Methods
Ethics statement
This study was approved by the Ethics Committee of China Medical University (Shenyang, China; Approval No. [2016]054). All healthy volunteers and patients who provided clinical specimens signed the written informed consent form. The need for written informed consent was waived by the Ethics Committee for patients who provided clinical data, pathological tissue sections, and follow-up data due to the retrospective nature of the study. All procedures were performed in studies involving human participants were in accordance with 1964 Declaration of Helsinki principles and its later amendments or comparable ethical standards.
Collection and synthesis of CTL epitopes
The HLA-A2.1-restricted CTL epitopes from the amino acid sequence of ECM1 (Table
1) were predicted using Immunological Epitope Database (IEDB) (
http://www.iedb.org). All peptides, including LA (ECM1
9-17, LVLTYLAVA) and YL (ECM1
43-51, YAAPPSPPL), were synthesized by Chinese Peptide Co., Ltd. (Shanghai, China) with a purity of > 95% as determined by high-performance liquid chromatography (HPLC) (Additional file
1: Fig. S1a). The molecular weight of the peptide product was verified by mass spectrometry (Additional file
1: Fig. S1b). Besides, the endotoxin content of peptides was below 0.07 EU/mg obtained with the Limulus amebocyte lysate assay (photometric method) (Additional file
1: Fig. S1c).
Table 1
Identification of potential HLA-A2.1-restricted CTL epitopes from ECM1
NP | 168 | 176 | 9 | PPGRPSPDN | 46,882.04 | − 11.09 |
KA | 90 | 98 | 9 | KLLPAQLPA | 15 | − 36.26 |
YV | 488 | 496 | 9 | YLSPGDEQV | 18.15 | − 34.22 |
IV | 409 | 417 | 9 | ILTIDISRV | 102.40 | − 38.65 |
AV | 8 | 16 | 9 | ALVLTYLAV | 174.81 | − 39.46 |
YL | 43 | 51 | 9 | YAAPPSPPL | 267.8 | − 40.34 |
LA | 9 | 17 | 9 | LVLTYLAVA | 334.48 | − 42.45 |
QP | 86 | 94 | 9 | LQQEKLLPA | 392.06 | − 41.10 |
Cell lines
Human non-triple-negative breast cancer cell line MCF-7 and human melanoma cell lines (SK-MEL-28 and A375) were cultured in the Dulbecco’s modified Eagle’s medium (DMEM; 12,800,017, Gibco, Gaithersburg, MD, USA) containing 10% foetal bovine serum (FBS; FND500, ExCell Bio, Inc., Shanghai, China); human triple-negative breast cancer cell line BT549 and human lung cancer cell line H2228 were cultivated in the Roswell Park Memorial Institute-1640 (RPMI-1640) medium (31,800,022, Gibco) with 10% FBS; human hepatocellular carcinoma cell line Hep G2 was cultured in the minimum essential medium (MEM) (12,500,062, Gibco), containing 10% FBS; human mammary epithelial cell line MCF-10A was cultivated in the MCF-10A complete medium (SCSP-575, The Cell Bank of Type Culture Collection, Chinese Academy of Sciences, Shanghai, China); T2 cells were cultured in the Iscove’s modified Dulbecco’s medium (IMDM) (12,440,053, Gibco) with 10% FBS. All the above-mentioned cells were cultured at 37 °C in presence of 5% CO
2. Human triple-negative breast cancer cell line MDA-MB-231 and human colorectal cancer cell line SW480 were cultivated in L15 medium (41,300,039, Gibco) containing 10% FBS at 37 °C in air. All the cells were harvested in logarithmic growth phase. The sequencing-based typing (SBT) method was used for identification of HLA-A alleles at genomic level by Beijing Bo Furui Gene Diagnosis Technology Co., Ltd. (Beijing, China) (Additional file
1: Table S1).
Animals
Mice were bred and housed under specific pathogen-free conditions at China Medical University. HLA-A2.1 transgenic mice (8-week-old) were purchased from Jackson Laboratory (003475, Bar Harbor, ME, USA); Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice (5-week-old, no. 406) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were carried out in accordance with the guidelines published by the Institutional Animal Care and Use Committee of China Medical University.
