In Vitro Generation and Life Span Extension of Human Papillomavirus Type 16-Specific, Healthy Donor-Derived CTL Clones

Abstract

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 CD8β-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, E711–20-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 E711–20-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.

CD4+CD25hi regulatory T‐cell frequency correlates with persistence of human papillomavirus type 16 and T helper cell responses in patients with cervical intraepithelial neoplasia

“…This low CTL detection rate may be due to the low sensitivity of the used assays available at the time of testing 23 or the overall low numbers of HPV16 specific CTL precursors in peripheral blood. 47 Direct analysis of the modulation of CD8 1 CTL responses against HPV or other viruses by isolated Tregs from patients with persistent HPV infection should be performed to prove whether HPV induced Tregs inhibit virus specific CTL responses. A role for Tregs and CD8 1 T-cells in cervical carcinoma and its metastatic spread was recently demonstrated in a study analyzing local Treg and CD8 1 Tcells in cervical carcinoma and pelvic lymph nodes.…”

Inducing Antitumor T Cell Immunity: Comparative Functional Analysis of Interstitial Versus Langerhans Dendritic Cells in a Human Cell Line Model

“…Ag-specific CD8 ϩ T cells were generated as described, with some minor modifications (22,23). In brief, mature MUTZ-3 IDC and MUTZ-3 LC, either derived from wild-type MUTZ-3 or IL-12-transfected MUTZ-3, were loaded with 1 g/ml peptide in the presence of 3 g/ml ␤ 2 -microglobulin (Sigma-Aldrich) for 2-4 h at room temperature and irradiated (40 Gy).…”

Cross‐typic specificity and immunotherapeutic potential of a human HPV16 E7‐specific CTL line

“…9 Our current results suggest that CTL to the E7 11--20 epitope would be useful for immunotherapy as CTL produced by in vitro stimulation protocols are effective against targets expressing endogenous antigen. This has been confirmed independently in a recent study by Schruers et al 29 who used DC pulsed with a modified HPV16 E7 [11][12][13][14][15][16][17][18][19][20] peptide (YMLDLQPETV) to generate CD8ϩ CTL clones from healthy donors. The clones killed targets expressing exogenous and endogenously processed E7 epitope presented either by CaSki cells or a cell line derived from an HPV16 positive biopsy of an HLA-A*0201 positive individual.…”

“…The cells were washed twice in cold 0.1% FCS/PBS before FITC-labeled rabbit anti-mouse IgG (Dakocytomation, Ltd., Denmark House, Ely, Cambridgeshire, UK) at a 1:20 dilution was added for a further 20 min at 4°C. The [11][12][13][14][15][16][17][18][19][20] YVLDLQPEAT HPV52 E7 [11][12][13][14][15][16][17][18][19][20] YILDLQPETT Analogue 31/52 YVLDLQPETT Analogue 52/31 YILDLQPEAT HPV16 E7 [11][12][13][14][15][16][17][18][19][20] P9 YMLDLQPEAT HPV16 E6 [29][30][31][32][33][34][35][36][37][38] TIHDIILECV Influenza A matrix protein M1 [58][59][60][61][62][63][64][65][66] GILGFVFTL cells were washed and fixed and the fluorescence analyzed on a FACScan (Becton Dickinson, Mountain View, CA). T...…”