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[Cancer Research 65, 5417-5427, June 15, 2005]
© 2005 American Association for Cancer Research

Immunology

Artificial Antigen-Presenting Cells Transduced with Telomerase Efficiently Expand Epitope-Specific, Human Leukocyte Antigen–Restricted Cytotoxic T Cells

Jakob Dupont1, Jean-Baptiste Latouche1, Chia Ma1 and Michel Sadelain1,2

1 Department of Medicine, Memorial Sloan-Kettering Cancer Center and the Joan and Sanford Weill Medical College of Cornell University and 2 Immunology Program, Sloan-Kettering Institute, New York, New York

Requests for reprints: Jakob Dupont, Department of Medicine, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: 212-639-8388; Fax: 212-717-3214; E-mail: gynbreast{at}mskcc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human telomerase reverse transcriptase (hTERT) is overexpressed in most human tumors, making it a potential target for cancer immunotherapy. hTERT-derived CTL epitopes have been identified previously, including p865 (RLVDDFLLV) and p540 (ILAKFLHWL), which are restricted by the human leukocyte antigen (HLA) class I A*0201 allele. However, it remains a major challenge to efficiently and consistently expand hTERT-specific CTLs from donor peripheral blood T lymphocytes. To bypass the need for generating conventional antigen-presenting cells (APC) on an autologous basis, we investigated the potential ability of fibroblast-derived artificial APCs (AAPC) to activate and expand HLA-A*0201-restricted CTLs. We show here that AAPCs stably expressing HLA-A*0201, human ß2-microglobulin, B7.1, intercellular adhesion molecule-1, and LFA-3, together with either p540 and p865 minigenes or the full-length hTERT, effectively stimulate tumoricidal, hTERT-specific CTLs. hTERT-expressing AAPCs stimulated both p540 and p865 CTLs as shown by peptide-specific cytolysis and tetramer staining, indicating that hTERT is processed by the AAPCs and that the two peptides are presented as codominant epitopes. The level of cytotoxic activity against a panel of tumors comprising hematologic and epithelial malignancies varied, correlating overall with the level of HLA-A2 and hTERT expression by the target cell. Starting from 100 mL blood, ~100 million hTERT-specific CTLs could be generated over the course of five sequential stimulations, representing an expansion of ~1 x 105. Our data show that AAPCs process hTERT antigen and efficiently stimulate hTERT-specific CTLs from human peripheral blood T lymphocytes and suggest that sufficient expansion could be achieved to be clinically useful for adoptive cell therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adoptive cellular therapy with CTLs represents an emerging and potentially powerful approach for the treatment of cancer and infectious diseases (1–3). This therapy involves the isolation of antigen-specific human leukocyte antigen (HLA)–restricted CTLs either from a patient or from a donor, which is followed by ex vivo expansion and T-cell infusion into the patient (1–3). Adoptive cellular therapies have been applied successfully to several hematologic malignancies, including relapsed chronic myelogenous leukemia after bone marrow transplant (BMT; ref. 4), high-grade B-cell lymphomas relapsed after BMT (5), and EBV-associated lymphomas after BMT (6). Recently, some patients with melanoma have shown objective responses following gp100 (7) or tyrosinase (8) specific T-cell infusions. Some patients with renal cell carcinoma (9) have also responded to therapy.

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is an attractive potential target antigen because of its expression in the vast majority of human tumors (10–13). Normal adult tissues, with few exceptions, do not express this protein (14). Telomerase is also targeted because of the critical role it plays in tumor development and survival (13, 15–20). The therapeutic strategies under investigation include functional inhibition by genetic and pharmacologic approaches (15, 16, 18, 19) as well as immunotherapy (20). hTERT contains several HLA-restricted-binding motifs that have been used to generate tumoricidal CTLs (21–23). Minev et al. found that immunization of patients with prostate cancer with two HLA-A*0201+-restricted peptides from hTERT, p540 (ILAKFLHWL) and p865 (RLVDDFLLV), developed hTERT-specific CTLs that specifically lysed a variety of HLA-A*0201+ cancer cell lines (24). Recently, HLA class II peptide epitopes from hTERT that can be used to induce T-helper cells have also been identified (25, 26). Thus, hTERT is a potential immunogenic antigen that could be used for tumor-specific CTL induction.

Current adoptive T-cell therapy targeting tumor antigens, however, is limited due to the difficulty in generating clinically relevant quantities of tumor-specific CTLs. The expansion of tumor-specific T cells requires potent antigen-presenting cells (APC), which include dendritic cells, macrophages, and B cells (27–29), to maximize T-cell stimulation. However, the isolation and expansion of these cells on an autologous basis, which is necessary to match the T cell's HLA restriction, are time-consuming processes that hinder the broad implementation of adoptive therapies. Novel approaches and user-friendly technologies for the rapid expansion of effective T cells are thus much desired and needed. Ideally, one would want a ready-to-use APC applicable to any patient, which permits selective expansion of potent T cells specific for any tumor antigen. Artificial APCs (AAPC) have thus been developed to generate clinically relevant quantities of CTLs for the purpose of adoptive therapy (30–36). AAPCs have been engineered to optimize the stimulation and expansion of CTLs by presenting antigen in the context of a specific HLA allele along with a variety of costimulatory molecules, including B7.1 (CD80), intercellular adhesion molecule-1 (ICAM-1; CD54), LFA-3 (CD58), and 4-1BB ligand (36). For viral antigens, such as the influenza and cytomegalovirus (CMV) matrix peptides, the AAPCs are as effective as autologous dendritic cells, cytokine-stimulated monocytes, and EBV-transformed B cells (30, 35). However, CTL responses to tumor antigens are more difficult to achieve than CTL responses to viral antigens (37–41); we therefore investigated the response elicited against hTERT starting from human peripheral blood T lymphocytes (PBL).

