In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones.
Human papillomavirus (HPV) type 16 infection is strongly associated with the development of cervical carcinoma (CxCa) in women. The HPV16-derived oncoproteins E6 and E7, responsible for both onset and maintenance of malignant transformation, are expressed constitutively in CxCa cells and represent tumor-associated Ags. As a result, E6 and E7 constitute potential targets for adoptive CTL-mediated immunotherapy of CxCa. However, the availability to date of well-characterized HPV16-specific, CxCa-reactive human CTLs is extremely limited. The current study describes the in vitro generation and isolation of HPV16 E7-specific, CxCa-reactive human CTL clones from low-frequency healthy donor-derived CD8beta-positive precursors. For this purpose, an in vitro CTL induction protocol was used involving mature monocyte-derived dendritic cells as stimulator cells loaded with an HLA-A2.1-restricted, E7(11-20)-derived high-affinity altered peptide ligand. A double tetramer-guided isolation procedure and subsequent limiting-dilution cloning resulted in Ag-specific CTL clones. Stringent CTL characterization clearly indicated Ag-specific, HLA-A2.1-restricted reactivity against different HPV16-transformed CxCa cell lines. To allow expansion of E7(11-20)-specific CTL clones to numbers required for prolonged in vitro as well as in vivo application, their life span was significantly extended by ectopic expression of human telomerase reverse transcriptase. Collectively, our results show that optimized CTL induction and stringent CTL selection procedures, followed by human telomerase reverse transcriptase-mediated life span extension will allow continued availability of low-frequency HPV16-specific, CxCa-reactive human CTL clones. This may enhance the prospects of HPV16-specific adoptive CTL immunotherapy in CxCa patients.
Schreurs
Marco W J
MW
Department of Pathology, Vrije Universiteit Medical Center, 1081 HV Amsterdam, The Netherlands.
Scholten
Kirsten B J
KB
Kueter
Esther W M
EW
Ruizendaal
Janneke J
JJ
Meijer
Chris J L M
CJ
Hooijberg
Erik
E
D017951:Antigen Presentation; D002454:Cell Differentiation / Q000276:immunology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D002469:Cell Separation; D002470:Cell Survival / Q000276:immunology; D002999:Clone Cells; D003602:Cytotoxicity, Immunologic; D018984:Epitopes, T-Lymphocyte / Q000276:immunology* / Q000378:metabolism; D015789:HLA-A2 Antigen / Q000276:immunology / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000201:enzymology / Q000276:immunology / Q000378:metabolism / Q000821:virology; D006801:Humans; D016130:Immunophenotyping; D020014:K562 Cells; D008024:Ligands; D009856:Oncogene Proteins, Viral / Q000276:immunology / Q000378:metabolism; D027383:Papillomaviridae / Q000276:immunology* / Q000378:metabolism; D050725:Papillomavirus E7 Proteins; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding / Q000276:immunology; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000096:biosynthesis; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism / Q000821:virology*; D019098:Telomerase / Q000502:physiology
D018984:Epitopes, T-Lymphocyte; D015789:HLA-A2 Antigen; D008024:Ligands; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D010446:Peptide Fragments; D016693:Receptors, Antigen, T-Cell, alpha-beta; C059731:oncogene protein E7, Human papillomavirus type 16; D019098:Telomerase