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The Journal of Immunology, 2003, 171: 2912-2921.
Copyright © 2003 by The American Association of Immunologists

In Vitro Generation and Life Span Extension of Human Papillomavirus Type 16-Specific, Healthy Donor-Derived CTL Clones 1

Marco W. J. Schreurs, Kirsten B. J. Scholten, Esther W. M. Kueter, Janneke J. Ruizendaal, Chris J. L. M. Meijer and Erik Hooijberg2

Department of Pathology, Vrije Universiteit Medical Center, 1081 HV Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 CD8{beta}-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cervical cancer is the second most common cause of cancer death in women worldwide. The vast majority of cervical carcinoma (CxCa) 3 is associated with infection by malignant human papillomavirus (HPV) serotypes, particularly HPV16 and HPV18 (1, 2). The HPV-derived oncoproteins E6 and E7 are exclusively responsible for both onset and maintenance of malignant transformation through inactivation of the p53 and retinoblastoma tumor suppressor genes, respectively (3, 4). Constitutive expression of E6 and E7 in CxCa tumor cells appears to result in the induction of HPV-specific cell-mediated immune responses in CxCa patients. HPV16-specific CD8+ CTLs have been detected in both PBLs and tumor-infiltrating lymphocytes isolated from CxCa patients, albeit only after repeated Ag-specific in vitro restimulation (5, 6, 7). Similarly, HPV16-specific CTLs could be generated from peripheral blood isolated from healthy donors, either with (7) or without (8) the use of dendritic cells (DCs) as APCs.

The apparent immunogenic potential of HPV16-derived E6 and E7 may allow CTL-mediated immunotherapeutic intervention in CxCa patients. Indeed, animal models have shown that HPV16-specific CTLs can eradicate HPV16-transformed tumor cells in vivo (9, 10, 11). Adoptive CTL transfer represents a potential immunotherapeutic strategy to intervene with malignant disease, including CxCa. Recent studies in melanoma patients have shown that adoptive transfer of ex vivo-selected and -expanded CTLs can result in transfer of their functional activity in vivo, leading to regression of the patient’s metastatic melanoma (12, 13). For adoptive immunotherapeutic purposes, the availability to date of well-characterized HPV16-specific CTL is extremely limited. So far, examples of HPV16-specific CTLs mainly represent poorly characterized, mostly bulk cultures with limited value for adoptive CTL therapy (6, 7, 8, 14).

The use of DCs as professional APCs for the induction of specific CTLs in vitro has boosted identification of new tumor Ags as well as the availability of tumor Ag-specific CTLs (reviewed in Refs.15, 16, 17). DCs loaded with different antigenic preparations, including synthetic peptides, recombinant protein, and apoptotic tumor cells have been shown to successfully induce specific human CTL in vitro from low-frequency healthy donor-derived precursors (15, 16, 17). Furthermore, the recent development of soluble fluorogenic MHC-peptide complexes (tetramers) has allowed identification and isolation of such CTL from bulk cultures (18). The current report describes a protocol for the in vitro DC-mediated induction and subsequent isolation of bona fide HPV16-specific, CxCa-reactive CTL clones from low-frequency healthy donor-derived precursors. For DC loading, a high-affinity altered peptide ligand (APL) (19, 20) was generated from the immunodominant HPV16-derived CTL epitope, the HLA-A2.1-restricted E711–20 epitope, identified previously by reverse immunology together with the E782–90 and E786–93 epitopes (8). However, results obtained in HLA-A2.1 transgenic mice suggest that the latter two epitopes are not processed or inefficiently processed from the E7 Ag (21, 22). We used an HLA-A2.1/E711–20 tetramer-directed isolation procedure to obtain E711–20-specific human CTL clones followed by extensive phenotypic and functional CTL characterization. To enable extensive in vitro characterization and future immunotherapeutical application in vivo of E711–20-specific human CTL clones, multiple rounds of CTL expansion are required. Unfortunately, the in vitro expansion of human T cells is hampered by corrosion of chromosomal telomeric ends, resulting in replicative senescence (23). However, current evidence indicates that the life span of CD4+ and CD8+ human T cells can be significantly extended by ectopic expression of human telomerase reverse transcriptase (hTERT) without malignant transformation (23, 24, 25, 26). In agreement with this, hTERT transduction resulted in a prominent life span extension of the E711–20-specific human CTL clones described herein.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The EBV-transformed B cell line JY and HLA-A2.1-transfected K562 cells (27) (K-A2; kindly provided by Dr. C. Britten (Tumorvakzinationzentrum, Mainz, Germany)) were cultured in Iscove’s medium (BioWhittaker, Verviers, Belgium) supplemented with 8% FCS (Perbio, Helsingborg, Sweden) and antibiotics (penicillin/streptomycin; Life Technologies, Paisley, U.K.). The HPV16-positive CxCa cell lines Caski (American Type Culture Collection, Manassas, VA), Siha (American Type Culture Collection), CxCa866 (28) (kindly provided by Dr. P. Stern (Christie Hospital NHS Trust, Manchester, U.K.)), and Siha transfected with HLA-A2.1 (Siha-A2; kindly provided by Dr. S. Man (University of Wales College of Medicine, Cardiff, U.K.)) were cultured in keratinocyte-serum-free medium (Life Technologies) supplemented with 5% FCS, bovine pituitary extract (Life Technologies), epidermal growth factor (Life Technologies), and antibiotics.

