Co-culture of tumor cells with apoptosis-resistant γδ-T cells: detection of tumor cell death.

(A) T lymphocytes alone (left plot) and HeLa target cells alone (center plot) have characteristic light scatter properties that allow each cell population to be distinguished, even when mixed together in coculture (right plot). (B) HeLa cells were cocultured for 4 hours with apoptosis-resistant γδ-T cells (TCR-γδ) or control αβ-T cells (TCR-αβ) at the indicated E:T ratios (0:1 to 20:1). Cocultured cells were then analyzed using FACS. Gating on the appropriate cell population (high forward scatter and high side scatter), uptake of annexin V-FITC by HeLa cells was determined and was taken as a measure of the ability of cocultured αβ- or γδ-T cells to induce apoptosis. Light microscopy and FACS using anti-CD3 mAbs were used to confirm that no tumor–lymphocyte aggregates remained after vortexing samples (not shown). Data are presented as histograms, and the percentage of HeLa cells staining with annexin V-FITC (and thus apoptotic) is indicated. These data are representative of experiments performed at least 3 times on materials obtained from 3 separate persons. (C) Measurement of HeLa target cell viability after coculture with apoptosis-resistant γδ-T cells or control αβ-T cells for longer periods at lower E:T ratios. Target HeLa cells were cultured alone or were cocultured with either human αβ- or γδ-T cells at a 1:1 E:T ratio for 18 hours. On the addition of ethidium bromide and acridine orange, cells were immediately viewed under fluorescence. As viewed using a 20× objective lens, tumor cells were readily distinguished from effector lymphocytes by size alone, permitting the enumeration of live (green) and dead (orange) tumor cells in each well. The percentage of tumor cells remaining viable was thus derived by dividing the number of green tumor cells by the number of green plus orange tumor cells ([green]/[green + orange]) in each well. Quantitations were performed in quadruplicate, with data presented as the mean viable tumor cells remaining per high-power field ± SD. Parallel determinations using trypan blue and a standard inverted microscope were also made and were in agreement with the results of these studies (not shown). These data are representative of experiments performed at least 3 times on materials obtained from 4 separate persons.