Patient material and cell lines
Patient tumor samples were obtained from biopsy specimens. A portion of each sample was subjected to formalin fixation and paraffin embedding, and the remaining tumor tissue was carefully manually dissociated in collagenase D (200 U ml-1) and DNase I (20 U ml-1) (Roche Life Sciences). The dissociated samples were suspended in the solution for a period of time, transferred to a tightly sealed plastic bag, incubated together, and periodically stirred for 30–60 minutes with a laboratory stirrer.
The study population included adult patients with a histologically or cytologically confirmed diagnosis of CRC from the Second Affiliated Hospital of Wenzhou Medical University between February 2017 and February 2019. All donors gave written informed consent for the collection of tissue specimens. This study involving human samples was approved by the Medical Ethics Committee of the Second Affiliated Hospital, Wenzhou Medical University (MEC numbers: LCKY2018-67), in line with the Helsinki Declaration, and always adhered to the privacy rights of human subjects. The procedure was performed in strict accordance with the protocol.
Cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Peripheral blood mononuclear cells (PBMCs) were the original material harvested for immunological monitoring or sample manufacturing; these cells were isolated by density gradient centrifugation using Ficoll-Hypaque (Amersham Biosciences) and collected from the buffy coat of peripheral blood samples from healthy donors or CRC patients, thereby producing immature dendritic cells (DCs). H522 cells were cultured under standard conditions described by the ATCC, and the cells were re-certified by short tandem repeat (STR) analysis at ATCC, while a cell bank for mycoplasma was generated.
Next-generation Sequencing (ngs)
For RNA sequencing (RNA-seq), RNeasy Mini Kit was utilized to extract RNA from fresh frozen tumor samples. The construction of RNA-seq libraries were performed using the TruSeq Stranded mRNA Library Prep kit (Illumina) (for cell suspensions). All libraries were sequenced paired-end 50 nt on an Illumina HiSeq 2500 sequencing platform by using two Illumina-based TruSeq SBS Kits v3-HS 50 cycles. Paired ends (100 nt) were sequenced using TruSeq SBS Kit v3-HS (Illumina) (200-cycles) to distribute the MZ-GaBa-018 cell line and its matched PBMC genome-wide sequencing library in 4 lanes.
For WES sequencing, DNA was extracted from the fresh-frozen tumor samples or cultured tumor cells using DNeasy Blood and Tissue Kit, purchased from Qiagen. Genomic DNA was cut, end-repaired, simultaneously connected to the bar-coded Illumina sequencing adapters, amplified, and size-selected. Whole-exome capture was performed using Agilent Sure Select Human All Exon 44-Mb version 2.0 bait set (Agilent Technologies), The resulting libraries were quantified by qPCR, and 76-base paired-end reads were pooled and sequenced using HiSeq 2000 or 2500 sequencers (Illumina).
Bioinformatics And Mutation Discovery
All mutations in a single patient were analysed using the Python programming language. DNA libraries required at least 150 × 106 paired-end 50 nt reads, while RNA libraries required at least 75 × 106 paired-end 50 nt reads. For mutation detection, DNA reads were consistent with the reference genomes hg19. Duplicate exomes from tumor and matched normal samples were analysed for single-nucleotide variants. Identification and screening of sites assumed to be homozygous genotypes in normal samples, thus preserving a high degree of trust in single-nucleotide variation. Further examination of the remaining sites was performed to determine the presumed homozygous or heterozygous mutation event. Screening suspicious sites to exclude potential false positives, and testing the sum of duplicates and duplicates respectively, and merging duplicates. We compared the genomic coordinates of the identified variants with the detailed known gene transcription coordinates in the UCSC, and further determined the relationship between the mutations and genes, transcription, expression values derived from RNA-seq, and the changes of potential amino acid sequences.
The HLA alleles of each patient were inferred from the WES data using OptiType(13) with the default settings after the reads were filtered by aligning to the HLA region using RazerS version 3.4.0(14).