Induction of epitope/DC-CTL
CD8+ T-cells were purified (> 95%) from a cell suspension harvested from non-adherent peripheral blood mononuclear cells (PBMCs), using a CD8a+ T Cell Isolation Kit (130–045-201, Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. CD8+ T cells were cultured in the ImmunoCult™-XF T Cell Expansion Medium (10,981, STEMCELL Technologies Inc., Vancouver, Canada) with ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (10,970, STEMCELL Technologies Inc.). DCs were resuspended in serum-free AIM-V medium (Gibco) and were stimulated with 50 μg/ml peptide twice with an interval of 24 h, treated with 30 ug/mL mitomycin C (A4452, APExBIO, Houston, TX, USA) for 30 min and washed. CD8+ T cells were stimulated with the above-described DCs at a ratio of 20: 1 on day 1 and day 6 to generate epitope/DC-CTL.
Cytotoxicity assay of epitope/DC-CTL
Target cells were labelled with 25 uM calcein-AM (C326, DOJINDO, Kumamoto, Japan) for 25 min. The effector cells (epitope/DC-CTL) and target cells were cocultured at different ratios (E: T = 5:1, 10:1, 20:1, 40:1) for 4 h. The supernatant was detected by multimode reader (Tristar, Berlin, Germany) with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The assay was performed in triplicate. The percentage of specific lysis was calculated as follows: (OD of experimental release—OD of spontaneous release)/(OD of maximum release—OD of spontaneous release) × 100% (OD, optical density). The target cells of the spontaneous release group were cultured in the medium alone, and the target cells of the maximal release group were coincubated with 2% Triton X-100.
Induction of epitope/DC-NK
NK cells were screened from PBMCs (DC-) by the Human NK Cell Isolation Kit (130–092-657, Miltenyi Biotec), and unlabelled cells were collected. NK cells were cultured in NK medium (DKW34-SCN1, Dakewe Biotech Co., Ltd., Shenzhen, China) and mixed with epitope-pulsed DCs at a ratio of 20:1 on day 1 and day 6 to generate epitope/DC-NK cells.
Cytotoxicity assay of epitope/DC-NK
Epitope/DC-NK-mediated lysis of tumour cells was analysed using a lactate dehydrogenase (LDH) release cytotoxicity assay kit (Beyotime Institute of Biotechnology, Shanghai, China) at different ratios (E: T = 5:1, 10:1, 20:1, 40:1) according to the manufacturer's instructions. LDH release values were normalized to spontaneous LDH release by detecting effector cells in the same culture medium. The cytotoxicity was calculated as follows: Cytotoxicity (%) = [(OD of experimental release – OD of spontaneous release of effector cells – OD of spontaneous release of target cells)/(OD of maximum release of target cells– OD of spontaneous release of target cells)] × 100.
In vivo study of immune effect
LA- or YL-ISA (Montanide ISA 51, SEPPIC, Paris, France) containing LA or YL was inoculated into inguinal region of HLA-A2.1 transgenic mice for three times (day -13, day -7, day -1). Natural saline (NS), LA, YL, and ISA were used as control groups. Six days following the 3rd injection (day 5), DC and splenocyte phenotypes were detected.
In vivo study of inhibitory effects on the growth of the transplanted tumours
NOD/SCID mice received a subcutaneous injection of 2.5 × 106 MDA-MB-231 cells, and then, they received 2 × 107 splenocytes of HLA-A2.1 transgenic mice intravenously. Splenocytes were injected three times, and the first immunization on HLA-A2.1 Tg mice started on day -13 and splenocytes were obtained on day 5. The second immunization started on day -10 and splenocytes were obtained on day 8, and the third immunization started on day -7 and splenocytes were obtained on day 11. Two batches of animals were included to assess the growing volume of xenograft tumours and the survival time. Paraffin-embedded sections of tumour tissues were prepared for immunohistochemistry (ICH). After staining with rabbit anti-CD8 alpha antibody (1:2000; ab217344, Abcam, Cambridge, UK) or NK1.1 monoclonal antibody (1:100; MA1-70,100, Carlsbad, CA, USA), the infiltration of CD8+ T and NK cells was detected. The tumours were measured in a blinded fashion.
In vivo study of inhibition of tumour metastasis
A metastasis model was established by tail vein injection of 2.5 × 106 MDA-MB-231-luc cells in NOD/SCID mice. Then, those mice intravenously received 2 × 107 splenocytes of HLA-A2.1 transgenic for three times. In vivo fluorescence imaging was conducted on day 24. Lung tumour burden was assessed at the end of the experiment (day 25) by an in vivo imaging system and the organs were fixed. Lung metastatic tumour nodules were visually identified and further confirmed by haematoxylin and eosin (H&E) staining. Paraffin-embedded sections of lung tissue were prepared for IHC and stained with rabbit anti-CD8-α antibody or NK1.1 monoclonal antibody. Two batches of animals were included to assess the metastasis and the survival time.
