• Open Access

Antitumor effect of new multiple antigen peptide based on HLA-A0201-restricted CTL epitopes of human telomerase reverse transcriptase (hTERT)

Authors


To whom correspondence should be addressed.

E-mail: shimingyang@yahoo.com or haojia2004@yahoo.com.cn

Abstract

The development of peptide vaccines aimed at enhancing immune responses against tumor cells is becoming a promising area of research. Human telomerase reverse transcriptase (hTERT) is an ideal universal target for novel immunotherapies against cancers. The aim of this work was to verify whether the multiple antigen peptides (MAP) based on HLA-A0201-restricted CTL epitopes of hTERT could trigger a better and more sustained CTL response and kill multiple types of hTERT-positive tumor cells in vitro and ex vivo. Dendritic cells (DC) pulsed with MAP based on HLA-A0201-restricted CTL epitopes of hTERT (hTERT-540, hTERT-865 and hTERT-572Y) were used to evaluate immune responses against various tumors and were compared to the immune responses resulting from the use of corresponding linear epitopes and a recombinant adenovirus-hTERT vector. A 4-h standard 51Cr-release assay and an ELISPOT assay were used for both in vitro and ex vivo analyses. Results demonstrated that targeting hTERT with an adenovector was the most effective way to stimulate a CD8+ T cell response. When compared with linear hTERT epitopes, MAP could trigger stronger hTERT-specific CTL responses against tumor cells expressing hTERT and HLA-A0201. In contrast, the activated CTL could neither kill the hTERT-negative tumor cells, such as U2OS cells, nor kill HLA-A0201 negative cells, such as HepG2 cells. We also found that these peptide-specific CTL could not kill autologous lymphocytes and DC with low telomerase activity. Our results indicate that MAP from hTERT can be exploited for cancer immunotherapy.

Despite multiple approaches for prevention and therapy, cancer remains a major cause of death in the world. Most approaches, including radiotherapy and chemotherapy, kill malignant cells but also affect normal cells, resulting in side effects that limit the use of these treatments. With the rapid increase in knowledge of the immune system and its regulation, immunologic approaches have been explored to target and eliminate cancer cells.[1, 2] Dendritic cell (DC)-based immunotherapy, which has the advantages of inducing strong immune responses, few side-effects and wide applicability, is a promising therapy for malignant tumors.

Tumor-associated antigen (TAA)-loaded DC is an ideal tumor immunotherapy. Over the past few decades, dozens of TAA have been described.[3] However, the expression of most TAA is restricted to a few tumor types. In addition, the appearance of antigen-loss mutations in tumor cells in the face of immune pressure is well described.[4, 5] To circumvent these issues, a new class of TAA, termed “universal tumor antigens,” has been proposed.[6] These TAA are thought to not only trigger T cell reactivity against a broad range of tumor types but also to play critical functional roles in tumor growth and development. An ideal universal TAA ought to have the following characteristics: (i) it should be expressed in the vast majority of human cancers, but rarely expressed in normal tissues; (ii) it should be indispensable in the process of tumorigenesis to avoid antigen variation or depletion; (iii) it should include peptide sequences that bind to MHC molecules; and (iv) it should be recognized by the T cell repertoire in an MHC-restricted manner to elicit specific T cell responses.[7, 8]

Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, is highly expressed in over 85% of cancer cells to maintain the immortality of the cancer cells but is not active in most normal somatic cells. Therefore, hTERT could be an ideal candidate for a universal TAA. Unlike most other TAA, the expression of hTERT in tumor cells is associated with tumor growth, development and invasion.[9] Targeting hTERT might also minimize immune escape because the inhibition of telomerase activity in hTERT-positive tumor cells leads to telomere shorting and tumor cell death by apoptosis.[10, 11]

CTL are considered to be the chief mediators of tumor immune surveillance and act by recognizing TAA as cognate peptides bound to MHC molecules expressed on the surface of tumor cells. A major achievement in the field of tumor immunology over the past 20 years has been the clear demonstration that CTL epitopes binding to the MHC rather than integral TAA induce CTL reactions. These peptides are usually 8–10 amino acids long, with 2–3 primary anchor residues that interact with the MHC class I molecules and 2–3 amino acid residues that bind to the T cell receptor.[12] Therefore, the identification of CTL epitopes using TAA has become a critical step in the development of peptide-based immunotherapy for cancer.