Neoantigen Candidates Identification
Non-synonymous somatic mutations, including SNVs, insertion, deletion and frame shift, were used to predict neoantigens by MuPeXI(15), which is a pipeline for neoantigen identification. First, 8-11-amino-acid long mutant peptides were generated for MHC I-restricted neoantigen prediction, and 12-15-amino-acid long mutant peptides were generated for MHC II-restricted neoantigen prediction. Then HLA binding affinity for each peptide was calculated by NetMHCpan4.0(16) and NetMHCIIpan3.2(17) for MHC I and MHC II molecules respectively. Expression level of mutant genes was derived from RNA-seq data in transcripts per million (TPM). The number of mismatches between the mutant and normal peptides was considered as the similarity to self-peptides. Mutant allele frequency was detected by the variant caller MuTect2(18). Each peptide was given a priority score based on HLA binding affinity, expression level, similarity to self-peptides and mutant allele frequency. Peptides with priority score larger than 0 were selected as neoantigen candidates.
Synthesis Of Long Peptidesand Final Vaccine Preparation
The peptides with purity greater than 95% were analysed by mass spectrometry and were synthesized by GL Biochem (Shanghai, China) using fluorenylmethoxycarbonyl chemistry and purified by reversed-phase high-performance liquid chromatography. The lyophilized peptides were dissolved in dimethyl sulfoxide and diluted to a concentration of 10 mM in phosphate buffer brine (pH 7.4), and continued to be stored as isocyanates at − 80 °C.
The construction process for tandem minigenes (TMGs) has been described previously(19). In each TMG construction, 12 small genes were included. A TMG plasmid was constructed by linearization with the restriction enzyme NsiI, and in vitro RNA transcription was performed by using each of the encoded linearized plasmids as a template with the mMESSAGE mMACHINE T7 Transcription Kit (Thermo Fisher Scientific); 20 mg of RNA was purified for cell transfection. The remaining PBMC cells after cryopreservation of removal of DC/T cells in this section were thawed and cultured, and minigene RNA was introduced into the PBMCs using a nuclear transfection kit (Amaxa Cell Line Nucleofector Kit V, Lonza). After 24 hours of transfection, these cells were used as stimulatory cells, and the above procedure was performed strictly in accordance with the manufacturer's instructions.
Induction of neoantigen-reactive T cells by coculture with CRC patient-derived peripheral blood lymphocytes (PBLs) in vitro
Autologous PBMCs from a patient were used to assess the in vitro immunogenicity of candidate neoantigens. We used a simple and rapid culture method to detect and monitor circulating antigen peptide-specific cytotoxic T lymphocyte precursors (CTL-P), which was only slightly modified in previous reports(20, 21). Peripheral blood was obtained from patients that had been undergoing postoperative chemoradiotherapy for more than 1 month. PBMCs were isolated and cultured. The human PBMCs were suspended in complete medium (RPMI 1640 medium containing 10% foetal bovine serum) for collection. PBMC-derived DCs were harvested on the sixth day after cultivation, adjusted for cell concentration using a complete medium for human DCs (RPMI 1640 complete medium supplemented with 500 U/ml rhGM-CSF and 10 ng/ml th-4), Frozen T cells collected in RPMI 1640 medium were suspended in 10% foetal bovine serum and co-culture for 3 stimulations. The cells were collected 7 days after the last stimulation, and positive selection with CD3 immunomagnetic beads (Miltenyi Biotec) was performed. The T cells were selected in strict accordance with the instructions provided by the manufacturer. In vitro gene transcription using the mMESSAGE mMACHINE UItra Kit (Thermo Fisher) kit was performed. Minigene RNA was transferred into PBMCs by a nuclear transfection kit (Amaxa Cell Line Nucleofector Kit V, Lonza). On day 10, the specific response of T cells to each peptide was assessed by ELISPOT assays. Comparing the response induced by the peptide to that induced by medium alone (no peptide) or the unrelated peptide VSV-NP43 − 69 (STKVALNDLRAYVYQGIKSGNPSILHI), and phytohemagglutinin was used as the positive control. T cell reactivity was assessed by DCs peptide pulses co-cultured with T cells in some cases. Finally, T cell activity was detected by an IFN-y ELISPOT, an ELISA kit and intracellular flow cytometry staining.