We show here that fibroblast-derived AAPCs transduced with cDNAs encoding human HLA-A*0201, B7.1, ICAM-1, and LFA-3 as well as the p540 or p865 peptide antigen from hTERT can be used to efficiently stimulate tumoricidal, hTERT-specific CTLs from several donors. Furthermore, stimulation of donor T cells with AAPCs expressing full-length hTERT generates tumoricidal T cells against both p540 and p865 epitopes, establishing that the murine AAPCs efficiently process and present both HLA-A*0201-restricted immunodominant epitopes to human T cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Vector construction. Bicistronic vectors encoding the encephalomyocarditis virus internal ribosomal entry site (42) and puromycin-N-acetyltransferase (30) were used to express the different peptide antigens used in this study. cDNAs were cloned into the NcoI and BamHI sites of the SFG vector (43). The human CD8{alpha} leader was fused to the peptide antigens to target them to the endoplasmic reticulum (30). These constructs were verified by DNA sequencing. Monocistronic vectors encoding the human ß2-microglobulin, ICAM-1, LFA-3, and B7.1 were described previously (30). The TIG vector encoding hTERT and enhanced green fluorescent protein (GFP) was described previously (44).

Peptide antigen selection and synthesis. For the hTERT protein, two nonamer peptides were selected: (a) 540-548 ILAKFLHWL and (b) 865-873 RLVDDFLLV. Both peptides have been determined previously to have HLA-A*0201-binding motifs and both peptides have proven immunogenicity (24). HLA-A*0201-binding efficiency was verified for these two peptides using two HLA-peptide-binding prediction models (45, 46). The DNA oligonucleotide sequences corresponding to the peptide antigens were synthesized by Sigma-Genosys (Woodlands, TX). The oligonucleotide fragments were cloned into the SGF vector using the NcoI and BamHI restriction sites.

Gene transfer procedure. 293 GPG packaging cells (47) were transfected with each plasmid by calcium chloride method (48). A total of 5 x 104 NIH 3T3 cells were plated in a 6-cm plate and cultured in the presence of penicillin (100 units/mL) and streptomycin (100 units/mL). They were then infected with cell-free retroviral supernatant (0.45 µm filtration, Acrodisc, Pall Corp., Ann Arbor, MI) in the presence of polybrene (Sigma, St. Louis, MO) at 8 µg/mL for 16 hours. Geneticin (Sigma) was added at 1.2 mg/mL to the medium for 2 weeks to select the cells expressing A*0201. Puromycin (Sigma) was added at 3 µg/mL to the medium for 1 week to select the cells expressing the vector-encoded peptide. If gene transfer was efficient (>95%), no cell purification was required. If gene transfer was less efficient, transduced cells were purified by using magnetic beads coated with monoclonal antibody (mAb) targeting human B7.1 (CD80), ICAM-1 (CD54), or LFA3 (CD58; Dynal, Oslo, Norway) or flow cytometry (Becton Dickinson, Franklin Lakes, NJ).

Construction of artificial antigen-presenting cells. NIH 3T3 mouse fibroblasts were transduced with retroviral vectors (30) encoding the human HLA-A*0201, B7.1 (CD80), ICAM-1 (CD54), and LFA-3 (CD58) proteins. The genetically engineered fibroblasts were also transduced with retroviral vector containing antigenic peptides or protein, so that the cells would present a single antigen in the context of a HLA-A*0201 molecule (Fig. 1). Flow cytometric analysis was done to assess the efficiency of expression of HLA-A*0201 and of the costimulatory molecules B7.1, ICAM-1, and LFA-3 on the surface of the AAPCs. The AAPCs were selected for HLA-A*0201 (neomycin resistance), B7.1 [by fluorescence-activated cell sorting (FACS)], ICAM-1 (by FACS), and LFA-3 (by FACS) expression. Expression levels of all were analyzed before coculture with T cells. Expression levels for these proteins at the time of coculture were 100%.



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  		Figure 1. Schematic representation of NIH 3T3–derived AAPCs. After retroviral transduction, the murine fibroblasts express human HLA-A*0201 and ß2-microglobulin as wells as the following costimulatory molecules: B7.1 (CD80), ICAM-1 (CD54), and LFA-3 (CD58). Finally, they contain the cDNA sequence for a tumor antigen.

 
Flow cytometry analysis. To analyze the phenotype of the AAPCs, fluorescent-labeled antibodies were used against human ß2-microglobulin, HLA-A2 (mAb 8B; gift of Dr. Bo Dupont, Memorial Sloan-Kettering Cancer Center, New York, NY), B7.1 (PharMingen, San Diego, CA), ICAM-1, and LFA-3 (Becton Dickinson). FACS analysis was done before infection with the bicistronic vector encoding puromycin-N-acetyltransferase and the minigenes encoding the different peptide antigens (hTERT). For FACS analysis, tumor cells were washed in PBS containing 2% fetal bovine serum (FBS). Prediluted anti-human HLA-A*02 antibody (4 µL, 10:1) was added to each sample and incubated at room temperature for 30 minutes. Goat anti-mouse FITC (GAM-FITC, 4 µL; Becton Dickinson) was added to the cells for incubation at room temperature for 30 minutes. Cells were washed with PBS containing 2% FBS.

Human leukocyte antigen genotyping. DNA obtained from peripheral blood mononuclear cells (PBMC) was typed for HLA class I and II alleles by standard methods at the Clinical Histocompatibility Testing Laboratory, Memorial Sloan-Kettering Cancer Center.

T-cell purification. PBMCs were isolated by density gradient centrifugation on lymphocyte separation medium (Accurate Chemical & Scientific Corp., Westbury, NY). T cells were purified as described (49). The T-cell–enriched population was collected. Briefly, after lysis of the sheep RBC (Colorado Serum Co., Denver, CO) and three washes in PBS with 2% FBS, B cells, natural killer cells, monocyte macrophages, and activated T cells were depleted with a mAb cocktail with mouse IgG mAbs directed against CD11b, CD16, and HLA DP, DQ, and DR (PharMingen) at 1 µg per 1 million cells for 30 minutes followed by a panning on Petri dishes coated with goat anti-mouse IgG as described (50). After three washes in PBS with 2% FBS, the T cells were resuspended at a final concentration of 10 million cells/mL. T cells were maintained in AIM V medium (Life Technologies, Rockville, MD) without serum. Penicillin (100 units/mL) and streptomycin (100 µg/mL) were added to all the cultures. T-cell purity, as assessed by FACS analysis, was ≥98%.

