title: Detection and quantification of CD8(+) T cells specific for HLA-A*0201-binding melanoma and viral peptides by the IFN-gamma-ELISPOT assay.
abstract: Blood lymphocytes from HLA-A*0201-subtyped melanoma patients and healthy controls were screened for the presence of T cells specific for HLA-A*0201-binding melanoma and viral peptide antigens by the enzyme-linked immunoSPOT (ELISPOT) assay. CD8(+) cells were tested for peptide-specific IFN-gamma release immediately after selection as well as after 2 weeks of in vitro stimulation. After in vitro stimulation, CD8(+) T cells specific for influenza were measured in all patients and controls, whereas these T cells could be detected among nonstimulated CD8(+) cells in only 52% of individuals. Similarly, T cells specific for EBV were more frequently measured among in vitro-stimulated than nonstimulated CD8(+) cells. In nonstimulated CD8(+) cells, T cells specific for MART-1/Melan-A, gp100, tyrosinase and CAMEL were present in 4 (33%), 1 (8%), 1 (8%) and 3 (25%) of 12 patients, respectively. Only MART-1/Melan-A-specific CD8(+) T cells were found in 1 (11%) of 9 healthy controls. CD8(+) T cells specific for MAGE-2 were not observed. After in vitro stimulation, CD8(+) T cells specific for MART-1/Melan-A could be demonstrated in 6 (46%) of 13 patients and 2 (20%) of 10 controls. CD8(+) T cells specific for gp100 were detected in 1 patient after in vitro stimulation. No CD8(+) T cells specific for tyrosinase, MAGE-2 or CAMEL could be measured after in vitro stimulation. These data show that the ELISPOT assay allows direct ex vivo detection of CD8(+) T cells specific for viral and melanoma antigens. Furthermore, the data show that the sensitivity of the ELISPOT assay to measure influenza- and EBV-specific CD8(+) T cells can be enhanced by a short in vitro stimulation step, whereas opposing effects on numbers of CD8(+) T cells specific for melanoma antigens have been observed.
authors.affiliation: Department of Clinical Oncology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Griffioen
authors.forename: M
authors.initials: M
authors.lastname: Borghi
authors.forename: P I
authors.initials: PI
authors.lastname: Schrier
authors.forename: S
authors.initials: S
authors.lastname: Osanto
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology*; D000956:Antigens, Viral / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002860:Chromium Radioisotopes; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D015789:HLA-A2 Antigen / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology; D058965:MART-1 Antigen; D008545:Melanoma / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology; D009363:Neoplasm Proteins / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology*; D014763:Viral Matrix Proteins / Q000276:immunology; D058951:gp100 Melanoma Antigen
chemical_list: D000951:Antigens, Neoplasm; D000956:Antigens, Viral; D002860:Chromium Radioisotopes; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D058965:MART-1 Antigen; C548667:MLANA protein, human; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; C548637:PMEL protein, human; D010446:Peptide Fragments; D014763:Viral Matrix Proteins; D058951:gp100 Melanoma Antigen; C098952:influenza virus membrane protein (58-66); D007371:Interferon-gamma