Our previous study indicated that a hTERT recombinant adenovirus vaccine and a peptide-nucleotide dual vaccine could also induce hTERT-specific CTL in vivo and in vitro.[13, 14] Several other groups have used the DNA or RNA of hTERT as vaccines and confirmed that these vaccines can induce T-helper cells and specific CTL responses against a broad spectrum of hTERT-positive tumors.[15-18] These results indicate that hTERT could be a universal TAA used in tumor immunotherapies. However, CTL epitopes must exist that can induce a specific CTL response to the protein of hTERT. In fact, the first epitope derived from hTERT was hTERT-540 reported by Vonderheide,[19] and it was found that CTL specific for hTERT-540, generated in vivo and in vitro, could kill a range of hTERT-positive tumor cell lines in a peptide-specific and MHC-restricted manner. Subsequently, several other immunogenic hTERT peptides, including hTERT-865, hTERT-973 and hTERT-572Y, have also been identified.[20] Although peptide vaccines have the advantage of easy synthesis and safe application, their small molecular weight, single structure and short half-life make them easily degradable, which results in weak immunogenicity and poor immune responses in vivo.[21-23]

Branched peptides, such as multiple antigen peptides (MAP), were invented by Tam ([24]), and they have been explored for uses in serodiagnosis, vaccines against bacteria, viruses and parasites, and as inhibitors of pathogenic infections.[24] MAP may be considered for use in the development of new strategies for antitumor immunotherapy targeting hTERT-positive tumor cells.

In the present study, we designed three tetrameric MAP vaccines based on HLA-A0201-restricted CTL epitopes of hTERT, including two high-affinity epitopes and one modified low-affinity epitope. The killing effects of CTL induced by these MAP vaccines and their corresponding linear peptides, as well as recombinant adenovirus-hTERT vectors, were studied using a standard 4 h 51Cr-release assay. The results demonstrated that although the killing activity of CTL induced by MAP vaccines of hTERT was lower than that induced by an adenovirus vaccine, it was much more potent than that induced by their corresponding linear peptides. These findings provide evidence for a potential clinical application of hTERT MAP vaccines in immunotherapy for cancers.

Materials and Methods

Mice and cell lines

C57BL/6 transgenic mice, which express a chimeric heavy chain of the MHC-I molecule, were kindly provided by Professor Ni from the Institute of Immunology of the Peoples' Liberation Army Third Military Medical University, Chongqing, China. Animal studies were performed with the approval of the local ethics committee of the Third Military Medical University.

The human colon carcinoma cell line SW480 (hTERT+, HLA-A0201+), the human gastric carcinoma cell line KATO-III (hTERT+, HLA-A0201+), the human osteogenic sarcoma cell line U2OS (hTERT, HLA-A0201+), the human breast cancer cell line MCF-7 (hTERT+, HLA-A0201+) and the human hepatocellular carcinoma cell line HepG2 (hTERT+, HLA-A0201) were purchased from the American Type Culture Collection (ATCC, University Boulevard, Manassas, VA, USA) and stored in our laboratory. HepG2/HLA-A0201 cells (hTERT+, HLA-A0201+) transfected with a plasmid encoding the chimeric heavy chain of the MHC-I molecule and U2OS/hTERT cells (hTERT+, HLA-A0201+) transfected with a plasmid vector containing the full-length cDNA of hTERT were constructed and stored in our laboratory. KATO-III, HepG2, MCF-7, U2OS and SW480 cells were all cultured in RPMI-1640 medium containing 10% FBS. All cells were kept at 37°C in a humidified atmosphere containing 5% CO2.

Recombinant adenovirus encoding human telomerase reverse transcriptase

The recombinant adenovirus vector encoding hTERT was constructed, amplified and identified as previously described.[13]

Synthetic peptides

Three HLA-A0201-restricted peptides derived from hTERT (hTERT-540 [ILAKFLHWL], hTERT-865 [RLVDDFLLV] and hTERT-572Y [YLFFYRKSV]) and their corresponding tetrameric MAP were synthesized by Shanghai Qiangyao Biotechnology (Shanghai, China) (Fig. 1a). One nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]), which served as a negative control, was synthesized by Beijing Scilight Biotechnology (Beijing, China). The molecular weights of the peptides were validated by mass spectrometry (API2000, PE). Lyophilized peptides were dissolved in DMSO, (Sigma, St. Louis, MO, USA) and stored at −20°C.

