PMC:3724992 / 2434-11711 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3724992","sourcedb":"PMC","sourceid":"3724992","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3724992","text":"Materials and methods\n\nCell lines and animals\nThe human TAP-deficient T2 cell line, BB7.2 cell line producing mAb against HLA-A2, human glioma cell line U251 and U87 (HLA-A2+), human breast cancer cell line MCF-7 (HLA-A2+) were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI-1640 medium containing 10 % FBS (Gibco, with endotoxin level ≤ 10 EU/ml), penicillin (200 U/ml), and streptomycin (100 μg/ml). All cell lines mentioned previously were kept at 37 °C in a humidified atmosphere containing 5 % CO2. HLA-A*0201/Kb transgenic (Tg) mice, 8–12 weeks-old, were purchased from The Jackson Laboratory (USA). Mice were bred and maintained in specific pathogen-free (SPF) facilities. Animal experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of Third Military Medical University.\n\nPeptide synthesis\nIn the present study, two programs BIMAS (http://www-bimas.cit.nih.gov/molbio/hla_bind/) and SYFPEITH (http://www.syfpeithi.de/Scripts/MHCServer.dll/EpitopePrediction.htm) were used to identify the candidate HLA-A2 restricted CTL epitopes from the neuritin antigen. The candidate peptides validated by epitope prediction were then synthesized by Fmoc chemistry (Sangon, China), and purified by HPLC to a purity of \u003e95 %. Lyophilized peptides were dissolved in DMSO at a concentration of 20 mg/ml and stored at −70 °C. The control peptide HBcAg (18–27) (FLPSDFFPSV) was synthesized and purified using the same methodology.\n\nPeptide-binding assay\nTo determine whether the candidate epitopes can bind to HLA-A*0201 molecules, up-regulation of peptide-induced HLA-A*0201 molecules on T2 cells was examined. Briefly, according to previous reference, 1 × 106 T2 cells were incubated with 50 μM of the synthesized peptides in serum-free RPMI 1640 medium supplemented with β2-microglobulin (Sigma) at a concentration of 3 μg/ml for 16 h at 37 °C, 5 % CO2 [18]. Expression of HLA-A*0201 on T2 cells was then determined with the FACS Calibur flow cytometer (Becton–Dickinson, USA), by staining with primary anti-HLA-A2 Ab derived from BB7.2 and FITC-labeled goat-antimouse IgG (BD Biosciences Pharmingen, USA) secondary antibody. The data were analyzed using Cell Quest software (Becton–Dickinson, USA). 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). Samples were measured in triplicate and then mean FI was calculated. An octapeptide mHpa (519–526) (FSYGFFVI) derived from mouse Hpa was served negative control.\n\nMeasurement of the peptide/HLA-A*0201 complex stability\nBriefly, T2 cells (106/ml) were incubated overnight with the candidate peptides, respectively, at a concentration of 20 μg/ml in serum-free medium supplemented with β2-microglobulin at a concentration of 3 μg/ml at 37 °C.\nThereafter, they were washed four times to remove free peptides, incubated with Brefeldin A (10 lg/ml) for 1 h to block cell surface expression of newly synthesized HLA-A2.1 molecules, washed and incubated at 37 °C for 0, 2, 4, 6, or 8 h. Subsequently, cells were stained with anti-HLA-A2 antibody from BB7.2 cells to evaluate the HLA-A2.1 molecule expression. For each time point, peptide induced HLA-A*0201 expression was evaluated by the formula mentioned above. Dissociation complex50 (DC50) was defined as the time required for the loss of 50 % of the HLA-A*0201/peptide complexes stabilized at time = 0.\n\nRT-PCR analysis of neuritin expression\nRT-PCR was used to analyze the expression of neuritin mRNA in cell lines. Total RNA was isolated from tumor cell lines using Tripure Isolation Regent Kit (Progema). Synthesis of cDNA was performed with 2 μg of total RNA with the aid of a reverse transcriptase Kit (Progema) and oligo(dT) primers. Two microliters RT product was amplified with PCR by using TaqDNA polymerase (Sangon, Shanghai) using standard procedures. The forward and reverse primer sequences were as follows: neuritin: sense primer, 5′-GTG CGA TGC AGT CTT TAA GTT-3′; anti-sense primer, 5′-GGG CTT TTC AGA CTG TTT GTT-3′; GAPDH: sense primer, 5′-GCA CCG TCA AGG CTG AGA AC-3′; antisense primer, 5′-ATG GTG GTG AAG ACG CCA GT-3′. 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 then run on a gel and visualized with ethidium bromide.