Screening of high cytotoxic tumor killer cells using a sensitive adherent target detachment assay

Screening of high cytotoxic tumor killer cells is of great importance in adoptive immunotherapy. Here, we describe a more sensitive assay, as compared to traditional 51Cr- or Calcein-release assay, to measure cytolytic activity of killer cells. This adherent target detachment (ATD) assay is carried out in microwells of Terasaki tissue culture trays using adherent tumor cells as targets. Target tumor cells are seeded at the concentration of 300-400 cells/well and incubated overnight to allow for the adhesion of cells to the plastic surface of the wells. Effector cells were added at various effector: target (E:T) ratios, and incubated for 24 h. During incubation, dead target cells became nonadherent and together with the added effector cells were removed by washing. The remaining viable adherent target cells were stained with acrydine orange contained in the quencher and optically counted by microcomputer. A notable dose-dependent killing on target tumor cells was reproducibly obtained by tested effector cells. Cytotoxic activities of most effector cells were significantly higher in the 24-h ATD assay than those of concordant 4-h target cell lysis test. Chloroquine (chq) inhibition test in the 24-h ATD assay showed negative or weak inhibition on cytotoxicity against adherent targets. Linear regression analysis also manifested the lack of a close correlation between 4- and 24-h target cell lysis tests, indicating these two assays measured different killing activities of the same effector cells. Because of its high sensitivity and reproducibility, this semiautomatic 24-h assay with computerized fluorescence measurement system could serve as a sensitive screening assay to select high cytotoxic tumor killer cells in adoptive immunotherapy.