Abstract
Identification of cytotoxic T lymphocyte (CTL) epitopes from tumor related antigens is a promising approach for malignant tumor immunotherapy. TC2N, a recently identified tumor associated antigen from human glioblastoma, is regarded as a promising target of tumor-specific immunotherapy. As one of the most widely used histocompatibility molecules in Chinese is HLA-A*0201, we were able to identify the TC2N peptides that are provided by this molecular type. A panel of antigenic peptides produced from TC2N were predicted by using a computer tool. The binding affinities of three peptides with the highest predicted score to the HLA-A*0201 molecule were evaluated after synthesis. In vitro and in vivo stimulation of the main T-cell response against the predicted peptides. The results demonstrated that TC2N (152-160) was able to release IFN-γ and lyse U251 cells in vitro as well as in vivo by eliciting peptide-specific CTLs. Our results indicated that peptide TC2N (152-160) (RLYGSVCDL) was a novel HLA-A2.1-restricted CTL epitope capable of inducing TC2N specific CTLs in vitro. As TC2N might qualify as a viable target for immunotherapeutic approaches for patients with GBM, we speculated that the newly identified epitope RLYGSVCDL would be of potential use in peptide-based, cancer-specific immunotherapy against GBM.
Introduction
Tumor-associated antigens (TAAs), selectively or preferentially expressed by tumors, can be regarded as anti-tumor vaccination [1–3]. TAA vaccination induced cancer eradication has been reported in several animal models [4–6]. Vaccination with TAAs related epitopes to elicit stimulate specific T-cells is a promising strategy in clinical trials [7–9]. Numerous antigenic peptides derived from TAAs have been expressed in the context of MHC molecules and recognized by cells in the available T-cell repertoire [10].
Gene encoding tandem C2 domains nuclear protein (TC2N) has recently been shown to function both as an oncogene and a tumor suppressor gene [11]. TC2N is located on human chromosome 14q32, belongs to the carboxyl-terminal type (C-type) tandem C2 protein family, and contains two C-terminal C2 domains (C2A and C2B) [12]. Given its tumorigenesis properties and its association with the immune system, it has been proposed as a potential target for the detection and treatment of various cancers [13]. In vitro and in vivo data demonstrated that TC2N interference could remarkably prevent glioma cell proliferation and tumor growth. High TC2N expression is significantly correlated with poor overall survival of glioma patients via enhancing tumor growth [14]. Therefore, TC2N may qualify as a viable target for immunotherapeutic approaches for patients with GBM [15]. HLA-A*0201 is one of the most common HLA allele in Asian populations, especially in the Chinese, and has about 98% expression in the HLA-A2+ Caucasians in North America [16]. Therefore, TC2N-derived HLA-A2.1-restricted CTL epitopes may be widely applied for specific immunotherapy against TC2N-positive tumor in clinic.
In this study, we identified three TC2N-derived peptides with high affinities to the HLA-A2.1 molecule by MHC peptide-binding assay and epitope prediction with computer algorithms. Then, the immunogenicity of these peptides was investigated with PBMCs from healthy donors in vitro and with HLA-A*0201 transgenic mice in vivo.
Materials and methods
Cell lines and animals
The American Type Culture Collection (ATCC, USA) provided the Human TAP-deficient T2 cell line, the BB7.2 cell line that produced mAb against HLA-A*0201, and the U251 cell line. All of the cell lines were maintained in RPMI 1640 with 10% FCS and 100 μg mL−1 of penicillin and streptomycin. Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FCS, 4 μg L−1 glucose, and 100 μg mL−1 penicillin/streptomycin was used to maintain the BB7.2 cell line. Every cell line was maintained in a humidified environment with 5% CO2 in the air at 37 °C. Anti-HLA-A2 antibody was produced using BB7.2 (106 T2 cells/100 μL hybridoma culture supernatant). We bought the anti-CD8 antibody from BD Biosciences Pharmingen in the United States. Transgenic (Tg) HLA-A*0201/Kb mice, aged 8–12 weeks, were acquired from The Jackson Laboratory (USA). Mice were raised and housed in facilities designated as pathogen-free (SPF). Animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Chongqing Medical University.
