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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

Selective expansion of a specific anti‐tumor CD8 + cytotoxic T lymphocyte clone in the bulk... 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Journal of Immunology Wiley

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

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References (31)

Publisher
Wiley
Copyright
Copyright © 1990 Wiley‐VCH Verlag GmbH & Co. KGaA, Weinheim
ISSN
0014-2980
eISSN
1521-4141
DOI
10.1002/eji.1830200417
pmid
1971794
Publisher site
See Article on Publisher Site

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 1010 cells which should allow testing of immunotherapeutic potential of these cells by adoptive transfer into melanoma patients.

Journal

European Journal of Immunology – Wiley

Published: Apr 1, 1990

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