In vitro expansion of Ag-specific T cells by HLA-A*0201-transfected K562 cells for immune monitoring
| Autor: | Teresa S. Rasalan, Jianda Yuan, Jedd D. Wolchok, Rajaram Ranganathan, Rodica Stan, Humilidad F. Gallardo, Katherine S. Panageas, Alan N. Houghton, Jian Wang, James W. Young, Yan Zhang |
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| Rok vydání: | 2006 |
| Předmět: |
Male
Cancer Research medicine.medical_treatment T-Lymphocytes Immunology Antigen-Presenting Cells Biology Transfection Interleukin 21 Immune system Cancer immunotherapy HLA-A2 Antigen medicine Immunology and Allergy Cytotoxic T cell Humans IL-2 receptor Genetics (clinical) Monitoring Physiologic Transplantation HLA-A Antigens ELISPOT Cell Biology Molecular biology Oncology Female Immunotherapy K562 Cells Multiple Myeloma Peptides CD8 K562 cells |
| Zdroj: | Cytotherapy. 8(5) |
| ISSN: | 1465-3249 |
| Popis: | 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.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.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 expanded100-fold over a 10-day culture period (peak at day 12).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. |
| Databáze: | OpenAIRE |
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