Epitope prediction
To find the potential HLA-A2 restricted CTL epitopes from the TC2N antigen, the programs SYFPEITH (http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm) was utilized in the current investigation. After the candidate peptides were confirmed by epitope prediction, Fmoc Chemistry (Sangon, China) synthesized and refined the material to a purity of >95% using HPLC. The lyophilized peptides were kept at −70 °C after being dissolved in DMSO at a concentration of 20 mg mL−1.
Peptide-binding assay
The ability of the potential epitopes to bind to HLA-A*0201 molecules was investigated by detecting the up-regulation of peptide-induced HLA-A*0201 molecules on T2 cells. In summary, 1 × 106 T2 cells were cultured for 16 h at 37 °C and 5% CO2 with 50 μM of the generated peptides in serum-free RPMI 1640 media supplemented with β2- microglobulin (Sigma) at a concentration of 3 μg mL−1. Using the FACS Calibur flow cytometer (Becton Dickinson, USA), the expression of HLA-A*0201 on T2 cells was then assessed by labeling the cells with primary anti-HLA-A2 Ab obtained from BB7.2 and secondary antibody labeled with FITC (goat anti-mouse IgG, BD Biosciences Pharmingen, USA). Becton Dickinson, USA's Cell Quest software was used to evaluate the data. The Fluorescence index (FI) was calculated as follows: FI = (mean FITC fluorescence with the given peptide – mean FITC fluorescence without peptide)/(mean FITC fluorescence without peptide). After measuring the samples three times, the mean FI was determined. Positive and negative controls were HLA-A2.1-restricted MAGE-2 CTL epitope KMVELVHFL (amino acid position in MAGE-2; 112-120) and Kb-restricted Hpa CTL epitope FSYGFFVI (amino acid position in Hpa; 519-526).
Analysis of TC2N expression with RT-PCR
In U251 cells, TC2N expression was examined using RT-PCR. Tumor cell lines' total RNA was extracted using the Progema Tripure Isolation Regent kit. Using oligo (dT)18 primers and a reverse transcriptase kit (Progema), 2 μg of total RNA were used to synthesize cDNA.Using TaqDNA polymerase (Sangon, Shanghai), 2 μL of the RT product was amplified using PCR according to conventional protocols. Oligonucleotide sequences of the primer sets used were as follows: TC2N (forward: 5′TGGCTGTACTGAGGATTATTTGC-3′, reverse: 5′- TGTGAAGGAGTTTCTTGTGTCC-3′); GAPDH (forward: 5′-ACAACTTTGGTATCGTGGAAGG-3′, reverse: 5′-GCCATCACGC C ACAGTTTC-3′). Commercial synthesis of both primers was carried out at Sangon Company in Shanghai, China. Thirty amplification cycles were run: 1 min at 94 °C; 1 min at 60 °C; and 1 min at 72 °C. Cycling was ceased with a final extension of 10 min at 72 °C. RT-PCR products were visualized with ethidium bromide.
Western blot analysis of TC2N expression
Proteins inside the cell extracts were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) via an 8% polyacrylamide gel, and subsequently transferred onto a nitrocellulose membrane for western blot analysis. The membrane was first incubated for 2 h at room temperature with anti-TC2N mAb, and then for 5% nonfat milk in PBS. Following washing, the membranes were incubated for 1 h at room temperature with a goat anti-mouse IgG antibody conjugated with alkaline phosphatase (Amersham Biosciences, Buckinghamshire, England). The ECL Western blot analysis system (Amersham Biosciences, Buckinghamshire, England) was used to detect immunoreactive bands.
