Enhanced anti-tumor effects of dendritic cells against glioblastoma by using cytoplasmic transduction peptide (CTP)-fused recombinant protein combined with anti-PD1 CURRENT STATUS: POSTED

Background Recent clinical trials utilizing antigen-pulsed dendritic cells have demonstrated increased survival of the vaccinated cancer patients. Besides, the cytoplasmic transduction peptide has not only excellent transcellular efficiency but also a strong tendency to remain in the cytoplasm after transduction without migrating into the nucleus. In this study, we investigated the effectiveness of immunotherapy against malignant gliomas using DCs pulsed with cytoplasmic transduction peptide (CTP)-fused recombinant protein combined with programmed cell death protein 1 blockade. Methods The expression of tumor associated antigen (WT1 and BIRC5) on glioblastoma target cells was confirmed by western blot. The effect of CTP-rhWT1 and/or CTP-rhBIRC5 on DCs was determined. The immunophenotypes of VaxDCs pulsed with CTP-rhWT1 and/or CTP-rhSBIRC5 was confirmed by flow cytometry and the cytokine production levels of T helper polarization were measured by enzyme-linked immunosorbent assay. The IFN-γ-enzyme linked immunospot assay and lactate dehydrogenase release assay were done to estimate the cytotoxic activity of CTLs stimulated by CTP-fused recombinant protein pulsed VaxDCs along with PD1 blockade against malignant glioma cells expressing WT1 and BIRC5. Results The CTP-rhWT1 and CTP-rhBIRC5 enhanced activating markers of DCs. Besides, the CTP-rhWT1 and CTP-rhBIRC5 combination resulted in Th1 cytokine polarization. The increase in number of IFN-γ-secreting cells paralleled with enhanced cytotoxicity of CTLs-stimulated by CTP-fused recombinant protein pulsed VaxDCs against glioblastoma target cells. Conclusions Our study suggested that treatment of CTP-fused recombinant protein along with PD1 blockade, which enhances cytotoxicity of DCs, could be an effective immunotherapy strategy for glioblastoma. cell death β-actin 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 monoclonal CD80, CD83, CD86, CCR7, MHC I, and MHC II. All antibodies 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 manufacturer’s in the reduction of antigen-specific IFN-γ production Our data indicated a stable IL-10 level for CTP-rhWT1 combined CTP-rhBIRC5. Combining, our CTP-fused recombinant protein showed a potential differentiation of Th1 effectors.


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) [15].

Cytotoxic T lymphocytes (CTLs) generation
The cytotoxic T lymphocytes (CTLs) were generated as previously described with several modifications [16]. 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 CTLstarget cancer interaction.

Enzyme-Linked ImmunoSpot (ELISPOT)
Cytotoxic T lymphocyte activity was examined by measuring the secreted IFN-ɣ cytokine using an IFNɣ ELISPOT assay kit (BD Biosciences). The ELISPOT assay was performed as previously described with several modifications [17]. Practically, ninety-six-well microplates were coated with the capturepurified anti-human IFN-ɣ antibody overnight at 4 0 C. 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 37 0 C in 5%CO 2 . 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).

IFN-γ secretion of CTP-recombinant human protein-specific CTLs against human glioblastoma cells
The U87 glioblastoma cell line and primary glioblastoma cells were used as the target cells for Developing a new antigen delivery tool, based on cross-presentation mechanism, for exogenous antigens in DCs by using CTP has been investigated [9, 21]. The present data also indicated that CTPfused recombinant protein not only increased the percentage of antigen-specific CTLs but also enhanced the function of these CTLs through Th1 immune response.
The antigenic effect of the single tumor-associated antigens (BIRC5 and WT1) has been mentioned in previous studies. In a clinical trial, WT1 vaccination was shown to trigger WT1-specific CTLs that suppressed cancer without damaging the normal tissues [22]. WT1 was recommended as the most promising cancer antigen [23]. Similarly, the functions of BIRC5, such as regulation of apoptosis, cell division, chemo-resistance, and tumor progression have also been explored [24], and this antigen has also been investigated for malignant glioma in the clinical study [25]. Therefore, these two antigens were chosen for our study. In our study, the presentation of WT1 and BIRC5 on target cells (U87 cell

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