Stimulation of antigen-specific CTLs. AAPCs were irradiated (1,500 Gy) and plated in a 24-well plate at a concentration of 105 cells/mL the day before stimulation in AIM V medium with 5% donor calf serum, 500 µL/well. T cells were resuspended in AIM V medium at a concentration of 2 x 106 cells/mL and the following day were added to the AAPCs and cultured for 8 to 10 days. Interleukin-2 (Chiron, St. Louis, MO) was added to the culture after day 7 (20 IU/mL every third day). T cells were restimulated 10 to 14 days after induction (30). Every third day, interleukin-2 at 20 IU/mL was added.

Cytotoxicity assays. Standard 51Cr release assay was done using TAP-deficient HLA-A*0201+ T2 cells loaded with the different hTERT peptides (10 µmol/L for 1 hour at 37°C) before labeling with 51Cr (for 1 hour at 37°C). Five thousand peptide-pulsed, 51Cr-labeled T2 cells were used per well in 96-well V-bottomed plates at different effector:target (E:T) ratios (1:100, 1:50, 1:25, 1:13, 1:6, 1:3, 1:2, 1:1) and incubated at 37°C for 4 hours. HLA-A*0201+ and HLA-A*0201– human tumor cell lines were also used as 51Cr-labeled targets. All human tumor cell lines were tested for expression of HLA-A*0201 and hTERT and used as positive and negative controls in the cytotoxicity assays. Finally, 51Cr-labeled HLA-A*0201+, hTERT– PBMCs were also used as an additional antigen specificity control.

Cell lines. The HLA-A*0201+ human tumor cell lines used were K562-A2 erythroleukemia (transfected with HLA-A*0201; gift of Dr. David Scheinberg, Memorial Sloan-Kettering Cancer Center, New York, NY), SKLY-18 lymphoma (gift from Dr. David Scheinberg), SK-Mel-29 melanoma (gift from Dr. Lloyd Old, Memorial Sloan-Kettering Cancer Center and the Ludwig Institute for Cancer Research, New York, NY), and LNCaP prostate carcinoma (American Type Culture Collection, Manassas, VA). The HLA-A*0201– human tumor cell lines used were K562 erythroleukemia (American Type Culture Collection), Raji B-cell lymphoma (American Type Culture Collection), PC3 prostate carcinoma (American Type Culture Collection), and 721.221 lymphoma (gift from Dr. Bo Dupont).

Human leukocyte antigen-peptide tetramer constructs. HLA-peptide tetrameric complexes were constructed for each peptide under investigation. The tetramers were constructed in the conventional way (51) using HLA-A*0201, ß2-microglobulin, and one of the peptide antigens (p540 or p865) from hTERT. These peptides were synthesized by Cell Essentials (Boston, MA).

Telomerase repeat amplification protocol assay. Human leukemic cell lines, both HLA-A*0201+ and HLA-A*0201–, were tested for expression of telomerase. The sensitive PCR-based method, known as telomerase repeat amplification protocol (TRAP) assay, was done (10).

Telomerase activity of each sample was detected by the extension of oligonucleotide, which serves as the substrate for the telomerase, and followed by [{gamma}-32P]ATP-labeled PCR amplification of the telomerase products (52) using the TRAPeze Telomerase Detection kit (Intergen, Purhase, NY). Tumor cells were collected and resuspended as 105 to 106 cells in 1x CHAPS lysis buffer [200 µL; 10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L MgCl2, 1 mmol/L EGTA, 0.1 mmol/L benzamidine, 5 mmol/L ß-mercaptoethanol, 0.5% CHAPS, 10% glycerol] containing RNase inhibitor to a final concentration of 100 to 200 units/mL. Samples were incubated on ice for 30 minutes and centrifuged at 14,000 x g for 20 minutes. Protein concentrations were determined by the Bradford method (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA), adjusted to 750 ng/µL, and used at 1.5 µg per assay.

For the radioactive PCR reaction, the extract of each sample (2 µL; corresponding to the adjusted protein concentrations) was combined with the 48 µL reaction mixture containing a [{gamma}-32P]ATP-labeled TS primer and 2 units Taq DNA polymerase. After a 30-minute telomerase extension reaction at room temperature, samples were subjected to PCR for 27 cycles followed by electrophoresis on 12% PAGE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of human telomerase reverse transcriptase p865-specific CTLs using artificial antigen-presenting cells. We first generated AAPCs expressing the hTERT p865 epitope (AAPCA*0201p865; Fig. 1) and sought to expand p865 peptide-specific CTLs from HLA-A*0201+ donors. Peripheral blood T cells from HLA-A*0201+ individuals were stimulated with AAPCs transduced with a bicistronic retroviral vector encoding the cDNA sequence for the HLA-A*0201-restricted p865 peptide (RLVDDFLLV) from hTERT (Fig. 2A). T cells from the same individual were simultaneously stimulated by AAPCA*0201, lacking the p865 minigene, as a control culture. Before stimulation, there were <0.01% p865-specific CD8+ T cells detectable by HLA-peptide tetramer FACS analysis. After five stimulations, 4.9 ± 1% p865 tetramer-positive CD8+ T cells were generated (n = 4 experiments with the same donor) using AAPCA*0201p865 (Fig. 2B), whereas <0.1% p865 tetramer-positive CD8+ T cells were generated using AAPCA*0201 (Fig. 2D).



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  		Figure 2. Induction of hTERT-specific CTLs using AAPCA*0201p865. A, SFG vectors containing minigene encoding the hTERT HLA-A*0201-restricted epitope p865. SD, splicing donor site; SA, splicing acceptor site; {Psi}+, extended packaging signal; pep., peptide sequence; puroR, puromycin resistance coding sequence. B, HLA-peptide tetramer specific for the hTERT p865 peptide analysis of a T-cell culture from a HLA-A*0201+ donor stimulated five times with AAPCs containing the p865 minigene. C, 51Cr release cytotoxicity assay using T cells from the same HLA-A*0201+ donor stimulated five times with AAPCs containing minigenes for the p865 peptide ({blacksquare} and {blacklozenge}) or a flu peptide ({blacktriangleup}) against HLA-A*0201+ T2 cells loaded with p865 peptide ({blacklozenge} and {blacktriangleup}) or the Db126 Wilms' tumor gene peptide ({blacksquare}). D, HLA-peptide tetramer specific for the hTERT p865 peptide analysis of a T-cell culture from the same HLA-A*0201+ donor stimulated five times with AAPCs lacking the p865 minigene.

