Sensitivity of B16 melanoma sublines to lymphokine-activated killer cells as determined by 51Cr-release and clonogenic assays

Abstract

The aim of this study was to compare the differential sensitivities of B16 melanoma sublines to LAK cells by means of the standard 51Cr release assay and a clonogenic assay, which measures both cell survival and proliferation. LAK cells, generated after 4 days incubation with 150 international units (IU)/ml of interleukin-2 (IL-2), showed both cytolytic and anti-proliferative activities against B16 targets. Using an 18 h 51Cr release assay, murine LAK cells showed the highest cytolytic activity against B16 parental cells compared to B16-F1, B16-F10, B16-FLR and B16-BL6 sublines at effector/target ( E/ T) ratios ranging from 6/1 to 100/1. Purified adherent LAK (A-LAK) cells showed greater cytolytic activity against B16 parental cells and other B16 sublines compared to LAK cells, but otherwise the pattern of reactivity was similar. Using a clonogenic assay, the surviving fraction of B16 parental cells co-cultivated with LAK cells decreased to 0 at an E/ T ratio of 50/1, while a 400/1 ratio was required to achieve a similar reduction of B16-F1, B16-F10, B16-FLR, and B16-BL6 sublines. No differences in subline sensitivity were seen with the 51Cr release assay, but these were observed using the clonogenic assay. An inverse linear relationship existed between % surviving fraction, as determined by the clonogenic assay, and cytolytic activity, as determined by the 51Cr release assay. Our data indicate that the clonogenic assay can detect differences in target cell sensitivity that otherwise are undetectable by the standard 51Cr release assay. The clonogenic assay may prove useful in delineating the long-term anti-adherent and anti-proliferative properties of effector cells from their cytolytic activity.