Figure 1.

Analogue structure of four-branched peptides of human telomerase reverse transcriptase (hTERT) and phenotypes of target cells. (a) Schematic representation of a multiple antigen peptide containing four peptide monomers: (a) MAP4-hTERT-540; (b) MAP4-hTERT-865; (c) MAP4-hTERT-572Y. (b) Expression of hTERT in target cells: (a) Expression of hTERT mRNA in target cells by RT-PCR and (b) hTERT protein expression in different target cells as determined by western blot. GAPDH or β-actin served as reference gene. (1) U2OS; (2) U2OS/hTERT; (3) KATO-III; (4) SW480; (5) MCF-7; (6) HepG2; (c) HLA-A0201 expression in various target cells: SW480, KATO-III, U2OS, MCF-7, HepG2, HepG2/HLA-A0201.

Dendritic cells generation from human peripheral blood mononuclear cells and mouse bone marrow

The DC from PBMC were generated using a procedure described previously.[25] In brief, PBMC were isolated from HLA-A0201+ donors by ficoll-hypaque density gradient centrifugation. The cells were allowed to adhere in culture flasks for 2 h at 37°C in RPMI-1640 with 10% FBS. Then, non-adherent cells were collected and frozen in freezing media (60% RPMI-1640, 30% FBS and 10% DMSO) for later use in CTL assays. Adherent cells were cultured in 6 mL of RPMI-1640 with 10% FBS containing 800 U/mL recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF, R&D Systems, McKinley Place NE, MN, USA) and 1200 U/mL recombinant human interleukin-4 (rhIL-4, R&D Systems). On days 3, 5 and 7, half of the media was refreshed without discarding any cells and fresh cytokine-containing (rhGM-CSF and rhIL-4) media was added. On day 8 of culture, 1000 U/mL of tumor necrosis factor-α (TNF-α, R&D Systems) was added to the media. On day 9, non-adherent cells obtained from these cultures were considered mature human PBMC-derived DC.

The procedure for generating mouse DC (mDC) has been described previously.[12] Briefly, bone marrow was flushed from the tibia and femur of C57BL/6-Tg mice and then depleted of erythrocytes with commercial lysis buffer (Sigma). The cells were washed twice in serum-free RPMI-1640 medium and cultured in culture flasks with RPMI-1640 medium containing 200 U/mL recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF, R&D Systems) and 400 U/mL recombinant murine interleukin-4 (rmIL-4, R&D Systems). On days 3, 5 and 7, half of the media was refreshed without discarding any cells and fresh cytokine-containing (rmGM-CSF and rmIL-4) media was added. On day 8 of culture, murine tumor necrosis factor-α (mTNF-α, R&D Systems) was added to the media. On day 9, non-adherent cells obtained from these cultures were considered mature bone marrow-derived DC.

The phenotype of mature DC generated from PBMC was detected by phycoerythrin (PE)-conjugated anti-human CD86, FITC-conjugated anti-human CD83 and FITC-conjugated anti-human CD1α using flow cytometry. Meanwhile, the phenotype of mature DC from mouse bone marrow was also detected by flow cytometry using FITC-conjugated goat anti-mouse CD11c, H-2Kb, MHC-II and CD86.

Flow cytometriy

Phenotypes of mature DC were performed by flow cytometry, as previously described.[26] In brief, DC generated from PBMC were immunostained with FITC conjugated mouse anti-human CD 1a, CD83 and PE-conjugated anti-human CD86 antibodies (eBioscience, Science Center Drive, San Diego, CA, USA) for 2 h at 4°C. Meanwhile, DC generated from mouse bone marrow were immunostained with FITC-conjugated goat antimouse CD11c, H-2Kb, MHC-II and CD86 antibodies (eBioscience) for 2 h at 4°C. The corresponding FITC IgG isotype control antibody (eBioscience) was used. These cells were then washed once with FACS buffer, resuspended and tested on a FACScan (Becton–Dickinson, Franklin Lakes, NJ, USA).

