title: In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring.
abstract: 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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
authors.forename: J
authors.initials: J
authors.lastname: Yuan
authors.forename: H F
authors.initials: HF
authors.lastname: Gallardo
authors.forename: T
authors.initials: T
authors.lastname: Rasalan
authors.forename: R
authors.initials: R
authors.lastname: Ranganathan
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.forename: K
authors.initials: K
authors.lastname: Panageas
authors.forename: R
authors.initials: R
authors.lastname: Stan
authors.forename: J W
authors.initials: JW
authors.lastname: Young
authors.forename: A N
authors.initials: AN
authors.lastname: Houghton
authors.forename: J D
authors.initials: JD
authors.lastname: Wolchok
mesh_terms: D000938:Antigen-Presenting Cells / Q000276:immunology*; D005260:Female; D015234:HLA-A Antigens / Q000235:genetics / Q000276:immunology*; D015789:HLA-A2 Antigen; D006801:Humans; D007167:Immunotherapy / Q000379:methods; D020014:K562 Cells; D008297:Male; D008991:Monitoring, Physiologic / Q000379:methods; D009101:Multiple Myeloma / Q000276:immunology* / Q000628:therapy; D010455:Peptides / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
chemical_list: D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D010455:Peptides