Safety review of epitopes
The bone marrow aspiration was prepared from marrow fluids obtained from HLA-A2.1 transgenic mice inoculated for 3 times and was then stained with Wright-Giemsa stain (BA4017, Baso Diagnostics Inc., Beijing, China). Liver, kidney, heart, lung, spleen, stomach, and intestine tissue sections were stained with H&E. Blood samples were tested in parallel by a haematology analyser. Liver function indexes and renal function indexes, such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid (UA), and creatinine (CRE), were measured by a biochemical analyser. The whole body and main organs were weighed to calculate the organ indexes.
HLA-2.1/ECM1-overexpressing transplanted tumour model
Female
HLA-A2.1 transgenic mice (6-week-old) received a subcutaneous injection of 1 × 10
6 ECM1
+/HLA-A
2+ E0771 into their right forelimbs. The mice were randomly assigned into the following groups: 1) control, 2) ISA, 3) YL-ISA, 4) LA-ISA, 5) ISA + TAK242, 6) YL-ISA + TAK242, and 7) LA-ISA + TAK242. TAK-242 (ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate; A3850, APExBio) is a novel small molecule that selectively inhibits Toll-like receptor 4 (TLR4) [
9]. Human TLR4 is homologous to mouse TLR4, and TAK-242 possesses similar inhibitory effects on mouse and human monocytes [
10]. TAK242 diluent in normal saline was injected into
HLA-A2.1 transgenic mice at a dose of 1 mg/kg 3 h before epitope-ISA vaccination. The dose of LA or YL was 1 mg/20 g body weight. ISA, LA-ISA or YL-ISA were inoculated into the inguinal region of
HLA-A2.1 transgenic mice in the corresponding groups on day 2, day 8, and day 14. Two batches of animals were included to evaluate the growing volume of transplanted tumours and the survival time. Paraffin-embedded sections of tumour tissue were prepared for IHC. The tumours were measured in a blinded fashion.
Statistical analysis
The SPSS 22.0 software (IBM, Armonk, NY, USA) was used to perform statistical analysis. ECM1 expression data, which were abnormally distributed, were compared between two groups using the Wilcoxon signed-ranks test. Categorical data were compared with the Pearson’s chi-square test or the Fisher’s exact test. Survival probabilities were estimated by the Kaplan–Meier method and assessed by the log-rank test. Other results were expressed as mean ± standard deviation (SD) and were compared with the independent samples t-tests. Probability values (P) < 0.05 was considered statistically significant.
See further methods in supplementary materials.
Discussion
Although the recent emergence of epitopes from mutated neoantigens [
15], mass spectrometry profiling of HLA-associated peptidomes [
16], and antigens associated with peptide processing [
17] have attracted extensive attention. These personalized epitope vaccines [
18] are likely more expensive than developing a conventional vaccine based on tumour-specific antigens or tumour-associated antigens (TAAs). Therefore, TAA-derived CTL epitope vaccines may foster the clinical development of antitumour vaccines. ECM1 is overexpressed in multiple tumours, enabling ECM1-based epitopes to be broadly applied in diverse types of cancer. Additionally, the high expression level of ECM1 in skin may cause a strong immunity and even induce Lichen sclerosis [
19,
20]; thus, ECM1, as a target, may elicit strong immune response rather than self-tolerance. Collectively, ECM1 is a more attractive immunotherapeutic target. We confirmed the feasibility of ECM1 as a universal TAA, identified the predominant HLA-A2.1-restricted epitopes derived from ECM1 (LA and YL), and verified their immunogenicity to induce DCs to elicit CD8
+ T cells and potent cytotoxicity against breast cancer in vitro and in vivo. Therefore, LA and YL showed promising clinical potential as candidate predominant CTL epitopes. Additionally, we found that LA could induce not only a strong CTL antitumour immunity, but also an NK cells’ immune response, which was different from previous studies. Both adaptive and innate immune responses of LA were enhanced and LA presented a stronger antitumour effect than YL. Meanwhile, LA/DC-NK-induced toxic killing ability could escape the restriction of the expression levels of MHC-I and ECM1. Tumour cells with the loss of MHC-I molecules could escape CTL-mediated cytotoxicity [
21], while could be susceptible to NK cell cytotoxicity [
22]. Hence, LA-induced NK activation could compensate for the deficiency of CTL cytotoxicity. Our previous study showed that the LT peptide could induce antitumour effect of NK cells [
23], rather than CTLs. The current study revealed that LA could induce the double-activation of CD8
+ T and NK cells, which was not reported in previous researches.