HLA-A0201 analysis of target cell lines (SW480, KATO-III, MCF-7, U2OS, HepG2 and HepG2/HLA-A0201) was also performed by flow cytometry, as previously described.[27] In brief, anti-HLA-A0201 mAbs BB7.2 (culture supernatant, which was stored in our laboratory) was used as the primary reagent. FITC-conjugated GAM-IgG-F(ab')z (JinQiao Biotechnology, Bejing, China) was used for the second incubation.

Generation of human telomerase reverse transcriptase -specific CTL in vitro

Mature human PBMC-derived DC were pulsed with 30 μmol/mL of MAP based on HLA-A0201-restricted CTL epitopes of hTERT (hTERT-540, hTERT-865 and hTERT-572Y) and their corresponding linear peptides for 4 h and recombinant adenovirus-hTERT vector for 24 h. The DC were then irradiated with 20 Gy to prevent outgrowth. Subsequently, the non-adherent autologous peripheral blood lymphocytes were co-cultured with the DC. After stimulation, 800 U/mL recombinant interleukin-2 (IL-2) was added. On day 7, and weekly thereafter, the autologous peripheral blood lymphocytes were restimulated with peptide-pulsed or adenovector-pulsed irradiated dendritic cells.

Generation of human telomerase reverse transcriptase-specific CTL ex vivo

C57BL/6 transgenic mice that were 12 weeks old were immunized three times once a week, by subcutaneous injection in the back with 2 × 106 syngeneic mature DC pulsed with the above peptides or recombinant adenovirus-hTERT vector. DC pulsed with HIVpol (476–484, ILLEPVHGV) were also subcutaneously injected in mice and served as a negative control. After 5 days, the spleens of the mice were removed and the splenocytes were harvested as effectors.

CD4+ T cell depletion

To identify whether CD4+ T cells play an important role in CD8+ T cell responses, CD4+ T cells were depleted in mice, as described previously.[28] For CD4+ T cell depletion, .C57BL/6 transgenic mice were injected each day with 100 μg of anti-CD4 murine antibody (Biolenged, Pacific Heights Blvd, San Diego, CA, USA) beginning 3 days before the first immunization, as previously reported.

Cytotoxicity assay

A standard 4-h 51Cr-release assay was used to evaluate the level of CTL activity. The target cells, SW480, KATO-III, U2OS, U2OS/hTERT, HepG2, HepG2/HLA-A0201 and MCF-7, were labeled with 100 μCi 51Cr in 1 mL of RPMI-1640 containing 10% FBS for 2 h at 37°C, 5% CO2. The labeled target cells were washed three times with RPMI-1640 medium without serum. Next, the target cells were plated in triplicate at a final concentration of 1 × 104 cells/well in 96-well V-bottom microtiter plates. The 51Cr-labeled target cells (100 μL) were then mixed with effector cells at effector-to-target ratios (E:T) of 10:1, 20:1, 40:1 and 80:1. After incubation for 4 h at 37°C, 5% CO2, 51Cr release was measured by quantifying the amount of 51Cr in 100 μL of supernatant using an automated gamma counter (Beijing Nuclear Instrument Factory, Beijing, China). Percent specific lysis was calculated as the percentage of specific 51Cr release using the following formula:

Specific lysis = (experimental release – spontaneous release)/(maximal release – spontaneous release) Χ 100%

Inferferon-gamma ELISPOT assay

CTL, generated by the method described above, were assayed in ELISPOT cultures (96-well coated microtiter plates, Dakewe Biotech, Shenzheng, China) for inferferon-gamma (IFN-γ) production. Briefly, effectors were plated in triplicate at a final concentration of 1 × 105 cells/well in 96-well nitrocellulose plates. Effector cells were stimulated with candidate peptides at a final concentration of 30 μM as well as adenovirus-hTERT vector. The plate was incubated at 37°C, 5% CO2 for 24 h. The plate was processed using a biotin labeled anti-mouse IFN-γ antibody, an enzyme labeling marker and an antimarker. Then, a freshly prepared developer was added and incubated in the dark at 37°C for 8 min (Quick Spot Mouse IFN-γ Precoated ELISPOT kit, Dakewe). Spots were quantified using the ELISPOT reader (BioReader 4000 Pro-X, BIOSYS, Germany).

Statistics

Statistical analysis was performed using the Student's t-test. The difference was considered statistically significant when P < 0.05. All statistical analyses were conducted with spss 17.0 software (SPSS Inc., Chicago, IL, USA).

