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


Authors: Yuan, J.; Gallardo, H. F.; Rasalan, T.; Ranganathan, R.; Wang, J.; Zhang, Y.; Panageas, K.; Stan, R.; Young, J. W.; Houghton, A. N.; Wolchok, J. D.
Article 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 × 10 6 PBMC, a 10 μg/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.
Keywords: controlled study; human cell; case report; sensitivity analysis; cd8 antigen; lymphocyte proliferation; t lymphocyte; t-lymphocytes; reproducibility; interleukin 2; melanoma; multiple myeloma; culture medium; in vitro study; validation study; transfection; tumor antigen; haplotype; hla matching; immunotherapy; gamma interferon; antigen specificity; peptides; tumor immunity; effector cell; reliability; monophenol monooxygenase; enzyme linked immunospot assay; immunosurveillance; culture technique; hla a antigen; chemokine receptor ccr7; matrix protein; antigen presenting cell; hla-a antigens; antigen-presenting cells; interleukin 15; concentration response; peripheral blood mononuclear cell; cd45ra antigen; memory t lymphocyte; padgem protein; immune monitoring; cd69 antigen; k562 cells; process optimization; monitoring, physiologic; cell kinetics; ag-specific t cells; intracellular cytokine staining; k562-a; t-cell expansion; tetramer staining
Journal Title: Cytotherapy
Volume: 8
Issue: 5
ISSN: 1465-3249
Publisher: Elsevier Science Ltd.  
Date Published: 2006-11-01
Start Page: 498
End Page: 508
Language: English
DOI: 10.1080/14653240600868262
PUBMED: 17050255
PROVIDER: scopus
DOI/URL:
Notes: --- - "Cited By (since 1996): 8" - "Export Date: 4 June 2012" - "CODEN: CYTRF" - "Source: Scopus"
Altmetric Score
MSK Authors
  1. Jedd D Wolchok
    669 Wolchok
  2. Katherine S Panageas
    331 Panageas
  3. James W Young
    257 Young
  4. Jianda Yuan
    100 Yuan
  5. Alan N Houghton
    273 Houghton
  6. Teresa Rasalan
    25 Rasalan
  7. Jane Jian Wang
    7 Wang
  8. Yan Zhang
    5 Zhang
  9. Rodica Stan
    10 Stan