Induction of peptide-specific CTL with synthetic TC2N peptides
Healthy donors who tested positive for HLA-A2.1 were used to collected PBMCs. These cells were then grown in RPMI 1640 supplemented with 10% FCS, 100 units/mL penicillin, and 100 μg mL−1 streptomycin. The synthetic peptides were pulsed into these cells at a final concentration of 10 μg mL−1. Every 7 d, the PBMCs were stimulated again with new media containing these peptides. On day 3 following each stimulation, recombinant interleukin 2 was added to the culture media at a concentration of 20 units/mL. On day 23, the CTL activity was evaluated.
ELISPOT assay
ELISPOT assay was performed using a commercially available kit (DIACLONE, France). Stimulator cells were T2 cells pulsed with the indicated concentration of synthetic peptides. Effector cells (1 × 105) and peptide-pulsed T2 cells (1 × 104) were seeded onto 96-well microplates supported by polyvinylidene Xuoride (PVDF) and coated with an IFN-γ specific monoclonal antibody. Cells were taken out of the plate and treatedin accordance with the manufacturer's instructions after 16 h of incubation at 37 °C. The ELISPOT reader, an automated image analysis device, was used to count T-cells secreting IFN-γ.
Cytotoxicity assay
U251 cells serving as target cells (1 × 106) were labeled with Na51CrO4 in 1 mL of RPMI 1640 supplemented with 10% FCS for 1 h at 37 °C in 5% CO2. After three rounds of washing, the 51Cr-labeled cells were plated in triplicate in 96-well V-bottom microtiter plates at a final concentration of 1 × 104cells/well. The 51Cr-labeled target cells were then exposed to different quantities of effector cells. The volume of each well was added to 200 μL, and it was incubated for 4 h at 37 °C with 5% CO2. To quantify the release of the 51Cr label, 100 μL of the supernatant was collected and then quantified using an automated gamma counter. The proportion of particular 51Cr release was used to calculate the percentage of specific cytotoxicity. The percentage of specific lysate was calculated as 100 × (experimental release – spontaneous release)/(maximum release – spontaneous release). Maximum release was determined from supernatants of cells that were lysed by the addition of 2% Triton X-100.
Analysis of in vivo immunogenicity
HLA-A*0201/Kb mice were immunized with 100 μg of different peptides produced in incomplete Freund's adjuvant (IFA) for three times once a week. Mice were injected with an IFA emulsion devoid of peptide as a control. After 3 d of last vaccination, autologous splenocytes (APCs) loaded with 10 μg mL−1 of the immunizing peptide were cultured with splenocytes from the injected animals in order to increase the CTLs in vitro with mIL-2. The stimulated cells were introduced to the targets as effector cells after 5 d.
Preparation of Trimera mice
The Institute of Animal of Beijing Medical University (Beijing, China) provided male severe combination immunodeficiency (NOD/SCID) and nonobese diabetes (BALB/c) (H-2d) mice at age of 6–10 weeks old. The mice were housed in pathogen-free animal facilities with regulated humidity and temperature, a 12-h light/dark cycle, and cyprofloxacin (20 g mL−1) in their food and drink during the trials. Prior to the studies, each animal was given at least one week to become acclimated. The care and usage of the animals was done in compliance with the Dutch Committee of Animal Experiments' regulations. Lethal doses of total body irradiation (day 0 at 3.5 Gy and day 3 at 9.5 Gy) were administered to recipient BALB/c mice. On days 4–6, 3 × 106 mixed bone marrow cells (in 0.2 mL PBS) from NOD/SCID mice were transferred into each irradiated recipient by i.v. injection. One day after bone marrow infusion, each recipient mouse was. injected (i.p.) with 2 × 108 human PBMCs (HLA-A2). All mice were kept under specific pathogen-free conditions, fed with sterile food and acid water containing cyprofloxacin (20 μ g mL−1).
Tumor challenge experiments
The Animal Protection Act criteria were followed in the approval of all animal protocols. Trimera mice were subcutaneously challenged (n = 10 per group) (s.c.) injection of 1 × 106 U251 cells to create a primary tumor model in the left flank. After a period of 10 days, 100 μg of different peptides produced in incomplete Freund's adjuvant (IFA) were injected subcutaneously (s.c.) into the base of the tail of Trimera mice. The volume of PBS given to control mice was the same. The mean lifetime and tumor volume of mice were measured. Two dimensions were used to measure the tumor volume, which was then computed as length/2 × width2.