Results

Human telomerase reverse transcriptase and HLA-A0201 expression in various target cells

In this study, we tested the hTERT expression in all target cell lines by RT-PCR and weston blot. The results demonstrated that hTERT expression was detected in SW480 colon carcinoma cells, KATO-III gastric carcinoma cells, MCF-7 breast cancer cells and HepG2 hepatocellular carcinoma cells. Meanwhile, hTERT expression was not detected in U2OS osteogenic sarcoma cells, but was detected in U2OS/hTERT osteogenic sarcoma cells that were transfected with a plasmid vector containing the full-length cDNA of hTERT (Fig. 1b). This was consistent with the results when we tested the hTERT expression in these target cells by immunohistochemistry.[13]

We then tested for HLA-A0201 expression in target cells by flow cytometry. The results revealed that the expression of HLA-A0201 in SW480, KATO-III, MCF-7 and U2OS cells was 99.87, 100.00, 97.97 and 100.00%, respectively. However, the HLA-A0201 expression in HepG2 cells was only 8.0%. After transfection with a plasmid encoding HLA-A0201, the HLA-A0201 expression of HepG2/ HLA-A0201 cells increased to 91.2% (Fig. 1c).

Identification of mature dendritic cells by flow cytometry

The phenotype of mature DC generated from PBMC or mouse bone marrow was identified by flow cytometry. The results demonstrated that expression of CD86, CD83, CD1α and HLA-A0201 in DC from PBMC was 99.77, 75.49, 67.17 and 98.74%, respectively (Fig. 2a), which indicated that these DC were mature and had a strong capacity for antigen presentation. We also found expression of H-2Kb (96.6%), CD11c (74.6%), CD86 (97.1%) and MHC-II (94.5%) to be strongly upregulated in the mature DC from mouse bone marrow (Fig. 2b).

Figure 2.

Phenotype of mature dendritic cells (DC). (a) molecular phenotypes in DC generated from PBMC. (b) molecular phenotypes in DC generated from mouse bone marrow.

Human telomerase reverse transcriptase specific multiple antigen peptides induce stronger antitumor responses than their corresponding linear peptides

To analyze immune responses against tumor cell lines in vitro, DC from HLA-A0201+ donors were pulsed with three HLA-A0201-restricted hTERT-derived linear peptides (hTERT-540, hTERT-865 and hTERT-572Y) or their corresponding tetrameric MAP (MAP4-hTERT-540, MAP4-hTERT-865 and MAP4-hTERT-572Y). A DC-pulsed recombinant adenovirus-hTERT vector served as the positive control, while DC pulsed with HIV polypeptides served as the negative control. The autologous peripheral blood lymphocytes were stimulated by the pulsed DC three times. We then used a standard 4-h 51Cr-release assay to analyze their ability to lyse various human target cells, such as KATO-III, SW480 and MCF-7 cells, which express both hTERT and HLA-A0201. The results demonstrated that the tetrameric MAP of hTERT-540, hTERT-865 and hTERT-572Y were able to induce 25% more lysis (< 0.05), as compared to their corresponding linear peptides, at the highest E/T ratio (80:1). However, the amount of lysis was lower than that induced by the adenovirus-hTERT vector (Fig. 3). We also tested the hTERT-specific CTL activity in C57BL/6 transgenic mice against KATO-III, SW480 and MCF-7 cells using a 4-h 51Cr-release assay (Fig. 4). The results also demonstrated that all of the hTERT-derived peptides and adenovirus-hTERT vector had significant killing effects on KATO-III, SW480 and MCF-7 cells. Targeting hTERT with an adenovector was more effective in stimulating a broad range of CD8+ T cell responses compared to peptide-based vaccines. However, when compared with hTERT linear peptides, hTERT-specific CTL responses induced by their corresponding tetrameric MAP were much stronger and the amount of specific lysis was increased by more than 18% (P < 0.05). These results are consistent with our in vitro study.

Figure 3.

Specific lysis of KATO-III, SW480 and MCF-7 tumor cells by CTL activated by human telomerase reverse transcriptase (hTERT)-derived peptides in vitro. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT-540-MAP vaccine or their corresponding linear peptide. (b) Specific lysis of CTL induced by specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT-865-MAP vaccine or their corresponding linear peptide. (c) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT-572Y-MAP vaccine or their corresponding linear peptide. Effector-to-target ratios are shown on the x-axis, and the y-axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence served as a negative peptide (NP). Results are given as the mean ± SD. Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric multiple antigen peptides (MAP) compared with the recombinant adenovirus-hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric MAP compared with their corresponding linear peptides.

