In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring

Cytotherapy. 2006;8(5):498-508. doi: 10.1080/14653240600868262.


Background: Development of a practical and sensitive assay for evaluating immune responses against cancer Ag has been a challenge for immune monitoring of patients. We have established a reproducible method using peptide-pulsed K562-A*0201 cells as APC to expand Ag-specific T cells in vitro. This method may be applied for monitoring T-cell responses in cancer immunotherapy clinical trials.

Methods: Autologous PBMC from HLA-A*0201+ healthy donors and patients with melanoma were stimulated with peptide-pulsed K562-A*0201 cells under varying conditions. We investigated (1) different culture conditions, including the requirements for serum and cytokines for expansion of CD8+ T lymphocytes; (2) a range of peptide concentrations for Ag loading; (3) phenotypic characterization of responding T cells; and (4) APC:responder ratios and their effects on T-cell expansion. We validated these conditions by ELISPOT and intracellular cytokine staining (ICS) assays using peptides from influenza, Epslein-Barr Virus (EBV) and tyrosinase.

Results: Conditions for optimal T-cell expansion using K562-A*0201 APC included input of 2 x 10(6) PBMC, a 10 microg/mL peptide concentration to pulse K562-A*0201 cells, a 1:30 APC:responder T-cell ratio and culture in 10% autologous plasma supplemented with IL-2 and IL-15. In these conditions, Ag-specific T cells expanded >100-fold over a 10-day culture period (peak at day 12).

Discussion: This bulk culture method is simple and reliable for expanding human Ag-specific T cells using peptide-pulsed K562-A*0201 cells. This HLA-matched APC line can be adapted to other HLA haplotypes, and has advantages for monitoring clinical trials of immunotherapy with limited availability of autologous APC and PBMC from patients.

Publication types

  • Clinical Trial
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigen-Presenting Cells / immunology*
  • Female
  • HLA-A Antigens / genetics
  • HLA-A Antigens / immunology*
  • HLA-A2 Antigen
  • Humans
  • Immunotherapy / methods
  • K562 Cells
  • Male
  • Monitoring, Physiologic* / methods
  • Multiple Myeloma / immunology*
  • Multiple Myeloma / therapy
  • Peptides / immunology*
  • T-Lymphocytes / immunology*
  • Transfection


  • HLA-A Antigens
  • HLA-A*02:01 antigen
  • HLA-A2 Antigen
  • Peptides