Bioinformatics, peptides, and HLA-A2.1 binding assay

The Rammensee (29) and Parker (30) peptide binding scores to HLA-A2.1, expressed in arbitrary units, were determined at http://syfpeithi.bmi-heidelberg.com/Scripts/MHCServer.dll/EpPredict.htm and http://www-bimas.dcrt.nih.gov/molbio/hla_bind/index.html, respectively. Peptides were synthesized with a free C terminus by solid-phase strategies on an automated multiple-peptide synthesizer (Syro II; MultiSyntech, Witten, Germany) using Fmoc chemistry. Peptides were >90% pure as analyzed by reverse-phased HPLC, dissolved in DMSO, and stored at -20°C. Peptide binding to HLA-A2.1 was determined using the JY-based peptide binding assay as described (31). Briefly, HLA-A2.1-presented peptides were stripped from the surface of JY cells by mild acid elution. After washing, JY cells were incubated with a mixture of fluorescein-labeled reference peptide (FLPSDC[fl]FPSV) and different concentrations of competitor peptide for 24 h at 4°C, followed by flow-cytometric analysis of fluorescence intensity. Binding capacity of competitor peptides was determined as the concentration required for 50% reference peptide-binding inhibition (IC50). Peptide IC50 values <5 µM are considered high-affinity binders to HLA-A2.1, whereas values of 5 > 15 µM represent intermediate-affinity peptides (31).

Abs, tetramers, and flow cytometry

FITC- or PE-labeled Abs directed against human CD2, CD3, CD4, CD8{alpha}, CD11a, CD16, CD25, CD27, CD28, CD45RA, CD45RO, CD54, CD56, CD57, CD69, TCR{alpha}{beta}, TCR{gamma}{delta}, CCR7 (all from BD Biosciences, Mountain View, CA), and CD8{beta} (Beckman Coulter, Marseille, France), and allophycocyanin-labeled anti-human nerve growth factor receptor (NGFR; Chromoprobe, Aptos, CA) were used for flow-cytometric analysis. Flow-cytometric analysis of TCR V{beta} expression was performed using Abs present in the {beta} Mark TCR V{beta} repertoire kit (Beckman Coulter). PE- and allophycocyanin-labeled HLA-A2.1 tetramers presenting the HPV16 E711–20, E711–20V, E786–93, and influenza A MP58–66 epitopes were prepared as described previously (32). Tetramer and/or Ab staining of cells was performed in PBS supplemented with 0.1% BSA and 0.01% azide (PBA) for 15 min at 37°C and/or 20 min on ice, followed by washing with PBA. Stained cells were analyzed on a FACSCalibur (BD Biosciences) using CellQuest software. To exclude dead cells, all flow-cytometric analyses were performed in the presence of 0.5 µg/ml propidium iodide (ICN Biomedicals, Zoetermeer, The Netherlands). Ab- and/or tetramer-directed flow sorting was performed (in the absence of azide and propidium iodide) on a FACStarPlus (BD Biosciences) using CellQuest software.

Primary CTL induction in vitro

Healthy donor-derived PBMC were isolated from an HLA-A2.1-positive buffy coat by density gradient centrifugation using Lymphoprep (Nycomed, Oslo, Norway). Isolation of resting CD8{beta}-positive CTL precursors from total PBMC was performed by positive selection on an automated magnetic sorting device (autoMACS; Miltenyi Biotec, Bergisch Gladbach, Germany). For this purpose, total PBMC were stained with anti-CD8{beta} mAb and microbead-conjugated anti-mouse IgG Abs (Miltenyi Biotec), followed by autoMACS sorting. The CD8{beta}-negative PBMC fraction was used to generate mature monocyte-derived DCs as described previously (33), with minor modifications. Briefly, plastic adherent PBMC (8 x 106/ml in Iscove’s supplemented with 1% human serum (HS; ICN Biomedicals) for 45–60 min at 37°C) were washed with PBS and cultured for 6–7 days in Iscove’s supplemented with 8% FCS, 1000 U/ml IL-4 (Sanquin, Amsterdam, The Netherlands), and 100 ng/ml GM-CSF (Schering-Plough, Kenilworth, NJ). Cytokines were refreshed at days 3 and 6, and DC maturation was induced by an additional 2-day culture in the presence of monocyte-conditioned medium, generated as described previously (34). Briefly, 50 x 106 PBMC were seeded on a human Ig-coated (30 µg/ml in PBS for 1 h at room temp) 10-cm bacteriological petri dish and incubated 30 min at 37°C. Adherent monocytes were washed with PBS and incubated for 24 h at 37°C in Iscove’s supplemented with 2% HS. Supernatant (monocyte-conditioned medium) was harvested, filtered and used at 25–50% v/v for DC maturation. Mature DCs were loaded with 25 µM peptide for 2–4 h at room temperature in Iscove’s supplemented with 1% HS in the presence of 3 µg/ml human {beta}2-microglobulin (Sigma-Aldrich, St. Louis, MO), washed twice with medium, and irradiated (40 Gy). Peptide-loaded DCs (1–2 x 105) were cultured for 10 days with 1 x 106 autologous CD8{beta}-positive CTL precursors and 1 x 106 irradiated (80 Gy) autologous PBMC as feeders in 1.5 ml of Yssel’s medium (35) supplemented with 1% HS, 10 ng/ml IL-6 (R&D Systems, Oxon, U.K.), and 5 ng/ml IL-12 (R&D Systems) per well of a 24-well culture plate (Nunc, Intermed, Denmark). On day 1, 10 ng/ml IL-10 (R&D Systems) was added to the cultures. From day 10 on, CTL cultures were restimulated weekly with fresh peptide-loaded mature DC (1 x 105) in the presence of 5 ng/ml IL-7 (R&D Systems). Two days after each restimulation, 20 U/ml IL-2 (Chiron, Amsterdam, The Netherlands) was added to the cultures. One day before each restimulation, a sample from each individual well was taken and analyzed by flow cytometry using the tetramers as indicated in Results. Tetramer-positive CTLs were isolated by tetramer-directed flow sorting and cloned by limiting dilution as previously described (35), and CTL clones were expanded in 24-well plates by weekly stimulation with an irradiated (80 Gy) feeder mix consisting of 1 x 106 allogeneic PBMC from two different donors and 1 x 105 JY cells/ml of Yssel’s medium supplemented with 1% HS, 100 ng/ml PHA (Murex Biotech, Dartford, U.K.), and 20 U/ml IL-2. The influenza A MP58–66-specific CTL clone used in Fig. 4 was isolated directly ex vivo from an HLA-A2.1-positive healthy donor. For this purpose, PBMC were stained with PE-labeled HLA-A2.1/MP58–66 tetramers followed by microbead-conjugated anti-PE Abs (Miltenyi Biotec) and autoMACS sorting (positive selection). The used MP58–66-specific CTL clone was subsequently obtained by limiting-dilution cloning and expansion as described above.



