Surface expression of CD1a (blue line) in Jurkat, MOLT4, and NALM6 cell lines. (G) CD1a antigen density in cell lines, primary coT-ALL samples, and primografts. (H) Cytotoxicity of CD1a CARTs and MOCK T cells against coT-ALL and B-ALL cell lines at the indicated E:T ratios in 16-hour assays (n = 4). (I) Absolute counts of alive eFluor-positive target cells measured according to FACS in 72-hour cytotoxicity assays at a 1:1 E:T ratio. (J) Representative FACS analysis of cytotoxicity with target cells labeled with eFluor 670 (shown in blue). (K) Enzyme-linked immunosorbent assay showing high-level production of the inflammatory cytokines IL-2, IFN-γ, and TNFα by CD1a CARTs exposed to Jurkat and NALM6 (negative control) cells in 16-hour assays at a 1:1 E:T ratio (n = 4). n.s., not significant. *P < .05, **P < .01, ***P < .001.