Figure 4

Determination of TCRγ4 or δ1 expression and binding/activity to normal cells of TCRγ4δ1-engineered αβT cells. (A) Cell surface expression of TCRδ1 and TCRγ4 chains on the TCRδ1- or TCRγ4-engineered αβT cells. TCRδ1- and TCRγ4-engineered αβT cells were separately stained with anti-human Vδ1-FITC antibody, and with rabbit anti-human TCRγ primary antibody and goat anti-rabbit IgG-FITC secondary antibody, and then assessed by flow cytometry. TCRγ4δ1 expression on the surface of TCRγ4δ1-engineered αβT cells served as a positive control. (B) Binding of TCRγ4δ1-Fc to normal cells including PBMCs, 1308.1.86, ccc-HEL-1 and 293T cells. After incubation with TCRγ4δ1-Fc, these normal cells were stained with FITC-conjugated goat anti-human IgG and then evaluated by flow cytometry. (C) Cytotoxicity of TCRγ4δ1-engineered αβT cells to PBMCs, 1308.1.86, ccc-HEL-1 and 293T. TCRγ4δ1-engineered αβT cells were incubated with these normal cells at an effector-to-target ratio of 10:1 for 6 h, and the cytolytic activity was detected by LDH assay. The data are representative of 3 independent experiments and expressed as the mean ± SD (ns, no significance). (D) Expression of IFN-γ in the TCRγ4δ1-engineered αβT cells. Mock- and TCRγ4δ1-engineered αβT cells were individually incubated with PBMCs, 1308.1.86, ccc-HEL-1 and 293T at an effector-to-target ratio of 3:1 for 6 h with BFA, and expression of intracellular IFN-γ was determined by flow cytometry. Shown is a representative FCM analysis of the IFN-γ and TCR chains. (PBMC, peripheral blood mononuclear cell.)