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  		FIGURE 4. A and B, Functional avidity analysis of four individual E711–20-specific CTL clones as determined in a standard chromium release assay using HLA-A2.1-positive JY cells as target (E:T ratio, 10:1), loaded with serial 10-fold dilutions of the E711–20 wild-type (A) or E711–20V APL (B) epitopes. C, Similar avidity analysis of an HLA-A2.1-restricted, influenza A MP58–66-specific CTL clone using the relevant MP58–66 ({blacktriangleup}) or an irrelevant E711–20 ({blacksquare}) epitope.

 
Chromium release and ELISPOT assays

Cytolytic activity of CTL clones was determined using a standard chromium release assay as described previously (36). Briefly, 1 x 106 target cells were labeled with 100 µCi of Na2[51Cr]O4 (Amersham, Bucks, U.K.) for 45 min at 37°C, washed extensively, and loaded with 1 µM peptide (when indicated) for 30 min at 37°C. Effector CTL clones were added to 2 x 103 target cells at the indicated E:T ratios in triplicate wells of a round-bottom 96-well plate (Nunc). After a 4-h incubation at 37°C, 50 µl of the supernatant was harvested, and its radioactive content was measured. The percentage specific lysis was defined as follows: [(experimental release - spontaneous release)/(maximum release - spontaneous release)] x 100%. CxCa cell lines CxCa866, Caski, and Siha(-A2) were cultured in the presence of 200 U/ml human IFN-{gamma} (R&D Systems) for 48–72 h before their use in a chromium release assay. Production of IFN-{gamma} by CTL clones was determined using an ELISPOT assay as described previously (37). Briefly, 1 x 104 effector CTL were incubated overnight with 1 x 104 target cells (loaded with 1 µM peptide when indicated) at 37°C in a multiscreen 96-well filtration plate (Millipore, Molsheim, France) coated with mAb 1-D1K (Mabtech, Nacka, Sweden). Plates were washed and incubated with biotinylated mAb 7-B6-1 (Mabtech) for 2–4 h at room temperature, followed by washing and incubation with streptavidin-alkaline phosphatase (Mabtech) for 1–2 h at room temperature. IFN-{gamma} spots were developed with an alkaline phosphatase conjugate substrate kit (Bio-Rad, Hercules, CA) and counted with an automated ELISPOT reader (Autoimmun Diagnostika, Strassberg, Germany).

RT-PCR analysis of TCR V{beta} repertoire

Total RNA was isolated from ~5 x 106 CTLs using RNAzol (Campro Scientific, Veenendaal, The Netherlands) according to the manufacturer’s instructions. Copy DNA was synthesized from 2–5 µg of RNA using oligo(dT) primers and reverse transcriptase (Life Technologies) in a volume of 20 µl according to the manufacturer’s instructions. PCR was performed using 12 mixtures of four to five primers (all primers used were a kind gift from Dr. T. Schumacher (The Netherlands Cancer Institute, Amsterdam, The Netherlands)) complementary to the variable TCR {beta}-chain and one primer complementary to the constant TCR {beta}-chain. PCR was performed on 1 µl of cDNA in the presence of 2 mM MgCl2, 15 µM each primer, dNTPs, and 2.5 U of Taq polymerase (Roche, Almere, The Netherlands) in a volume of 50 µl. When a band of the expected size was visible on an agarose gel stained with ethidium bromide, the PCR was repeated using each of the variable primers separately together with the constant TCR {beta} primer. V{beta}3 was subsequently confirmed by sequence analysis (BaseClear, Leiden, The Netherlands).

Retroviral hTERT transduction

The retroviral vector LZRS-hTERT-IRES-{Delta}NGFR was constructed by replacing the marker green fluorescent protein (GFP) for the coding sequence of a truncated, signaling incompetent form of the low-affinity NGFR ({Delta}NGFR) (38) and inserting the hTERT coding sequence (39) in the multiple cloning site in the original LZRS-mcs-IRES-GFP (32). The LZRS-hTERT-IRES-{Delta}NGFR construct was used to produce retroviral supernatant followed by retroviral hTERT transduction of CTL clones using protocols described previously (32, 40). Briefly, 5 x 105 CTLs, stimulated for 48 h with feeder mix as described above, were resuspended in 0.5 ml of retroviral supernatant supplemented with 20 U/ml IL-2 and transferred to a fibronectin (RetroNectin; Takara, Otsu, Japan)-coated well of a non-tissue-culture-treated 24-well plate (BD Biosciences). Plates were centrifuged for 90 min at 2000 rpm, followed by 5-h incubation at 37°C. The CTLs were subsequently harvested, washed with culture medium, and kept at 37°C overnight in culture medium supplemented with 20 U/ml IL-2. The next day, retroviral transduction as described above was repeated. Expression of hTERT was investigated after 48 h and later time points by flow-cytometric analysis of {Delta}NGFR marker gene expression using an NGFR-specific Ab. CTLs transduced with hTERT were stimulated weekly or biweekly in 24-well plates with feeder mix as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Selection of an HLA-A2.1-restricted high-affinity APL derived from HPV16 E7