Figure 4.

Specific lysis of KATO-III, SW480 and MCF-7 tumor cells by CTL activated by human telomerase reverse transcriptase (hTERT)-derived peptides ex vivo. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT-540-MAP vaccine or their corresponding linear peptide. (b) Specific lysis of CTL induced by specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT-865-MAP vaccine or their corresponding linear peptide. (c) Specific lysis of CTL induced by adenovector encoding full length hTERT, hTERT-572Y-MAP vaccine or their corresponding linear peptide. Effector-to-target ratios are shown on the x-axis, and the y-axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence served as a negative peptide (NP). Results are given as the mean ± SD. Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric multiple antigen peptides (MAP) compared with the recombinant adenovirus-hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric MAP compared with their corresponding linear peptides.

To further confirm hTERT specificity of the CTL, we took advantage of the HLA-A0201-positive, hTERT-negative osteogenic sarcoma cell line U2OS and U2OS/hTERT cells (hTERT+, HLA-A0201+) that were transfected with a plasmid vector containing hTERT. As shown in Figure 5, these hTERT-specific CTL could lyse U2OS/hTERT cells, whereas no obvious lysis of U2OS cells was detected even at the highest E/T ratio. To confirm HLA-A0201 restriction, we used the hTERT-positive, HLA-A0201-negative liver cancer cell line HepG2 and the hTERT-positive, HLA-A0201-positive cell line HepG2/HLA-A0201 in a 51Cr-release assay. The results showed that CTL generated from hTERT-540, hTERT-865 or hTERT-572Y peptide-pulsed DC could lyse HepG2/HLA-A0201 cells. However, no cytotoxicity was observed against the HLA-A0201-negative liver cancer cell line HepG2 even at the highest E/T ratio (Fig. 6).

Figure 5.

hTERT specificity of CTL generated from human telomerase reverse transcriptase (hTERT) vaccines in vitro and ex vivo. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, multiple antigen peptides (MAP) vaccines or their corresponding linear peptide-pulsed DC from PBMC against U2OS, U2OS/hTERT in vitro. (b) Specific lysis of CTL induced by adenovector encoding full length hTERT, MAP vaccines or their corresponding linear peptide-pulsed DC from mouse bone marrow against U2OS, U2OS/hTERT ex vivo. Effector-to-target ratios are shown on the x-axis, whereas the y-axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative peptide (NP). Results are given as the mean ± SD. Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric MAP is compared with the recombinant adenovirus-hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric MAP is compared with their corresponding linear peptides.

Figure 6.

HLA-A0201 restriction of CTL generated from human telomerase reverse transcriptase (hTERT) vaccines in vitro and ex vivo. (a) Specific lysis of CTL induced by adenovector encoding full length hTERT, multiple antigen peptides (MAP) vaccines or their corresponding linear peptide-pulsed DC from PBMC against HepG2, HepG2/HLA-A0201 in vitro. (b) Specific lysis of CTL induced by adenovector encoding full length hTERT, MAP vaccines or their corresponding linear peptide-pulsed DC from mouse bone marrow against HepG2, HepG2/HLA-A0201 ex vivo. Effector-to-target ratios are shown on the x-axis, whereas the y-axis shows percent specific lysis. CTL induced by one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative peptide (NP). Results are given as the mean ± SD. Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric MAP is compared with the recombinant adenovirus-hTERT vector. *Statistically significant values at P < 0.05 using a paired Student's t-test when tetrameric MAP is compared with their corresponding linear peptides.

Human telomerase reverse transcriptase-specific CTL are unable to lyse autologous lymphocytes or dendritic cells

It is reported that hTERT is abundantly present in most human tumor cells.([2, 8, 9]) However, hTERT is also expressed in some normal somatic cells, such as stem-cells, activated lymphocytes, basal keratinocytes and DC. In our previous study, we tested the expression of hTERT in DC by western blot and immunohistochemistry and we found low-level expression in mature DC.[13, 14] Therefore, in the present study, we used a 51Cr-release assay to investigate whether hTERT-specific effectors were able to lyse autologous lymphocytes and DC. The results indicated that the CTL primed by these peptides or adenovector-pulsed DC could neither lyse autologous lymphocytes and human PBMC derived DC in vitro nor autologous lymphocytes and mouse bone marrow-derived DC ex vivo (Fig. 7).

