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.
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 E7(11-20) 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 E7(11-20) peptide-pulsed dendritic cells. Cytolytic activity was determined by a standard 4-h (51)Cr-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 E7(11-20) 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.


CONCLUSIONS
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.
Schoell
W M
WM
Division of Gynecologic Oncology, University of Miami, Miami, Florida, 33136, USA. wolfgang.schoell@kfunigraz.ac.at
Mirhashemi
R
R
Liu
B
B
Janicek
M F
MF
Podack
E R
ER
Penalver
M A
MA
Averette
H E
HE
D003713:Dendritic Cells / Q000276:immunology*; D005260:Female; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D009856:Oncogene Proteins, Viral / Q000276:immunology*; D027383:Papillomaviridae / Q000276:immunology*; D050725:Papillomavirus E7 Proteins; D013045:Species Specificity; D013602:T-Lymphocytes, Cytotoxic; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000628:therapy*
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; C059731:oncogene protein E7, Human papillomavirus type 16