Analysis of effector functions after stimulation of bi-specific T cells through the virus-specific TCRs and CD19-CARs. (A) Cytotoxicity. HLA-B*0702_CMVpp65 bi-specific T cells were tested for lysis of K562 (●), K562 transduced with CD19 (■), and K562 transduced with HLA-B*0702 and pulsed with CMVpp65 peptide (▴; top left panel). Bi-specific T cells lysed CD19⁺ tumor cell lines (top right panel), primary CLL cells from 2 patients (bottom left panel) and primary ALL cells from 2 patients (bottom right panel). In assays against primary tumor cells, K562 cells were used as a negative control (triangles in the bottom panels). (B) Intracellular cytokine staining. HLA-B*0702_CMVpp65 bi-specific T cells stimulated for 4 hours with an equal number of the indicated K562 transfectants, permeabilized, and stained for intracellular IFN-γ, TNF-α, and IL-2, or left unstimulated. Stimulation with PMA/ionomycin served as a positive control. Data are shown for B*0702_CMVpp65 bi-specific T cells and are representative of 5 experiments with bi-specific T cells from different donors. (C) Time-course analyses of intracellular IFN-γ staining of the bi-specific T cells. T cells were stimulated either with the indicated K562 transfectants or with PMA/ionomycin for 4-12 hours, permeabilized, and stained for intracellular IFN-γ. The mean and SEM of the percentage of T cells that stained positive for IFN-γ is shown for 5 independent experiments with bi-specific T cells from 3 donors (N.S., not significant). (D) IFN-γ, TNF-α, and IL-2 secretion. T cells were stimulated with the indicated K562 transfectants at a 1:1 ratio, and culture supernatants were harvested at 24 hours and analyzed by Luminex assay. Data are pooled from 7 independent experiments with bi-specific T cells from 7 donors (mean and SD).