Statistical analysis
Results were from three independent experiments and each sample for each experiment was set with triplicate wells. The statistical significance of difference between each group was assessed with a one-way ANOVA followed by the Dunnett's post hoc test by using SPSS 10.0. A difference was considered as significant at the significance level of P < 0.05.
Results
Prediction of putative CTL epitopes restricted with HLA- A*0201
To predict the putative HLA-A*0201-restricted CTL epitopes of TC2N, SYFPEITHI program was used to scan the complete amino acid sequence of this antigen. Three highest-scored 9-amino-acid peptides were chosen as candidates for further assess (Table 1). These peptides were chemically synthesized, purified, and identified. The molecular weight of each peptide determined by mass spectrometry assay was similar to its theoretical molecular weight, and the purities of these peptides were all >95% (data not shown).
Predicted TC2N epitopes binding to HLA-A2.1
| Position | Length | Sequence | SYFPEITHI score |
| 152-160 | 9 | RLYGSVCDL | 27 |
| 347-355 | 9 | KISVCHAEL | 23 |
| 385-393 | 9 | LTLSFFVKV | 23 |
MHC peptide-binding
The binding affinity and stability of these peptides to HLA-A2.1 were determined by using antigen processing-deficient T2 cells because their enhanced HLA-A2.1. As shown in Table 2, all of the peptides synthesized were bound to HLA-A2.1 molecules with various affinity. Of three selected peptides, TC2N (152-160) up-regulated the HLA-A2.1 molecular expression and showed high affinity to HLA-A2.1, whereas TC2N (347-355) and TC2N (385-393) showed low affinity to the molecule. The results suggested that TC2N (152-160) peptide could bind to HLA-A2.1 and might be a promising candidate for epitope prediction.
HLA-A2-binding affinity of peptides
| Name | Position | Length | Sequence | FI |
| TC2N1 | 152-160 | 9 | RLYGSVCDL | 1.73 |
| TC2N2 | 347-355 | 9 | KISVCHAEL | 0.62 |
| TC2N3 | 385-393 | 9 | LTLSFFVKV | 0.59 |
| MAGE-2 | 112-120 | 9 | KMVELVHFL | 1.78 |
| Hpa 519-526 | 519-526 | 8 | FSYGFFVI | 0.39 |
Expression of TC2N in target cells
The expression of TC2N mRNA and protein of cell lines was analyzed by RT-PCR and Western blot. As shown in Fig. 1, TC2N mRNA and protein were detected in U251 cells. However, TC2N mRNA and protein could not be detected in BV2 cells and autologous lymphocytes. The results suggested that TC2N expressed in target U251 cells, but not in other cells.
Expression of TC2N in cell lines by RT-PCR and Western blot.
The tumor cells were homogenized, and total RNA was isolated using Tripure Isolation Regent Kit. PCR amplification was performed with specific oligonucleotides and PCR products were demonstrated through electrophoresis on 1% agarose gel with ethidium bromide staining. For Western blot analysis, proteins in the cell extracts were separated by SDS-PAGE and were then analyzed with anti-TC2N mAb. 1: U251 cells; 2: BV2 cells; 3: autologous lymphocytes
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2024.00002
Enzyme-linked immunospot (ELISPOT) assay
Peptide-specific T cells were monitored by measuring IFN-γ-producing cells with ELISPOT assay. As shown in Fig. 2, TC2N (152-160) peptides elicited a robust peptide-specific T cell response by virtue of their ability to generate increased frequencies of IFN-γ-producing T cells. These data suggested that TC2N peptide could generate IFN-γ secretion CTLs.
ELISPOT assay.
