Visualization and microscopic quantification of HCMV-peptide-specific cytotoxic T lymphocytes using tetramer binding.
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.
Bissinger
A L
AL
Institut für Medizinische Virologie, Universitätsklinikum Tübingen, Elfriede-Aulhom-Strasse 6, D-72072 Tübingen, Germany. lennart.bissinger@med.uni-tuebingen.de
Brugger
J
J
Grigoleit
G U
GU
Gründemann
C
C
Hebart
H
H
Einsele
H
H
Jahn
G
G
D000595:Amino Acid Sequence; D002469:Cell Separation; D003587:Cytomegalovirus / Q000235:genetics / Q000276:immunology*; D005434:Flow Cytometry; D015234:HLA-A Antigens; D015789:HLA-A2 Antigen; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000235:genetics / Q000276:immunology; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000235:genetics / Q000276:immunology
D015234:HLA-A Antigens; C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D009842:Oligopeptides; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa