ROR1-CAR transduced CD8⁺ T cells obtained from B-CLL patients specifically recognize autologous tumor cells. (A) Phenotype and transgene expression of a ROR1-CAR and a GFP-transduced CD8⁺ T-cell clone obtained from a B-CLL patient. Data are representative for results obtained in 3 B-CLL patients. (B) ROR-CAR CD8⁺ T cells from 3 B-CLL patients specifically recognize autologous primary B-CLL and ROR1-positive tumor cells by chromium release assay. Cytotoxicity data are presented as mean values of triplicate wells. The standard deviation of each triplicate was ≤ 3% in all cases (B,F). (C) Secretion of INF-γ (left axis), TNF-α and IL-2 (right axis) of ROR1-CAR transduced CD8⁺ T cells obtained from a healthy donor and a B-CLL patient in response to primary (autologous) B-CLL and ROR1-positive tumor cell lines after incubation for 24 hours at an E:T ratio of 2:1 quantified by Luminex Assay. (D) Proliferation of ROR1-CAR expressing CD8⁺ T cells obtained from a healthy donor and a B-CLL patient after stimulation with primary (autologous) B-CLL and ROR1-positive tumor cell lines assessed by CFSE dilution after incubation for 72 hours at an E/T ratio of 2:1. Specific proliferation (black solid line) versus background proliferation (gray histogram). (E) Detection of a side population of Hoechst 33342 effluxing B-CLL cells by flow cytometry after staining of primary B-CLL cells with Hoechst 33342 (left dot plot). Expression of ROR1 on SP and non-SP B-CLL cells (center histograms). Inhibition of Hoechst 33342 efflux by addition of 50μM verapamil (right dot plot). Data are representative for results obtained in 3 B-CLL patients. (F) Specific and equivalent elimination of sort-purified SP and non-SP primary B-CLL cells by ROR1-CAR transduced CD8⁺ T cells by chromium release assay at an E/T ratio of 20:1.