Visualization and microscopic quantification of HCMV-peptide-specific cytotoxic T lymphocytes using tetramer binding |
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Authors: | Bissinger A L Brugger J Grigoleit G U Gründemann C Hebart H Einsele H Jahn G |
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Institution: | Institut für Medizinische Virologie, Universit?tsklinikum Tübingen, Elfriede-Aulhom-Strasse 6, D-72072 Tübingen, Germany. lennart.bissinger@med.uni-tuebingen.de |
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Abstract: | To monitor the frequencies of virus-specific cytotoxic T lymphocytes (CTLs), FACS analyses were performed detecting lymphocyte-specific surface molecules and tetramer binding, as marker for peptide-specificity. Aim of this investigation was to establish an alternative protocol for the quantification of virus-specific CTLs using tetramer binding and microscopic analyzing. The frequencies of HCMV-pp65-peptide-specific CTLs in the blood of eight different HLA-A*0201-positive, HCMV-IgG antibody-positive donors were analyzed with both methods. Using FACS analyses, a median of 0.8% and, using the microscopic analyses, a median of 3.0% was detected in the CD3+CD8+ cells. After enrichment of HCMV-pp65-peptide-specific CTLs using the interferon-gamma secretion assay followed by expansion in cell culture, a median of 90.6% using FACS analyses and a median of 87.1% using the microscopic analyses was detected. Thus, the staining protocol presented in this investigation is an alternative approach to detect and to quantify virus-specific CTLs in low as well as in high frequencies. |
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