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

Abstract

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) beta and gamma probes. Identical configuration of the nonfunctional gamma and functional beta 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 alpha/beta 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 10(10) cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.

Loss of oncostatin M receptor β in metastatic melanoma cells

“…Metastatic melanoma cell lines were established from tumorinvaded lymph node fragments as described previously (Gervois et al, 1990;Dreno et al, 2002;Labarriere et al, 2002). Melanoma cell lines were established before injection of autologous TILs plus IL-2 or IL-2 only.…”

Studies of the mechanism of cytolysis by tumour-infiltrating lymphocytes

“…In malignant melanoma, it has been shown that TIL are able to recognize tumour-associated antigens, and in some cases to lyse the malignant cells [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. These findings provided a basis for the use of in vitro cultured TIL in adoptive immunotherapy in an effort to enhance the in vivo accumulation of tumour-lytic lymphocytes [29,31].…”

Characterization of fresh (uncultured) tumour-infiltrating lymphocytes (TIL) and TIL-derived T cell lines from patients with renal cell carcinoma

“…Blocking experiments using MoAb recognizing the CD3 antigen or the a/^TCR inhibited cytotoxicity at the effector cell level, demonstrating that these cell surface structures were involved in the cytolytic process. Southern blotting analysis revealed an oligoclonal pattern of /3-chain TCR gene rearrangements on melanoma [7,32] or ovarian carcinoma [26] TIL T cell lines with aulologous tumour-specific cytotoxicity. Culture of mixtures of melanoma or ovarian carcinoma TIL and tumour cells in low [5,7] or high [6,29 31] concentrations of rIL-2 resulted in rapid expansion of the T cells up to 3500-fold or higher, without the need for a single restimulation with autologous tumour celts [7].…”