 
The T-cell culture stimulated with AAPCA*0201p865 had peptide-specific cytolytic activity against TAP-deficient HLA-A*0201+ T2 cells pulsed with the p865 peptide in a 51Cr release assay (Fig. 2C). The p865-stimulated T cells exhibited 92% lytic activity of T2 cells pulsed with the p865 peptide, whereas 14% killing was noted of T2 cells loaded with a control HLA-A*0201-restricted peptide for the Wilms' tumor gene (RMFPNAPYL) at an E:T ratio of 50:1. T cells from the same donor stimulated by AAPCA*0201flu containing the minigene for the HLA-A*0201-specific, influenza matrix protein–derived peptide58-66 (GILGFVFTL) generated 4% killing of T2 cells pulsed with p865 peptide at an E:T ratio of 50:1.

Activation of human telomerase reverse transcriptase p540-specific CTLs using artificial antigen-presenting cells. Having generated AAPCs expressing the hTERT p540 epitope (AAPCA*0201p540), we sought to expand p540-specific CTLs from HLA-A*0201+ donors. Peripheral blood T cells from HLA-A*0201+ individuals were stimulated with AAPCA*0201p540, transduced with a bicistronic retroviral vector encoding the cDNA sequence for the HLA-A*0201-specific p540 peptide (ILAKFLHWL) from hTERT (Fig. 3A). T cells from the same individual were simultaneously stimulated by AAPCA*0201, lacking the p540 minigene, as a control culture. Before stimulation, there were <0.01% p540-specific CD8+ T cells detectable by HLA-peptide tetramer FACS analysis. After four stimulations, 4.7 ± 1.9% p540 tetramer-positive CD8+ T cells were generated (n = 4 experiments with the same donor) using AAPCA*0201p540 (Fig. 3B), whereas 0.1% p540 tetramer-positive CD8+ T cells were generated using AAPCA*0201 lacking the minigene (Fig. 3D).



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  		Figure 3. Induction of hTERT-specific CTLs using AAPCA*0201p540. A, SFG vectors containing minigene encoding the hTERT HLA-A*0201-restricted epitope p540. B, HLA-peptide tetramer sp ecific for the hTERT p540 peptide analysis of a T-cell culture from a HLA-A*0201+ donor stimulated four times with AAPCs containing the p540 minigene. C, 51Cr release cytotoxicity assay using T cells from the same HLA-A*0201+ donor stimulated four times with AAPCs containing minigenes for the p865 peptide ({blacksquare} and {blacklozenge}) or a flu peptide ({blacktriangleup}) against HLA-A*0201+ T2 cells loaded with p865 peptide ({blacklozenge} and {blacktriangleup}) or the Db126 Wilms' tumor gene peptide ({blacksquare}). D, HLA-peptide tetramer specific for the hTERT p540 peptide analysis of a T-cell culture from an the same HLA-A*0201+ donor stimulated four times with AAPCs lacking the p540 minigene.

 
The T-cell culture stimulated with AAPCA*0201p540 had peptide-specific cytolytic activity against TAP-deficient HLA-A*0201 T2 cells pulsed with the p540 peptide in a 51Cr release assay (Fig. 3C). The p540 T-cell culture had 82% killing of T2 cells pulsed with the p540 peptide, whereas 21% killing was noted of T2 cells loaded with a control HLA-A*0201-restricted peptide for the Wilms' tumor gene (RMFPNAPYL) at an E:T ratio of 50:1. T cells from the same donor stimulated by AAPCA*0201flu generated 4% killing of T2 cells pulsed with p540 peptide at an E:T ratio of 50:1.

Activation of human telomerase reverse transcriptase–specific CTLs using AAPCA*0201hTERT-GFP. To assess the ability of fibroblast-derived AAPCs to process hTERT protein for presentation of HLA-A*0201-restricted peptides, we transduced AAPCs with a retroviral vector encoding the full-length hTERT (Fig. 4B and C). We next sought to expand p865 and p540 hTERT peptide-specific CTLs from HLA-A*0201+ donors. T cells from HLA-A*0201+ individuals were stimulated four times with AAPCA*0201hTERT-GFP or AAPCA*0201 as a control culture. Before stimulation, there were <0.01% p540- or p865-specific CD8+ T cells detectable by HLA-peptide tetramer FACS analysis. After four stimulations, 2.1 ± 0.4% p865 and 1.8 ± 0.3% p540 tetramer-positive CD8+ T cells were generated (n = 3 experiments) using AAPCA*0201hTERT-GFP (Fig. 4A), whereas 0.1% p865 or p540 tetramer-positive CD8+ T cells were generated using AAPCA*0201 lacking the hTERT gene (Fig. 4D).



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  		Figure 4. Induction of hTERT-specific CTLs using AAPCA*0201hTERT-GFP. A, HLA-peptide tetramer specific FACS for either the hTERT p540 or p865 peptide analysis of a T-cell culture from a HLA-A*0201+ donor stimulated four times with AAPCs containing the full-length hTERT gene with GFP. B, SFG bicistronic vector containing cDNA encoding the full-length hTERT protein with GFP. hTRT, hTERT sequence; GFP, green fluorescent peptide sequence. C, FACS analysis of NIH 3T3 cells, gating on GFP, after transduction with the bicistronic hTERT-GFP vector reveals 94% transduction. D, HLA-peptide tetramer specific for either the hTERT p540 or p865 peptide analysis of a T-cell culture from the same HLA-A*0201+ donor stimulated four times with AAPCs lacking the full-length hTERT-GFP gene. E, 51Cr release cytotoxicity assay using T cells from the same HLA-A*0201+ donor stimulated four times with AAPCs containing the full length hTERT-GFP gene ({blacksquare}, {blacklozenge}, and x) or a flu peptide ({blacktriangleup} and {bullet}) against HLA-A*0201+ T2 cells loaded with p865 peptide ({blacklozenge} and {blacktriangleup}), p540 peptide ({blacksquare} and {bullet}), or the Db126 Wilms' tumor gene peptide (x).

