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Summary The expression of the gp100 antigen is generally thought to be confined to cells of the melanocytic lineage, which makes the protein a suitable melanoma-specific marker. Strikingly, after screening a panel of normal tissues, tumour samples and cell lines of nonmelanocytic origin, we found transcripts encoding gplOO in virtually every tissue and cell line tested. In contrast, tyrosinase and MART-1/MelanA transcripts were detected only in cells of the melanocytic lineage. However, no gpl 00 protein could be detected by either Western blotting or cytotoxicity assays. Therefore, at the protein level, gp100 remains exclusive for cells of melanocytic origin despite its transcription in many cell types. The major implication of this finding is that screening of patient material for gp100 expression should preferrably be performed by antibody staining. Reverse transcriptase polymerase chain reaction (RT-PCR) can be employed, provided that it is performed in a tightly controlled, semiquantitative setting.Keywords: gp100; RT-PCR; melanoma; renal cell carcinoma; tumour-associated antigens The molecular cloning of tumour-associated antigens has provided new tools for the immunotherapy of cancer (reviewed in Van den Eynde and Brichard, 1995). Now it has become feasible to immunize cancer patients against these antigens to stimulate specifically a cellular anti-tumour response (Marchand et al, 1995). To select patients eligible for immunotherapeutic protocols, the antigenic profiles of the patients' tumours must be characterized. This is usually achieved by reverse transcriptase polymerase chain reaction (RT-PCR) using RNA obtained from tumour samples when available. An alternative source of tumour cells is whole blood, which often contains numerous circulating residual tumour cells (Brossart et al, 1995;Hoon et al, 1995). A suitable antigen to target in melanoma is gplOO because it is thought to be specific for cells of melanocytic lineage and it is adequately expressed in melanoma cells. Expression in non-melanocytic cells, at least as measured by antibody reactivity (Vennegoor et al, 1988) or Northern analysis (Kawakami et al, 1994), is virtually absent. In a routine RT-PCR screening of a number of human tumour samples and normal tissues, we noticed to our surprise that gplOO transcripts were present in almost all materials tested, whereas the protein was not detectable. The renal cell carcinoma cell lines (RCC) LE-9104-RCC, LE-921 l-RCC and LE-9415-RCC and the melanoma cell line Mel 603 were established in our laboratory. The RCC cell line SK-RC-7 was kindly provided by Dr E Oosterwijk (Department of Urology, Nijmegen University, The Netherlands). The RCC cell lines MZ-1851-RCC and Camejo and melanoma cell lines MZ-2-mel and MZ-7.4-mel were generously provided by Dr B Seliger (J Gutenberg University, Mainz, Germany). The breast carcinoma cell lines (BRCA) MCF-7 and SK-BR-3 were a gift from Dr

Tumor Ag NY-ESO-1 is an attractive target for immunotherapy of cancer, since both CD8+ CTL and CD4+ Th cells against NY-ESO-1 have been described. Moreover, NY-ESO-1 as well as the highly homologous tumor Ag LAGE-1 are broadly expressed in various tumor types. Interestingly, the NY-ESO-1 and LAGE-1 genes also encode for proteins translated in an alternative open reading frame. These alternatively translated NY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1 genes, respectively, have been demonstrated to be immunogenic, since CTL specific for these proteins have been isolated from melanoma patients. In this study a panel of advanced melanoma patients was screened for the presence of Th cells specific for the alternatively translated tumor Ags NY-ESO-ORF2 and CAMEL. PBMC of melanoma patients were stimulated for 4 days with mixes of overlapping peptides covering the entire NY-ESO-ORF2 and CAMEL protein sequences and were tested for the release of type 1 (IFN-γ) and type 2 (IL-13) cytokines in ELISPOT assays. In three of 15 patients, T cells specific for two CAMEL peptides (CAMEL71–92 and CAMEL81–102) could be detected. From one of these patients, CD4+ T cell clones specific for CAMEL81–102 could be generated. These clones recognized a naturally processed epitope presented in both HLA-DR11 and HLA-DR12 and produced high levels of IL-4, IL-5, and IL-13. In conclusion, this study shows the presence of Th cells specific for the alternatively translated tumor Ag CAMEL in melanoma patients and is the first report that describes the isolation of tumor Ag-specific CD4+ Th 2 clones.