Detection and quantification of CD8+ T cells specific for HLA-A*0201-binding melanoma and viral peptides by the IFN-?-elispot assay

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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-γ 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.

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