Interactions of the allogeneic effector leukemic T cell line, TALL-104, with human malignant brain tumors

Abstract

TALL-104 is a human leukemic T cell line that expresses markers characteristic of both cytotoxic T lymphocytes and natural killer cells. TALL-104 cells are potent tumor killers, and the use of lethally irradiated TALL-104 as cellular therapy for a variety of tumors has been explored. We investigated the interactions of TALL-104 cells with human brain tumor cells. TALL-104 cells mediated increased lysis of a panel of brain tumor cells at low effector-to-target ratios over time. We obtained evidence that TALL-104 cells injured glioma cells by both apoptotic and necrotic pathways. A 7-amino actinomycin D flow cytometry assay revealed that the percentages of both apoptotic and necrotic glioma cells increased after TALL-104 cell/glioma cell coincubations. Fluorescent microscopy studies and a quantitative morphologic assay confirmed that TALL-104 cell/glioma cell interactions resulted in tumor cell apoptosis. Cytokines are secreted when TALL-104 cells are coincubated with brain tumor cells; however, morphologic analysis assays revealed that the soluble factors contained within clarified supernates obtained from 4 h coincubates added back to brain tumor cell cultures did not trigger the glioma apoptosis. TALL-104 cells do not express Fas ligand, even upon coincubation with glioma targets, which suggests that the Fas/Fas ligand apoptotic pathway is not likely responsible for the cell injury observed. We obtained evidence that cell injury is calcium dependent and that lytic granule exocytosis is triggered by contact of TALL-104 cells with human glioma cells, suggesting that this pathway mediates glioma cell apoptosis and necrosis.

Fig. 1

Detection of apoptotic and necrotic cells by the 7AAD assay. Images show carboxyfluorescein diacetate succinimidyl ester-labeled glioma cells that were and those that were not coincubated with TALL-104 cells at a 10:1 E:T for 4 h. A. 10-08-MG cells. B. 10-08-MG + TALL-104 cells. C. 14-07-MG cells. D. 14-07-MG + TALL-104 cells. E. 04-11-MG cells. F. 04-11-MG + TALL-104 cells.

Fig. 2

Detection of glioma cell apoptosis caused by TALL-104 cells. TALL-104 cells were coincubated with 04-11-MG, 10-08-MG, and 14-07-MG glioma cells for 0, 2, and 4 h. The percentages of apoptotic glioma cells were determined from Hoechst dye–stained fragmented or condensed nuclei visualized by fluorescence microscopy. The mean percentages ± standard error are given from counts taken from 4 separate experiments.

Fig. 3

Confirmation of apoptosis induction in glioma cells by a fluorescent microscopy technique and by an in vitro morphologic assay. A. Three CellTracker Green–labeled TALL-104 cells (smaller cells) in contact with a CellTracker Orange–labeled glioma cell (larger cell). B. Hoechst 33342 dye–labeled nuclei of the same cells demonstrating that the glioma cell has a fragmented nucleus. C. H&E-stained brain tumor cell monolayer cultured in the absence of TALL-104 cells demonstrating that the normal, nonapoptotic brain tumor cells are large, contain abundant cytoplasm, and have large oval nuclei. A mitotic figure in a glioma cell at metaphase is seen (arrowhead). D. A view of an H&E-stained brain tumor cell monolayer after coincubation with TALL-104 cells for 4 h. The coincubation of TALL-104 cells with the human brain tumor cells resulted in an increase in apoptotic tumor cells with a concurrent decrease in the number of brain tumor cells with mitotic figures. Cells identified as apoptotic (thin arrows) demonstrated classic morphological changes: either condensed nuclei, fragmented nuclei, or shedding of apoptotic bodies and membrane blebbing. TALL-104 cells were visualized in direct contact with apoptotic brain tumor cells (thick arrows). The inset shows H&E staining of TALL 104 cells.

Fig. 4

Calcium-dependent lysis of glioma cells by TALL-104 cell effectors is demonstrated. Three human gliomas (04-11-MG, 10-08-MG, and 14-07-MG) were placed into 4-h cytotoxicity assays with (□) or without (▪) 10 mM ethylene glycol-bis (2-amino ethyl) N′N′N′,N′-tetraacetic acid (EGTA) in the assay medium. At a 10:1 E:T ratio, a complete or partial inhibition of glioma cell lysis occurred when EGTA, a calcium chelator, was present. The mean percent lysis ± standard error is given.

Fig. 5

Evidence that granule exocytosis occurs during the interaction between TALL-104 and glioma cells. A. Calcein-loaded 04-11-MG glioma cells before (upper panel) and then at 5000 s after the addition of TALL-104 cells (lower panel). The arrows in each of the panels point to the locations of adherent fluorescent cells initially present that lost fluorescence during the experiment. B. Fluorescence traces from individual cells shown in A. The upper trace is an example of a cell that did not release calcein; the second, third, and bottom three panels monitor individual cells that displayed calcein release at 900 s, 1500 s, and 3100 s, respectively, after the addition of the TALL-104 cells. C. Time course of hitting for the experiment shown in A and B. Approximately 25% of cells were hit over the 5000-s (83-min) duration of the experiment.