The E711–20 epitope YMLDLQPETT represents the best-studied HLA-A2.1-presented, HPV16-derived epitope against which CTL-mediated reactivity has been found in both CxCa patients and healthy donors (6, 7, 8, 14). Consequently, this epitope may prove a suitable target for CTL-mediated immunotherapy of CxCa, including the generation of specific CTL clones followed by adoptive transfer into CxCa patients. For this purpose, we set out to generate E711–20-specific healthy donor-derived CTL clones by primary in vitro CTL induction. Because previous work in both mice and humans has indicated that epitope affinity for its presenting MHC class I molecule positively correlates with CTL induction efficiency (41, 42), APLs of the E711–20 epitope were designed to improve binding affinity. E711–20 APLs containing the dominant C-terminal HLA-A2.1 anchor residues valine (V), leucine (L), isoleucine (I), or methionine (M), absent in the wild-type epitope, were screened for HLA-A2.1 affinity by bioinformatics. Table I indicates that the calculated binding affinity of the E711–20 epitope, depicted as Rammensee and Parker scores in arbitrary units, can be increased substantially by substitution of the C-terminal threonine (T) into either a V or an L. The use of C-terminal I or M residues affected the calculated affinity only marginally, resulting in selection of the E711–20V and E711–20L APLs for further study. Analysis of actual binding affinity to HLA-A2.1 was performed with both E711–20V and E711–20L APLs and indicates a 2-fold decrease of the IC50 value for the E711–20V APL (3 µM; high affinity) compared with the wild-type E711–20 epitope (6 µM; intermediate affinity) (Fig. 1), whereas the IC50 value for the E711–20L APL decreased to 3.5 µM (data not shown). Consequently, the E711–20V APL was selected for CTL induction.


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  		Table I. Calculated HLA-A2.1 binding of HPV16 E711-20 wild-type and APL epitopes

 


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  		FIGURE 1. Binding affinity to JY-expressed HLA-A2.1 of the E711–20 wild-type ({blacksquare}) and E711–20V APL ({blacktriangleup}) epitopes as determined in a competition assay. Increasing amounts of competitor (test) peptide are used to inhibit HLA-A2.1 binding of a FITC-labeled reference peptide to determine the concentration of competitor peptide required to inhibit 50% reference peptide binding (IC50).

 
Generation and isolation of HPV16 E7-specific CTL clones from healthy donor-derived precursors

As professional APC, DCs have the unique capability to activate naive Ag-specific T cells both in vivo and in vitro (15, 16, 17). To generate CxCa-reactive, HPV16 E711–20-specific CTL clones, mature monocyte-derived DCs loaded with the above selected E711–20V APL were used as stimulator cells. The employed in vitro CTL induction procedure, as described in Materials and Methods, represents a procedure adopted from previously published protocols (15, 16) involving continued stimulations with mature peptide-loaded DCs in the presence of different cytokines to allow specific CTL expansion. Mature DCs were derived as described previously (33) and displayed typical mature DC veiled morphology and phenotype including expression of CD80, CD86, CD83, and HLA-DR (data not shown). Precursor CTLs were selected as responder cells from total PBMC based on the expression of CD8{beta}. Because CD8{beta} is only expressed by CTLs, selection of these cells as responders may avoid potential aspecific in vitro activation and expansion of CD8{alpha}+ NK cells or CD4+ Th cells.

Stimulation of 18 individual cultures per healthy donor (1 x 106 CD8{beta}+ precursors as starting material per individual culture) with peptide-loaded DCs was repeated on a weekly basis, and each stimulation was monitored by tetramer-guided flow-cytometric analysis of a sample taken from each individual culture (Fig. 2, A–C). As depicted in Fig. 2A, all tetramer-guided analyses were performed on cells present within a CD8+, propidium iodide-negative gate to ensure analysis of live CTLs. Moreover, tetramer staining was performed by the simultaneous use of two individual HLA-A2.1 tetramers presenting the same E711–20V epitope, labeled with two different fluorochromes. Such an approach was chosen to avoid false-positive interpretation of potential aspecific staining with either of the single tetramers, especially when low numbers of specific, tetramer-positive cells are present. Additionally, CTL clones for validation of specific and background staining by individual tetramer preparations were not available to us at the time. Fig. 2B indicates tetramer analysis of a representative culture of 54 cultures analyzed in three individual HLA-A2.1-positive healthy donors (18 cultures per donor) after the first DC stimulation, showing absence of any double-positive cells. Similar results were obtained after the second DC stimulation (data not shown). In contrast, low numbers of E711–20V tetramer double-positive cells could be detected after the third DC stimulation in 2 of 54 individual cultures (0.02–0.07% of live CTLs), both derived from the same healthy donor (data not shown) and both increasing to 0.2% of live CTLs after the fourth stimulation (Fig. 2C). Double tetramer-positive cells increased to 0.6% of live CTLs after the fifth stimulation of both individual cultures (data not shown). Next to the low percentages of specific CTLs obtained after multiple DC stimulations, the low number of positive cultures obtained in one donor and the absence of positive cultures in the other two donors further illustrate the low precursor frequency of HPV16 E711–20-specific CTLs. Moreover, these results point out the difficulty of obtaining such CTLs in vitro despite the use of the high-affinity E711–20V APL.



