Example 6
Three or four days after G418 selection (0.5 mg/mL), retroviral vector-transduced primary T cells were cultured in complete RPMI media containing 25 U/mL human IL-2 for an additional 2-3 days. Viable lymphocytes were recovered by centrifugation over HISTOPAQUE®-1083 (Sigma, St. Louis, MO) and used as effector cells. Lysis of target cells (RMA, RMA/Rae-1R, RMA/H60, EG7, RMA-S, RMA-S/Rae-1p, and YAC-1) was determined by a 4-hour 51Cr release assay (Sentman, et al. (1994) supra). To block NKG2D receptors, anti-NKG2D (clone: CX5, 20 μg/mL) was included in those assays. The percentage of specific lysis was calculated as follows:
% Specific lysis=[(Specific51Cr release−spontaneous51Cr release)/(Maximal51Cr release-spontaneous51Cr release)]×100.
