Recognition of various target cells and the antigenic peptide by CTL clones derived from TIL586. T cell clones were generated by limiting dilution (1 cell/well) from the TIL586 cell line and were further expanded in AIM-V medium containing 6,000 IU/ml IL-2. GM-CSF secretion by CTL clone 586TILC1 (left) and clone 4 (right) was measured after coculturing with a normal melanocyte cell line NHEM680 (HLA-A31⁺), 586EBV B cells pulsed with the ORF3P peptide or irrelevant peptide, 397mel or 586mel cells.

Construction of deletions and subclones of the TRP-2 gene and T cell recognition. The full-length cDNA of TRP-2 which comprises the 1,557-bp open reading frame is shown. Nucleotides are numbered from the first nucleotide from the 5′ untranslated region of TRP-2 cDNA. A series of deletion constructs and subcloning of DNA fragments were made. T cell recognition of each construct was determined after coculturing CTL clone 4 with COS-7 cotransfected with the DNA fragments shown above and the HLA-A31 gene.

Characterization of the antigenic peptide recognized by CTL clone 4. (A) GM-CSF release by T cells at different peptide concentrations. 586EBV (A31+) were pulsed with the TRP_197–205 peptide ( ) and T2 (non-A31) cells were pulsed with the TRP_197–205 ( ) at various peptide concentrations for 90 min. ORF3P as a control peptide was pulsed onto 586EBV B cells (--▵--). GM-CSF release by CTL clone 4 was determined after coincubation with 586EBV B cells pulsed with TRP_197–205 and ORF3P, and T2 cells pulsed with TRP_197–205. (B) Sensitization of the target cells for lysis by CTL clone 4 at different peptide concentrations. 586EBV B cells were incubated with TRP_197–205 ( ), an irrelevant peptide ORF3P (--▵--), and T2 cells pulsed with TRP_197–205 ( ) at various peptide concentrations. After peptide incubation, target cells were labeled for 30 min. Following washes, cytolytic activity of CTL clone 4 at an E/T of 40:1 was measured after a 4 h incubation of T cells with target cells. (C) Lysis of the target cells by CTL clone 4 at different E/T. Target 586EBV cells were separately incubated with TRP_197–205 ( ) or the irrelevant peptides ORF3P (--▵--), and target T2 cells were incubated with the TRP_197–205 peptide ( ) for 90 min. 586mel ( ) and 397mel ( ) were used as positive and negative controls, respectively.

Identification of TRP-2 as a tumor antigen recognized by CTL clone 4. GM-CSF release by CTL clone 4 was measured after coculture with COS-7 cotransfected with the HLA-A31 cDNA along with genes encoding MART-1, gp75/TRP-1, gp100, tyrosinase, p15, and TRP-2. Control stimulator cells included 586mel, 397mel, COS-7 alone, and COS-7 transfected with the HLA-A31 cDNA.

Antigenic peptide and partial coding sequence of TRP-2. The partial nucleotide and amino acid sequences of the TRP-2 gene are shown. The length and 3′ terminus of the DNA fragments in pTD4, pTA, pTD3, and pTK are indicated by arrows and the restriction sites for ApaI, PstI, and KpnI, are marked. The antigenic peptide sequence recognized by CTL clone 4 is in bold and underlined.