The study revealed that LA could cross CD8
+ T cells through MHC-I presentation pathway in DCs to induce CD8
+ T cells, which was consistent with the classical pathway of cross-presentation of exogenous peptides [
24]. Exogenous antigens are typically associated with immature DCs in vivo, and it is essential to promote DCs to tend to mature states for further cross-presentation [
25]. We found that LA-ISA could stimulate immature DCs towards maturation and exhibited a favourable DC immunogenicity.
LA could be taken up by phagocytosis- and receptor-mediated internalization into DCs by active transport, which was consistent with pathway of classical exogenous antigen uptake of DCs [
26]. The phagosome-to-cytosol pathway and vacuolar pathway are major mechanisms of cross-presenting antigens [
24]. The current study confirmed that vacuolar pathway contributed more to cross-priming to LA in DCs than phagosome-to-cytosol pathway. The evidence for this comes from the fact that LA could be internalized into phagosomes, secreted by vesicles, reached lysosomes, and transferred into the endoplasmic reticulum by TAP. However, these epitopes, which are involved in the phagosome-to-cytosol pathway, could not mediate DC cross-priming. In addition, the confocal microscopy and UPLC-QTOF-MS displayed LA in form of a LA/MHC-I complex presented on DC surface. Although LA could bind directly to the HLA-A2 molecule on DC surface, the possibility of directly binding to the HLA molecule on cell surface is limited [
27]. This may be because the majority of DCs are in an immature state with insufficient expression of MHC-I molecules in vivo [
28]; thus, the intracellular presentation pathway is the main approach for LA to induce immunity [
29‐
31]. The results further confirmed that the LA/MHC-I complex on DC surface could activate the TCR of CD8
+ T cells to upregulate the phosphorylation level of ZAP70, a key downstream signal of the CD3-ζ chain [
32]. Previous studies on cross-presentation of exogenous antigens by DCs mainly concentrated on microorganisms (such viruses), proteins, and long peptides [
33‐
35], rather than short peptides. The current study depicted the mechanistic routine of short peptides represented by LA via DC cross-presentation.
We, for the first time, identified that DCs were necessary for LA to induce the NK cells immune response in the process of exploring the mechanism by which LA activates NK cells. Previous studies have shown a cross-talk occurs between DCs and NK cells. A number of contact-dependent factors could promote DC-NK cross-talk, such as some ligands in DCs and receptors in NK cells [
28,
36]. The results demonstrated that MICA/B expression was significantly upregulated in DCs treated with LA. Because MICA/B could stimulate natural killer group 2 member D (NKG2D) on the NK cell surface to activate NK cells [
37], we speculated that MICA/B could be the key signal in the LA-mediated NK cell activation. LA-loaded DCs with MICA/B monoclonal antibody pretreatment could not activate NK cells, which further confirmed our hypothesis. Meanwhile, the p38 MAPK pathway could be activated by LA among MICA/B-regulating related pathways [
12‐
14]. LA-loaded DCs could not activate NK cells when p38 MAPK of DCs was blocked; thus, activation of p38 MAPK could be the mechanism by which LA upregulated MICA/B expression. Our previous study indicated that peptides could regulate he p38/MAPK pathway to increase MICA/B expression in hepatocellular carcinoma cells [
23], whereas the CTL epitope upregulating MICA/B expression on DCs to activate NK cells has not been reported.
