Activation and proliferation of T cells by autologous CLL cells before and after infusion of Ad-CD154–transduced CLL cells.
T cells were isolated from the blood mononuclear cells collected from patient 003 prior to (Pre Rx) and 6 months after infusion of Ad-CD154–transduced CLL cells (Post Rx). Autologous CLL B cells were infected with Ad-CD154 for 24 hours at a multiplicity of infection of 300. Transduced cells were treated with mitomycin C (60 μg/mL) for 1 hour and then washed prior to using them as stimulator cells (Ad-CD154-CLL) for autologous MLRs with the isolated T cells. (A) Patient T cells (1 × 106 cells/well) with or without autologous stimulator cells (Ad-CD154-CLL) (5 × 105cells/well) were added to wells of a 96-well plate in serum-free AIM-V medium containing recombinant IL-2 at 25 U/mL. The murine anti-HLA mAb W6/32 was added to a final concentration of 10 μg/mL to some wells, as indicated. Plates were incubated for 4 days at 37°C prior to harvesting the supernatants. IFN-γ concentration in the supernatants was determined by standard capture ELISA. The graph depicts the mean IFN-γ concentration of triplicate wells. The error bars indicate the SD about the mean. (B) Patient T cells (1 × 106cells/well) with or without autologous stimulator cells (Ad-CD154-CLL) (5 × 105 cells/well) were added to wells of a 96-well plate as described in panel A. The plates were incubated for 84 hours at 37°C prior to adding 3H-thymidine to each well at 1 μCi per well. The cells were harvested 12 hours later and the incorporated 3H-thymidine assessed by scintillation counting. The mean cpm values of triplicate wells are indicated ± SD.