Effectors were induced from the PBMCs of four HLA-A2.1 healthy donors through three sequential rounds of stimulation with every peptide at the concentration of 10 μg mL−1 once a week. The IFN-γ secretion was then assessed on day 23. Experiments performed in triplicate showed consistent results. Data were represented as means ± SD. Compared with controls, *P < 0.05
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2024.00002
Induction of CTLs efficiently in vitro
PBMCs from HLA-A2.1+ donors were stimulated with synthetic peptides for CTL induction. Of these peptides, TC2N (152-160) peptide were able to elicit TC2N-specific CTLs with high efficiency, which could lyse target cells expressing TC2N and HLA-A2.1 (Fig. 3). These data suggested that TC2N peptide could generate CTLs efficiently in vitro.
Specific lysis of TC2N-derived peptide elicited CTLs.
HLA-A2.1 healthy donors' peripheral blood monocysteine-derived macrophages (PBMCs) were stimulated by peptide at a concentration of 10 μg mL−1 once a week for three times. On day 23, 4-h 51Cr-release assays were performed to test their cytotoxic activities against target cells. The effector-to-target (E/T) ratios are 25:1, 50:1, or 100:1. The percentage of cytotoxicity was calculated as follows: percentage of lysis= (sample cpm- spontaneous cpm)/(maximum cpm- spontaneous cpm) × 100%. Experiments performed in triplicate showed consistent results. Data were represented as means ± SD. Compared with control, *P < 0.05 versus the
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2024.00002
Inhibition of the recognition of effectors
To identify whether the peptides induced effectors recognized target cells in an HLA-A2-restricted manner and whether the effectors were CD8+ T lymphocytes, the mAbs against HLA-A2 and CD8 mAbs were used to inhibit recognition by effectors. In addition, HLA-B0702 mAbs and CD4 mAbs were served as negative controls. The results indicated that the anti-HLA-A2 antibody and anti-CD8 antibody could significantly inhibit the cytotoxicity of the effectors. However, negative controls could not attenuate the cytotoxicity of the effectors (Fig. 4). These data suggested that the induced effectors lysed target cells in an HLA-A2-restricted manner, and the induced effectors were mainly from CD8+ T lymphocytes.
Antibody inhibition assay by anti-HLA-A2 or anti-CD8 antibody.
Peptide-coated T2 target cells were incubated with or without anti-HLA-A2 antibody from BB7.2 cells for 1 h at 4 °C. Moreover, effectors induced by different TC2N-derived peptides were also incubated with or without anti-CD8 antibody for 1 h at 4 °C. The cytotoxic activities of CTLs against T2 cells were analyzed at various E/T ratios by 51 Cr release assay. Experiments performed in triplicate showed consistent results. Data were represented as means ± SD. Compared with control, *P < 0.05
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2024.00002
In vivo induction of epitope-specific CTLs in vivo
We explored whether peptides could generate immunity in vivo. HLA-A*0201/Kb mice were immunized with 100 μg of various peptides prepared in incomplete Freund's adjuvant (IFA). The cytolytic assay showed that CTLs from TC2N (152-160) immunized mice could lyse TC2N and HLA-A2.1 positive cells with high efficiency (Fig. 5). These results suggested that the peptides could also achieve higher immunogenicity in vivo.
Generation of epitope-specific CTLs in vivo.
HLA-A*0201/Kb mice were immunized with 100 μg of various peptides prepared in incomplete Freund's adjuvant (IFA) and boosted once a week for three times. As a control, mice were injected with an IFA emulsion without peptide. 7 days after immunization, splenocytes from injected animals were cultured and used as effector cells. The cytotoxic activities of CTLs were determined against target cells at various E/T ratios using 51Cr release assay. Experiments performed in triplicate showed consistent results. Data were represented as means ± SD. Compared with controls, *P < 0.05
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2024.00002
Epitope immunization inhibited tumor growth
To test whether epitope immunization had antitumor function in vivo, the tumor volume was observed. Trimera mice (n = 10 per group) were immunized subcutaneously (s.c.) at the base of the tail with 100 μg of peptide prepared in incomplete Freund's adjuvant (IFA). This immunization was performed three times, with an interval of 1 week. As shown in Fig. 6, the tumor volume expanded rapidly 15 days after tumor challenge in the control groups. However, the tumor volume expanded steadily compare with control groups in TC2N (152-160) epitope immunization group. These data demonstrated that TC2N (152-160) epitope immunization could notably inhibit tumor growth.