Generation Of Neoantigen-specific T Cells In Hla-a2.1/k-transgenic Mice
All experimental procedures and animal protocols were approved by the AAALAC-accredited Animal Studies Committee of Wenzhou Medical University (AEC numbers:WYDW2019-0924) and were in compliance with all relevant ethical regulations. All procedures are designed in accordance with the ethical standards of the national research council to minimize the suffering and number of animals. All animals used in this study were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Environmental conditions were a temperature of 20 ± 2 °C, the humidity of 55% ±10%, the lighting of 350 lux (at bench level) and a 12/12 h light/dark cycle and All the mice were fed the standard rodent diet with free access to water. The health of the animals was monitored twice daily during the feeding period. No adverse events were observed. All parts of the report follow the ARRIVE Guidelines for animal studies(22).
For the in vivo efficacy evaluation studies, we used eight 6-8-week-old female HLA-A2.1/Kb-Tg mice (mean body weight: 20 ± 2 g), which were raised under standard conditions and cared for in accordance with the animal care facility guidelines(23). Tumor neoantigen peptides from patients with HLA-A0201 were selected and synthesized to vaccinate HLA-A2.1/Kb transgenic mice subcutaneously at 0,7 days. 50 mg poly (I: C) was mixed with 100 mg polypeptide each time. Seven days after the last immunization, the spleen of mice was extracted by aseptic operation, and the residual blood was rinsed with sterile saline, soaked in RPMI1640 medium, and the single-cell suspension was obtained by gently grinding the needle core of aseptic syringe on 400 mesh steel net, and filtered through steel net to remove large tissue blocks. After centrifuging 1000 × g of the filtered single-cell suspension for 5 minutes, the supernatant of the culture medium was discarded and suspended in the low permeable NH4Cl solution (0.15 M NH4Cl, 1 M KHCO3, 0.1 mM Na2EDTA, pH 7.2) for 3 minutes to destroy the red blood cells, and then centrifuged and washed twice with RPMI1640 culture medium. The spleen cells of the mice without red blood cells were suspended in RPMI1640 medium containing 10% fetal bovine serum, counted and placed in 6-well plates. Mouse spleen cells were cultured in vitro and stimulated again with corresponding peptide (20 µM). After 7 days, the effector cells were collected and analysed by ELISPOT, intracellular staining and specific killing activity in tumor-bearing mice.
An ELISPOT assay (R&D Systems, Minneapolis, MN) was performed using a commercial kit. T cells sorted by CD3 immunomagnetic beads were suspended in RPMI 1640 medium supplemented with 10% foetal bovine serum and used as reacting cells, and the cell concentration was adjusted to 5 × 106/ml, and the cell suspension was directly transferred to an ELISPOT assay plate (100 µl/well) coated with an anti-IFN-y antibody, PBMC (mutant type (Mut) minigenes), PBMC (wild-type (WT) minigenes) 15 Gy irradiation was used as a stimulating cell and was added to the detection well (100 ul/well). IFN-y-secreting T cell colony was detected by the method described in the IFN-y ELISPOT assay kit instructions. The frequency of nocturnal peptide (80) activation in cytokine-secreting cells was determined by an IFN-γ ELISPOT kit (Dakewei). In this research, multiple culture protocols were used to analyse T cell-mediated immune responses. Peptide-stimulated PBMCs were added to two wells for approximately 18 hours or DCs pulsed with a peptide were added and cocultured with the T cells. The plate was washed first, and then a diluted test antibody (1:100 dilution) was added and incubated at 37 °C for 1 hour. After washing the plate, streptavidin-HRP diluted to the above mentioned year-on-year dilution was added and incubated at the same temperature for 1 hour, and a 3-amino-9-ethylcarbazole (AEC) solution was prepared according to the manufacturers specifications, and added per well. The plate was allowed to incubate at room temperature for approximately 15–20 minutes in darkness, after which time development was stopped by the addition of deionized water. The plates were scanned with an ELISPOT CTL Reader (Cellular Technology Inc) and analysed by ElisPot software (AID), and spots two-times larger than those in the peptide-free (culture medium) control were considered to be T cell reactive positive.