 
The T-cell culture stimulated with AAPCA*0201hTERT-GFP had peptide-specific cytolytic activity against TAP-deficient HLA-A*0201+ T2 cells pulsed with either p865 or p540 peptide in a 51Cr release assay (Fig. 4E). The T-cell culture had 23% and 30% killing of T2 cells pulsed with the p865 and p540 peptides, respectively, with only 12% killing of T2 cells loaded with the control Db126 WT1 peptide at an E:T ratio of 30:1. T cells from the same donor stimulated by AAPCA*0201flu generated 3% and 4% killing of T2 cells pulsed with p865 and p540 peptides, respectively, at an E:T ratio of 30:1, establishing that the hTERT-expressing AAPCs did stimulate specific CTLs against both epitopes.

Stimulation of human telomerase reverse transcriptase–specific CTLs from multiple donors. We next explored the hTERT AAPCs system by stimulating T cells from multiple donors. Three different HLA-A*0201+ individuals were identified and PBLs were isolated. The T cells were subsequently stimulated with either AAPCA*0201p865, AAPCA*0201p540, AAPCA*0201hTERT-GFP, or the control AAPCA*0201. hTERT-specific CTLs were obtained from all three HLA-A*0201+ individuals when stimulating with the AAPCs four or five times (Fig. 5). After four stimulations of donor 1 T cells, AAPCA*0201p865and AAPCA*0201p540 generated 1.5% and 1.7% tetramer-positive cells, respectively. The same donor 1 T cells stimulated four times with AAPCA2.1hTERT-GFP generated 2.7% and 2.1% p865- and p540-specific CTLs, respectively. Four stimulations with AAPCA*0201p865 and AAPCA*0201p540 of donor 2 T cells generated 1.9% and 7.9% tetramer-positive cells, respectively. The same donor 2 T cells stimulated four times with AAPCA*0201hTERT-GFP generated 1.7% and 1.4% p865- and p540-specific CTLs, respectively. Five stimulations with AAPCA*0201p865 and AAPCA*0201p540 of donor 3 T cells generated 5.9% and 2.1% tetramer-positive cells, respectively. The same donor 3 T cells stimulated four times with AAPCA*0201hTERT-GFP generated 1.9% and 1.5% p865- and p540-specific CTLs, respectively. The level of p865 and p540 tetramer positivity for control culture T-cell populations stimulated with AAPCA*0201 lacking the p865 or p540 minigenes was 0.1%.



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  		Figure 5. Stimulation of three HLA-A*0201+ donors with AAPCA*0201p540, AAPCA*0201p865, and AAPCA*0201hTERT-GFP. HLA-peptide tetramer analysis targeting the p540 or p865 peptides from hTERT in T-cell cultures from three different HLA-A*0201+ donors stimulated with AAPCs containing the p540 or p865 minigenes or the full-length hTERT-GFP genes.

 
Expansion of CTL populations using artificial antigen-presenting cells. We did repeated stimulations of T-cell populations from donors to quantify the absolute number of hTERT-specific T cells that could be generated with the use of the AAPCs. T-cell populations from HLA-A*0201+ individuals were stimulated with AAPCA*0201p865. During the first cycle of stimulation, cell numbers increased from 0.7 x 106 to 3.1 x 106 (4.8 ± 1-fold increase) for T cells stimulated with the AAPCA*0201p865 compared with 0.7 x 106 to 1.3 x 106 for cells stimulated with empty AAPCA*0201 (1.9 ± 0.3-fold increase). The expansion experiment was repeated thrice with a single donor. The p865 tetramer-positive T cells expanded from <700 to 80 ± 18 x 106 cells over five stimulations and 109 days (88,000- to 140,000-fold increase; Fig. 6).



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  		Figure 6. hTERT-specific CTL expansion with AAPCA*0201p865. AAPCs induce a potent expansion of T cells from HLA-A*0201+ individuals. AAPCA*0201p865 stimulation causes an average 4.8 ± 1-fold increase in absolute cell number per stimulation. AAPCA*0201p865 stimulation causes a 3,000-fold increase in absolute cell number over five stimulations. p865 tetramer-positive T cells expanded from <700 to 80 ± 18 x 106 cells over five stimulations and 109 days (>110,000-fold increase). Results are based on three experiments.

 
Human telomerase reverse transcriptase expression by human tumor cell lines. To assess the level of hTERT antigen expression by human tumor cell lines, we did a TRAP assay on the lines. hTERT expression by the eight human tumor cell lines was measured by TRAP assay (Fig. 7A) using the radioactive quantitative protocol. High levels of telomerase activity were noted in seven of eight human tumor cell lines tested: K562 (erythroleukemia), K562-A2 (HLA-A*0201 transduced), SKLY-18 (lymphoma), LNCaP (prostate cancer), 721.221 (lymphoma), PC3 (prostate cancer), and Raji (Burkitt's lymphoma), whereas one cell line, SK-Mel-29 (melanoma), showed lower telomerase activity. Heat-inactivated control samples were also run for each tumor cell line, and it was apparent that there was an absence of telomerase activity in all of the heat-inactivated samples. The positive control sample (provided in the TRAPeze kit) also had excellent telomerase activity in the TRAP assay.



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  		Figure 7. Tumor HLA-A*0201 and hTERT expression. A, telomerase activity measures for eight human tumor cell lines by TRAP assay [A, Raji; B, 721.221; C, K562-A2; D, K562; E, SKLY-18; F, SK-Mel-29; G, PC3; H, LNCaP; I, positive control)]. Each TRAP assay was done on a normal (+) and a heat-inactivated (–) tumor sample. A positive control (I) is included in the assay and a loading standard is included in the gel. B, HLA-A*0201 expression by the eight human tumor cell lines as assessed by FACS analysis with a primary mouse and human HLA-A2.1 antibody and a secondary GAM-FITC antibody.