Epitope immunization inhibited tumor growth.
Trimera mice (n = 10) were given subcutaneous challenges. (s.c.) injection of 1 × 106 U251 cells to create a primary tumor model in the left flank. After 10 days, Trimera mice (n = 10 per group) were immunized subcutaneously (s.c.) at the base of the tail with 100 μg of peptide prepared in incomplete Freund's adjuvant (IFA). This immunization was performed three times, with an interval of 1 week. The tumor volume was observed. Three repeated experiments showed consistent results. Data were represented as means ± SD. Compared with controls, *P < 0.05
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2024.00002
Epitope immunization improved the lifespan of tumor bearing mice
To test whether epitope immunization had antitumor function in vivo, the lifespan of the mice was observed. Trimera mice (n = 10 per group) were immunized subcutaneously (s.c.) at the base of the tail with 100 μg of peptide prepared in incomplete Freund's adjuvant (IFA). This immunization was performed three times, with an interval of 1 week. As shown in Fig. 7, death occurred only 20 days after tumor challenge in TC2N (152-160) epitope immunization. However, all of the control mice treated with PBS died within 30 days. In addition, the mean lifespan of mice in the group with TC2N (152-160) epitope immunization was prolonged remarkably, with approximately half of the mice living beyond 25 days. These data demonstrated that TC2N (152-160) epitope immunization had an effective antitumor effect.
Epitope immunization inhibited tumor growth in mice.
Trimera mice (n = 10) were given subcutaneous challenges. (s.c.) injection of 1 × 106 U251 cells to create a primary tumor model in the left flank. After 10 days, 100 μg of peptide produced in incomplete Freund's adjuvant (IFA) was subcutaneously (s.c.) injected at the base of the tail of Trimera mice (n = 10 per group). This immunization was performed three times, with an interval of 1 week. The mean lifespan of the mice was observed. Three repeated experiments showed consistent results. Data were represented as means ± SD. Compared with controls, P < 0.05
Citation: European Journal of Microbiology and Immunology 14, 1; 10.1556/1886.2024.00002
Discussion
Glioblastoma patients have been treated with a variety of approaches inrecent years, including radiation therapy, chemotherapy, and surgical excision [17]. However, the expectancy of patient is short. Identification of tumor-associated antigens has shown to be a successful strategy in tumor treatment in recent years [18]. Peptide specific immunotherapy employing antigens specific to tumors is one tactic [19]. Many human tumor-associated antigens recognized by distinct CTLs have been identified by the integration of immunology and molecular biology approaches [20]. High-through screening could be efficiently accomplished by identifying CTL epitopes through the use of computer algorithms [21].
TC2N gene is located on chromosome 14a32.12 and encodes a C2 domain containing protein that belongs to the carboxyl terminal type (C-type) tandem C2 family of proteins [22]. Recently, TC2N was identified as a novel oncogene that accelerated tumorigenesis by suppression of p53 signaling in lung cancer [23]. Subsequently, the same group reported TC2N as a potent suppressor of PI3K-AKT signaling in breast cancer, suggesting its tumor suppressor activity in breast cancer [24]. These studies highlighted TC2N as a potential player in lung and breast cancers. Univariate and multivariate analyses identified TC2N as a novel independent prognostic factor of gliomas. The mRNA level of TC2N was significantly higher in glioma tissues, which correlated with poorer overall survival [25]. However, the immunotherapy strategy based on TC2N has not been explored.