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  		FIGURE 2. Flow-cytometric HLA-A2.1 tetramer (T A2)-guided analysis of healthy donor-derived CD8{beta}-positive precursors stimulated with E711–20V-loaded mature DC. A, Inclusion criterium (R2 gate) for tetramer analysis: propidium iodide-negative, CD8+ T cells. B and C, Double-tetramer (T A2 11-20V-PE and -allophycocyanin (apc), presenting the 11–20V APL) analysis of an individual CTL bulk culture after the first DC stimulation (B) and after the fourth DC stimulation (C) (percentage of double-tetramer-positive cells is indicated in the upper right corner). D–F, Double-tetramer analysis of CTL clones after tetramer-guided flow sorting followed by limiting-dilution cloning using tetramers T A2 11–20V-PE and -allophycocyanin (apc) (D), T A2 11–20-PE (presenting the wild-type 11–20 epitope) and T A2 11–20V-apc (E), and T A2 11–20-PE and an irrelevant T A2 86–93-apc (F).

 
Based on the binding of both E711–20V tetramers shown in Fig. 2C, specific CTLs were sorted after the fourth and fifth DC stimulations by flow cytometry, followed by limiting-dilution cloning and expansion of CTL clones. As shown in Fig. 2D, CTL clones reacting with both E711–20V tetramers could be isolated by double tetramer-directed flow sorting. A comparable binding capacity of both tetramers on a per-cell basis is illustrated by the diagonal cell distribution of the cells plotted in Fig. 2D. A similar staining could be observed when the E711–20V tetramer was combined with the tetramer presenting the E711–20 wild-type epitope, indicating full TCR cross-reactivity between the E711–20V APL and E711–20 wild-type epitopes regarding tetramer binding (Fig. 2E). In contrast, binding to HLA-A2.1 tetramers presenting the E786–93 epitope (Fig. 2F) or the influenza A matrix protein-derived MP58–66 epitope (data not shown) could not be detected, further emphasizing TCR specificity for the HPV16 E711–20 epitope.

Phenotype and functional activity of HPV16 E7-specific CTL clones

Multiple CTL clones generated and isolated as described above were subsequently used for phenotypical (n = 4) and functional (n = 6) analysis. Table II shows the expression levels of T cell-associated surface molecules as determined by flow-cytometric analysis. According to the resulting phenotype CD8+CD27-CD28-CD45RA+/-CD45RO+CCR7-TCR{alpha}{beta}+, the isolated E711–20-specific CTL clones can be defined as CD8+ type 1 effector memory CTL expressing an {alpha}{beta}TCR (43, 44).


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  		Table II. Phenotype of HPV16 E711-20-specific CTL clonesa

 
Functional activity of E711–20-specific CTL clones was investigated using standard chromium release and IFN-{gamma} ELISPOT assays (Fig. 3). Fig. 3A indicates lytic activity against HLA-A2.1-positive EBV-transformed B cells exogenously loaded with the relevant E711–20 wild-type epitope, but not with an irrelevant E786–93 epitope. Importantly, HPV16-transformed CxCa866 cells, isolated and expanded from a biopsy taken from an HLA-A2.1-positive CxCa patient (28), were specifically lysed by E711–20-specific CTL clones, suggesting recognition of endogenously processed E7 Ag as well (Fig. 3A). Similar results were obtained in an ELISPOT assay using the same targets, showing Ag-specific production of IFN-{gamma} (Fig. 3B).



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  		FIGURE 3. Lytic activity of an E711–20-specific CTL clone, representative of six individual clones analyzed, in a standard chromium release assay (A, C, and D) and an IFN-{gamma} ELISPOT assay (B), both performed as indicated in Materials and Methods. A and B, The targets used were HLA-A2.1-positive JY cells loaded with the relevant E711–20 epitope ({blacksquare}) or an irrelevant E786–93 epitope (•), and the HLA-A2.1, HPV16-transformed CxCa line CxCa866 ({blacktriangleup}). C, The targets used were HLA-A2.1-transfected K562 cells (K-A2) loaded with the relevant E711–20 epitope ({blacksquare}) or an irrelevant E786–93 epitope (•). D, The targets used were HPV16-transformed CxCa lines Caski ({blacktriangleup}) (HLA-A2.1-positive) and Siha (•) (HLA-A2.1 negative), and HLA-A2.1-transfected Siha cells ({blacksquare}) (Siha-A2). CxCa cell lines CxCa866, Caski, and Siha(-A2) were pretreated for 48–72 h with 200 U/ml IFN-{gamma}.

 
To provide more extensive characterization of Ag recognition and specificity, an additional set of target cells were used in chromium release assays. HLA-A2.1-transfected K562 cells (27), devoid of any other MHC class I molecule, were only lysed upon exogenous loading with the relevant E711–20 wild-type epitope (Fig. 3C). These results further emphasize epitope specificity and rule out NK-like lytic activity. Fig. 3D shows specific lysis of the broadly used HLA-A2.1-positive, HPV16-transformed CxCa cell line Caski, but not the HLA-A2.1-negative, HPV16-transformed CxCa cell line Siha. However, Siha cells were sensitized for lysis upon transfection with the HLA-A2.1 restriction element, providing formal proof for HLA-A2.1-restricted recognition of endogenously processed E7 Ag by E711–20-specific CTL clones. Collectively, the data presented in Fig. 3 clearly show that our strategy involving the use of APL-loaded mature DCs for E711–20-specific CTL induction and restimulation allows the generation of bona fide CTLs recognizing exogenously loaded and, more importantly, endogenously processed wild-type E711–20 in an HLA-A2.1-restricted fashion.