Figure 7.

The effect of human telomerase reverse transcriptase (hTERT)-specific CTL on lymphocytes or dendritic cells (DC). Specific lysis of CTL generated from adenovector encoding full length hTERT, multiple antigen peptides (MAP) vaccines or their corresponding linear peptide against autologous lymphocytes and DC in vitro (a) and ex vivo (b). Effector-to-target ratios are shown on the x-axis, whereas the y-axis shows percent specific lysis. CTL induced using one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative peptide (NP). Results are given as the mean ± SD.

Human telomerase reverse transcriptase-specific CD8+ T cell responses require CD4+ T cell help in immunized mice

It was known that CTL could produce the TH1 cytokine IFN-γ. Therefore, we enumerated the hTERT-specific CD8+ T cells by ELISPOT. Mice vaccinated with MAP4-hTERT-540, MAP4-hTERT-865 and MAP4-hTERT-572Y had increased numbers of IFN-γ positive cells than did mice vaccinated with their corresponding linear peptides (< 0.05). However, the number of IFN-γ-producing effectors was significantly reduced in CD4-depleted mice regardless of whether the mice were MAP vaccinated or linear peptide vaccinated (Fig. 8). This result demonstrates that CD4+ T cells play an essential role in the priming of naive CD8 responses.

Figure 8.

CD4 T cell depletion reduced inferferon-gamma (IFN-γ)-producing effectors in human telomerase reverse transcriptase (hTERT) peptide immunized mice. Mice vaccinated with MAP4-hTERT-540, MAP4-hTERT-865 and MAP4-hTERT-572Y had increased numbers of IFN-γ positive cells compared to mice vaccinated with the corresponding linear peptides (hTERT-540, hTERT-865 and hTERT-572Y). However, the number of IFN-γ-producing effectors was significantly reduced in CD4-depleted mice regardless of whether the mice were vaccinated with multiple antigen peptides (MAP) or linear peptides. Phytohemaglutinnin (PHA) was used as a positive control and one nonapeptide from the HIV sequence (HIVpol [476–484] [ILLEPVHGV]) served as a negative control. *Statistically significant values at P < 0.05 using a paired Student's t-test compared with the corresponding controls. **Statistically significant values at P < 0.01 using a paired Student's t-test compared with the corresponding controls.

Discussion

In this study, we used TNF-induced mature DC pulsed with recombinant adenovirus-hTERT vector and hTERT-derived peptides, including tetrameric MAP and their corresponding linear peptides, to test their respective immunogenicity. We found that adenoviral vectors encoding hTERT were more effective in stimulating a broad range of CD8+ T cell responses compared to peptide vaccines both in vitro and ex vivo. Previous trials, as well as trials currently in progress, using recombinant viruses that express tumor antigens have reported similar results.[29-31] Adenovirus, vaccinia and lentivirus vectors have also been used. This strategy is likely to be the most potent, but also the most challenging from a manufacturing and quality control perspective. Stronger immune responses may be induced against viral vector antigens than against weaker tumor antigens.[32]

When compared with the vector-based vaccine that uses an adenovirus or lentivirus as a vector, peptide-based vaccines are simple, safe, stable and economical, as well as easy to produce and modify. However, there are unique disadvantages, which include HLA restriction, low immunogenicity and a low level of memory responses. Currently, many strategies are being explored to increase the immunogenicity of peptide vaccines, such as modifying the peptide sequence with acidic residues to increase the interaction with the HLA, using immunoadjuvants,[33] and employing carrier proteins. In the present study, we selected three hTERT-derived epitopes (two high affinity epitopes, hTERT-540 and hTERT-865, and one modified low affinity epitope, hTERT-572Y) to investigate their respective immunogenicity. Our results demonstrated no statistical differences among the three linear peptides. hTERT-572Y is a modified peptide from hTERT-572. The immunogenicity of peptide hTERT-572 was greatly enhanced by a single amino acid (Arg → Tyr) substitution at position one, which augmented its relative affinity for the HLA-A0201 molecule as we previously reported.[34]

