Expression of CD19-specific chimeric immunoreceptor in UCB-derived T cells leads to CD19-dependent cytokine production. (A) Western blot of lysates of lane 1 unmodified Jurkat cells, lanes 2 and 3 two UCB-derived T-cell clones genetically modified with the plasmids CD19R/ffLucHyTK-pMG and CD19R/HyTK-pMG, respectively, and lane 4 CD19R⁺ Jurkat cell under nonreducing (i) and reducing (ii) conditions and stained with mAb specific for CD3-ζ. (B) Flow cytometry staining of UCB-derived CD8⁺ genetically modified T-cell clone with goat-derived polyclonal FITC-conjugated Fc-specific antibody (bold line) and nonspecific control antibody. (C) CD19-specific activation of genetically modified UCB-derived T cells for IFN-γ cytokine production. As a positive control, UCB-derived T cells were stimulated in CM with PMA (10 ng/mL) and ionomycin (1 μM). Background cytokine production was determined from T cells incubated in CM. IFN-γ was measured by CBA after 12 hours of culture.