 
HLA-A*0201 expression by human tumor cell lines. To evaluate the relative HLA expression by the panel of human tumor cell lines, we did FACS analysis with a HLA-A*02-specific antibody. HLA-A*0201 expression was determined for all human tumor cell lines (Fig. 7B). HLA-A*0201 expression was detected on four human tumor cell lines: K562-A2 (transduced), SKLY-18, LNCaP, and SK-Mel-29. The mean fluorescent index (MFI) was noted for all cell lines. The highest HLA-A*0201 expression was noted on SK-Mel-29 (MFI = 80.3), and the lowest HLA-A*0201 expression was noted on LNCaP (MFI = 11.5). The four HLA-A*0201– cell lines were K562, 721.221, Raji, and PC3, with MFI ranging from 1.9 to 3.9.

Tumoricidal activity of human telomerase reverse transcriptase–specific CTLs stimulated by artificial antigen-presenting cells. To assess the tumoricidal activity of AAPC-stimulated hTERT-specific CTLs, we did 51Cr release assays against a panel of human tumor cell lines with variable hTERT and HLA expression. 51Cr release assay of hTERT-specific CTLs against human tumor cell lines was done. Eight human hTERT-overexpressing tumor cell lines were identified by TRAP assay (Fig. 7A). Of these cell lines, four (K562-A2.1 transduced, SK-Mel-29, SKLY-18, and LNCaP) were found to be HLA-A*0201+ and four (K562, 721.221, Raji, and PC3) were found to be HLA-A*0201–. These eight human tumor cell lines were targeted in the 51Cr release assay by CTL populations stimulated with either AAPCA*0201p865, AAPCA*0201p540, AAPCA*0201hTERT-GFP, or the control AAPCA*0201.

Using a T-cell culture containing 2.2% tetramer-positive p540 cells, 51Cr release assay revealed that all four HLA-A*0201+, hTERT+ tumor cell lines were lysed (Fig. 8A), whereas the HLA-A*0201–, hTERT+ cell lines were not lysed. Ninety-two percent and 72% cytotoxicity were seen against SKLY-18 and K562-A2, respectively, whereas 63% and 57% of SK-Mel-29 and LNCaP were lysed, respectively, at an E:T ratio of 50:1. The HLA-A*0201–, hTERT+ cell lines were all lysed at ≤10% at the same E:T ratio.



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  		Figure 8. Tumoricidal activity of AAPC-stimulated, hTERT-specific CTLs. 51Cr release assay of T cells stimulated with AAPC containing the hTERT p540 minigene (A), the p865 minigene (B), the full-length hTERT-GFP genes (C), the flu peptide minigene (D), and empty vector (E) targeting a panel of human tumor cell lines. All eight cell lines are hTERT+. Four of the cell lines are HLA-A*0201+: {blacksquare}, K562-A2 transduced; {blacktriangleup}, SK-Mel-29; {bullet}, SKLY-18; {blacklozenge}, LNCaP. Four of the cell lines are HLA-A*0201–: {square}, K562; {triangleup}, 721.221; {circ}, Raji; {lozenge}, PC3.

 
Using a T-cell culture containing 2.5% tetramer-positive p865 cells, 51Cr release assay revealed that all four HLA-A*0201+, hTERT+ tumor cell lines were lysed (Fig. 8B), whereas the HLA-A*0201–, hTERT+ cell lines were not lysed. One hundred percent and 62% cytotoxicity were obtained against K562-A2 and SKLY-18, respectively, whereas 38% and 32% of SK-Mel-29 and LNCaP were lysed, respectively, at an E:T ratio of 50:1. The HLA-A*0201–, hTERT+ cell lines were all lysed at ≤10% at the same E:T ratio.

Using a T-cell culture containing 2.4% and 3.0% tetramer-positive p540 and p865 cells, respectively, stimulated with the use of AAPCA*0201hTERT-GFP, 51Cr release assay revealed that all four HLA-A*0201+, hTERT+ tumor cell lines were lysed (Fig. 8C), whereas the HLA-A*0201–, hTERT+ cell lines were not lysed. Seventy percent and 44% cytotoxicity were obtained against K562-A2 and SKLY-18, respectively, whereas 20% and 24% of SK-Mel-29 and LNCaP were lysed, respectively, at an E:T ratio of 30:1. The HLA-A*0201–, hTERT+ cell lines were all lysed at ≤10% at the same E:T ratio.

T cells from the same individual stimulated five times with AAPCA*0201flu or AAPCA*0201 were included in the 51Cr release assay against the human tumor cell lines. Neither T-cell culture had significant cytolytic activity against either HLA-A*0201+ or HLA-A*0201– tumor cell lines (Fig. 8). Finally, T cells from a HLA-A*0201+ donor were stimulated four times with either AAPCA*0201p540, AAPCA*0201p865, or AAPCA*0201hTERT-GFP and were included in the 51Cr release assay against the hTERT+ tumor cell line K562-A2 and against hTERT– PBMCs from a HLA-A*0201+ donor. The hTERT+ K562-A2 cells were lysed by all three T-cell cultures, whereas the hTERT– PBMCs were not lysed (Fig. 9). This speaks to the antigen-specific nature of the cytolysis produced by the T-cell cultures stimulated by the hTERT-containing AAPCs.



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  		Figure 9. Cytolytic activity of AAPC-stimulated, hTERT-specific CTLs against hTERT+ and hTERT– targets. 51Cr release assay of T cells stimulated four times with AAPC containing the hTERT p540 minigene (A), the p865 minigene (B), and the full-length hTERT-GFP genes (C) targeting hTERT+, HLA-A0201+ K562-A2 transduced ({blacksquare}) or hTERT–, HLA-A0201+ PBMC ({circ}).