Intracellular Detection Of Ifn-γ
The detection method used to evaluate intracellular IFN-γ production was performed as described above. Briefly, in RPMI 1640 medium supplemented with 10% foetal bovine serum, frozen T cells collected previously were suspended and adjusted to a cell concentration of 4 × 106/ml, and 0.5 ml was added to each of the above collected peptide-sensitized autologous DCs, and co-cultured at 37 °C, 5% carbon dioxide (T:DC ratio of 10:1), and the same stimulation was provided three times per week. Recombinant IL-2 (rhIL-2; 20 U/ml) was added every 3 days during the culture, the medium was replaced with fresh medium every 2–3 days, and the cells were expanded according to need. The cells were collected 7 days after the last stimulation, and CD3 immunomagnetic beads (Miltenyi Biotec) were used for positive selection of T cells. The sorted cells were subjected to intracellular cytokine staining analysis and fixed in 4% paraformaldehyde until flow-cytometry analysis (FACScan™ or FACSVantage™ SE; BD Biosciences). Flow cytometry data were analysed by CellQuest software (BD Biosciences).
In Vitro T Cell Cytotoxicity Assay
The cytotoxic activity of neoantigen-specific tumour-specific cytotoxic T cells (CTLs) can be tested by a Carboxy fluorescein Succinimidyl Ester (CFSE)/propidium iodide-labelling cytotoxicity assay. HLA-A0201+T2 cells loaded with a mutant polypeptide and HLA-A0201+SW480 cells transfected with the corresponding mutant polypeptide minigene were used as target cells. For CFSE labeling, target cells were labelled for 10 minutes in PBS containing 4 mM CFSE (Invitrogen) at 37 °C. Labelling was terminated by adding a 10x volume of PBS and washing thoroughly with PBS. Cells were seeded in 24-well plates, and the CFSE-labelled cells at different effector:target cell ratios (E:T ratios) were incubated with T cells for 6 hours. Then, propidium iodide (Sigma-Aldrich) was added directly to detect cell death rate, and the samples were analysed by flow cytometry.
Cytotoxicity assays were determined by CCK8 assay(24). The formula for determining percent specific lysis was as follows: cell cytotoxicity = (1 − (Ae − Ab)/(Ac − Ab)) × 100%, where Ae is the absorbance of the experimental group, Ac is the absorbance of the control group, and Ab is the absorbance of a blank well.
Adoptive Immunotherapy In Tumor-bearing Nude Mice
For the adoptive transfer model, we used 6-8-week-old female C57BL/6nu/nu mice (mean body weight: 20 ± 2 g). Splenocytes from vaccinated Tg mice in each group were stimulated with 20 µM HAVCR2-F39V, SEC11A-R11L and ULK1-S248L for 7 days. MAD-MB-468-hPEBP4 tumor cells (5 × 106) were injected into the C57BL/6nu/nu mice breast fat pads, forming 100% homogeneous tumors. Three days later, the mice were injected intravenously with splenocytes (1 × 108 cells per mouse) from vaccinated HLA-A2.1/Kb transgenic mice in each group that were stimulated with 20 µM HAVCR2-F39V, SEC11A-R11L and ULK1-S248L for 7 days as described in the section detailing the protocol used for the cytotoxicity test. This adoptive infusion was performed two times 1-week intervals, and intraperitoneal injection of 2000 U of hIL-2 every 2 days. Control mice accepted transgenic mice splenocytes by immunization with non-pulsed DCs or injected with only IL-2. The size of the tumor (two dimensional) was measured three times per week, and the HLA-A2.1/Kb mice were sacrificed on day 80 after tumor inoculation.
The two-tailed Student’s t-test was used to determine statistical significance of the differences between means. Tumor sizes were compared between groups using the Mann–Whitney U-test. Survival analyses were plotted using Kaplan-Meier curves. Values of P < 0.05 were considered as statistically significant. All statistical tests were performed using SPSS manager software (v21; IBM Inc.)