Selective expansion of a specific anti‐tumor CD8+ cytotoxic T lymphocyte clone in the bulk culture of tumor‐infiltrating lymphocytes from a melanoma patient: Cytotoxic activity and T cell receptor gene rearrangements

In vitro culture of metastatic melanoma fragments with 150 units of recombinant interleukin 2 resulted in the successive expansion of CD4+ and then CD8+ tumor‐infiltrating lymphocytes (TIL) throughout a 2‐month period. TIL cultured for 43 days and consisting of 95% CD8+ and 10% CD4+ T lymphocytes were cloned by limiting dilution (LD). Thirteen CD8+ and thirty‐one CD4+ clones were obtained, indicating that the frequency of clonogenic CD8+ proliferative T lymphocytes was much lower than that of their CD4+ homologues. When LD was performed in the presence of autologous melanoma cells the frequency of CD8+ clones was increased by factor 4. The DNA from TIL of day 43 bulk culture and of six CD8+ clones was hybridized with T cell receptor (TcR) β and γ probes. Identical configuration of the nonfunctional γ and functional β TcR genes was found in “bulk culture” and cloned TIL. The CD8+ clones therefore derived from a clonal population of CD8+ cells which had expanded in vitro before the LD. All the CD8+ clones tested were strongly cytotoxic for autologous melanoma cells but did not kill autologous fibroblasts or concanavalin A blasts, or any of the 10 allogeneic tumor targets tested, including 5 melanomas, 2 breast cell lines, 1 neuroblastoma, K‐562 and the Epstein‐Barr virus‐transformed cell line used as a feeder. Furthermore, specific killing was inhibited by monoclonal antibodies against CD3, CD8, TcR α/β and against class I major histocompatibility complex antigens indicating that these cytotoxic T lymphocyte clones recognized autologous tumor cells through the TcR, in an HLA class I‐restricted manner. These data show that it is feasible to obtain tumor‐specific cytotoxic T lymphocytes from melanoma TIL with a simple culture technique and that a single clone could be expanded to more than 1010 cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.