Establishment of an immortalized T-cell line from lung cancer tissue: phenotypic and functional analyses.

Abstract

In order to investigate host defense against solid tumors, valuable information could be provided by ex-vivo analyses of functional immune cells in tumor tissues. However, available sources of fresh tumor-infiltrating T cells (TIL) are usually very limited, and it is often difficult to establish TIL lines. In this study, we analyzed the phenotypic and functional characteristics of TIL, using an immortalized cell line prepared by cotransfection of human c-myc and c-Ha-ras. A human T-cell line was established by cotransfecting c-Ha-ras and c-myc oncogenes to T lymphocytes freshly isolated from human lung large-cell carcinoma tissue. The phenotypes were assessed by flow cytometry. T-cell receptor (TCR) V alpha- and beta-usage was analyzed by polymerase chain reaction (PCR) and Southern blot. Cytotoxic activity against autologous and allogeneic tumor cell lines was examined by standard 51Cr-release cytotoxicity assays. Cytokine production by the established T-cell line, 904-T1, in response to stimulation by autologous tumor cells was assayed by using an enzyme-linked immunosorbent assay. 904-T1 (CD3+, CD8+, CD56+, CD16-, CD161-, TCR V alpha 9, 13, and V beta 1, 5) displayed a broad range of MHC-nonrestricted tumoricidal activity against various human tumor cell lines, but did not lyse autologous B cells transformed by Epstein-Barr virus. The cytotoxicity of 904-T1 was not mediated by a T-cell antigen receptor or by Fas-ligand, but by perforin-based cytolytic pathways, and was enhanced by interleukin (IL)-12. 904-T1 cells produced large amounts of interferon (IFN)-gamma, but not tumor necrosis factor (TNF)-alpha or IL-4 in response to autologous tumor cells, and produced high levels of IFN-gamma and TNF-alpha, and a substantial level of IL-4 following stimulation with anti-CD3 monoclonal antibody. Our results suggest that 904-T1 cells were natural killer T (NKT)-like cells with regard to their non-specific killing, cytokine repertoire, and sensitivity to IL-12, although the repertoire of the TCR variable region was not compatible with that of NKT cells.