Cytoplasmic transduction peptide (CTP)-fused recombinant protein synthesis
CTP-fused recombinant human Wilm’s tumor gene 1 (CTP-rhWT1) and CTP-fused recombinant human survivin (CTP-rhBIRC5) were synthesized at JW CreaGene (Seongnam, Korea). The purity of each protein was confirmed to be >95% by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Synthetic protein was dissolved in dimethyl sulfoxide (DMSO) according to the manufacturer’s recommendations and stored at -70°C until use.
Target cells and western blotting
Surgical specimens from glioblastoma patients were obtained for research purposes following approval by the Chonnam National University Hwasun Hospital Ethics Panel. Samples were gathered by the Neurosurgery Department and kept at -800C until used. The U87 human glioblastoma cell line (Gibco-BRL, Gaithersburg, MD, USA) was obtained for cell culture. These cells were routinely grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) at 37°C in a humidified atmosphere containing 95% air and 5% CO2.
The bicinchoninic acid (BCA) assay kit (Thermo Scientific, USA) was used to measure protein concentration. Then, SDS- PAGE was used to separate the protein of interest which was transferred onto a polyvinylidene difluoride (PVDF) membrane and soaked in a blocking solution (5% non-fat dry milk in TBST (tris-buffered saline, Tween 20) for 1 h. The membrane was then probed with the primary antibodies for Wilm’s tumor gene 1 (WT1; Abcam, Cambridge, United Kingdom), survivin (BIRC5; Santa Cruz Biotechnology, CA, USA), programmed cell death ligand-1 (PD-L1; Santa Cruz Biotechnology, CA, USA), and β-actin (Santa Cruz Biotechnology, CA, USA) at 4°C overnight, and then incubated with horseradish peroxidase-conjugated a goat anti-rabbit or anti-mouse polyclonal IgG secondary antibodies (Ab frontier, Korea). Chemiluminescent detection was performed using immobilon western chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA). The β-actin was used as an internal control. The expression levels of WT1, BIRC5 and PD-L1 were determined with Amersham Imager 600 (GE Healthcare).
Dendritic cell maturation and CTP-fused recombinant protein pulsed DCs
To confirm effect of CTP-rhWT1 and CTP-rhBIRC5 in combination with anti-PD1 in vitro, dendritic cells were used for checking the function of CTP-fused recombinant protein in stimulating cytotoxic T cells through pulsing CTP-fused recombinant protein with DCs. Human CD14+ monocytes, obtained from the peripheral blood of healthy human donors, were used for the experiment. Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS was used for cell culture with the addition of granulocyte-macrophage colony stimulating factor (GM-CSF; 50 ng/mL) and IL-4 (20 ng/mL). The immature DCs were differentiated from CD14+ monocytes after 6 days. Then, the immature DCs were differentiated into mature DCs (VaxDCs) by using a cocktail of cytokines such IFN-α (3,000 IU/mL), IFN-γ (10 ng/mL) and poly (I:C) (20 µg/mL), LPS (1 µg/ mL) and were loaded with CTP-rhWT1 and CTP-rhBIRC5 (5 µg/ml) after 2hrs [9, 14]. On day 8, mature DCs were harvested and cryopreserved in liquid nitrogen until used.
Immunophenotyping and polarization cytokine production of DCs
The VaxDCs characteristics were evaluated by immunophenotyping. The expression of activating markers on the dendritic cells was compared using flow cytometry analysis at three stages: immature dendritic cells, dendritic cells after 48 hours maturation with and without CTP-fused recombinant protein (CTP-rhWT1 and CTP-rhBIRC5). Immature DCs were used as a negative control. At day 8, cells were obtained and stained for maturation markers, antigen presenting receptor and co-stimulatory molecules. Mainly, cell staining was performed using fluorescein isothiocyanate (FITC) or phycoerythrin (PE) conjugated monoclonal antibodies against CD40, CD80, CD83, CD86, CCR7, MHC I, and MHC II. All antibodies were purchased from eBioscience (San Diego, CA, USA).
The supernatant collected from the co-culture of DCs and CD40L-transfected J558 cells was subjected to ELISA. To evaluate DCs function after pulsing with CTP-rhWT1 and CTP-rhBIRC5, the cytokine levels for polarized helper T cells such as IL-12p40 and IL-10 were estimated. These cytokines were measured using ELISA kits following the manufacturer’s protocols (BD Biosciences) .
Cytotoxic T lymphocytes (CTLs) generation
The cytotoxic T lymphocytes (CTLs) were generated as previously described with several modifications . In general, the magnetic activated cell sorting system (MACs) was used to separate the CD3 lymphocyte populations. Then, these cells were stimulated by CTP-rhWT1and CTP-rhBIRC5 pulsed VaxDCs with or without anti-PD1, respectively. On day 3, IL-2 (5 ng/mL) and IL-7 (10 ng/mL) cytokines were added. After that, the CTLs were harvested and re-stimulated with CTP-rhWT1and CTP-rhBIRC5 pulsed VaxDCs at second times (day 15) and third times (day 22). Anti-PD1 (BioXcell, USA) was added during each stimulation. Two or three days from the last re-stimulation, an Enzyme-Linked ImmunoSpot (ELISPOT) assay and lactate dehydrogenase (LDH) release cytotoxicity assay were performed. The anti-PD1 was used for blockade in both DCs-CTLs interaction and CTLs-target cancer interaction.
Enzyme-Linked ImmunoSpot (ELISPOT)
Cytotoxic T lymphocyte activity was examined by measuring the secreted IFN-ɣ cytokine using an IFN-ɣ ELISPOT assay kit (BD Biosciences). The ELISPOT assay was performed as previously described with several modifications . Practically, ninety-six-well microplates were coated with the capture-purified anti-human IFN-ɣ antibody overnight at 40C. Then, RPMI medium supplemented with FBS was added to saturate the treated antibody. CTLs stimulated by VaxDCs pulsed with CTP-rhWT1and CTP-rhBIRC5 were co-cultured with the target cells (U87 glioblastoma cell line and primary glioblastoma cells) at a ratio 1:10 with or without Anti-PD1. Co-cultured cells were added to triplicate wells in 10% FBS-RPMI medium and incubated 24 hours at 370C in 5%CO2. Then, cells were incubated for 2 hours with the biotinylated detection anti–human IFN-ɣ antibody and 1 hour with the streptavidin-HRP. After washing, spots were revealed by using an AEC substrate reagent set (BD Bioscience) and measured with an automatic CTL Immunospot Analyzer (Cellular Technology Ltd., USA).
LDH release cytotoxicity assay
For in vitro experiment, the CytoTox 96 nonradioactive cytotoxicity assay (CytoTox 96, Promega, USA) was performed to estimate the cytotoxic activity of CTLs according to the manufacturer’s instructions. CTLs stimulated with CTP-rhWT1and CTP-rhBIRC5 pulsed VaxDCs acted as the effector cells. The U87 cell line and primary cells (5×103 cells/well) were used as the target cells. The W6/32 monoclonal antibody (1 μg/mL) (mAb; a gift from Dr. Bin Gao, ICH, London, United Kingdom) was used to block MHC-A, B, C antigen presentation on the target cells. The stimulated CTLs were co-cultured with the target cells at a ratio 1:10 in the 96-well uncoated plates (Costar, USA) for 4h in 37°C and 5% CO2. Anti-PD1 (10 µg/mL) was added during co-culture. Then, supernatants were collected for lactate dehydrogenase concentration determination. The mean percentage of specific lysis was calculated as following: % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] × 100
All statistical analyses were performed with SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was used to across multiple groups. P < 0.05 was considered statistically significant.