An important step in the identification of T-cell epitope was the identification that ligands of a certain MHC-molecule containing chemically related amino acids in certain positions, which lead to the definition of a peptide motif for every MHC allele. The four steps of this method are as follows: (a) the amino acid sequence of a candidate antigen derived computer-based epitope prediction, (b) peptide-binding assay to determine the affinity of the predicted peptide with MHC molecule, (c) the generation of primary T-cell response against the predicted peptides in vitro, and (d) identification of the CTLs against target cells endogenously expressing the antigen [26–28].
In this study, we firstly predicted three candidate epitopes from TC2N antigen by using HLA-A2.1-restricted epitope prediction algorithms based on supermotif and quantitative motif methods. Secondly, we analyzed three predicted candidate epitopes with peptide-binding assay to determine the affinity of each epitope with HLA-A2.1. The results demonstrated that RLYGSVCDL had high affinity to HLA-A2.1, whereas KISVCHAEL and LTLSFFVKV had low affinity to HLA-A2.1 molecule. Thirdly, we used 51Cr-release assay to determine the peptide specific CTL response. These results demonstrated that the cytotoxic activity against targets could be inhibited by anti-HLA-A2.1 mAbs and anti-CD8 antibody. Lastly, RLYGSVCDL epitope immunization could notably inhibit tumor growth and improve the lifespan of tumor bearing mice. Thus, the finding indicated that peptide RLYGSVCDL was a novel HLA-A2.1-restricted CTL epitope capable of inducing TC2N -specific CTLs.
In conclusion, our results proved that TLDTLTAFY peptide derived from TC2N might be capable of inducing HLA-A2.1-restricted CD8+ CTL, which would be applied in glioblastoma expressing TC2N and HLA-A2.1.
Funding
The project was supported by Chongqing Science and Health Project (2023MSXM028 and 2023MSXM072).
Authors' contribution
Conceptualization: ZY; Methodology: HZ and XX; Manuscript Preparation: JZ.
Conflict of interest
None of the authors have any conflict of interest.
References
- 1.↑
Yang B, Jeang J, Yang A, Wu TC, Hung C-F. DNA vaccine for cancer immunotherapy. Hum Vaccin Immunother. 2014;10(11):3153–64.
- 2.
Banerjee K, Kumar S, Ross KA, Gautam S, Poelaert B, Nasser MW, et al. Emerging trends in the immunotherapy of pancreatic cancer. Cancer Lett. 2018 Mar 28;417:35–46.
- 3.
Pan RY, Chung WH, Chu MT, Chen SJ, Chen HC, Zheng L, et al. Recent development and clinical application of cancer vaccine: targeting neoantigens. J Immunol Res. 2018 Dec 19;2018:4325874.
- 4.↑
González FE, Gleisner A, Falcón-Beas F, Osorio F, López MN, Salazar-Onfray F. Tumor cell lysates as immunogenic sources for cancer vaccine design. Hum Vaccin Immunother. 2014;10(11):3261–9.
- 5.
Salmaninejad A, Zamani MR, Pourvahedi M, Golchehre Z, Hosseini Bereshneh A, Rezaei N. Cancer/testis antigens: expression, regulation, tumor invasion, and use in immunotherapy of cancers. Immunol Invest. 2016 Oct;45(7):619–40.
- 6.
Fiedler K, Lazzaro S, Lutz J, Rauch S, Heidenreich R. mRNA cancer vaccines. Recent Results Cancer Res. 2016;209:61–85.
- 7.↑
Ribas A, ButterWeld LH, Glaspy JA, Economou JS. Current developments in cancer vaccines and cellular immunotherapy. J Clin Oncol. 2003;21:2415–2432.
- 8.
Finn OJ. Cancer vaccines: between the idea and the reality. Nat Rev Immunol. 2003;3:630–641.
- 9.
Mine T, Gouhara R, Hida N, Imai N, Azuma K, Rikimaru T, et al. Immunological evaluation of CTL precursor-oriented vaccines for advanced lung cancer patients. Cancer Sci. 2003;94:548–556.
- 10.↑
Novellino L, Castelli C, Parmiani G. A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother. 2005;54:187–207.
