Generation of Tumor-Specific Cytotoxic T Lymphocytes by Stimulation with HPV Type 16 E7 Peptide-Pulsed Dendritic Cells: An Approach to Immunotherapy of Cervical Cancer

Abstract

Objective. The aim of this study was to generate HPV-16 E7 peptide-specific cytotoxic T lymphocytes (CTLs) in vitro for future adoptive immunotherapy of cervical cancer.Methods. Peripheral blood mononuclear cells (PBMC) were isolated from HLA-A2+ healthy donors. The PBMCs were incubated with HPV-16 E711–20 peptide and varying cytokines in the primary culture. Restimulation was performed weekly with peptide-pulsed, irradiated autologous PBMCs. Alternatively, the PBMCs were depleted of abundant CD4+ cells and stimulated with HPV-16 E711–20 peptide-pulsed dendritic cells. Cytolytic activity was determined by a standard 4-h 51Cr-release assay.Results. After 6 weeks in culture, we were able to establish peptide-specific CTL lines in one of seven donors by incubating PBMCs with HPV-16 E711–20 peptide. When we employed autologous peptide-pulsed dendritic cells to stimulate CD8+ cell-enriched PBMCs, we obtained CTL lines in four of seven donors. The primed CTLs were able to lyse the HLA-A2+ and HPV-16+ cervical cancer cell line Caski. SiHa, an HLA-A2-, but HPV 16+, cervical cancer cell line could be lysed only after transfection with HLA-A2. In addition, a high cytotoxicity (>80%) was obtained against peptide-pulsed, but not unpulsed, targets such as autologous Ebstein–Barr virus-immortalized B cells or allogeneic lipopolysaccaride-stimulated PBMCs. DCs were clearly the most potent of all tested antigen presenting cells to stimulate a CTL response in a proliferation assay.Conclusion. HPV-16 E7 peptide-specific CTLs could be generated in vitro. A practical protocol to expand the CTLs to a sufficient number for an application in a clinical trial is in progress.