We also found that tetrameric MAP could trigger stronger hTERT-specific CTL responses compared to their corresponding linear peptides, and the amount of specific lysis was significantly increased in vitro and ex vivo. The result is consistent with a previous study of ours in which we found that MAP of human heparanase, another universal TAA, could elicit a much more potent immune response against tumors than their corresponding linear peptides.[35] The MAP for peptide-based vaccines designed by Tam and coworkers were used to increase immunogenicity and to overcome the ambiguity and requirement of synthetic peptides to be conjugated to a carrier protein.[36-38] Vogel et al.[39] obtained better quality antisera than peptide protein conjugates when HIV-1MN multiple antigen peptides were used. The MAP system has been shown to deliver not only multiple B-cell but also T-cell epitopes and has already been shown to be valuable in vaccine development for infectious diseases and tumors. For example, Ciesielski et al.[40] examined the cellular immune effects of MAP of the unique EGFRvIII epitope in a rat glioma model and found that MAP vaccine also induced a specific lytic antitumor CTL immune response against F98 glioma cells expressing EGFRvIII. Akira et al.[41] previously showed that immunization with pRL1a MAP efficiently induced in vitro CTL generation and inhibited in vivo RL1 tumor growth. The pRLla MAP enhanced immunogenicity and generated CTL against the pRLla peptide. MAP consist of concentrated epitopes and possess an immunologically silent lysine core. There are two possible reasons for increased immunogenicity. First, the conformation of the epitope in the MAP format might contribute to the longevity and strength of the responses induced by MAP. Second, MAP retain an unnatural polymeric structure compared to a linear peptide, which might aid in resistance to proteolytic degradation.[42] The persistence of the immunogen in the systemic circulation might induce longer lasting immune responses. As immunogens, MAP have additional benefits, such as simplicity in design and synthesis, versatility for investigating various immune responses, and the ability to generate site-specific antibodies in the laboratory. The mechanism for hTERT epitope generated from hTERT MAP is still unclear. Lysine adjacent to the COOH termius of the epitope peptide could provide the target site for proteases with trypsin-like activity present in the cytosol.[43] The epitope peptide would be subsequently produced by deleting lysine. Additional studies are needed to address this question.

In addition, we used an IFN-γ ELISPOT to enumerate the antigen-specific effectors secreting IFN-γ and found that the hTERT-specific CD8+ T cells secreting IFN-γ were strongly reduced in CD4 depleted mice. It is well known that CD4+ T helper cells are critical in the development of immune responses. CD4+ T helper cells can release IFN-γ to activate antigen-presenting cells, resulting in the upregulation of molecules, such as MHC-I, which contribute to increased antigen presentation to CTL.[44] Several studies show that CD4+ T cells are essential, not only for the generation of CD8+ T cell responses, but also for the persistence of CTL.[45, 46] Although the exact mechanism has yet to be elucidated, these results offer inspiration to find a more effective method for cancer immunotherapy.

In the development of cancer vaccines, we must pay attention to safety concerns. Although hTERT is abundantly present in most human tumor cells, it is also expressed in some normal cells and tissues, such as stem-cell precursors of the bone marrow, spermatogonia in the testis, activated lymphocytes and basal keratinocytes. In this study, we used a 51Cr-release assay to investigate the ability of effectors to lyse autologous lymphocytes and DC and found that hTERT-derived MAP were unable to lyse autologous lymphocytes and DC in vitro or ex vivo. Robert et al.[47] also find that induction of tumor-lytic hTERT-specific T cells in vivo by vaccination does not result in a detectable decline in hematopoietic potential despite the expression of hTERT and major histocompatibility complex class I in bone marrow progenitors and stem cells. Tumor immunity does not involve autoimmunity in normal tissues that share the target.

In conclusion, we demonstrated that DC pulsed with MAP based on HLA-A0201-restricted CTL epitopes could trigger stronger antitumor immune response than their corresponding traditional linear peptides in vitro and ex vivo. This vaccine format, either as a single agent or in combination, offers great promise for cancer treatments.

Acknowledgment

This work was supported by grants from the National Nature Science Foundation of China (81071845) and the Chongqing Science Fund for Distinguished Young Scholars (CSTC, 2009BA5045).

Disclosure Statement

The authors have no conflicts of interest to declare.

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