 
Correlation of CTL tumoricidal activity and tumor expression of human telomerase reverse transcriptase and human leukocyte antigen. The tumoricidal activity of hTERT-specific CTLs was examined in relation to the level of hTERT and HLA-A*0201 expression by the tumor cell line (Table 1). The 51Cr release assay experiments revealed that the HLA-A*0201– cell lines were not lysed by the p865 CTLs, whereas HLA-A*0201+ tumor cell lines that overexpressed hTERT were lysed. The level of cytolytic activity correlated with the level of antigen and HLA expression by the tumor cell line. K562-A2 and SKLY-18 were lysed by p865 CTL at high levels (100% and 58%, respectively) at an E:T ratio of 25:1. Both cell lines had high expression of hTERT and HLA-A*0201. Meanwhile, tumoricidal activity was lower against SK-Mel-29 and LNCaP (34% and 38%, respectively). SK-Mel-29 had lower hTERT expression and LNCaP had lower HLA-A*0201 expression than the other cell lines tested.


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  		Table 1. Relative telomerase activity, HLA-A*0201 expression, and cytolytic activity of p865-specific T cells against human tumor cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed the ability of AAPCs to generate telomerase-specific tumoricidal CTLs from several HLA-A*0201+ individuals. These results establish that AAPCs represent a ready and replenishable supply of APCs for stimulation of tumor-specific peripheral blood CTLs. We show that AAPCs engineered to express the p865 and p540 peptides derived from hTERT can both stimulate and expand functional antigen-specific CTL populations as shown by tetramer FACS analyses and 51Cr release assays. The p540- and p865-specific CTLs had cytolytic activity against peptide-loaded T2 cells and against tumor cell lines expressing their endogenous hTERT antigen. The hTERT-specific CTLs lysed peptide-pulsed targets and tumor cells in a HLA-restricted manner.

There is debate about whether the p540 peptide is naturally processed by tumors. Ayyoub et al. (53) reported that p540-specific clones failed to recognize telomerase-positive HLA-A*0201+ tumor cells. However, three other groups (20, 21, 24) reported excellent tumoricidal activity of p540-specific CTLs. Lev et al. (54) recently reported the isolation of an antibody that recognizes the p540 antigen in the context of HLA-A*0201 and that this antibody recognizes this complex on tumor cells, which also supports the assertion that p540 protein is naturally processed by tumor cells. Vonderheide et al. (20) recently published their clinical trial of p540 peptide-loaded autologous dendritic cell vaccination of cancer patients. The p540 CTLs induced in cancer patients had cytolytic activity against human tumor cells, and there was evidence of antitumor activity in the study, suggesting that the p540 antigen is a relevant immunotherapeutic target. Our findings further establish that the hTERT p540 peptide is presented by human tumors and can be a target for CTL-mediated tumor lysis.

Parkhurst et al. (55) recently published a report arguing against the utility of the p540 antigen as an immunologic target. The investigators immunized patients with p540 peptide and then harvested PBMCs from the patients. The p540-specific CTLs, generated with in vitro peptide-pulsed PBMC stimulation, did not have cytolytic activity against telomerase-expressing tumors. These results differ from the results in our study in which p540 CTLs have clear tumoricidal activity. The major difference between their study and ours is that they derived their p540 CTL from patients vaccinated with p540 peptide, whereas we used unvaccinated normal donors. T cells from patients may be different from normal individuals in terms of their ability to be stimulated against the p540 antigen and in terms of their tumoricidal activity. Furthermore, peptide vaccination of patients might alter the function of the p540 CTLs harvested from the patients. Finally, our AAPCs might well provide a more potent stimulation signal to p540 CTLs than the peptide-pulsed PBMCs used in the Parkhurst et al. study. In their study, no p540-specific T cells could be generated, with in vitro stimulation, from the patients without vaccination with the p540 peptide.

The AAPCs provided potent stimulation of hTERT-specific CTLs. Over the course of 109 days (and five stimulations with AAPCA*0201p865), p865 CTLs expanded from <700 total cells to 80 x 106 cells, a potentially relevant number of CTLs for adoptive cell therapy. Several clinical trials of adoptive cellular therapy targeting tyrosinase, MART-1, gp-100, and Melan-A antigens or autologous tumor have used from 0.25 x 108 to 7 x 1010 total T cells to treat cancer patients in early-phase clinical trials (7–9, 56, 57). In this study, we have generated nearly 1 x 108 hTERT-specific CTLs following repeated stimulations. Importantly, the p865 CTLs maintained high cytolytic activity against pulsed targets and tumor cells after five cycles of stimulation. More than 90% of the T cells in the culture were CD8+ after five cycles of stimulation with AAPCs expressing B7.1, LFA-3, and ICAM-1.

Because the AAPCs are constructed from murine fibroblasts, the possibility exists that the AAPCs will present mouse antigens within the context of HLA-A*0201, which could lead to the induction of CTLs specific for murine antigen. This assumption may be supported by the fact that hTERT-specific CTLs generally represent <10% of the total cells sustained in the culture after multiple stimulations. This observation is true for the hTERT antigens and WT1 antigen.3 In contrast, viral antigens from CMV or flu (influenza virus matrix protein) induce much higher percentages (~30-70%) of antigen-specific CTLs in the same AAPC system (30, 35). Furthermore, the T-cell repertoires against the autoantigens hTERT and WT1 and the viral antigens are different. The modest precursor frequency of hTERT-specific CTLs may reflect the weak proliferation compared with CMV or flu. The affinity/avidity of the T-cell receptors is also different. In the case of autoantigens, central and peripheral tolerance plays a role, whereas for viral antigens the response is a memory response that induces more potent proliferation. Thus, perhaps high affinity antigens, such as CMV or flu, overcome competition from the murine antigens for presentation on HLA more efficiently than autoantigens. The hTERT-specific T cells generated by the AAPCs have roughly 2 orders of magnitude less avidity for their targets than their positive control (flu) specific T cells. It is possible to increase the percentage of antigen-specific CTLs by sorting the cells after staining with tetramer and isolating the positive cells using magnetic beads loaded with anti-PE antibody (Miltenyi system). We (35) and others (33) have applied this approach to obtain higher percentages of antigen-specific CTLs that could be further expanded with AAPCs or anti-CD3/CD28 mAbs.

