Cytotoxicity of AbTCR or AbTCR-CSR against AML cells. (A) A cartoon depicting the design of the AbTCR and the costimulatory receptor CSR only, which, together, form the AbTCR-CSR. AbTCR is the primary signal, CSR is the secondary signal. The combination is known as ARTEMIS 2.0. ESK2 AbTCR or ESK2 AbTCR-CSR T cells (B-C) were incubated with the indicated leukemia target cells at an E:T ratio of 1:1, overnight. The cytotoxicity was measured by luciferase-based assay. Each data point was the average of triplicate cultures ± standard deviation, and representative of 3 similar experiments with different donors. To test whether the cytotoxicity of AbTCR-CSR T cells required the primary signal, (D) AML-14, (E) HL-60, or (F) SKOV3/A2 cell lines were labeled with CFSE, washed, and incubated with mock T cells, CSR cells (ESK2 is replaced with an irrelevant Fab AbTCR as the recognition receptor), ESK2 AbTCR-CSR T cells no. 18, or ESK2 AbTCR-CSR T cells no. 34 at an E:T ratio of 1:1, overnight. The cells were harvested, washed, and stained with mAb to CD33 and subjected to flow cytometry. The analysis was performed by gating on larger tumor cells based on forward and side scatters, and the percentage of CD33⁺ cells was shown in the CFSE⁺ target population.