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o 1996 Wiley-Liss, Inc.Uveal melanoma is the most common primary intra-ocular tumor in adults (Egan et al., 1988). These tumors spread hematogeneously and preferentially to the liver. A 50% overall incidence of metastasis occurs within 15 years after initial treatment by enucleation or radiotherapy of the tumorcontaining eye (Kath et al., 1993). After clinical diagnosis of hepatic metastases, median survival is extremely poor: between 2 and 7 months (Kath et al., 1993). The biological and molecular properties of the successive steps involved in uveal melanoma progression and metastasis could be studied in detail once established uveal melanoma cell lines were available. A limited number of cell lines obtained from primary uveal melanoma have indeed been described (Kan-Mitchell et al., 1989; Aubert et al., 1993; Massarelli et al., 1994; De Waard-Siebinga et al., 1995), but not from metastatic uveal melanoma.In this study, we report on the successful establishment of 2 primary (EOM-3, EOM-29) and 3 metastatic weal melanoma cell lines (OMM-1, OMM-2, OMM-3) in our institute. These cell lines were characterized by immunocytochemistry, light and electron microscopy and cytogenetic analyses. The relative growth rate was determined by bromodeoxyuridin (BUdR) incorporation. In addition, a previously described primary uveal melanoma cell line, OCM-1 (Kan-Mitchell et al., 1989), was further characterized by extended karyotypic analysis.during follow-up of the patient, metastatic disease was suspected, a biopsy was taken for diagnosis and for cell culture. All patients had given full informed consent. Between January 1992 and May 1993, cells of the primary tumors of 16 patients and of the metastatic tumors of 4 patients were processed for culture. The patients with primary uveal melanoma ranged in age from 22 to 87 (mean: 56.8); 9 were female and 7 were male; 8 patients had a choroidal and 8 patients had a ciliary-body melanoma; 3 patients had a spindle-cell tumor, nine a mixedcell tumor and 4 had an epithelioid-cell tumor; tumor diameter ranged from 3 to 22 mm (mean: 11.6 mm). The patients were followed until May 1995; 2 patients died of metastatic disease and one is alive, 6 months after detection of a metastatic lesion in the subcutis. From all 4 patients with metastatic disease, tumor material was obtained from sub-cutaneous lesions. From one patient the primary as well as the metastatic tumor were available. Culturing methodsThe tumor material was processed and cultured within 1 to 3 hr after dissection, as described in Luyten et al. (1 993). In brief, the tumor-containing eye or the metastatic lesion was dissected under sterile conditions. A full-thickness biopsy was taken and transported in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 1% penicillin/streptomycin (full DMEM). The tumor material was suspended with a small pair of scissors and strained through a linen cloth by continuous irrigation with full DMEM. The resulting suspension of single cells, and to a lesser extent of small clumps of tum...

The CTL response to the HLA-A*0201-restricted, HIV-1 p17 Gag77–85 epitope (SLYNTVATL; SL9) has been extensively studied in patients. Although this reactivity is exceptionally prominent in chronically infected patients and inversely correlated to viral load, SL9-specific CTLs (SL9-CTLs) are rarely detected in acute infection. To explore the cellular basis for this unusual manifestation, SL9-CTLs primed ex vivo from naive circulating CD8+ T cells of healthy, seronegative donors were generated and characterized. SL9 appeared to differ from other well-studied A*0201-restricted epitopes in several significant respects. In contrast to published reports for influenza and melanoma peptides and the HIV gag IV9 epitope studied here in parallel, SL9-CTLs were primed by immature but not mature autologous dendritic cells. Highly activated SL9-CTLs produce sufficient autocrine mediators to sustain clonal expansion and CTL differentiation for months without CD4+ T cells or exogenous IL-2. Moreover, SL9-CTLs were sensitive to paracrine IL-2-induced apoptosis. IL-2 independence and sensitivity to paracrine IL-2 were also characteristic of SL9-CTLs immunized by dendritic cells transduced by a nonreplicating lentiviral vector encoding full-length Gag. In vitro-primed SL9-CTLs resembled those derived from patients in degeneracy of recognition and functional avidities for both SL9 and its natural mutations. Together, these data show that SL9 is a highly immunogenic, help-independent HIV epitope. The scarcity of SL9-CTLs in acute infection may result from cytokine-induced apoptosis with the intense activation of the innate immunity. In contrast, SL9-CTLs that constitutively produce autocrine help would predominate during CD4-diminished chronic infection.