Importantly, this study reveals that murine fibroblast-derived AAPCs that have been transduced with the full-length hTERT cDNA have the ability to express and process the hTERT protein as well as present the immunodominant p865 and p540 peptides in a HLA-A*0201-restricted manner. Thus, the AAPCA*0201hTERT-GFP can stimulate and expand both p865 and p540 tetramer-positive CTLs, whereas AAPCA*0201, which lacks the full-length hTERT, does not. These findings extend our findings with the CMV pp65 (35) to a human tumor antigen. Furthermore, the AAPCA*0201hTERT-GFP-stimulated CTLs have cytolytic activity against pulsed targets and against HLA-matched, hTERT-expressing tumors. These findings suggest that the cellular machinery of fibroblast-derived AAPCs transduced with any tumor antigen cDNA could be used to identify and/or present immunodominant peptides restricted by the cotransduced HLA type and allow for the induction of tumor-specific CTLs without prior knowledge of the immunogenic epitopes of the antigen.

We investigated the tumoricidal activity of hTERT-specific CTLs against a panel of human tumor cell lines and found variable levels of cytolysis (Table 1). Although the variability could be accounted for by alloreactivity or differential sensitivity to specific tumoricidal signals, it is noteworthy that tumor sensitivity seemed to be proportional to the hTERT activity by TRAP assay and HLA-A*0201 expressed by the cell line. We found that SK-Mel-29 and LNCaP were consistently lysed to a lesser extent than the other HLA-A*0201+ and hTERT-expressing cell lines tested. In fact, SK-Mel-29 had much lower telomerase activity by TRAP assay than the other cell lines, and LNCaP cells had much lower HLA-A*0201 expression than the other cell lines tested. Lower hTERT activity and lower HLA-A*0201 expression by LNCaP cells could thus account for the lesser cytolysis. This result is consistent with findings indicating that tumors can escape immune surveillance by down-regulating HLA (58–60) or antigen (39, 61). It is also notable that the hematologic tumor cells (K562-A2 and SKLY-18) were lysed more effectively than the solid tumors (SK-Mel-29 and LNCaP). Interestingly, K562-A2 was consistently lysed more effectively by p865 CTLs than SKLY-18 tumor cells, whereas SKLY-18 was consistently lysed more effectively by p540 CTLs in contrast to K562-A2. It is thus possible that K562-A2 cells process or present the p865 antigen more effectively, whereas SKLY-18 tumor cells better process or present the p540 antigen. This finding suggests that different tumor types may differentially process and/or load potential epitopes onto their HLA molecules.

Altogether, our results obtained with fibroblast-derived AAPCs transduced with retroviral vectors encoding the p865 or p540 hTERT epitopes or the full-length hTERT cDNA establish that this system is a practicable approach for the induction of possibly clinically relevant numbers of hTERT-specific CTLs. Furthermore, our finding that the murine cells can process full-length hTERT protein and load the immunodominant p865 and p540 peptides onto HLA-A*0201 suggests a general approach for stimulating CTLs against dominant, cryptic, or unknown epitopes, which could be useful for epitope discovery as well as for adoptive immunotherapy.


   Acknowledgments
 
Grant support: NIH grants CA-59350 and CA-23766, American Society of Clinical Oncology Career Development Award 2003 (J. Dupont), Charles H. Revson Foundation (J. Dupont), and Damon Runyon-Lilly Clinical Investigator Award (J. Dupont).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Eric Pamer and the Tetramer Core Facility at Memorial Sloan-Kettering Cancer Center for p865 and p540 tetramer synthesis.


   Footnotes
 
Note: J-B. Latouche is currently at Laboratoire de Génétique Moléculaire, Institut National de la Sante et de la Recherche Medicale U614-IFRMP, Centre Hospitalier Universitaire, Faculté de Médecine et de Pharmacie, 22 Bvd. Gambetta, 76183 Rouen, France.

3 Unpublished observation. Back

Received 8/18/04. Revised 3/22/05. Accepted 4/ 6/05.


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 Introduction
 Materials and Methods
 Results
 Discussion
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A. N. Hasan, W. J. Kollen, D. Trivedi, A. Selvakumar, B. Dupont, M. Sadelain, and R. J. O'Reilly
A Panel of Artificial APCs Expressing Prevalent HLA Alleles Permits Generation of Cytotoxic T Cells Specific for Both Dominant and Subdominant Viral Epitopes for Adoptive Therapy
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R. Zappasodi, M. Di Nicola, C. Carlo-Stella, R. Mortarini, A. Molla, C. Vegetti, S. Albani, A. Anichini, and A. M. Gianni
The effect of artificial antigen-presenting cells with preclustered anti-CD28/-CD3/-LFA-1 monoclonal antibodies on the induction of ex vivo expansion of functional human antitumor T cells
Haematologica, October 1, 2008; 93(10): 1523 - 1534.
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L. Wenandy, R. B. Sorensen, L. Sengelov, I. M. Svane, P. thor Straten, and M. H. Andersen
The Immunogenicity of the hTERT540-548 Peptide in Cancer
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S. M. Domchek, A. Recio, R. Mick, C. E. Clark, E. L. Carpenter, K. R. Fox, A. DeMichele, L. M. Schuchter, M. S. Leibowitz, M. H. Wexler, et al.
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Cancer Res., November 1, 2007; 67(21): 10546 - 10555.
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H. Zhang, K. M. Snyder, M. M. Suhoski, M. V. Maus, V. Kapoor, C. H. June, and C. L. Mackall
4-1BB Is Superior to CD28 Costimulation for Generating CD8+ Cytotoxic Lymphocytes for Adoptive Immunotherapy
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 Molecular Cancer TherapeuticsHome page
M. A. Purbhoo, Y. Li, D. H. Sutton, J. E. Brewer, E. Gostick, G. Bossi, B. Laugel, R. Moysey, E. Baston, N. Liddy, et al.
The HLA A*0201-restricted hTERT540-548 peptide is not detected on tumor cells by a CTL clone or a high-affinity T-cell receptor
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