To clarify the mechanism of p38 MAPK pathway activation by LA, we further analysed RNA-seq data and found that the molecular functions of immune-related differentially expressed genes included signalling pattern recognition receptor activity, peptide antigen binding, etc. These results suggested that TLRs, playing critical roles as pattern recognition signal receptors in the innate immune response on DC surface [
38], might be a direct target for LA to induce NK activation through DCs. RNA-seq-based transcriptome analysis also demonstrated that the expression levels of TLR4 were downregulated most among the TLRs and the downstream signals of TLR4 (
MYD88 and
TRAF) were moderately upregulated in DCs with LA treatment. It also showed the most stable binding between TLR4 and LA by MOE. In addition, TAK-242 could significantly inhibit the upregulation of Rael (a ligand for mouse NKG2D with a very similar structure to MICA/B) expression in DCs, the increased frequencies of NK cells, and the improved antitumour effects in HLA-A2.1
+/ECM1
+ mouse breast cancer cell-bearing mice treated with LA. Therefore, we speculated that LA could be used as a TLR4 agonist to initiate TLR4 intracellular signals to further activate the p38 MAPK pathway [
39‐
41]. YL did not affect NK activation, possibly because the fact that TLR4 with a deep hydrophobic pocket is more likely to bind hydrophobic molecules, such as LPS. It is extremely difficult for YL with a hydrophilic property to bind to TLR4 relative to LA with a hydrophobic property (Additional file
1: Fig. S6d, e) to initiate intracellular signals for NK activation. The increased expressions of MYD88, TRAF6, MICA/B, and p38 phosphorylation level, and the elevated cytotoxicity of NK cells were attenuated even disappeared DCs were pretreated with TAK242, an inhibitor of TLR4, and pulsed with LA. Previous studies demonstrated that the MHC-I/epitope complex could directly activate NK cells via the KIR3DS1 receptor [
42]. To further investigate the role of KIR3DS1 signals on NK cell activation, we used an anti-KIR3DS1 antibody to block the KIR3DS1 receptor on NK cells. The results showed that LA-loaded DCs could still activate NK cells (Additional file
1: Fig. S10), indicating that the HLA-A2.1/LA complex might not activate NK cells via the KIR3D receptor. Therefore, LA-activated NK cells through mediating the TLR4-p38 MAPK pathway to upregulate the MICA/B expression on DC surface to further interact specifically with NKG2D on NK cell surface through ligand-receptor coupling for the DC-NK crosstalk.
CTL epitopes of low-molecular weight possess an inferior stability and a short half-life in vivo, making it difficult to induce robust immune responses [
43]. In addition, the strategy of utilizing cytokine-induced DCs to prepare CTL epitope-DC vaccines in vitro has a number of disadvantages, such as complicated operation and a long production period [
44]. In this study, we found that the use of peptide alone (LA or YL) did not produce significant antitumour effects (Additional file
1: Fig. S8), which may be associated with its rapid degradation in vivo (Fig.
5j). Some strategies have been used to overcome these disadvantages, including introduction of fatty acid ligands [
45], substitution of amino acid cleavage sites [
46], and sustained-release preparations [
47]. Besides, peptide mixed with adjuvants is a rational option to compensate for the immune response deficiency of peptide application alone [
48]. ISA is an ideal immune stimulatory adjuvant [
49]. We prepared formulations of ISA-51 [
50] and LA or YL, which could enhance the stability of epitopes in vivo, to immunize
HLA-A2.1 transgenic mice for the collection of splenic cells to further inoculate into tumour-bearing NOD/SCID mice for immunological reconstitution. LA-ISA/YL-ISA significantly inhibited the formation and proliferation of xenograft tumours and pulmonary metastases. Additionally, LA could spontaneously activate CD8
+ T and NK cells to infiltrate into tumour tissues to further exert stronger antitumour effects than YL, which was consistent with the in vitro results
. Moreover, we validated the antitumour effect on HLA-A2.1
+/ECM1
+ murine breast cancer cell-bearing mouse model. These results indicated that LA-ISA based on the double-activation of CD8
+ T and NK cells could elicit a more potent antitumour immune response than traditional tumour vaccines based on CTL epitopes and has a promising clinical application in the future.
Immune toxicity has become an essential consideration of immunotherapy [
51]. In the present research, we found that LA-induced specific CTLs had nonimmune toxicity to PBMCs. NK cells, as the main member of innate immune cells, could accurately distinguish between "self" and "nonself", thereby precisely killing tumour cells rather than normal autologous cells. Hence, LA-induced CTLs had nonimmune toxicity to normal cells, such as PBMCs and DCs. Although studies reported that the in vivo application of ISA-51 could cause allergic reactions [
52,
53], the assay on
HLA-A2.1 transgenic mice immunized with LA-ISA and immunologically reconstituted NOD/SCID mice injected with splenocytes showed a promising safety and no immune toxicity in the normal tissues or organs.
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