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

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. © 2001 Wiley‐Liss, Inc.