Functional avidity of HPV16 E7-specific CTL clones

Functional CTL avidity involves the amount of TCR triggering required for functional activity, thereby reflecting the amount of presented specific peptide/MHC complexes required for optimal CTL function. Analysis of functional avidity of HPV16 E711–20-specific CTL clones was performed by using HLA-A2.1-positive target cells loaded with decreasing amounts of specific peptide in a chromium release assay (Fig. 4). As such, four individual CTL clones were analyzed using target cells loaded with decreasing amounts of the E711–20 wild-type epitope (Fig. 4A) or the E711–20V APL (Fig. 4B). The results indicate CTL-mediated recognition of the E711–20 wild-type and E711–20V APL epitopes with half-maximal lysis observed around the 10 and 1 nM range, respectively. The one log increased avidity of E711–20V recognition by the CTLs most probably reflects the higher affinity of the E711–20V APL for HLA-A2.1 as compared with the E711–20 wild-type epitope (Fig. 1). The observed half-maximal lysis by E711–20-specific CTL clones in the low nanomolar range reflects an intermediate-avidity recognition of Ag (45). For comparative purposes, the avidity of Ag recognition by an MP58–66-specific CTL clone is depicted in Fig. 4C. CTL responses against the influenza A MP58–66 epitope are usually of high avidity (46). Indeed, the half-maximal lysis of MP58–66-loaded target cells by MP58–66-specific CTLs is not reached within the peptide titration range used in Fig. 4, reflecting high-avidity Ag recognition.

TCR gene usage of HPV16 E7-specific CTL clones

The success rate of E711–20-specific CTL induction described in the current study is relatively low, despite the use of mature DCs loaded with a high-affinity APL. As a result, the expected variety of isolated CTL clones regarding TCR gene usage may be restricted. To investigate this, individual E711–20-specific CTL clones isolated from the culture depicted in Fig. 2C were analyzed for TCR V{beta} gene usage by RT-PCR. A total of 12 groups of PCR primers containing four to five individual V{beta} family-specific primers each and the same constant TCR region-specific primer were used for RT-PCR analysis (Fig. 5A). The results depicted in Fig. 5A indicate that primer group 12 convincingly amplifies a V{beta} gene segment of the expected size from E711–20-specific CTL-derived RNA. RT-PCR analysis performed with the five individual V{beta} primers present in group 12 only resulted in a PCR product of the expected size with one of the variable region primers, specific for V{beta}3 (data not shown). Expression of V{beta}3 was subsequently confirmed by sequence analysis (data not shown) and flow cytometry using a V{beta}3-specific mAb (Fig. 5B). Comparative RT-PCR analysis of seven individual E711–20-specific CTL clones isolated from the culture depicted in Fig. 2C is shown in Fig. 5C and clearly indicates uniform expression of V{beta}3 by all clones analyzed. These results strongly suggest that the tetramer-positive cells depicted in Fig. 2C represent a clonal CTL population derived from a single CD8{beta}-positive precursor.



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  		FIGURE 5. V{beta} TCR gene usage by E711–20-specific CTLs using RT-PCR (A and C) and flow-cytometric (B) analysis. A, RT-PCR on total RNA isolated from a representative E711–20-specific CTL clone using a single constant TCR region-specific primer combined with 12 groups of primers representing four to five V{beta}-specific primers each, as specified in Materials and Methods. M, DNA marker. B, V{beta}3 expression by a representative E711–20-specific CTL clone determined using a FITC-labeled, V{beta}3-specific mAb together with T A2 11–20-allophycocyanin (apc) tetramer staining. C, Comparative RT-PCR on seven individual E711–20-specific CTL cloned using a constant TCR region-specific primer combined with a V{beta}3-specific primer. M, DNA marker; the dash (–) represents control PCR without reverse transcription.

 
Telomerase-mediated life span extension of HPV16 E7-specific CTL clones

Given the difficulties in obtaining tumor-reactive, E711–20-specific CTLs, continued availability and expansion to sufficient amounts of such specificities seem to be of the utmost importance for extended in vitro and in vivo use. One strategy to ensure continued availability of such specificities of clones is described in the section above regarding TCR gene usage of HPV16E7-specific CTL clones, and represents the isolation of the gene fragments encoding the E711–20-specific TCR, i.e., the process of molecular immortalization. Alternatively, the recently described process of T cell life span extension, i.e., cellular immortalization, may ensure continued CTL availability and CTL expansion to sufficient amounts (23, 24, 25, 26). Ectopic expression of the catalytic subunit of telomerase, hTERT, efficiently compensates for replicative CTL senescence induced by corrosion of telomeric ends of chromosomes. To extend the life span of the isolated E711–20-specific CTL clones, retroviral hTERT transduction was performed and subsequently monitored on the basis of {Delta}NGFR marker gene expression (Fig. 6A). As compared with nontransduced counterparts (Fig. 6A; t = 0), hTERT-IRES-{Delta}NGFR expression can be detected in >10% of transduced CTLs 1 wk after transduction (A). Without selection of hTERT-IRES-{Delta}NGFR-positive cells, but with the requirement of weekly CTL stimulation using feeder mix, the fraction of hTERT-transduced CTLs increases to ~100% in the 10 wk following retroviral transduction (Fig. 6A). Similar results were obtained upon hTERT transduction of human influenza A-specific CTL clones (data not shown). These results illustrate the replicative advantage of hTERT-transduced E711–20-specific CTL clones in agreement with previous studies (23, 24, 25, 26). The recently described elevated expression levels of the antiapoptotic Bcl-2 proto-oncogene observed upon hTERT transduction of human T cells may account for the growth advantage observed in Fig. 6A (26).