\n\nWestern blot analysis of neuritin expression\nFor Western blot analysis, proteins in the cell extracts were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) through an 8 % polyacrylamide gel and were then transferred onto a nitrocellulose membrane. The membrane was incubated with 5 % non-fat milk in PBS and later with anti-neuritin MAb for 2 h at room temperature. After washing, the membranes were incubated with an alkaline phosphatase-conjugated goat anti-mouse IgG antibody (Amersham Biosciences, Buckinghamshire, England) for 1 h at room temperature. Immunoreactive bands were detected using the ECL Western blot analysis system (Amersham Biosciences, Buckinghamshire, England).\n\nDendritic cell generation from human peripheral blood precursors\nPBMCs were isolated from healthy HLA-A2+ donors by Ficoll–Hypaque density gradient centrifugation (TBC company, Tianjing, China) and then seeded into culture flasks in RPMI-1640 medium supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 % FBS. After monocytes adhered (incubation for 2 h), the nonadherent cells were collected and frozen in freeze medium (60 % RPMI-1640 and 30 % FBS, 10 % DMSO) for later use in CTL assays. The adherent cells were cultured for 5 days in RPMI-1640 containing 1,000 U/ml of granulocyte–macrophage colony-stimulating factor (R\u0026D Systems, Inc., Minneapolis, MN) and interleukin-4 (IL-4; R\u0026D Systems, Inc.) and were the cultured for an additional 2 days in the presence of 1,000 U/ml of tumor necrosis factor α (R\u0026D Systems, Inc.) to induce final maturation. After 7 days of culture, the mature DCs were harvested and analyzed for DC typical phenotypes by FACS analysis.\n\nInduction of peptide-specific CTL with synthetic peptides\nBriefly, DCs were loaded with different peptides at a final concentration of 100 μg/ml for 4 h and were then irradiated with 20 Gy, which prevented all outgrowths in the control cultures. Autologous T cells were restimulated every 7 days with the previously mentioned peptide-pulsed DCs to generate peptide-specific CTLs. Recombinant interleukin 2 (IL-2) at a concentration of 20 U/ml was added to the culture medium on day 3 after every stimulation. Cytotoxic T lymphocyte activity was then assessed on day 23 by a 4 h 51Cr release assay. Effectors generated from negative peptide-pulsed DCs were used as controls.\n\nELISPOT assay\nIFN-γ secretion of effectors was assayed by enzyme-linked immunospot (ELISPOT). Multiscreen 96-well assay plates (Dakewe, Shenzhen, China) were precoated overnight at 4 °C with anti-IFN-γ antibody according to the manufacturer’s instruction. After washing with PBST (PBS-0.05 % Tween 20), plates were blocked for 1 h at 37 °C with PBS/1 % BSA. Cytotoxic T lymphocyte effectors from human HLA-A2+ donors were plated in triplicate wells at a density of 1 × 105/100 μl in RPMI-1640 medium. Plates were cultured overnight, washed extensively with PBST, and incubated with anti-IFN-γ mAb for 1 h at 37 °C. After washing, goat anti-biotin antibodies (Dakewe) were added, and the plates were incubated for 1 h at 37 °C. Thirty microliters of activator solution (Dakewe) was added to develop spots, and after 10–30 min, the plates were washed with distilled water to stop the reaction. After being air-dried, the number of spots in each well was counted using the Bioreader 4000 PRO-X (Bio-Sys; Germany).\n\nCytotoxicity assay\nTo evaluate the levels of CTL activity, a standard 4-h 51Cr release assay was used. Briefly, target cells were incubated with 51Cr (100 μCi per 1 × 106 cells) for 2 h in a 37 °C water bath. After incubation with 51Cr, target cells were washed three times with PBS, resuspended in RPMI-1640 medium, and mixed with effector cells at a 25:1, 50:1 or 100:1 of effector to target (E/T) ratio. Assays were performed in triplicate for each sample at each ratio in a 96-well round-bottomed plate. After a 4-h incubation, the supernatants were harvested, and the amount of released 51Cr was measured with a gamma counter. The percent specific lysis was calculated according to the following formula:\nSpecific lysis = (experimental release − spontaneous release)/(maximal release − spontaneous release) × 100 %\n\nAnalysis of in vivo immunogenicity\nHLA-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.\n\nStatistical analysis\nResults were expressed as mean ± SEM. Analysis of Student’s t test were performed to determine effects of the treatments. 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