- 11.↑
Pastan I, Hassan R. Discovery of TC2N and exploiting it as a target for immunotherapy. Cancer Res. 2014;74:2907–2912.
- 12.↑
Johnson MD, Vito F, O'Connell MJ. TC2N expression in the leptomeninges and meningiomas. The J Histochem Cytochem. 2008;56:579–585.
- 14.↑
Liu Z, Rao M, Poiret T, Nava S, Meng Q, von Landenberg A, et al. TC2N as a novel biomarker and immunotherapeutic target in human glioblastoma. Oncotarget. 2017;8(46):80208–80222.
- 15.↑
Illei PB, Alewine C, Zahurak M, Cowan ML, Montgomery E, Hassan R, et al. TC2N expression in advanced gastroesophageal cancer represents a novel target for immunotherapy. Appl Immunohistochem Mol Morphol. 2016;24:246–252.
- 16.↑
Shieh DC, Lin DT, Yang BS, Kuan HL, Kao KJ. High frequency of HLA-A*0207 subtype in Chinese population. Transfusion. 1996;36:818–821.
- 17.↑
Eisenbach L, Bar-Haim E, El-Shami K. Antitumor vaccination using peptide based vaccines. Immunol Lett. 2000;74:27–34.
- 18.↑
Coulie PG, Brichard V, Van Pel A, Wolfel T, Schneider J, Traversari C, et al. A new gene coding for a differentiation antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med. 1994;180:35–42.
- 19.↑
Cheever MA, Disis ML, Bernhard H, Gralow JR, Hand SL, Huseby ES, et al. Immunity to oncogenic proteins. Immunol Rev. 1995;145:33–59.
- 20.↑
Parmiani G, Castelli C, Dalerba P, Mortarini R, Rivoltini L, Marincola FM, et al. Cancer immunotherapy with peptide-based vaccines: what have we achieved? Where are we going? J Natl Cancer Inst. 2002;94:805–818.
- 21.↑
Brinkman JA, Fausch SC, Weber JS, Kast WM. Peptide-based vaccines for cancer immunotherapy. Expert Opin Biol Ther. 2004;4:181–198.
- 22.↑
Hao XL, Han F, Zhang N, Chen HQ, Jiang X, Yin L, et al. TC2N, a novel oncogene, accelerates tumor progression by suppressing p53 signaling pathway in lung cancer. Cell Death Differ. 2019 Jul;26(7):1235–1250.
- 23.↑
Hao XL, Han F, Zhang N, Chen HQ, Jiang X, Yin L, et al. TC2N, a novel oncogene, accelerates tumor progression by suppressing p53 signaling pathway in lung cancer. Cell Death Differ. 2019 Jul;26(7):1235–1250.
- 24.↑
Hao XL, Gao LY, Deng XJ, Han F, Chen HQ, Jiang X, et al. Identification of TC2N as a novel promising suppressor of PI3K-AKT signaling in breast cancer. Cell Death Dis. 2019 May 29;10(6):424.
- 25.↑
Dou Y, Xu H, Wu X, Liu P. Tac2-N promotes glioma proliferation and indicates poor clinical outcomes. Tohoku J Exp Med. 2021 Nov;255(3):247–256.
- 26.↑
Tang Y, Lin Z, Ni B, Wei J, Han J, Wang H, et al. An altered peptide ligand for naive cytotoxic T lymphocyte epitope of TRP-2(180-188) enhanced immunogenicity. Cancer Immunol Immunother. 2007;56:319–329.
- 27.
Zhu B, Chen Z, Cheng X, Lin Z, Guo J, Jia Z, et al. Identification of HLA-A*0201-restricted cytotoxic T lymphocyte epitope from TRAG-3 antigen. Clin Cancer Res. 2003;9:1850–1857.
- 28.
Chen A, Wang L, Zhang J, Zou L, Jia Z, Zhou W, et al. H-2 Kd-restricted hepatitis B virus-derived epitope whose specific CD8+ T lymphocytes can produce gamma interferon without cytotoxicity. J Virol. 2005;79:5568–5576.
