Herpes Simplex Virus-1-Specific Human Cytotoxic T Lymphocytes Are Induced in Vitro by Autologous Virus-Infected Mononuclear Cells

A new technique for in vitro activation of cytotoxic T lymphocytes (CTLs) specific for herpes simplex virus type 1 (HSV-1) is described. Autologous phytohemagglutinin (PHA)-activated, HSV-1-infected peripheral blood mononuclear cells (PBMC) were used, after fixation with 1% paraformaldehyde, to activate virus-specific CTLs in short-term cultures. The same unfixed PBMC were used as target cells in the cytotoxicity assay. By using this technique high levels of HSV-1-specific cytotoxic activity (50.06 ± 16.76% at 30:1 effectontarget ratio) were repeatedly obtained in 24 experiments using PBMC from 16 HSV-1 antibody-positive healthy donors, while no cytotoxic activity was observed using PBMC from 3 HSV-1 antibody-negative donors. HSV-1-induced CTLs were shown to be virus-specific as they did not lyse autologous, PHA-activated PBMC infected with influenza A virus or autologous Epstein-Barr virus (EBV) lymphoblastoid cell line (LCL), while they were able to lyse both HSV-1-infected, autologous PHA-activated PBMC and EBV-LCL. HSV-1-specific cytotoxicity was mediated by T lymphocytes, since depletion of CD3-positive cells from the effector population completely removed the killing of HSV-1-infected target cells. CD8-positive CTLs were primarily involved in the killing of HSV-1-infected targets since depletion of CD8-positive cells caused a strong reduction of virus-specific cytotoxic activity while elimination of CD4-positive lymphocytes increased killing capacity. Finally, this technique has proven to be highly reproducible, easy to perform, and thus suitable for clinical investigations.