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" |
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