Increased frequency of antigen-specific CD8+ cytotoxic T lymphocytes infiltrating an Epstein-Barr virus–associated gastric carcinoma
Kiyotaka Kuzushima, … , Tohru Kiyono, Tatsuya Tsurumi
Kiyotaka Kuzushima, … , Tohru Kiyono, Tatsuya Tsurumi
Published July 15, 1999
Citation Information: J Clin Invest. 1999;104(2):163-171. https://doi.org/10.1172/JCI6062.
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Article

Increased frequency of antigen-specific CD8+ cytotoxic T lymphocytes infiltrating an Epstein-Barr virus–associated gastric carcinoma

Abstract

Gastric adenocarcinomas carrying Epstein-Barr virus (EBV) are known to be accompanied by massive lymphocyte infiltration. To characterize the tumor-infiltrating lymphocytes (TILs), we isolated and cultured such cells from a surgically resected EBV-associated gastric carcinoma. They were found to be positive for CD3, CD8, T-cell receptor β chain, and cytotoxic molecules. The isolated TILs consisted of human leukocyte antigen (HLA) class I–restricted CD8+ cytotoxic T lymphocytes (CTLs), which killed autologous EBV-transformed cells (but not phytohemagglutinin blast cells) and recognized HLA-A24 as restriction molecules. However, the TILs did not recognize known EBV antigenic peptides presented by HLA-A24 molecules, nor HLA-A24+ fibroblasts infected with vaccinia recombinant virus expressing each of the EBV latent proteins. EBV+ gastric carcinomas do not express conventional target proteins of EBV-specific CTLs, and the data suggest that some cellular proteins may be involved in the strong T-cell response to EBV-associated gastric carcinoma. In addition, our data suggest that class I–restricted, antigen-specific CD8+ CTLs are specifically expanded within EBV+ gastric carcinoma tissue.

Authors

Kiyotaka Kuzushima, Shigeo Nakamura, Tsuneya Nakamura, Yoshitaka Yamamura, Naoaki Yokoyama, Masatoshi Fujita, Tohru Kiyono, Tatsuya Tsurumi

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Characterization of TILs. (a) The TILs recognized and killed autologous ...
Characterization of TILs. (a) The TILs recognized and killed autologous LCLs, but not PHA blast cells. HLA-A24+ allogeneic LCLs (donors 1 and 2) were killed more efficiently than HLA-A24– LCLs (donors 3 and 4). (b) The TILs killed HLA-A*2402–transfected EBV+ C1R cells more efficiently than untransfected C1R cells. (c) Cytotoxicity of TILs is largely mediated by the CD8+ population. Aliquots of TILs were reacted with anti-CD19–, anti-CD4–, and anti-CD8–coated magnetic beads, and unbound cells were resuspended in the same volume and used as effector cells. Autologous LCLs were used as targets. The effector/target ratio was 20:1 before the magnetic bead treatment. Results are mean ± SD in a–c. (d) TILs did not recognize known antigenic peptides presented by HLA-A24 molecules. The peptide derived from EBNA3A (RYSIFFDY), EBNA3B (TYSAGIVQI), or LMP2A (TYGPVFMSL) was pulsed on 51Cr-labeled HLA-A24+ PHA blast cells used as targets. Filled and dotted bars indicate EBV-transformed cells and PHA blast cells, respectively. (e) TILs did not recognize any EBV latent proteins presented by HLA-A24 molecules. HLA-A24+ fibroblasts were infected with each of the vaccinia recombinants expressing the EBV latent proteins. E1ΔGA is a recombinant virus that expresses a glycine-alanine repeat-deleted form of EBNA1. LP represents the EBV leader protein. Filled bars indicate results using EBV-specific CTLs as effectors. Dotted bars indicate results using the TILs. The effector/target ratio was 20:1 in a, d, and e, and 10:1 in b.