Generation of human cytomegalovirus-specific cytotoxic T-lymphocytes in a short-term culture.

A method to generate human cytomegalovirus (HCMV)-specific CTL (cytotoxic T lymphocytes) from human peripheral blood mononuclear cells is described. This assay is unique in comparison with other methods reported to date, because it only requires a short-term (6 days) coculture of PBM and autologous infected fibroblasts without the addition of exogenous IL-2 (interleukin-2) and nevertheless is sensitive enough to determine HCMV-specific killing in a short (6 h) 51Cr-release assay using autologous HCMV-infected fibroblasts as targets. The virus-specific killing is mediated by CTL of the CD8 phenotype and it can be inhibited by a HLA class I monoclonal antibody. The sensitivity of the assay can be significantly enhanced by pretreating the targets with interferon-gamma (IFN-gamma) prior to infection with HCMV. HCMV-specific 51Cr-release is more than doubled when the IFN-gamma pretreated targets are used. This increase is mostly due to enhanced sensitivity of the fibroblasts to killing mediated by CD8-positive CTL, but some killing can be attributed to CTL of the CD4 phenotype.