Functional characterization of CD8(+) antigen-specific cytotoxic T lymphocytes after enrichment based on cytokine secretion: comparison with the MHC-tetramer technology

Scand J Immunol. 2000 Dec;52(6):544-9. doi: 10.1046/j.1365-3083.2000.00810.x.


Cell therapy with antigen-specific T cells holds promise for various diseases including cancer and viral infections. The powerful enrichment procedure based on major histocompatibility complex (MHC)-tetramers, however, is of limited applicability so far. Therefore, the recently developed cell surface affinity matrix technology that allows direct identification and enrichment of life antigen-specific T cells based on cytokine secretion was evaluated in this respect. To this end, CD8(+) T cells directed against the HLA-A(*)0201-restricted melanoma-associated peptide Melan-A (aa26-35) were generated by combining stimulation of peptide-pulsed autologous dendritic cells (DC) with antigen-independent expansion with anti-CD3/anti-CD28 monoclonal antibodies (MoAb). Antigen-specific cytotoxic T lymphocyte (CTL) were detected based on stimulation-induced interferon (IFN)-gamma and interleukin (IL)-4 secretion and enriched > 100-fold using the cell surface affinity matrix technology. The resulting IFN-gamma- and IL-4-secreting CTL lines contained > 80% and > 70% cytokine positive T cells, respectively. They exhibited a cytotoxic activity against Melan-A expressing target cells that was significantly higher as compared to nonpurified CTL. Direct staining of enriched CTL with HLA-A2-Melan-A-tetramers revealed a high correlation between the results obtained from the cell surface affinity matrix technology and those obtained from tetrameric complexes. Altogether, our study demonstrates that cytokine-driven enrichment based on the cell surface affinity matrix technology enables selective isolation of functionally active antigen-specific CTL that may be used for an adoptive T cell transfer in immunotherapy.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, Neoplasm
  • Cell Line
  • Cell Separation / methods*
  • Cell- and Tissue-Based Therapy*
  • Cytokines / metabolism*
  • Humans
  • Interferon-gamma / metabolism
  • Interleukin-4 / metabolism
  • MART-1 Antigen
  • Major Histocompatibility Complex*
  • Neoplasm Proteins / immunology*
  • T-Lymphocyte Subsets / cytology
  • T-Lymphocyte Subsets / immunology
  • T-Lymphocytes, Cytotoxic / cytology
  • T-Lymphocytes, Cytotoxic / immunology*


  • Antigens, Neoplasm
  • Cytokines
  • MART-1 Antigen
  • MLANA protein, human
  • Neoplasm Proteins
  • Interleukin-4
  • Interferon-gamma