THE PATTERN OF CYTOKINE GENE-EXPRESSION ON FRESHLY EXCISED HUMAN METASTATIC MELANOMA SUGGEST A A STATE OF REVERSIBLE ANERGY OF TUMOR-INFILTRATING LYMPHOCYTES

Expression of an extended panel of cytokine genes was investigated by reverse polymerase chain reaction (PCR) in 10 freshly excised melanoma metastases infiltrated by lymphocytes (TIL). cDNA encoding for CD3-de lta and tyrosinase could be amplified in all samples, confirming the p resence of T lymphocytes and melanoma cell. Cytokine genes possibly tr anscribed by both cell types, such as GM-CSF, IL-6 and IL-10 could be amplified from 5, 2 and 2 samples respectively. In contrast, IL-1 beta and TNF-alpha mRNA were never detectable, IL-1 alpha, IL-3 and IL-7 m RNA could be observed only in one case each. Transcripts encoding for TGF-beta 1 were observed in 8 samples, while TGF-beta 2 and 3 mRNA wer e detectable in only 2 specimens. mRNA encoding for cytokine genes typ ically transcribed by antigen-stimulated T lymphocytes, such as IL-2, IL-4 and IFN-gamma were rarely or never detectable (none, none and 1 o f the samples respectively). In one case, where no cytokine gene trans cription was detectable at the time of surgery, we addressed the quest ion of the antigenicity of the tumor and of the functional competence of TIL. A primary tumor cell line was generated and cultured TIL were induced to transcribe IL-2 and IFN-gamma genes by incubation with the autologous irradiated tumor cell line, but not with autologous EBV-tra nsformed cells. In these conditions, tumor-specific cytotoxic T lympho cytes (CTL) could be generated only after 3 weekly re-stimulations. In contrast, if autologous irradiated EBV-transformed cells were added t o the cultures, specific CTL could be detected after one single tumor stimulation. Thus, signs of active responsiveness in terms of lymphoki ne gene mRNA are seldom detectable in melanoma metastases. Tumor-speci fic responses, however, including IL-2 and IFN-gamma gene expression a nd generation of CTL can be produced in vitro from specimens in which no cytokine gene mRNA is detectable ex vivo. (C) 1994 Wiley-Liss, Inc.