CD4(+) T cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro

Purpose: Most melanoma cell lines express HLA class II antigens constitutiv ely or can be induced to do so with interferon gamma (IFN gamma). We have p reviously demonstrated that peptide-specific CD4(+) T cells proliferate in response to HLA-class-II-antigen-mediated peptide presentation by melanoma cells in vitro and produce interleukin-10 (IL-10) and (IFN gamma). We asked whether the responding T cells kill the tumor cells and, if so, whether di rect cell contact was required. Methods: Two HLA class II+ melanoma cell li nes derived from metastases were co-cultured with a human CD4+ T cell clone specific for influenza hemagglutinin peptide (HA). T cells, melanoma, and HA were co-cultured for 48 h. Melanoma cells with and without HA and/or T c ells served as controls. After 36 h, the medium was removed for cytokine an alysis by enzyme-linked immunosorbent assay (ELISA). Twelve hours later non -adherent cells were washed away and the adherent melanoma cells were tryps inized and counted. Dual-chamber culture plates were used to determine whet her cell contact and/or exposure to cytokine were required for tumor cell d eath. Results: Melanoma cell counts were over 80% lower in wells containing T cells than in wells with melanoma and peptide alone (P < 0.05). ELISA of supernatants revealed production of IFN gamma and IL-10 by the responding T cells. Direct T cell contact with tumor cells was not required for tumor cell death, as melanoma cells were killed when they shared medium but had n o contact with T cells responding to peptide presentation by HLA-class-II-a ntigen-positive melanoma cells in a separate chamber. Blocking antibody to IFN gamma but not IL-10 prevented melanoma cell death at levels of cytokine similar to that present in co-culture assays. Conclusions: Peptide-specifi c CD4(+) T cells kill melanoma cells in vitro when they recognize peptide p resented by the tumor cell in the context of HLA class II antigen. Direct c ell contact is not required, suggesting that it is a cytokine-mediated even t. Immunotherapy, using primed CD4(+) T cells and peptide, may be beneficia l in patients whose tumors express HLA class II antigens or can be induced to do so with IFN gamma.