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  		FIGURE 6. Flow-cytometric (A) and functional (B) analysis of E711–20-specific CTLs after retroviral transduction with LZRS-hTERT-IRES-{Delta}NGFR, representative of six individual CTL clones transduced. A, An hTERT-transduced E711–20-specific CTL clone was monitored for hTERT-{Delta}NGFR expression up to 10 wk after transduction using an NGFR-specific, allophycocyanin-labeled mAb. B, Lytic activity of wild-type, nontransduced ({blacksquare}, •) and hTERT-transduced ({square}, {circ}) E711–20-specific CTLs, representative of six individual CTL clones transduced, in a standard chromium release assay using as target HLA-A2.1-positive JY cells loaded with the relevant E711–20 epitope ({blacksquare}/{square}) or an irrelevant E786–93 epitope (•/{circ}).

 
A total of six individual E711–20-specific CTL clones were hTERT transduced and displayed the replicative advantage over nontransduced counterparts as described above, resulting in a ~100% hTERT-IRES-{Delta}NGFR-positive population after 6–12 wk of culture without selection. The transduced CTL clones could be maintained in vitro for over 6 mo, whereas their nontransduced counterparts were routinely lost after a 2- to 4-mo culture period (data not shown). The observation that hTERT-transduced HPV16 E7-specific CTLs thrive somewhat better in vitro and their prolonged in vitro life span indicate a significant increase in the expansion potential of CTLs upon hTERT transduction.

To investigate the effect of hTERT transduction on the phenotype and function of HPV16 E7-specific CTLs, a direct comparison was made between wild-type and transduced CTL clones. The phenotype of hTERT-transduced CTLs remained similar (data not shown) as depicted in Table II for their nontransduced counterparts. Moreover, the functional activity of hTERT-transduced CTL clones remains unaffected as shown in Fig. 6B. As compared with their nontransduced counterparts, the lytic activity of an hTERT-transduced E711–20-specific CTL population (>95% hTERT-IRES-{Delta}NGFR positive) is of similar magnitude and dependent on the presentation of the relevant Ag. Collectively, life span extension of E711–20-specific CTL clones by hTERT transduction represents a valuable strategy to ensure continued availability of such low-frequency CTLs.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adoptive CTL immunotherapy represents a promising immunotherapeutical strategy to fight malignant disease as indicated by the results obtained so far in different preclinical mouse models (reviewed in Ref.47). Moreover, recent clinical studies have shown that objective regressions of human melanoma can be induced in patients upon adoptive transfer of tumor-specific CTL clones (12, 13). The constitutive expression of HPV16-derived oncoproteins E6 and E7 confers immunogenic potential to the majority of CxCa tumors and thus could be targeted by CTL-mediated active or adoptive immunotherapy. However, the low precursor frequencies of HPV16 E6- and E7-specific CTLs observed among human PBMC may render active vaccination against CxCa less attractive, as illustrated by the limited success obtained so far with synthetic peptide-based vaccines in a clinical setting (48, 49). One of the primary conditions for successful adoptive immunotherapy is the availability of tumor-reactive T cells, well characterized both phenotypically and functionally. The current availability of human CTLs specific for HPV16 as well as reactive against CxCa is extremely limited and hampers the efforts to explore adoptive CTL therapy of CxCa patients. Consequently, we set out to circumvent this restriction by deriving HPV16-specific, CxCa-reactive CTL clones in vitro from healthy donor-derived precursors.

It is well appreciated that mature DCs have the unique capability to activate Ag-specific CTLs from naive precursors in vitro (15, 16, 17). In accordance with previous work (33), mature immunostimulatory DCs were generated and used to induce HPV16-specific CTLs from low-frequency precursors in vitro. Existing protocols regarding primary CTL induction in vitro were adopted with modifications, as described in Materials and Methods, to allow the induction and isolation of such low-frequency CTLs. Modifications of interest represent the use of purified CD8{beta}-positive CTL precursors as responder cells to exclude the activation of NK(T) and/or Th cells, the continued use of mature DCs for effective CTL restimulation, and a compilation of different cytokine combinations previously used to allow activation and expansion of Ag-specific CTLs (15, 16). Furthermore, we are the first to include the use of a high-affinity APL derived from the immunodominant E711–20 epitope to improve and prolong the stability of DC-presented MHC/peptide complexes. Detection of E711–20-specific CTLs in vitro required three E711–20V APL-loaded mature DC stimulations and was observed in only 2 of 54 induction cultures analyzed in three healthy donors. These results illustrate the low frequency of HPV16-specific CTLs in the periphery, in contrast to other virus-specific CTLs including CMV, EBV, and influenza A (50, 51, 52). The underlying mechanisms for this phenomenon are currently unclear and require further investigation, but may involve holes in the T cell repertoire induced in early life by related nonmalignant HPV serotypes, or perhaps even molecular mimicry as suggested previously (53).

