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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
Author(s) -
Gervois Nadine,
Heuze Françoise,
Diez Elisabeth,
Jotereau Francine
Publication year - 1990
Publication title -
european journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.272
H-Index - 201
eISSN - 1521-4141
pISSN - 0014-2980
DOI - 10.1002/eji.1830200417
Subject(s) - cytotoxic t cell , biology , cd8 , microbiology and biotechnology , clone (java method) , t cell receptor , tumor infiltrating lymphocytes , antigen , immunology , t cell , immune system , in vitro , biochemistry , dna
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) 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 10 10 cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.