The simultaneous use of two HLA-A2.1 tetramers presenting the same epitope but labeled with different fluorochromes represents a novel approach for specific CTL detection and isolation. This resulted in highly specific CTL isolation from bulk cultures containing low numbers (0.2–0.6%) of E711–20-specific CTLs as illustrated by the almost exclusive presence of E711–20-specific CTLs in cultures expanded from wells that were seeded with 100 as such-sorted cells (data not shown). A representative phenotypical analysis of expanded E711–20-specific CTL clones indicated a predominant type 1 effector memory classification, proposed previously to exhibit a high degree of antitumor reactivity (43, 44). Indeed, analysis of their functional activity clearly indicates both lytic activity and production of IFN-{gamma} upon tumor cell recognition. The use of an extended set of target cells in the chromium release assays depicted in Fig. 3 provides formal proof for E711–20-specific and HLA-A2.1-restricted recognition of endogenously processed E7 Ag expressed at physiological levels. This is in contrast to previous reports using as targets peptide-loaded cells, recombinant vaccinia virus-infected cells, and the HPV16-transformed Caski cell line (6, 8, 14). Because the E711–20 epitope was identified by reverse immunology (8), and Caski cells appear sensitive for aspecific lysis (54), results obtained with CTLs generated with E711–20 peptide-loaded DCs require careful interpretation. All E711–20-specific CTL clones analyzed in the current report were shown to recognize their respective MHC/peptide complex with intermediate avidity, reaching half-maximal lytic activity in the low nanomolar range of peptide concentration. This is in agreement with the results obtained by Youde et al. using the CxCa patient-derived C6 clone (Ref.14 ; Dr. S. Man, personal communication). In contrast, the influenza A MP58–66-specific CTL clone used in this study displays high-avidity Ag recognition. Different factors contribute to functional CTL avidity, including TCR affinity for its respective MHC/peptide complex (45).

Our preliminary results so far fail to indicate increased TCR affinity of the MP58–66-specific CTL clone as compared with the E711–20-specific CTL clones (data not shown), as determined by tetramer decay kinetics (55). However, other factors may be involved including additional receptor-ligand interactions and downstream signaling processes, which are currently under investigation. We and others (14) have failed to detect any high-avidity E711–20-specific CTL clones. Peptide-based CTL restimulation may favor the outgrowth of lower avidity CTLs in vitro (54). However, the odd chance that mixed avidity precursors have ended up in the same induction culture is basically ruled out by the low precursor frequencies of E711–20-specific CTLs. Moreover, analysis of TCR V{beta} gene usage revealed the shared expression of a single V{beta} segment by all E711–20-specific CTL clones analyzed, suggesting a monoclonal origin. Adoptive transfer experiments in preclinical animal models will be required to investigate whether the observed intermediate avidity of the current E711–20-specific CTL clones is sufficient for CxCa eradication in vivo. The recently described retroviral TCR display technology may be included to improve on the avidity of E711–20-specific CTL clones (56).

The results presented in this article show that HPV16 E7-specific, CxCa-reactive CTL clones can be generated in vitro from low-frequency healthy donor-derived precursors. Such CTL clones hold promise for adoptive transfer in vivo to contribute to eradication of CxCa tumors in patients, provided that sufficient CTL expansion can be reached in vitro. Replicative senescence, a decrease in proliferative potential resulting from corrosion of telomeric ends of chromosomes (57), imposes severe restrictions on T cell expansion in vitro (23, 24, 25, 26). However, ectopic expression of human telomerase (hTERT) in human T cells can effectively compensate for this, mediating extension of the life span of CD4+ and CD8+ T cells (23, 24, 25, 26). In agreement with these results, we have been able to significantly extend the life span of multiple E711–20-specific CTL clones by retroviral hTERT transduction, allowing continued expansion of such transduced CTLs for a culture period of >6 mo. In contrast, their nontransduced counterparts could be maintained in culture for only 2–4 mo, severely restricting CTL expansion to relatively large numbers. Experiments performed on human fibroblasts have shown that hTERT expression alone does not result in malignant transformation, but requires the additional expression of at least two other premalignant features including the H-ras and SV40 large T oncogenes (58). The hTERT-transduced E711–20-specific CTL clones described herein remained dependent on (bi-)weekly growth stimulation during a continued culture period of >6 mo. This is in agreement with a previous study showing that CTLs fail to proliferate in the absence of growth stimulation despite hTERT transduction (59). Importantly, hTERT-transduced E711–20-specific CTL clones displayed both an unaltered phenotype and functional activity as compared with nontransduced CTLs. Collectively, our and other in vitro results so far might indicate the safe use of hTERT-transduced CTLs (23, 24, 25, 26). However, more extensive analyses of the potential adverse affects may be required to allow their use in a clinical setting. These may include cytogenetic analysis, analysis of insertional mutagenesis, and follow-up of preclinical in vivo adoptive transfer experiments in animal models. When these requirements are met, the use of hTERT-transduced human CTL clones may boost the clinical evaluation of adoptive CTL immunotherapy of CxCa and other malignancies.


   Acknowledgments
 
We thank the following persons for reagents: Dr. H. Spits, Dr. G. Nolan, and A. Q. Bakker for retroviral constructs; Dr. C. Bordignion for the {Delta}NGFR cDNA; Dr. P. Stern for CxCa866 cells; Dr. S. Man for Siha-A2 transfectants; Dr. C. Britten for K562-A2 transfectants; Dr. R. Weinberg for hTERT cDNA; and Dr. T. Schumacher for a set of TCR {alpha} and {beta} primers. We are also grateful to Dr. H. Bontkes and D. Kramer for fruitful discussions.


   Footnotes
 
1 This study was supported by Grants VUMC2001-2503 from the Dutch Cancer Society and 901-10-124 from the Netherlands Organization for Scientific Research. Back

2 Address correspondence and reprint requests to Dr. Erik Hooijberg, Department of Pathology, Vrije Universiteit Medical Center, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail address: erik.hooijberg{at}vumc.nl Back

3 Abbreviations used in this paper: CxCa, cervical carcinoma; HPV, human papillomavirus; DC, dendritic cell; APL, altered peptide ligand; hTERT, human telomerase reverse transcriptase; NGFR, nerve growth factor receptor; HS, human serum. Back

Received for publication March 28, 2003. Accepted for publication July 14, 2003.


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 Results
 Discussion
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